33rd Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2018)

Poster Presentations

Co-Stimulatory Ligand-Receptor Interaction

P397 Ectopic Tim-3 expression on T regulatory cells leads to lymphoproliferation and T cell activation

Hridesh Banerjee, Héctor Nieves-Rosado, Lawrence P. Kane, Ph.D.

University of Pittsburgh, Pittsburgh, PA, USA

Correspondence: Lawrence P. Kane (lkane@pitt.edu)

Background

T cell (or transmembrane) immunoglobulin and mucin domain 3 (Tim-3) is a transmembrane protein that has been associated with both inhibitory and co-stimulatory function in T cells. In tumor-infiltrating (TI) T cells and during chronic infection, Tim-3 has been seen to be expressed in terminally exhausted T cells and a significant proportion of regulatory T cells (Treg). However, what role Tim-3 plays in Treg is still unclear. Another factor complicating the role of Tim-3 is that along with Tim-3, other checkpoint receptors such as PD-1 are also upregulated in TI-Treg and very little is known about crosstalk between various checkpoint receptors in effector T cells and Treg.

Methods

To investigate the role of Tim-3 in Treg, we used two mouse models, a constitutive Tim-3/Treg model (Foxp3-YFP-Cre x flox-stop-flox Tim-3) and a tamoxifen-inducible Treg/Tim-3 model (Foxp3-CreERT2 x flox-stop-flox Tim- 3).Basic characterisation of the immune system specifically the lyymphoid compartment and T cells including Treg cells was carried out. Functional assays on T regulatory cells was also done to look at effect of TIM-3 expression on T reg cells.

Results

At ten weeks after Tim-3 induction, Tim-3 transgenic mice had larger spleens and lymph nodes. This phenotype was observed to be milder in younger mice. Lymphoid organs in constitutive Tim-3 transgenic mice showed systemic lymphoid hyperplasia. T cells in these mice displayed a more activated phenotype. Overall frequency, numbers and phenotype of Treg cells in the peripheral lymphoid organs were also altered in constitutive Tim-3 transgenic mice. In the inducible Tim-3 mice however, we do not find systemic lymphoid hyperplasia but changes in numbers and phenotype of Treg were consistent with constitutive Tim-3 transgenic mice. Ectopic Tim-3 expression on Treg was also associated with changes in Treg function both in vitro and in vivo.

Conclusions

TIM-3 is sufficient to change the basic regulatory function of T reg cells, thereby studying how checkpoint therapies effect T reg in tumormicroenvironment and chronic infection may lead us to better Understanding the role of Tim-3 in Treg, and could contribute to novel therapeutic approaches for diseases such as cancer and chronic infection.

P398 Activation of the T Cell costimulatory protein CD137 using multivalent bicyclic peptides

Kristen Hurov, Punit Upadhyaya, Jessica Kublin, Xueyuan Zhou, Julia Kristensson, Rachid Lani, Gemma Mudd, Katerine van Rietschoten, W. Frank An, Johanna Lahdenranta, Liuhong Chen, Gavin Bennett, Kevin McDonnell, Nicholas Keen, Peter U. Park, PhD

Bicycle Therapeutics, Lexington, MA, USA

Correspondence: Peter U. Park (peter.park@bicycletx.com)

Background

CD137 (4-1BB/TNFRSF9) is a costimulatory receptor belonging to the TNF receptor superfamily. It was originally cloned as an inducible gene from stimulated helper and cytotoxic T cells and has since been shown to also be expressed on natural killer (NK) cells. Agonistic anti-CD137 antibodies have shown potent, often curative anti-tumour activity in preclinical models. These effects are mainly mediated by cytotoxic T cells and generate long lasting, memory responses. Two human anti-CD137 antibodies, binding to the extracellular domain of CD137, urelumab and utomilumab are currently undergoing clinical testing. Urelumab has shown several single-agent, partial responses, but its use has been hampered by hepatoxicity, whilst utomilumab has shown little or no single agent activity.

Methods

Bicycles® are a new class of drugs - fully synthetic, constrained bicyclic peptides that combine the attributes of three therapeutic modalities (antibodies, small molecules, and peptides) by delivering high affinity, good PK, and rapid clearance. Their small size (1.5-2 kDa) delivers advantages in tumour penetration, and rapid renal elimination may avoid the liver and GI toxicity often associated with other drug modalities, including certain antibodies. We hypothesised that a fully synthetic Bicycle CD137 agonist with rapid renal clearance, minimal liver interaction and no Fc receptor interaction may induce CD137 mediated anti-tumour activity while avoiding liver toxicity. We screened for CD137 binders with a library of 10e12 Bicycles using phage display and following phage and chemical optimization, a high affinity lead BCY3814 (KD ~30 nM) was selected.

Results

BCY3814 binds to the human CD137 ligand-binding site. In common with many TNF receptors, CD137 activation requires receptor crosslinking, thus multivalent binders would be expected to recapitulate the action of its natural trimeric ligand. We generated more than 50 different bi-, tri- and tetra-valent variants of BCY3814 with chemical linkers and hinges of various lengths and rigidity using different sites of attachments, while maintaining a compact size (<15 kDa). We developed molecules exhibiting a wide range of potency in a cell-based CD137-dependent reporter assay. In addition, these molecules activate human T cells in vitro as monitored by increased cytokine release. Selected CD137 multimers are being tested in a humanized CD137 mouse model to demonstrate T cell activation and anti-tumour activity, without the liver toxicity reported for urelumab.

Conclusions

We hypothesise that such molecules could be promising, novel cancer immunotherapy candidates and importantly, they pave the way for development of synthetic agonists of other TNF receptors.

P399 Induction of tumor-specific immune responses and modulation of the tumor micro-environment by TLR9 agonist lefitolimod in murine syngeneic tumor models

Kerstin Kapp, PhD¹, Barbara Volz¹, Detlef Oswald¹, Burghardt Wittig, MD, PhD², Manuel Schmidt, MSc¹

¹Mologen AG, Berlin, Germany; ²Advisor to Mologen AG, Berlin, Germany

Background

Preclinical and ongoing clinical studies support the application of TLR9 agonists for immunotherapy. The immune surveillance reactivator (ISR) lefitolimod is in advanced clinical development for single-agent maintenance treatment in metastatic colorectal cancer (phase III, IMPALA) and extensive disease small cell lung cancer (phase II, IMPULSE). Lefitolimod activates plasmacytoid dendritic cells to secrete interferon-alpha, followed by a broad activation of cells of the innate and adaptive immune system. Lefitolimod therefore provides the necessary and sufficient signals for the initiation of an immunotherapeutic anti-tumor response.

Methods

It was evaluated, if lefitolimod is able to induce local and systemic anti-tumor immune responses in the murine syngeneic colon carcinoma CT26 and the breast cancer EMT-6 models. The presence and activation state of CD8+ T cells within tumor infiltrating cells was determined via flow cytometry. Tumor antigen-specific T cells were analyzed via IFN-gamma ELISpot using spleen cells stimulated with either tumor cells or the peptide AH1, derived from an immunodominant antigen of CT26 cells.

Results

Intratumoral administration of lefitolimod resulted in a beneficial modulation of the tumor micro-environment (TME) characterized by increased infiltration of activated CD8+ T cells, which showed an up-regulation of Granzyme B. Notably, an increase of IFN-gamma secreting CD8+ T cells within the spleen was detected after re- stimulation with the tumor-specific AH1 peptide antigen or CT26 tumor cells. This beneficial TME modulation and antigen-specific effects were associated with a markedly reduced tumor growth in the CT26 model. The anti-tumor effect was even more pronounced in the EMT-6 model, where nine out of ten mice showed complete tumor regression. The 9 tumor-free mice subsequently rejected both, the initially used EMT-6 as well as CT26 tumor cells in re-challenge studies, in contrast to age-matched naïve mice. This indicates that treatment with lefitolimod induces a sustained, long-lasting immune memory against shared antigens of both tumor types.

Conclusions

Treatment of tumors with lefitolimod resulted in a beneficial modulation of the TME with an increase in anti-tumor effector cells. A strong systemic immune response as well as a sustained immune memory against different tumors was induced. These data indicate that lefitolimod provides the essential requirements for use as mono- immunotherapy or as an optimal combination partner of other immunotherapeutic drugs like checkpoint inhibitors in immuno-oncological trials.

P400 Tumor-localizing NKp30/ICOSL vIgD fusion proteins direct effective dual CD28/ICOS T cell costimulation to B7-H6+ tumor cells in vitro and tumors in vivo

Steven Levin, PhD^2*, Lawrence Evans, BS², Erika Rickel², Katherine Lewis, PhD², Daniel Demonte², Martin Wolfson, BS², Stacey Dillon, PhD², Ryan Swanson, BS², Kristine Swiderek, PhD², Stanford Peng, MD, PhD²

¹Alpine Immune Sciences, Inc., Seattle, WA, USA; ²Alpine Immune Sciences, Seattle, WA, USA

Correspondence: Steven Levin (steve.levin@alpineimmunesciences.com)

Background

Background: Although checkpoint inhibitor therapies have significantly improved outcomes in multiple cancers, complete and durable responses remain infrequent, possibly attributable to a lack of adequate T cell costimulation and/or activating signals. Novel therapeutic proteins which confer T cell costimulation may be particularly effective anti-tumor therapies, particularly in combination with checkpoint inhibitors. But at the same time, localization of such costimulatory activity to tumors, such as via a tumor-specific targeting antigen, may be simultaneously important to maintain tolerability of such agonist therapeutics. B7-H6, a cell surface immunoglobulin superfamily (IgSF) member which binds the NKp30 receptor, appears to be expressed specifically in multiple tumor types, and may serve as such a tumor-specific antigen. Novel therapeutic proteins which localize costimulatory agonist domains to B7 H6 may therefore be capable of significant antitumor efficacy yet may be safely administered systemically by preferentially localizing agonist activity to the B7-H6 tumor microenvironment.

Methods

Methods: Using our platform technology, which is based on the directed evolution of IgSF members, NKp30/ICOSL variant immunoglobulin domain (vIgD) fusion proteins were created from NKp30 vIgDs with high affinity against B7-H6 and ICOSL vIgDs, which dually agonize the T cell costimulatory receptors ICOS and CD28. These tumor- localizing vIgD proteins were evaluated in vitro in T cell costimulation assays with target cells with or without B7- H6, and in vivo in a B7-H6+ CT26 mouse tumor model.

Results

Results: NKp30/ICOSL vIgD-Fc fusion proteins conferred effective B7-H6-dependent costimulation to T cells in vitro, with enhanced T cell proliferation and cytokine production (IFN-gamma, TNF-alpha, IL-2) in response to B7- H6-expressing but not B7-H6-negative target cells. Isolated ICOSL and NKp30 vIgDs alone were not efficacious. Importantly, NKp30/ICOSL vIgD-Fc fusion proteins demonstrated anti-tumor efficacy in a B7 H6+ mouse tumor model, especially when administered in combination with a PD-1 inhibitor (Figure 1).

Conclusions

Conclusions: Tumor-localizing NKp30/ICOSL vIgDs confer potent T cell costimulation via CD28 and ICOS dependent upon the tumor antigen B7-H6 and elicit encouraging efficacy against B7-H6+ tumors in vivo, including in combination with PD-1 inhibitors. Such fusion proteins may provide particularly effective therapeutics for B7- H6+ tumors either as monotherapy or in combination with checkpoint blockade. These findings further suggest tumor-localized immunomodulation is possible and may improve cancer outcomes.

Ethics Approval

All animal procedures were approved by the appropriate Institutional Animal Care and Use Committee overseeing the vivarium where the studies were conducted (Alpine Immune Sciences and Charles River Laboratories), and followed the guidelines set forth in the 8th Edition of the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

Fig. 1 (abstract P400).

See text for description

P401 Blockade of T cell immunoreceptor with Ig and ITIM domains (TIGIT) leads to increased proliferation of bone marrow T cells from patients with acute myeloid leukemia (AML)

Yoko Kosaka, PhD¹, Adam Lamble, MD², Fei Huang, PhD³, Evan Lind, PhD¹

¹Oregon Health & Science University, Portland, OR, USA; ²Seattle Children’s Hospital, Portland, OR, USA;³Janssen Pharmaceutical R&D, Spring House, PA, USA

Correspondence: Evan Lind (linde@ohsu.edu)

Background

Background: The success of immunotherapeutic checkpoint blockade in cancer has led to great interest in finding novel targets that play a pivotal role in immune responses. One such molecule is T cell immunoreceptor with Ig and ITIM domains (TIGIT), which has been shown to be inhibitory and expressed by nonresponsive and suppressive T cells in the tumor microenvironment.

Methods

Methods: In the present study, we investigate the role of TIGIT on immune suppression of T cell responses in bone marrow microenvironment of patients with AML. Bone marrow aspirates were subjected to T cell proliferation assays using stimulation though TCR with or without accompanying TIGIT blockade. Samples were also subjected to high parameter mass-spec based flow cytometry and both mutational and transcriptional profiling by deep sequencing and clinical parameters (age, sex, blast count, ELN risk stratification) were recorded.

Results

Results: Of 57 total samples tested, 24 (42.1%) showed a profound defect in T cell proliferation in response to anti-CD3 stimulation (<5% of T cells responding to stimulation). Of these 24 that showed the most functional impairment, 12 (50%) had at least a 2-fold and 6 (25%) at least a 5-fold increase in the frequency of dividing T cells with the addition of an anti-TIGIT blocking antibody.

Conclusions

Conclusions: These results indicate that in many samples, TIGIT blockade can partially overcome functional suppression of T cells in AML bone marrow, and suggest that TIGIT is involved in mediating immune defects in AML. A better understanding of the role of TIGIT in the AML microenvironment will provide insight in determining whether TIGIT blockade could represent an effective therapy in AML.

Acknowledgements

This work was also funded in part by generous support from the Leukemia and Lymphoma Society of America Beat AML project (PIs Brian Druker MD/Jeffrey Tyner PhD).

Ethics Approval

All human experiments are approved under IRB protocol #00004422 Marc Loriaux MD, PI.

P402 DuoBody-CD40x41BB conditionally enhances immune activation by crosslinking of CD40- and 4-1BB positive cells

Alexander Muik, PhD¹, Friederike Gieseke¹, Isil Altintas, PhD², Saskia Burm², Mustafa Diken², Christian Grunwitz², Sebastian Kreiter², David Satijn, PhD², Danita Schuurhuis², Ozlem Tureci², Ugur Sahin², Esther Breij, PhD²

¹BioNTech AG, Mainz, Germany; ²Genmab B.V., Utrecht, Netherlands

Correspondence: Alexander Muik (Alexander.Muik@biontech.de)

Background

Immune checkpoint inhibitor antibodies that can (re-)activate anti-tumor immunity show great promise for the treatment of cancer. Similarly, therapeutic agents that boost anti-tumor immunity by direct activation of immunostimulatory molecules may provide clinical benefit. In this context, targeting tumor necrosis factor (TNF) receptor superfamily members, which deliver essential co-stimulatory activity for immune responses, gained attention. We hypothesized that simultaneous engagement of the T-cell co-stimulatory molecule 4-1BB and CD40 on antigen-presenting cells (APCs) using a bispecific antibody could be an elegant and potent mechanism to induce conditional activation of both CD40-positive immune cells and 4-1BB positive T cells.

Methods

DuoBody-CD40x4-1BB (GEN1042) is an IgG1 Fc-silenced bispecific antibody that was obtained by controlled Fab- arm exchange of humanized parental CD40- and 4-1BB-specific monoclonal antibodies. The binding characteristics and functional activity of DuoBody-CD40x4-1BB were analyzed in vitro using flow cytometry, cell-based reporter assay systems and primary human lymphocyte assays. To evaluate the capacity of DuoBody-CD40x4-1BB to induce proliferation of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment, ex vivo TIL expansion assays were conducted using freshly isolated human tumor specimen.

Results

DuoBody-CD40x4-1BB induced activation of both CD40 and 4-1BB intracellular signaling, which was dependent on simultaneous binding to CD40- and 4-1BB positive cells as measured by reporter assays. The monospecific control antibodies did not show agonist activity. This demonstrates that the bispecific antibody confers conditional activity upon receptor cross-linking. Using human primary T cells and monocyte-derived dendritic cells, both obtained from human healthy donor PBMCs, antigen-specific T-cell assays were conducted in vitro. DuoBody- CD40x4-1BB enhanced T-cell proliferation as well as concomitant pro-inflammatory cytokine secretion. Enhanced T-cell proliferation was again dependent on binding of DuoBody-CD40x4-1BB to both CD40 and 4-1BB. Importantly, DuoBody-CD40x4-1BB did not enhance proliferation of T cells that were not pre-activated by TCR stimulation. Furthermore, in ex vivo cultures of fresh human tumor tissue resections DuoBody-CD40x4-1BB increased the expansion of TILs up to 10-fold over control antibody treatment.

Conclusions

In summary, DuoBody-CD40x4-1BB is a bispecific antibody that crosslinks CD40 and 4-1BB positive cells, thereby inducing conditional stimulation and subsequently co-stimulatory activity. In the context of cancer, DuoBody-CD40x4-1BB can enhance anti-tumor immunity by (re-)activating tumor-specific T cells, either intratumorally or in the tumor-draining lymph nodes. The unique mechanism of action, its conditional agonist activity, distinguishes DuoBody-CD40x4-1BB from agonistic antibodies targeting CD40 or 4-1BB. Therefore, DuoBody-CD40x-4-1BB represents a novel therapeutic agent with potential for treatment of solid tumors.

Ethics Approval

The use of tumor tissue resections was approved by BioNTech AG’s Ethics Board, approval number 837.309.12 (8410-F).

P403 Preliminary results from a first-in-human phase 1 study of the CD40 agonist monoclonal antibody (mAb) CDX-1140

Rachel Sanborn, MD¹, Michael S. Gordon, MD², Mark O’Hara, MD³, Nina Bhardwaj, MD, PhD⁴, Yi He, PhD⁵, Tracey Rawls⁵, Tibor Keler, PhD⁵, Michael Yellin, MD⁵

¹Providence Portland Medical Center, Portland, OR, USA; ²Scottsdale Healthcare Hospital, Scottsdale, AZ, USA; ³Hospital of the University of PA, Philadelphia, PA, USA; ⁴Icahn School of Medicine Mt. Sinai, New York, NY, USA; ⁵Celldex Therapeutics, Inc., Hillsborough, NJ, USA

Correspondence: Michael S. Gordon (msgordon@u.arizona.edu)

Background

Agonist CD40 mAbs can mediate antitumor immunity through multiple mechanisms, including enhancing tumor antigen presentation, activation of tumoricidal macrophages, and direct growth inhibition/killing of CD40- expressing tumor cells. To fully exploit these mechanisms may require the mAb to be dosed at levels that provide significant tumor and tissue penetration, without dose-limiting-toxicities (DLT) from systemic CD40 activation. Our agonist CD40 mAb, CDX-1140, was selected based on its unique and linear dose-dependent in vitro and in vivo activity and is postulated will achieve maximum agonist activity at dose levels associated with good systemic exposure. CDX-1140 is a fully human IgG2 agonist anti-CD40 mAb that activates dendritic cells (DCs) and B cells in an Fc receptor independent manner and has potent antitumor activity against CD40-expressing cancer cells. In addition, CDX-1140 does not block the natural CD40-CD40L interaction; combination of CDX-1140 with added soluble CD40L is synergistic in the activation of immune cells suggesting a potential to enhance in vivo CD40L dependent immune responses. In toxicology studies, CDX-1140 demonstrated potent CD40-mediated pharmacological effects without significant toxicities.

Methods

A phase 1 dose-escalation study of CDX-1140 (CDX1140-01; NCT03329950) is underway in patients with advanced tumors who have exhausted standard-of-care treatment options. The primary endpoint is determining the safety profile and maximum tolerated dose. Secondary endpoints include pharmacokinetics, immunogenicity, clinical and biological outcome assessments. Baseline and on-study biopsies will be used to explore the pharmacodynamic effects of CDX-1140 in the tumor microenvironment (TME). The dose escalation (DE) portion evaluates CDX-1140, given every 4 weeks, at doses from 0.01 to 3 mg/kg; the first 2 cohorts are single-patient cohorts and all subsequent DE cohorts are conducted utilizing a 3+3 design. Tumor-specific expansion cohorts will further explore the activity of CDX-1140. This study will also evaluate CDX-1140 in combination with CDX-301 (rhFLT3L), a DC growth factor that markedly increases DC numbers, including the CD141+ subset which are critical to an antitumor immune response and are often scarce within the TME.

Results

To date, CDX-1140 cohorts at 0.01 (n=2), 0.03 (n=1), and 0.09 (n=3) mg/kg have been completed without any drug-related serious adverse events, infusion reactions, or DLTs reported. The only drug related toxicity has been grade 1 fatigue (n=2) . Expected pharmacodynamic effects, including transient, dose-dependent decreases in lymphocyte counts and dose-dependent increases in serum IL-12p40 and TNF-Alpha, have been observed.

Conclusions

The early data suggest that CDX-1140 has the expected immune activating and safety profile.

Ethics Approval

The study was approved by University of Pennsylvania, approval number 828733; Mount Sinai School of Medicine, approval number IRB-18-00213; Providence Health and Services, approval number 201700532 and Western Institutional Review Board, approval number 115925

P404 The discovery and characterization of PTZ-522 (ASP1951), a fully-human, high affinity agonistic anti-GITR tetravalent monospecific monoclonal antibody

Cynthia Seidel-Dugan, PhD³, Sonja Kleffel¹, Sandra Abbott¹, Heather Brodkin¹, Daniel Hicklin¹, Nels Nielson², Christopher Nirschl¹, Rebekah O'Donnell¹, Andreas Salmeron¹, Philipp Steiner, PhD¹, Christopher Thomas¹, William Winston¹

¹Potenza Therapeutics, Inc, Cambridge, MA, USA; ²Adimab, LLC, Lebanon, NH, USA; ³Potenza Therapeutics, Cambridge, MA, USA

Correspondence: Cynthia Seidel-Dugan (cseideldugan@potenzatherapeutics.com)

Background

Multiple studies have demonstrated that tumors establish an immunosuppressive microenvironment (TME) to escape immune surveillance and promote tumor development. Tumor-infiltrating lymphocytes (TILs) become suppressed in the TME so their proliferative capacity and effector functions are impaired. Members of the TNF Receptor (TNFR) family and their ligands modulate the proliferation, differentiation, and activation of immune effector cells. Glucocorticoid-induced TNFR-related (GITR) is a receptor belonging to the TNFR family with costimulatory activity. In preclinical studies, GITR agonists increase effector T cell proliferation and function, and decrease the tumor infiltration, stability, and/or survival of Tregs, resulting in a more pro-inflammatory TME. In multiple syngeneic mouse tumor models, treatment with GITR agonists demonstrates compelling anti-tumor activity. Based on these promising preclinical data, a number of GITR agonist agents are being tested in the clinic.

Methods

Functional and structural studies have demonstrated that optimal activation of human GITR requires an adequate clustering of the receptor with trimeric GITR ligand (GITRL). Traditional bivalent agonistic antibodies are not as efficacious as trimeric GITRL and are expected to require FcR mediated cross-linking for full activity, which introduces potentially undesired FcR activation, cytokine release, and/or elimination of key effector cells expressing GITR. Potenza Therapeutics has identified PTZ-522 (also known as ASP1951), a novel, tetravalent monospecific (TM) anti-GITR agonist antibody designed to overcome these potential liabilities.

Results

PTZ-522 is a hinge-stabilized IgG4 antibody which binds with high affinity to human and cynomolgus monkey GITR. PTZ-522 has agonistic activity in engineered cell assays and primary T cells from peripheral blood of healthy donors. The TM-formatted antibody PTZ-522 is more active in cell assays than the same antibody in a bivalent format (522-IgG4) and has similar or greater activity than trimeric GITRL. Moreover, this activity was observed in the absence of any FcR cross linking. Analysis of GITR expression on T cells using human tumor samples as well as PBMCs demonstrates that GITR is more highly expressed on TILs, highlighting the different immune phenotype of cells found in the tumor microenvironment. The TM-formatted antibody PTZ-522 increases proliferation and inflammatory cytokine production by GITR expressing TILs. Increased activity in the TIL assay is also observed with the combination of PTZ-522 with a PD-1 inhibitor. Mechanistically, PTZ-522 binding to T cells results in rapid GITR activation and internalization not observed with bivalent antibodies.

Conclusions

These data support the continued development of PTZ-522 as a novel GITR agonist for the treatment of cancer.

P405 Agonistic IgM antibodies targeting immunostimulatory TNFRSF family members GITR and OX40 enhance immune responses beyond that of IgGs

Angus Sinclair, PhD, Dalya Rosner, PhD, Beatrice Wang, Tasnim Kothambawala, Ling Wang, Susan Calhoun, Avneesh Salini, Sachi Rahman, Ramesh Baliga, PhD, Bruce Keyt

IGM Biosciences, Mountain View, CA, USA

Correspondence: Angus Sinclair (angus.m.sinclair@gmail.com)

Background

Tumor necrosis factor receptor (TNFR) superfamily T cell immunostimulatory receptors OX40 and GITR require trimerization to induce agonistic signaling. Anti-OX40 and anti-GITR therapeutic IgG antibodies have demonstrated limited anti-tumor activity in the clinic, perhaps due to inefficient multimerization through FcγR engagement in the tumor microenvironment. We generated and evaluated the functional properties of agonistic anti-OX40 and anti- GITR IgM antibodies and have compared their activities to corresponding IgGs.

Methods

Agonistic anti-OX40 and anti-GITR IgG1 and IgM antibodies were generated using variable regions inserted into the same light chains and either the IgG or IgM heavy chain framework. Antibody binding was evaluated using antigen ELISAs or by flow cytometry with human T cells. Activated human T cells were used to evaluate antibody induced proliferation and cytokine release. Effects of these antibodies on regulatory T cell (Treg) suppression of T effector proliferation and cytokine release were measured.

Results

Agonistic anti-OX40 and anti-GITR IgM antibodies demonstrated superior binding potencies to recombinant antigens (>10 fold in molar comparisons) compared to IgG antibodies, which were amplified at low antigen density. Improved binding was due to the enhanced avidity of the multivalent IgMs compared to bivalent IgGs. As OX40 and GITR require trimerization with ligand or antibodies for function, the agonistic properties of the IgMs and IgGs were compared. In NF-kB-luciferase reporter assays driven by either GITR or OX40, a significant increase in potency was observed with the IgM in both EC50 and max activity compared to IgG in presence or absence of cross-linking agents. In primary human T cell activation assays, IgMs significantly enhanced T cell proliferation and cytokine secretion compared to IgGs. OX40 and GITR are also expressed on immunosuppressive Tregs. In co- culture of Tregs with T effector cells, IgM antibodies significantly reversed the suppressive activity of Tregs on T cell proliferation and inflammatory cytokine secretion, whereas IgGs had little to no effect.

Conclusions

We have discovered IgM antibodies significantly bind to and signal more efficiently through OX40 and GITR than corresponding IgGs, even in presence of cross linking agents. Efficient multimerization and agonism of OX40 and GITR with IgM antibodies may therefore enhance the anti-tumor immunostimulatory effects of immunotherapeutic antibodies targeting these T cell agonists.

Acknowledgements

We thank Sarah Wadsworth for her assistance with some of the studies.

P406 ALPN-202, a combined PD-L1/CTLA-4 antagonist and PD-L1-dependent CD28 T cell costimulator, elicits potent intratumoral T cell immunity superior to and differentiated from PD-L1 inhibitor monotherapy

Ryan Swanson, BS, Mark Maurer, BS, Chris Navas, BS, Chelsea Gudgeon, BS, Katherine Lewis, PhD, Martin Wolfson, BS, Sherri Mudri, BS, Kayla Susmilch, MS, Joe Kuijper, Stacey Dillon, PhD, Steven Levin, PhD, Kristine Swiderek, PhD, Stanford Peng, MD, PhD

Alpine Immune Sciences, Seattle, WA, USA

Correspondence: Ryan Swanson (ryan.swanson@alpineimmunesciences.com)

Background

ALPN-202 is a variant CD80 vIgD™-Fc fusion protein blocking the PD-L1 and CTLA-4 checkpoints while providing PD-L1-dependent T cell activation via CD28. This strategy delivers potent T cell costimulation, which is currently missing from checkpoint inhibitor only regimens, and may be critical for the generation of clinical anti- tumor responses, seeking to broadly improve cancer outcomes. ALPN-202 has previously demonstrated preclinical anti-tumor activity superior to PD-L1 inhibition, but the specific mechanism(s) of superiority remain unreported.

Methods

In a hPD-L1-transduced MC38 tumor model treated with ALPN-202 or durvalumab, an approved PD-L1 inhibitor, anti-tumor responses were evaluated by serial tumor volume measurements, and intratumoral immune responses were assessed by RNA-Seq, flow cytometry, and immunoSEQ TCR repertoire analysis (Adaptive Biotechnologies).

Results

Multiple doses of ALPN-202 elicited anti-tumor responses superior to durvalumab as judged by tumor volume measurements. Efficacy was importantly also observed with single ALPN-202 doses administered intraperitoneally or intratumorally. RNA-Seq and flow cytometric analyses of tumors revealed higher T cell, NK cell, macrophage, and dendritic cell markers after ALPN-202 treatment vs. durvalumab, along with higher T cell effector transcripts, including IL-2, IFN-gamma, granzyme B, and T-bet. TCR repertoire analysis demonstrated increased clonality and richness in response to ALPN-202, two characteristics previously not reported in response to PD-(L)1 or CTLA-4 inhibition alone.

Conclusions

ALPN-202 elicits intratumoral immune responses superior to PD-L1 inhibition alone, including T cell infiltration and T cell effector molecules. This suggests it may translate into clinical anti-tumor responses in cancers currently resistant to checkpoint inhibition alone and/or may improve outcomes in cancers when administered in combination with existing therapies. Ongoing studies seek to further define such potential and specific clinical indications and modalities to guide upcoming clinical trials.

P407 CTX-471, a novel agonistic antibody targeting CD137, eradicates very large tumors in vivo by selectively reprogramming the tumor microenvironment without causing hepatic toxicity

Ugur Eskiocak, PhD¹, Wilson Guzman, BS¹, Nora Zizlsperger, PhD¹, Benjamin Wolf¹, Christine Cummings¹, Thomas Daly¹, Puru Nanjappa¹, Lauren Milling¹, Xianzhe Wang¹, Lucy Liu¹, Samantha Ottinger¹, Jason Lajoie¹, Michael Schmidt¹, Robert Tighe, BS²

¹Compass Therapeutics, Cambridge, MA, USA; ²Compass Therapeutics LLC, Cambridge, MA, USA

Correspondence: Robert Tighe (robert.tighe@compasstherapeutics.com)

Background

CD137 (4-1BB) is a member of the TNFR superfamily that provides costimulatory signals to activated cytotoxic lymphocytes. Agonistic antibodies against CD137 have shown promising therapeutic activity in mouse tumor models. However, hepatic toxicity has been observed in animals and humans with a few anti-CD137 antibodies [1,2]. Recent advances in our understanding of TNFR agonist antibodies implicate epitope, affinity, and IgG subclass as important contributors to function [3,4]. Here we describe the preclinical characterization of CTX-471, a fully human IgG4 agonist of CD137 with a differentiated pharmacology and toxicology profile.

Methods

CTX-471 was identified based on epitope binning and antigen-binding assays. The in vitro bioactivity of CTX-471 was measured in a human IFN-γ release assay. The in vivo efficacy of CTX-471 was assessed in multiple syngeneic mouse tumor models that included various mechanistic endpoints: FACS analysis of TILs, effector cell depletion, tumor histology, and Fc receptor profiling. The efficacy of CTX-471 was further evaluated in mice bearing very large (~500 mm3) CT26 tumors. Finally, the toxicity profile of CTX-471 was evaluated in mice and cynomolgus monkeys.

Results

CTX-471 binds to a unique epitope shared by human, cynomolgus monkey, and mouse CD137. In vitro, CTX-471 increased IFN-γ production by human T cells in an FcγR-dependent manner, displaying an intermediate level of activity between two clinical-stage anti-CD137 antibodies. In Vivo, CTX-471 exhibited curative monotherapy activity in CT26, A20, and EMT-6 models. When compared to known anti-CD137, OX-40, PD-1, PD-L1, and CTLA-4 antibodies, only an affinity-optimized version of CTX-471 showed the ability to eradicate very large tumors. All mice cured by CTX-471 rejected tumors upon rechallenge. CTX-471 profoundly reprogramed the TME, leading to an influx of inflammatory cells, decreased T cell exhaustion, Treg depletion, and TAM modulation, while having very little effect on the peripheral immune system. Tumor models with abundant expression of FcγR’s responded more strongly to CTX-471 treatment, and Fc silencing mutations attenuated efficacy. In mice and monkeys, extremely high doses of CTX-471 (up to 100 mg/kg weekly for 4 weeks) were well-tolerated, with no signs of hepatic toxicity.

Conclusions

CTX-471 displays a favorable and well-differentiated efficacy-safety profile that is attributed to a unique epitope, optimized affinity, and FcγR-dependent activity. To our knowledge, CTX-471’s level of monotherapy efficacy against very large tumors is unprecedented for an IO antibody. IND-enabling toxicology studies are underway, and a Phase 1 trial is planned for the first-half of 2019.

References

1. Bartkowiak T, Jaiswal AR, Ager CR, et al. Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27–dependent pathway. Clinical Cancer Research. 2018 Apr;24(5):1138–51.

2. Segal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist Anti- CD137 monoclonal antibody. Clinical Cancer Research. 2016;23(8):1929–36.

3. Yu X, Chan HTC, Orr CM, et al. Complex interplay between epitope specificity and isotype dictates the biological activity of anti-human CD40 antibodies. Cancer Cell. 2018;33(4):664-675.e4. 4. Chodorge M, Züger S, Stirnimann C, et al. A series of fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency. Cell Death Differ. 2012;19(7):1187-95.

Ethics Approval

All animal studies were approved by Compass Therapeutics Institutional Animal Care and Use Committee under protocol CTX-016-01.

P408 Preclinical identification of the pharmacologically active dose range of the tumor targeted 4-1BB agonist MP0310 based on tumor regression, receptor occupancy and CD8 T lymphocyte expansion

Elmar Vom Baur, PhD, MBA, MEng, Ivana Tosevski, PhD, Laurent Juglair, MSc, Alexander Link, PhD, Guy Lemaillet, Heïdi Poulet, Christian Reichen, PhD, Patricia Schildknecht, MSc, Joanna Taylor, MSc, Alexander Titz, MSc, Waleed Ali, Doris Schaible, Mirela Matzner, Christof Zitt, PhD, Jörg Herbst, PhD, DABT, Keith Dawson, PhD, Julia Hepp, PhD, Dan Snell, PhD, Michael T. Stumpp, PhD, Victor Levitsky, MD, PhD, Hong Ji, MD, PhD, Ivana Tosevski, PhD

Molecular Partners AG, Schlieren, Switzerland

Correspondence: Elmar Vom Baur (elmar.vombaur@molecularpartners.com)

Background

In animal models, agonistic antibodies targeting the T cell costimulatory receptor 4-1BB (CD137) have shown promise as anti-tumor agents, but clinical studies have shown only limited signs of efficacy as well as dose-limiting hepatotoxicity with one of the candidates. To avoid systemic toxicities and to direct immune activation to the tumor, we have generated the tumor-targeted 4-1BB agonist MP0310. MP0310 is a multi-domain DARPin® molecule comprising domains binding to 4-1BB, fibroblast activation protein (FAP), and human serum albumin, the latter for half-life extension. FAP binding targets MP0310 activity to tumors as FAP is highly expressed in many solid tumors and crucially, activation of 4-1BB by MP0310 is dependent on FAP-mediated clustering of 4-1BB. Previously we have shown, in vitro and in vivo, that MP0310 is at least as potent as the agonistic 4-1BB antibodies and, in contrast to the antibodies, does not induce hepatotoxicity or exacerbate graft versus host disease in humanized mouse models. Also, no systemic T cell activation has been observed in cynomolgus monkeys. The present study, in support of the clinical development of MP0310, is to establish the pharmacologically active dose range of MP0310 in a mouse model based on parameters such as receptor occupancy, CD8+ T cell expansion and anti-tumor activity.

Methods

NSG mice were implanted subcutaneously with HT-29 tumor cells and subsequently were injected with human PBMCs. Mice were then treated with a suboptimal dose of a tumor associated antigen binding T cell engager and with a variant of MP0310 containing a mouse FAP binding domain (mMP0310) at doses from 0.0128 to 40mg/kg. Tumor volume and potential biomarkers of T cell activation were measured.

Results

mMP0310 strongly increased intra-tumoral CD8 T cell expansion in a dose-dependent manner compared to monotherapy with the T cell engager. Maximal increases in intra-tumoral CD8 T cell numbers and the ratio of CD8 versus CD4 cells correlated with 100% receptor occupancy (RO) reached at a dose of 1.6mg/kg. Only marginal activity on T cells was seen at a dose of 0.0128mg/kg correlating with a 4-1BB RO level below 10%. In addition, mMP0310 enhanced tumor regression induced by the T cell engager in a manner suggesting dose-dependency starting at 0.32mg/kg

Conclusions

The novel FAP-targeted 4-1BB agonist mMP0310 has been shown to enhance T cell activation and antitumor activity without producing toxicity. Dose dependency of pharmacodynamic activities related to 4-1BB RO has been demonstrated and biomarkers for clinical development have been identified

P409 Pharmacodynamic activity of MEDI1873, a Glucocorticoid-Induced tumor necrosis factor family-related protein (GITR) agonist molecule, administered intravenously to patients with advanced solid tumors

Nicholas Durham, PhD², Nathan Standifer, PhD², Jennifer Cann, PhD², Christopher Morehouse, MD², Li Yan², Kristina Kovacina², Xu Liu², Jia Li², Yuling Wu², Katie Streicher, PhD², Paolo Vicini², Ayesh Perera², Rakesh Kumar, PhD², Raid Aljumaily, MD³, Aung Naing, MD, FACP⁴, Ashish Chintakuntlawar, MBBS, PhD⁵, Naiyer Rizvi, MD⁶, Helen Ross, MD⁵, Michael Gordon, MD⁷, Jeffrey Infante⁸, Crystal Denlinger⁹, Ani Balmanoukian, MD¹⁰

¹Ashfield Healthcare; ²MedImmune, Gaithersburg, MD, USA; ³Oklahoma University College of Medicine, Oklahoma City, OK, USA; ⁴University of Texas, Houston, TX, USA; ⁵Mayo Clinic, Rochester, MN, USA; ⁶Columbia University Medical Center, New York, NY, USA; ⁷HonorHealth, Scottsdale, AZ, USA; ⁸Sarah Cannon Research Institute, Spring House, PA, USA; ⁹Fox Chase Cancer Center, Philadelphia, PA, USA; ¹⁰The Angeles Clinic, Los Angeles, CA, USA

Correspondence: Nicholas Durham (durhamn@medimmune.com)

Background

MEDI1873 is a novel GITR-ligand/IgG1 agonist fusion protein that binds to the co-stimulatory molecule GITR, which is upregulated on activated T cells. This Phase 1 study (NCT02583165) evaluated safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity in patients with advanced solid tumors.

Methods

MEDI1873 was administered IV Q2W in sequential dose escalation (DE) cohorts: 2 single patient cohorts (1.5 and 3 mg), followed by six 3+3 DE cohorts (7.5, 25, 75, 250, 500 and 750 mg). Antitumor response was assessed using RECIST v 1.1. Pharmacodynamic cohorts of patients with non-small cell lung cancer, head and neck squamous cell carcinoma (HNSCC) or colorectal cancer receiving 75 or 250 mg were evaluated for intratumoral CD8, FOXP3, GITR and tumoral PD-L1 expression by immunohistochemistry using matched biopsies from pretreatment and day 29. Peripheral blood from all patients was monitored for type-2 IFN cytokine levels, gene expression, and lymphocyte phenotype by flow cytometry up to day 43.

Results

As of 1 March 2018, 40 patients have been dosed in the DE (28) and pharmacodynamic (12) cohorts. MEDI1873 elimination half-life was ~2.0 days. MEDI1873 pharmacokinetics was dose-proportional over a range of 1.5 to 500 mg. Antidrug antibodies were rare and had minimal impact on pharmacokinetics. MEDI1873 engaged GITR as evidenced by >50% reductions in peripheral GITR-expressing memory CD4+ T cells using a competitive target engagement assay. The duration of GITR+ T-cell suppression correlated with MEDI1873 dose. At doses ≥75 mg, peripheral IFNγ, IP-10, I-TAC and MIG were elevated on days 2 and 3. This was followed by increased Ki67+CD4+ T cells on day 15. Additionally, patients with high baseline GITR levels (≥ median) had a sustained elevation of Ki67+CD4+ T cells to Day 43. Of 8 patients with evaluable paired tumor biopsies, one patient with HPV+ HNSCC and the highest baseline GITR on CD4+ T cells showed a 2x increase in intratumoral CD8+ and tumoral PD-L1+ cells. Intratumorally, MEDI1873 induced a ≥25% reduction in GITR+/FOXP3+ T cells in 5 of 5 evaluable patients. Patients with prolonged stable disease (≥40 weeks) had an average 2x elevation in Ki67+CD4+ peripheral T cells compared to all other patients.

Conclusions

MEDI1873 engages GITR on circulating blood cells resulting in increased peripheral IFNγ production and CD4+memory T-cell proliferation coupled with a decrease in intratumoral GITR+/FOXP3+ cells. These pharmacodynamic effects are consistent with the compound’s mechanism of action and may be enhanced in patients with high baseline GITR expression.

Trial Registration

ClinicalTrials.gov [NCT02583165]

Ethics Approval

Multicenter study conducted at 11 sites:(1) Pinnacle Oncology Hematology, 9055 E Del Camino Dr., Scottsdale, AZ 85358, USA [PI: Michael Gordon; IRB Registration No. IRB00002349](2) Mayo Clinic, AZ, 5777 E. Mayo Boulevard, Phoenix, AZ 85054, USA [PI: Helen Ross; IRB Registration No. IRB00000020](3) The Angeles Clinic and Research Institute, 11818 Wilshire Boulevard, Los Angeles, CA 90025, USA [PI: Ani Balmanoukian; IRB Registration No. IRB00002349](4) Mayo Clinic, MN, 200 First Street SW, Rochester, MN 55905, USA [PI: Asish Chintakuntlawar; IRB Registration No. IRB00000020](5) H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA [PI: Scott Antonia; IRB Registration No. IRB00000790](6) Sarah Cannon Research Institute, 250 25th Avenue North, Nashville, TN 37203, USA [PI: Howard Burris; IRB Registration No. IRB00001035](7) Columbia University Medical Center, 161 Fort Washington Avenue, Herbert Irving Pavilion Mezzanine, New York, NY 10032, USA [PI: Richard Carvajal; IRB Registration No. IRB00006882](8) Greenville Health System, 120 Dillon Drive, Spartanburg, SC 29307, USA [PI: Ki Chung; IRB Registration No. IRB00002227](9) Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA [PI: Crystal Denlinger; IRB Registration No. IRB00002349](10) Oklahoma University, 1000 Stanton L Young Blvd, Oklahoma City, OK 73117, USA [PI: Raid Aljumaily; IRB Registration No. IRB00001035](11) MD Anderson, 1515 Holcombe Blvd, Houston, TX 77030, USA [PI: Aung Naing; IRB Registration No. IRB00002203]

Cytokines in Anti-Tumor Immunity

P410 MV-626, a potent and selective inhibitor of ENPP1 enhances STING activation and augments T-cell mediated anti-tumor activity in vivo

Jason Baird, PhD¹, Gregory Dietsch, PhD, DABT², Vincent Florio, PhD², Michael Gallatin, PhD², Clayton Knox, MD², Joshua Odingo, PhD², Marka Crittenden, MD, PhD¹, Michael J. Gough, PhD¹

¹EACRI Providence Portland Medical Center, Portland, OR, USA; ²Mavupharma, Kirkland, WA, USA

Correspondence: Michael J. Gough (michael.gough@providence.org)

Background

STING is an endogenous sensor of cGAMP, which is synthesized by cGAS following detection of cytoplasmic DNA. STING activation leads to interferon production and activation of inflammatory pathways that facilitate cytolytic T cell priming. STING agonists administered intratumorally show potent anti-tumor efficacy in a range of preclinical models; several agonists are in clinical development. Radiation therapy also increases cytoplasmic DNA levels in cancer cells, resulting in STING activation and secretion of inflammatory cytokines. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the phosphodiesterase that negatively regulates STING by hydrolyzing cGAMP. MV-626, a highly potent and selective ENPP1 inhibitor with 100% oral bioavailability in rats and mice, blocks cGAMP hydrolysis and increases STING activation in cells where cGAS is active. We hypothesize that by conditionally enhancing STING activation, ENPP1 inhibitors will facilitate development of anti-tumor cellular immune responses, particularly following radiation therapy.

Methods

The effects of ENPP1 inhibition on STING activation using cGAMP or DNA treatment of cells were assessed. Panc02-SIY tumors were implanted in C57BL/6 mice and randomized to receive 20Gy CT-guided radiation therapy, 5 daily ip doses of MV-626, or both MV-626 and radiation. Mice were followed for outcome, tumor antigen specific T cell responses and changes in the tumor immune environment. Additional studies were conducted in mice bearing MC38 tumors.

Results

In vitro, MV-626 blocks ENPP1-mediated hydrolysis of cGAMP and enhances STING activation by DNA-mediated cGAS activation or exogenous cGAMP. Therapeutic doses of MV-626 were well tolerated in mice, with no evidence of toxicity or clinically-significant increases in systemic cytokine levels. Systemic administration of MV- 626 monotherapy caused tumor growth delay. MV-626 combined with radiation therapy significantly increased overall survival, and most animals achieved durable tumor cures. Additional studies in the MC38 model confirmed MV-626 activity. Studies characterizing effects of MV-626 in the tumor microenvironment are underway.

Conclusions

These data demonstrate that a potent, selective ENPP1 inhibitor augments STING activation and enhances immune responses to tumors. We demonstrate for the first time that, in combination with radiation therapy, ENPP1 inhibition improves outcomes and cures tumors in preclinical models through changes in the tumor immune environment. These translational studies represent a novel approach to enhancing tumor directed immune response following radiation, and provide a foundation for clinical development of an ENPP1 inhibitor as a cancer immunotherapy.

P411 An IL15/IL15Rα heterodimeric Fc-fusion engineered for reduced potency demonstrates an optimal balance of in vivo activity and exposure

Matthew Bernett, PhD¹, Rajat Varma, PhD¹, Christine Bonzon, PhD¹, Liz Bogaert, PhD¹, Rumana Rashid, PhD¹, Ke Liu, PhD¹, Irene Leung, PhD¹, Suzanne Schubbert, PhD¹, Sung-Hyung Lee, PhD¹, Daniel Kirouac, PhD², Fei Hua, PhD², Nicole Rodriguez, PhD¹, Yoon Kim, PhD¹, Kendra Avery, PhD¹, Connie Ardila¹, Nargess Hassanzadeh- Kiabi, PhD¹, Umesh Muchhal, PhD¹, Seung Chu, PhD¹, Gregory Moore, PhD¹, John R. Desjarlais¹

¹Xencor Inc., Monrovia, CA, USA; ²Applied BioMath, LLC, Oakland, CA, USA

Correspondence: John R. Desjarlais (jrd@xencor.com)

Background

IL15 and IL2 are similar cytokines that bind to the IL2Rβ/γc receptor complex and induce the proliferation of lymphocytes. Their therapeutic potential has been well established in animal models and human trials. As potential drugs, both IL2 and IL15 are extremely potent and suffer from low tolerability and very fast clearance that limits therapeutic window. To engineer a more druggable version of IL15, we created various IL15/IL15Rα heterodimeric Fc-fusions (IL15/IL15Rα-Het-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and prolong half-life.

Methods

We engineered IL15/IL15Rα-Het-Fc by fusing IL15 to one side of a heterodimeric Fc, and the sushi domain of IL15Rα to the other. Fc-fusions were tuned for optimal activity by engineering amino acid substitutions in IL15 - at the IL2Rβ or γc interface - that reduced in vitro potency. In vitro proliferation of lymphocytes in normal human PBMCs was monitored by counting Ki67+ cells after incubation with Fc-fusions for 4 days and by measuring signaling in a STAT5 phosphorylation assay. In vivo activity was evaluated using a huPBMC-NSG mouse model by measuring the extent of human leukocyte engraftment by flow cytometry and IFNγ. Tolerability, immune stimulation, and pharmacokinetics were evaluated in non-human primates (NHP). A computational PK/PD model was developed and trained on available data to quantify relationships between affinity, dose, and biological activity.

Results

IL15/IL15Rα-Het-Fc were produced with good yield and purity. The Fc-fusions enhanced proliferation of CD8+ T and NK cells in vitro. Variants with substitutions at the IL2Rβ and/or γc interface reduced potency up to ~700-fold compared to wild-type IL15/IL15Rα-Het-Fc. Treatment of huPBMC-NSG mice with IL15/IL15Rα-Het-Fc promoted enhanced T cell engraftment and elevated IFNγ. NHP studies indicated half-lives of several days for potency-reduced IL15/IL15Rα-Het-Fc, which are significantly longer than the <1 hr half-life of IL15. In both in vivo settings, a marked inverse correlation of pharmacodynamics and clearance was observed, with reduced potency variants allowing higher, more tolerated doses and enhanced lymphocyte proliferation due to more sustained exposure. Our mechanism-based PK/PD model was used to predict optimal drug affinities, balancing potency vs. target-mediated clearance, and will be used to facilitate prediction of human PK/PD and regimen design. A lead candidate XmAb24306 was selected based on combined experimental observations and modeling predictions, and has been selected for clinical development.

Conclusions

Multiple IL15/IL15Rα heterodimeric Fc-fusions were engineered for reduced potency and evaluated in vitro and in vivo. We identified a variant, named XmAb24306, that optimally balanced potency and exposure.

P412 Tumor cell-intrinsic defects in STING pathway signaling

Blake Flood, BS¹, Leticia Corrales², Thomas Gajewski, MD, PhD¹

¹University of Chicago, Chicago, IL, USA; ²Aduro, Berkeley, CA, USA

Correspondence: Blake Flood (blakeflood@uchicago.edu)

Background

Our laboratory has previously shown that immunogenic tumors spontaneously activate the innate immune system through the STING pathway. The STING pathway senses cytosolic DNA, which activates a signal transduction pathway culminating in phosphorylated IRF3 that translocates to the nucleus where it acts as a transcription factor to induce several genes including IFN-β. STING signaling and IFN-β receptor signaling in tumor-infiltrating immune cells, in turn, are required for optimal priming of CD8+ T cells against tumor antigens. Based on this notion, STING agonists have been pursued as a pharmacologic approach to activate the pathway. However, whether tumor cells themselves also can experience STING pathway activation through to IFN-β production has been unclear.

Methods

We stimulated various cell populations present in the tumor microenvironment as well as multiple tumor cell lines with STING agonists to test their ability produce IFN-β, and analyzed each step in STING pathway signaling. Further biochemical techniques including Western blotting and intracellular immunofluorescence were used to carefully analyze each step of the STING pathway in tumor cells or controls.

Results

We observed that tumor cells themselves usually fail to produce IFN-β in response to STING agonists or cytoplasmic DNA, arguing that loss of activation of this pathway might occur regularly as a component of oncogenesis. Surprisingly, we found that most tumor cells retain expression of each gene in the STING pathway, and that STING signal transduction was intact up to and including nuclear translocation of IRF3 in most instances. However, ChIP assays demonstrated that IRF3 was unable to bind the IFN-β promoter but could still bind the promoters of other genes. B16 melanoma cells, in particular, demonstrated a concurrent deficiency in NF-κB signaling downstream of STING pathway activation.

Conclusions

These results suggest that defective IRF3 DNA binding to the IFN-β locus may be a frequent alteration in cancer. Uncovering the detailed molecular mechanism of this effect could lead to new therapeutic interventions to restore the STING pathway in cancer cells.

P413 The FAP-IL2v immunocytokine is a versatile combination partner for cancer immunotherapy

Valeria Nicolini¹, Inja Waldhauer¹, Anne Freimoser-Grundschober¹, Federica Cavallo², Sara Colombetti¹, Marina Bacac¹, Gonzalo Acuna¹, Jehad Charo, PhD¹, Stefan Evers, PhD¹, Volker Teichgraeber, MD¹, Pablo Umana, PhD¹, Christian Klein, Dr rer nat¹

¹Roche Innovation Center Zurich, Schlieren, Switzerland; ²University of Turin, Turin, Italy; ³ROCHE Innovation Center Zurichoche, Schlieren, Switzerland

Correspondence: Christian Klein (christian.klein.ck1@roche.com)

Background

FAP-IL2v (RG7461) is a novel FAP-targeted immunocytokine based on a novel IL-2 variant (IL2v) with abolished binding to CD25 (IL2Ra) to overcome preferential expansion of Tregs, activation induced cell death and to reduce IL-2 toxicities due to CD25 binding. Binding of FAP-IL2v to the intermediate affinity IL-2Rbg heterodimer is retained resulting in induction of activation and expansion of immune cells, particularly NK cells and cytotoxic CD8 T-cells. These properties make FAP-IL2v a promising partner for combination with checkpoint inhibitors, ADCC- competent/enhanced therapeutic and T-cell bispecific antibodies (TCBs).

Methods

muFAP-IL2v, a murinized surrogate of FAP-IL2v, was tested in combination with muPD-L1 and muCD40 specific surrogate antibodies and the T-cell bispecific muCEA-TCB surrogate in syngeneic orthotopic pancreatic Panc02 or Panc02-CEA models, in C57BL/6 or human CEA transgenic C57BL/6 mice, respectively. The combination of muFAP-IL2v with a anti-ratHER2 muIgG2a antibody was evaluated in the fully immunocompetent BALB-neuT genetically engineered mouse model for breast cancer, whereas the combination of FAP-IL2v with the glycoengineered Type II anti-CD20 antibody obinutuzumab was evaluated in the WSU-DLCL2 xenograft model in hCD16 transgenic Scid mice.

Results

In the pancreatic orthotopic Panc02 model in C57BL/6 mice, FAP-IL2v can boost the activation of pre-existing antigen specific T-cells in combination with anti-PD-L1 checkpoint inhibition. In the same model FAP-IL2v further enhances the efficacy of PD-L1 checkpoint inhibition when combined with an agonistic CD40 antibody resulting in long term survival in the majority of animals, and in the induction of immunological memory as evidenced by protection from tumor cell re-challenge. Furthermore, FAP-IL2v is able to enhance the activity of 1) an ADCC- competent HER2 antibody in the BALB-neuT genetically engineered mouse model and 2) the ADCC-enhanced CD20 antibody obinutuzumab in the aggressive non-Hodgkin’s lymphoma model WSU-DLCL2 in human CD16 transgenic SCID mice. Finally, the activity of the T-cell bispecific antibody CEA-TCB was enhanced by combination with FAP-IL2v in the syngeneic pancreatic orthotopic Panc02 model stably expressing CEA in human CEA transgenic C57BL/6 mice.

Conclusions

The presented efficacy studies support the role of the FAP-targeted immunocytokine FAP-IL2v as a versatile combination partner for cancer immunotherapy and serve to inform the selection of combination partners for clinical studies. Particularly, they demonstrate FAP-IL2v's potential in combination with the PD-L1 checkpoint inhibitor atezolizumab, ADCC-competent antibodies e.g. trastuzumab, cetuximab or obinutuzumab, and T-cell bispecific antibodies such as CEA-TCB. Based on these data FAP-IL2v is currently being tested in Phase 1b clinical trials in combination with atezolizumab, trastuzumab and cetuximab.

P414 Efficacy of anti-PD-L1/IL-15 fusion protein in multiple tumor models

Stella Martomo, PhD, Dan Lu, MA, Zhanna Polonskaya, Xenia Luna, Kevin McCracken, Jeegar Patel

Kadmon Corporation, New York, NY, USA

Correspondence: Jeegar Patel (Jeegar.patel@kadmon.com)

Background

Therapeutic antibodies targeting immune checkpoint inhibitors such as PD-1/PD-L1 effectively expand and reactivate T cells in patients, leading to durable objective response rates in select cancers. However, a substantial number of patients fail to respond or become resistant to these therapies. Thus, combination therapies which include these agents as well as therapies targeting alternative effector cell types are needed. IL-15 promotes survival and cytotoxicity of both CD8 T and NK cells in the absence of the concomitant expansion of Treg population, making it a strong candidate for immunotherapy. Direct administration of IL-15 proved to be clinically challenging; durability of responses likely hindered by the short half-life and toxicity. To capitalize on the anti-tumor potential of IL-15, we generated a therapeutic fusion protein (KD033), combining a proprietary high affinity human-PD-L1 antibody (or mouse-PD-L1 surrogate antibody (KD033-surrogate)) with human IL-15. Initial assessment of this fusion antibody showed enhanced tolerability relative to a non-targeted IL-15 fusion antibody and potent anti-tumor activity.

Methods

Mouse syngeneic tumors were grown to 100 mm3 prior to a single IV administration of KD033-surrogate. Immune cell activation in cynomolgus monkeys was assessed following IV administration of KD033 (Day 1 and 15). Where applicable, tumor volumes were measured and immune cell infiltration and modulation was evaluated by immunohistochemistry, flow cytometry and Luminex.

Results

To assess broad anti-tumor potential of our molecule, single dose of KD033-surrogate was tested against a panel of 12 murine syngeneic tumors. Pronounced tumor growth inhibition was observed in multiple tumor types. In CT-26, colorectal tumor model, KD033-surrogate treatment achieved complete tumor regression in multiple animals, and consistent with generation of immune memory, tumors in these animals failed to regrow following CT-26 re- challenge. Interestingly, KD033-surrogate demonstrated synergistic response when co-administered with anti-PD-1 antibody, suggesting that KD033 could be effectively combined with other checkpoint modulators. Supporting the IL-15-dependent mechanism of action, KD033 (in monkeys) or KD033-surrogate (in mice) increased peripheral blood CD8, NK, NKT and/or gamma delta T (CD3+CD4-CD8-) cells. Additionally, an increase in tumor CD8 cells was observed in mice treated with KD033-surrogate compared to the non-targeted IL-15 fusion antibody.

Conclusions

KD033 treatment led to a robust activation of multiple effector cell types associated with a potent and durable anti-tumor activity. Based on the therapeutic activity and improved safety of the fusion protein, Kadmon is developing KD033 with the aim of clinical testing in 2019.

P415 Preclinical characterization of IL-2 Superkines engineered with biased CD8+ T cell stimulating properties

Fahar Merchant, PhD¹, Shafique Fidai¹, Aaron Ring²

¹Medicenna Therapeutics Inc., Toronto, Canada; ²Yale University School of Medicine, New Haven, CT, USA

Correspondence: Fahar Merchant (fmerchant@medicenna.com)

Background

Interleukin-2 (IL-2) is a cytokine immunotherapy approved by the FDA in 1992 that shows rare, but dramatic activity in metastatic renal cell carcinoma and melanoma. However, IL-2 therapy is hampered by limited efficacy, severe toxicities, and a short circulating half-life that necessitates frequent administration. These limitations may be overcome by engineering IL-2 variants with extended half-life and decreased reliance on the IL-2 accessory receptor CD25 that is believed to mediate toxicity and unwanted stimulation of Tregs.

Methods

A series of novel IL-2 ‘Superkines’ were engineered with biased potency towards the intermediate affinity IL-2 receptor (heterodimer of CD122 and CD132) and fused to a low-effector function Fc moiety for extended serum half-life. These Superkines were assayed for in vitro signaling potency on IL-2 receptor reporter cells lines and human peripheral blood mononuclear cells (PBMCs). Selected muteins were evaluated for their efficacy in syngeneic mouse models and for their in vivo PK and safety.

Results

A novel Fc-fused IL-2 mutein, MDNA109-Fc, was found to have a unique biased activation profile for cells expressing the intermediate affinity receptor, through a unique mechanism of action involving >1000 times increased affinity for CD122 vs. wild-type IL-2, while having similar affinity for CD25. This change improved IL-2 receptor dimerization in human cell lines and greatly enhanced phospho-STAT5 signaling and proliferation of CD8 lymphocytes vs. Tregs, leading to a 43-fold increase in potency at stimulating CD8+ T cells vs. wild-type IL-2. MDNA109-Fc also had improved potency on CD4+ Foxp3- T cells and NK cells. In vivo, this potency bias translates to an increased splenic CD8/Treg ratio. In addition, MDNA109-Fc demonstrated improved tumor growth inhibition over wild-type IL-2 in the aggressive B16F10 melanoma model. MDNA109-Fc was characterized at multiple doses and with several administration methods, demonstrating a greatly extended serum half-life that enabled a semiweekly to weekly subcutaneous dosing schedule in mice, paired with a good safety profile in vivo.

Conclusions

MDNA109-Fc is an improved interleukin-2 agent with a unique biased activation profile targeting effector versus immunosuppressive immune cells, and improved efficacy in a melanoma model. Unlike other next-generation IL-2 molecules in development, MDNA109-Fc specifically targets CD122, resulting in potent activation of effector T cells relative to Treg. MDNA109-Fc could improve the therapeutic potential of an effective, but limited use IL-2 immunotherapy by improving its efficacy, safety, and dosing convenience, a profile that may synergize well with immune checkpoint therapy.

P416 Short-course IL-15 given as a continuous infusion leads to massive expansion of NK cells: Implications for combination therapy with anti-tumor antibodies

Milos Miljkovic, MD, MSc¹, Sigrid Dubois, PhD¹, Thomas Fleisher, MD², Jennifer Albert, RN¹, Thomas Waldmann, MD¹, Kevin C. Conlon, MD¹

¹National Cancer Institute, Bethesda, MD, USA; ²NIH Clinical Center, Bethesda, MD, USA

Correspondence: Kevin C. Conlon (conlonkc@mail.nih.gov)

Background

Successful development of cytokines as immunotherapeutics for the treatment of cancer requires defining the optimal treatment regimen[1]. Post-infusional reactions limited dose escalation and immune activation in the first- in-human clinical trial of recombinant human IL-15 (rhIL-15) given as a 30-minute intravenous bolus (IVB)[2].

Ten-day treatment schedules of subcutaneous injection (SC) and continuous intravenous infusion (CIV-10) were better tolerated at 2 mcg/kg, with the CIV-10 schedule producing noteworthy increases in CD8 lymphocytes and NK cells[3, 4]. We report the results of the 5-day (CIV-5) rhIL-15 regimen, with a safety profile and stimulation of effector cells comparable to CIV-10, dosed up to 5 mcg/kg without dose-limiting toxicities.

Methods

Eleven patients were treated at 3 (n=4), 4 (n=3), and 5 mcg/kg/day (n=4, Table 1) with the CIV-5 regimen in a standard phase I dose-escalation study of rhIL-15 for patients with refractory metastatic cancers.

Results

There were no dose-limiting toxicities, but two patients did not complete cycle 1 for reasons unrelated to rhIL-15 (NSAID-induced SIADH and infectious gastroenteritis). The most common adverse events were fever, chills, fatigue, nausea, transient liver function test abnormalities, anemia, and thrombocytopenia (Tables 2-3). The best response was stable disease. Impressive expansion of NK cells was seen at all dose levels (21 to 44-fold, mean 33- fold) as well as an increase in CD8 cells (1.6-8.9-fold, mean 3.8-fold). The mean increase was greatest at 4 mcg/kg: NK cells 42-fold, and CD8 cells 4.8-fold. This effector lymphocyte expansion exceeded results seen with other rhIL- 15 dosing regimens or other IL-15 formulations (Table 4). The emergence of pulmonary capillary leak symptoms and slower patient recovery from toxicities at 5 mcg/kg dose level, without a further rise in immune cell subsets, led to our choice of 4 mcg/kg as the highest CIV-5 dose to be tested in new combination treatment trials.

Conclusions

The shorter duration of the CIV-5 rhIL-15 regimen and its safety profile may make outpatient administration via an ambulatory infusion pump feasible. The massive expansion of NK cells and increases in CD8 cells it produced were greater than other IL-15 regimens. NK cells are key mediators of antibody-dependent cell cytotoxicity (ADCC); in mice, the increase of NK cell number following CIV IL-15 was associated with increased ADCC of anti-tumor antibodies and their efficacy[5]. Our upcoming trials of CIV-5 rhIL-15 with obinutuzumab or avelumab will leverage the observed massive NK cell expansion to augment the ADCC and therefore their efficacy as anti-tumor antibodies.

References

1. Conlon, K.C., M.D. Miljkovic, and T.A. Waldmann, Cytokines in the Treatment of Cancer. J Interferon Cytokine Res, 2018. [epub ahead of print]2. Conlon, K.C., et al., Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during first-in-human clinical trial of recombinant human interleukin-15 in patients with cancer. J Clin Oncol, 2015. 33(1): p. 74-82.3. Conlon, K.C., et al., Abstract 1596: Phase I trial of recombinant human Interleukin 15 (rhIL-15) administered as continuous intravenous infusion (CIV) for 10 days (240 hours) in patients with refractory metastatic cancers. Cancer Research, 2017. 77(13 Supplement): p. 1596-1596.4. Miller, J.S., et al., A First-in-Human Phase I Study of Subcutaneous Outpatient Recombinant Human IL15 (rhIL15) in Adults with Advanced Solid Tumors. Clinical Cancer Research, 2018. 24(7): p. 1525-1535.5. Miljkovic, M.D., et al., IL-15-enhanced antibody-dependent cellular cytotoxicity mediated by NK cells and macrophages: Implications for immunotherapy of T-cell lymphoma [abstract], In: Proceedings of the 2018 AACR Meeting on Advances in Malignant Lymphoma. 2018, Philadelphia (PA): AACR: Boston, Massachusetts.

Ethics Approval

The study was approved by the National Cancer Institute's Institutional Review Board, approval number 385250.

Table 1 (abstract P416). See text for description

Table 2 (abstract P416). See text for description

Table 3 (abstract P416). See text for description

Table 4 (abstract P416). See text for description

P417 SYTX80-013-A: an engineered IL-2 for the treatment of solid tumors with superior pre-clinical efficacy and safety evidence

Marcos Milla, PhD¹, Jerod Ptacin, PhD¹, Carolina Caffaro¹, Hans Aerni, PhD¹, Lina Ma², Kristine San Jose¹, Michael Pena¹, Robert Herman¹, Yelena Pavlova¹, David Chen¹, Laura Shawver², Lilia Koriazova¹, Ingrid Joseph¹

¹Synthorx, Inc., La Jolla, CA, USA; ²Synthorx.com, La Jolla, CA, USA

Correspondence: Marcos Milla (mmilla@synthorx.com)

Background

Aldesleukin, a recombinant form of IL-2, is the first approved immuno-oncology drug leading to complete, durable remissions in metastatic melanoma and renal cell carcinoma patients. Yet, its use is very limited because of vascular leak syndrome (VLS), a severe dose-limiting adverse event stemming from the engagement of the high affinity IL-2 receptor alpha chain in group 2 innate lymphoid cells, eosinophils and vascular endothelial cells. IL-2’s high potency for activation of CD4+ regulatory T cells (Tregs) that suppress T cell-mediated tumor killing responses further reduces its therapeutic window.

Methods

N/A

Results

We applied our Expanded Genetic Alphabet technology platform to the engineering of SYTX80-013-A: a site-directed, singly pegylated form of IL-2 completely lacking IL-2 receptor (IL-2R) alpha chain engagement yet retaining normal binding to the intermediate affinity beta-gamma IL-2R signaling complex present at the surface of natural killer (NK) and CD8+ tumor-killing cells. SYTX80-013-A potently induces pSTAT5, Ki67 and the proliferation of peripheral NK and CD8+ effector T cells in vivo in mice. Remarkably, dosing of SYTX80-013-A in those animals has minimal effect on molecular and clinical markers of VLS, even at high dose levels. In the mouse CT-26 and B16F10 syngeneic tumor models, SYTX80-013-A induces NK and CD8+ T cell tumor infiltration with marked elevation of CD8+/Treg TIL ratios. In non-human primates, SYTX80-013-A can be dosed for maximal elevation in lymphocytes (pharmacodynamic marker) with negligible elevation in eosinophils (toxicology marker).

Conclusions

This demonstrates that SYTX80-013-A is a re-programmed IL-2 that changes the pharmacological profile of that cytokine from low lymphocyte/high eosinophil to high lymphocyte/no eosinophil induction: an IL-2 with a therapeutic window. We are now advancing this molecule into GLP toxicology studies, in preparation for FIH studies in 2019.

P418 Pre-clinical investigation of NKTR-255, a polymer-conjugated human IL-15 with a potent NK cell dependent anti-tumor efficacy

Takahiro Miyazaki, MS, Murali Addepalli, PhD, Arunasree Lanka, PhD, Amol Murkar, MSc, Ravikumar Nutakki, Palakshi Obalapur, PhD, Peiwen Kuo, PhD, Phi Quach, BS PhD, Mekhala Maiti, PhD, Laurie VanderVeen, PhD, Ping Zhang, MS PhD, Loui Madakamutil, Jonathan Zalevsky, PhD

Nektar Therapeutics, San Francisco, CA, USA

Correspondence: Takahiro Miyazaki (tmiyazaki@nektar.com)

Background

IL-15 is a cytokine that activates and provides survival benefit to T and NK cells and has potential as an immunotherapeutic agent for the treatment of cancer. Exploiting the therapeutic value of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic properties and tolerability. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the pharmacological properties of NKTR-255 on NK cells and the effect of NKTR-255 in NK cell-dependent tumor models.

Methods

For in vivo NK cell characterization, mice received single IV doses of 0.03, or 0.3 mg/kg of NKTR-255. Blood and spleen samples were collected to assess the NK population and function. Flow cytometry was used to measure pSTAT5 and Ki-67 in NK cells. Purified splenic NK cells were co-cultured with YAC-1, a mouse T lymphoma cell line, to measure cytotoxic function. In the CT26 model, 1x105 cells were administered intravenously on Day 0, treatment was initiated on Day 1 at 0.3, 1, or 3 mg/kg, and on Day 13 lungs were scored for metastases. In the orthotopic 4T1 model, 5x105 cells were implanted in the mammary fat pad on Day 0, treatment was initiated on Day 5 at 0.3 mg/kg, and on Day 14, metastases were determined from culture of single lung cell isolates.

Results

In vitro, NKTR-255 showed a dose-dependent phosphorylation of STAT5 and enhancement of cytotoxic function in mouse NK cells. NKTR-255 administration increased thebpSTAT5+ populations, the Ki67+ populations and the absolute number of NK cells. In addition, NKTR-255 provided sustained effects of NK cell activation, as determined by enhanced Granzyme B and CD16 expression and cytotoxic function. In the disseminated CT26 model, NKTR-255 treatment resulted in a significant increase of NK cells in lung and a dose-dependent reduction in the number of lung metastases in a NK cell-dependent manner. In the physiological 4T1 metastasis model, NKTR- 255 also showed a significant anti-metastatic effect although it did not affect primary tumor growth.

Conclusions

NKTR-255 is a powerful immune stimulator of NK cells that provides a dose-dependent effect in the proliferation and activation of NK cells. This property of NKTR-255 translates into enhanced anti-metastatic activity in mouse lung metastasis models. These results indicate that NKTR-255 has the therapeutic capacity to be an anti-tumor agent that enhances NK cell expansion and survival.

Ethics Approval

All animal care and procedures were ethically approved and performed according to AAALAC accredited Nektar Therapeutics IACUC guidelines.

P419 NKTR-214 in combination with radiation produces a potent in situ vaccine in the syngeneic B78 melanoma model

Alexander Pieper, BS¹, Alexander Rakhmilevich, MD, PhD¹, Jacob Slowinski, Mr¹, Amy Erbe, PhD¹, Jacquelyn Hank, PhD¹, Zachary Morris, MD, PhD¹, Deborah Charych, PhD², Paul Sondel, MD, PhD¹

¹University of Wisconsin Madison, Madison, WI, USA; ²Nektar Therapeutics, San Francisco, CA, USA

Correspondence: Alexander Pieper (aapieper@wisc.edu)

Background

NKTR-214 is an engineered agonist of the IL2 pathway, biased to the CD122 receptor resulting in sustained signaling and increased CD8/Treg ratios in human and murine tumors. NKTR-214 has shown promising clinical results by enhancing systemic anti-tumor responses. Radiation therapy (RT) alone rarely generates an effective in situ vaccination due, in part, to poor persistence of activated tumor-specific lymphocytes. However, RT can increase tumor immunogenicity by local release of immune stimulatory cytokines, immunogenic tumor cell death, and phenotypic changes that enhance immune susceptibility of tumor cells surviving RT. NKTR-214 may sustain, expand, and drive the systemic anti-tumor response initiated by RT leading to tumor clearance and tumor specific immunologic memory.

Methods

C57BL/6 mice were inoculated with B78 melanoma cells on the right flank. Once average tumor volumes reached 125mm3 (~4 weeks), mice were randomized and treated with 12 Gy external beam local RT to this tumor site (defined as treatment day 0). Cohorts of mice were then treated with one of the following: 1) intravenous (IV) IL-2 (0.47 mg/kg), qdx5 starting on day 5; or 2) intra-tumoral (IT) IL2 (0.47 mg/kg), qdx5 starting on day 5; or 3) IV NKTR-214 (0.8 mg/kg) q9dx3 starting on day 5; or 4) buffer alone, q9dx3 starting on day 5. Tumor growth was monitored biweekly. All mice with complete response (CR) were rechallenged at day 90 with a second inoculation of B78 melanoma to test for immunologic memory.

Results

Both RT and NKTR-214 alone slowed tumor growth compared to the buffer alone group; however, neither RT nor NTKR-214 alone caused tumor regression. In contrast, the combination of RT + NTKR-214 resulted in significant tumor regression (p<0.01). The rate of complete response (CR) was significantly greater with RT + NKTR-214 compared to RT + IV IL-2 (80% CR vs. 16% CR, p<0.05). RT + NKTR-214 also performed better than RT + IT IL- 2 causing significantly more tumor regression (p<0.01) and a higher CR rate (80% CR vs. 60% CR). The combination of RT + NKTR-214 resulted in stronger immunologic memory than RT + IT IL-2 as more mice receiving RT + NKTR-214 rejected a second B78 inoculation (100% rejection vs. 55% rejection, p<0.01).

Conclusions

Previously, IT IL-2 was required to activate and sustain tumor-specific lymphocytes generated from RT of B78. Here we showed that this effect of in-situ vaccination can be realized through IV administration of systemic NKTR- 214 coupled with standard RT.

P420 Outpatient staccato pulse intravenous Interleukin-2 in metastatic melanoma

Walter Quan, MD¹, Leah Gutierrez, RN BSN², Erin Johnson¹, Francine Quan, RN MSN OCN³

¹Loma Linda University, Loma Linda, CA, USA; ²Western Regional Medical Center, Goodyear, AZ, USA; ³Loma Linda University Beaumont, Tempe, AZ, USA

Correspondence: Walter Quan (wquan@llu.edu)

Background

Daily single intravenous Interleukin-2 (IL-2) infusions (pulses) have been developed to decrease toxicity while maintaining anticancer activity of this molecule against melanoma. Such IL-2 schedules have previously been shown to elicit Lymphokine Activated Killer cell (LAK) activity [1]. Hank has demonstrated in vitro that LAK generated by IL-2 then subsequently exposed to additional IL-2 displayed enhanced cytotoxicity [2]. In patients receiving IL-2 therapy, a rebound lymphocytosis occurs approximately 2-3 days later. The staccato schedule was developed to administer an additional IL-2 pulse during the time of rebound lymphocytosis.

Methods

In this retrospective study, twenty-two patients with metastatic melanoma were treated with IL-2 18 Million IU/M2 intravenously over 15-30 minutes on days 1-3 and 21.6 Million IU/M2 intravenously over 15-30 minutes on day 5 on an outpatient basis. Cycles were repeated every 3 weeks.

Results

Patient characteristics: 9 males/13 females, median age-55 (range: 21-74), median ECOG performance status-1 (0-1); common metastatic sites: lymph nodes (17), lungs (15), subcutaneous (12), bone (6), liver (4). Prior systemic therapy: Ipilimumab (8); Interferon (7); Pembrolizumab or Nivolumab (7); Interleukin-2 (5); oral targeted therapy (4); none (4). Most common toxicities were nausea/emesis, decreased appetite, sinus/catarrhal symptoms, myalgia/arthralgia, peripheral swelling, and rigors. No patients required hospitalization for toxicity of therapy. One patient (5%) has had a complete response (ongoing at 12.5+ months) while ten other patients (45%) had partial responses (total response rate =50%; 95% CI: 28-72%). Two of the patients with partial responses have been rendered free of disease following surgical resection of their residual cancer. Responses occurred in lung, bones, lymph nodes, pancreas, peritoneum, breast, small bowel, and subcutaneous sites. Median response duration is 10.1 months.

Conclusions

Outpatient staccato pulse intravenous Interleukin-2 has activity in melanoma.

References

1. Mitchell MS, Kempf RA, Harel W, Shau H, Boswell WD, Lind S, Dean G, Moore J, Bradley EC. Low-dose cyclophosphamide and low-dose interleukin-2 for malignant melanoma. Bull NY Acad Med 1989; 65:128-144.

2. Hank JA, Weil-Hillman G, Surfus JE, Sosman JA, Sondel PM. Addition of interleukin-2 in vitro augments detection of lymphokine-activated killer activity generated in vivo. Cancer Immunol Immunother 1990; 31:53-59.

Ethics Approval

The study was approved by Loma Linda University's Institutional Review Board, approval number 5180218.

P421 Combination of Pegilodecakin (AM0010) with Docetaxel improves immune cell-mediated anti-tumor response in mouse 4T1 tumor model

Navneet Ratti, BS, MBA, Rakesh Verma, PhD, Martin Oft, MD

ARMO BioSciences, a wholly owned subsidiary of Eli Lilly and Company, Redwood City, CA, USA

Correspondence: Navneet Ratti (navneet.ratti@armobio.com)

Background

Pegilodecakin is a PEGylated-recombinant hIL-10 that has single agent and combination efficacy with chemotherapy and checkpoint inhibitors across multiple cancers. Pegilodecakin stimulates the survival, proliferation and cytolytic ability of the CD8⁺ T-cells. Clinical studies with Pegilodecakin have reported 41% ORR in combination with anti-PD1 in 2^nd line NSCLC. Pegilodecakin induced expansion of PD1⁺Lag3⁺CD8⁺ T-cells correlates with clinical response. Microtubule inhibiting molecules are used as chemotherapeutic agents but combination efficacy with immuno-oncology therapies is not well understood. Here we report the enhanced immune responses and efficacy of AM0010 when combined with Docetaxel.

Methods

Pegilodecakin is active, but immunogenic in mice. Therefore, B-cell deficient mice were employed for in-vivo studies. 5x10³ 4T1 cells were inoculated subcutaneously and allowed to reach a median tumor volume of 100 mm³ prior to treatment. Mice received Pegilodecakin alone at 0.5mpk/qd and/or Docetaxel alone at 40mpk/qw. Tumor size and body weights were monitored twice weekly. Immune cells were phenotyped by flow cytometry. Sera were analyzed for cytokines.

Results

The control cohort reached the terminal tumor size by Day 39 PI. Compared to control, Tumor Growth Inhibition percentage (TGI) was 80.91% on Pegilodecakin, 21.39% on Docetaxel and 97.04% on the combination cohort.Docetaxel cohort showed body-weight loss in mice, which was alleviated on Pegilodecakin+Docetaxel. Systemic metastases were only observed in control and Docetaxel cohorts.In the tumors, Pegilodecakin showed an increase of 82-fold in tumor infiltrating T-cells (TILs), 622-fold increase in PD1⁺Lag3⁺CD8⁺ T-cells and a 545-fold increase in proliferative Ki67⁺PD-1⁺Lag-3⁺CD8⁺ T-cells compared to the control cohort.Docetaxel showed an 11- fold increase of TILs but no significant changes in further subsets (CD8⁺/PD1⁺Lag3⁺CD8⁺/Ki67⁺PD1⁺Lag3⁺CD8⁺ T-cells).Pegilodecakin+Docetaxel showed the largest increase in TILs (>400-fold), PD1⁺Lag3⁺CD8⁺ (>1300-fold) and proliferating Ki67⁺PD1⁺Lag3⁺CD8⁺ TILs (1641-fold).Serum IFNG was increased on Pegilodecakin+Docetaxel (6.03pg/mL), compared to 3.39pg/mL on Pegilodecakin, 0.30pg/mL on Docetaxel and 0.72pg/mL in untreated mouse. IFNG was undetectable in control mice at 3 weeks and not available at the terminal endpoint.

Conclusions

Pegilodecakin stimulated T-cell mediated tumor regression of 4T1 breast cancers was increased on Pegilodecakin/Docetaxel. Tumor regression correlated with presence and proliferation of PD1⁺Lag3⁺CD8⁺ T-cells in the tumor. Tumor regression and TIL activation was most enhanced on Pegilodecakin+Docetaxel. The immune stimulation of the combination therapy is further reflected in the systemic increase of IFNG in the combination arm compared to monotherapy. These results provide rationale to clinically test a combination Docetaxel with Pegilocakin in tumors with low T-cell infiltration and resistance to available immunotherapies.

P422 A polymer-associated human IL-15 (NKTR-255) has optimized biological activity and unique mechanisms of action on CD8 T Cells and NK Cells

Tanya Robinson, PhD¹, Shweta Hegde, Research Assistant¹, Sarai Rivas, BS¹, Takahiro Miyazaki, MS², Kimberly S. Schluns, PhD¹

¹University of Texas MD Anderson Cancer Center, Houston, TX, USA; ²Nektar Therapeutics, San Francisco, CA, USA

Correspondence: Tanya Robinson (TORobinson@mdanderson.org); Kimberly S. Schluns

Background

IL-15 has anti-tumor activity but with limited efficacy due to its unfavorable pharmacokinetic properties and tolerability. Nektar Therapeutics has developed a polymer-conjugated human IL-15 (NKTR-255) that exhibits a prolonged in vivo half-life and enhanced potency, which is currently being examined as a potential cancer immunotherapeutic agent. Since responses by IL-15 can be mediated by transpresentation via the IL-15Rα, as soluble IL-15/IL-15Rα complexes, or by cis-presentation, we investigated the role of IL-15Rα in driving NKTR-255 responses by naïve and memory CD8 T cells and NK cells in mice.

Methods

The effects of NKTR-255 were examined by the adoptive transfer of CFSE-labeled naïve ovalbumin-specific CD8 T cells (OT-I) or established memory OT-I T cells followed by systemic administration of NKTR-255. To assess responses by central and effector memory T cell subsets, sorted CD44hi memory phenotype CD8 T cells were transferred into wild-type (Wt) recipients followed by NKTR-255 treatment. Additionally, NK cell responses to NKTR-255 were analyzed in IL-15Rα bone marrow (BM) chimeras by BrdU incorporation.

Results

Naïve CD8 OT-I T cells transferred into Wt and IL-15Rα-/- mice proliferated at similar levels and acquired a central memory phenotype in response to NKTR-255. Interestingly, naive IL-15Rα-/- OT-I T cells had a deficient response to NKTR-255 but not to rhIL-15 or soluble IL-15 complexes. Additionally, proliferation by memory IL-15Rα-/- OT- I T cells in response to NKTR-255 was partially impaired compared to Wt OT-I cells. Sorted memory CD8 T cells maintained their proportion of CD62L+ and - subsets after NKTR-255-stimulated proliferation. Since IL-15Rα expression is essential for NK cell development, BM chimeras were generated with either IL-15Rα-/- or Wt BM in Wt recipients. In this model system, similar levels of BrdU were incorporated in IL-15Rα-/- and Wt NK cells after treatment with NKTR-255.

Conclusions

These findings suggest naive CD8 T cells are critically dependent on cis-presentation of NKTR-255, while memory CD8 T cells are only partially dependent. For both naive or memory CD8 T cells, transpresentation of NKTR-255 was not required. In contrast to CD8 T cells, NK cell responses to NKTR-255 are not dependent on cis-presentation. Overall, these findings highlight the potential of polymerized IL-15 to modify IL-15Rα dependency leading to different mechanisms of action on CD8 T cells and NK cells and unique therapeutic effects.

Ethics Approval

All animal procedures were conducted in accordance with the animal care and use protocols (00000851-RN01) approved by the IACUC at the UT MD Anderson Cancer Center.

P423 Safety, pharmacokinetics and pharmacodynamic effects of ALKS 4230 in patients with advanced solid tumors from the ongoing dose escalation portion of a first in human (FIH) study

Ulka Vaishampayan, MD¹, Vamsidhar Velcheti, MD FACP², David McDermott, MD³, Mayer Fishman, MD, PhD⁴, Chris Hoimes, MD⁵, Daniel Cho, MD⁶, Lei Sun, Ph.D⁷, Juan Alvarez, PhD⁸, Heather Losey, PhD⁷, Rose Marino, MD⁷, Emily Putiri, PhD⁷, Sean Rossi⁷, Lisa Von Moltke, MD⁷, William Slichenmyer, MD⁹, Marc Ernstoff, MD¹⁰

¹Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA; ²Cleveland Clinic, Pepper Pike, OH, USA; ³Beth Israel Deaconess Medical Center, Boston, MA, USA; ⁴Moffitt Cancer Center, Tampa, FL, USA; ⁵University Hospital, Cleveland, OH, USA; ⁶New York University, New York, NY; ⁷Alkermes, Inc., Waltham, MA, USA; ⁸Merck, Boston, MA, USA; ⁹Alacrita, Waltham, MA, USA; ¹⁰Roswell Park Cancer Institute, Buffalo, NY, USA

Correspondence: Lei Sun (Lei.Sun@alkermes.com)

Background

ALKS 4230 is a fusion of circularly permuted IL-2 and IL-2 Receptor (IL-2R) α designed to selectively activate the intermediate-affinity IL-2R, comprised of IL-2Rβ and γ, for activation of cytotoxic CD8+ T cells and NK cells. ALKS 4230 has previously been shown to have enhanced antitumor activity relative to IL-2 in murine models.

Methods

In the ongoing FIH Phase 1 study in patients with advanced solid tumors, ALKS 4230 is administered as a 30 minute intravenous infusion once daily for 5 consecutive days repeating in treatment cycles of 14 days (first cycle) or 21 days (subsequent cycles). The primary objectives are to investigate ALKS 4230 safety and tolerability and to determine the maximum tolerated dose and recommended Phase 2 dose. Other assessments include pharmacokinetics, lymphocyte sub-population expansion, immunogenicity, and anti-tumor activity.

Results

Twenty-four patients have received ALKS 4230 at doses ranging from 0.1 to 3 μg/kg/day. Patients with multiple tumor types were enrolled, including 5 with prostate carcinoma, 4 with renal cell carcinoma, and 3 with melanoma.

Patients had a median of 3 (range 1-8) prior lines of systemic therapy. The most common treatment emergent adverse events (AEs) seen in ≥ 60% of patients were fever and chills. Grade 3 treatment-related AEs seen in 1-2 patients occurred at the 3 μg/kg/day dose level and included neutropenia, leukopenia, jaundice, febrile neutropenia, lymphopenia, diarrhea, cholangitis, hyperbilirubinemia and hypoalbuminemia. There were no Grade 4 or 5 AEs. Systemic exposure to ALKS 4230 increased with increasing dose and serum ALKS 4230 concentrations at 3 μg/kg/day have exceeded the EC50 values for NK and CD8+ T cell activation determined in in vitro pharmacology studies. Treatment with ALKS 4230 resulted in a dose-dependent increase in circulating NK and CD8+ T cells with an approximately 4-fold and 2-fold expansion at 3 μg/kg/day, respectively, and minimal, non-dose dependent change in Tregs. Transient, dose dependent elevations in serum IL-6 levels occurred 4-6 hours post-dose and were associated with transient fever and chills but not cytokine storm. No objective responses have been seen, and dose escalation is ongoing.

Conclusions

ALKS 4230 was well tolerated at the doses tested, with treatment-related AEs that were manageable and transient. The 3 μg/kg/day dose level induced expected immunologic effects, supporting the rationale for assessing combination therapies with ALKS 4230, as well as continued dose escalation in the monotherapy setting.

Acknowledgements

Study was sponsored by Alkermes, Inc. The authors gratefully acknowledge the patients and their families who participated in this study.

Trial Registration

Trial Registration at Clinicaltrials.gov: NCT02799095

Ethics Approval

The study was approved by Beth Israel Deaconesses Medical Center Investigation Review Board (IRB), approval number 16-229, Roswell Park Cancer Institute IRB, approval number MOD00002327 / PH 285316, Cleveland Clinic IRB, approval number 16-804, Western IRB, approval number 1166122, New York University IRB, approval number i15-01394, University Hospitals IRB, approval number 16-804, and Chesapeak IRB approval number 00000790.

P424 NKTR-214, an engineered IL-2, selectively depletes intratumoral Tregs and expands immunotherapy- induced effector T cell responses

Meenu Sharma, PhD¹, Hiep Khong, PhD¹, Faisal Fa'ak, MD¹, Brent Chesson¹, Barbara Pazdrak¹, Laura Maria S Kahn¹, Louise Janssen, MSc¹, Uddalak Bharadwaj¹, Binisha Karki¹, Zhilan Xiao, MD¹, Yared Hailemichael, PhD¹, Manisha Singh, PhD¹, Christina Vianden, MSc¹, David Tweardy¹, Salah Eddine Bentebibel¹, Cara Haymaker, PhD¹, Chantale Bernatchez¹, Adi Diab, MD¹, Ute Hoch, PhD², Jonathan Zalevsky, PhD², Willem W. Overwijk, PhD¹

¹UT MD Anderson Cancer Center, Houston, TX, USA; ²NEKTAR Therapeutics, San Francisco, CA, USA

Correspondence: Willem W. Overwijk (woverwijk@mdanderson.org)

Background

High dose IL-2 has been used in treatment of metastatic melanoma and renal cell carcinoma. However, expansion of suppressive Tregs and physiologic toxicities associated with IL-2 has limited its use in anti-cancer therapies. NKTR- 214 is an engineered IL-2 cytokine that provides sustained activation of the IL-2 pathway through controlled release of IL-2 with a bias to the IL-2 receptor CD122 (IL-2Rbeta gamma), so that it can selectively enhance CD8+ T cell over regulatory T cells (Tregs). We tested this idea by assessing the therapeutic synergy of NKTR-214 with CTLA-4 and PD-1-based checkpoint blockade therapy or with peptide-vaccination in CT26 colon carcinoma and B16 melanoma models. We investigated impact of treatment on proliferation and apoptosis of effector CD8+ T cells and immunosuppressive CD4+Foxp3+ Tregs, as well as effector cytokines and chemokines in tumor and peripheral tissues. In vivo cytokine neutralization experiments and in vitro assays revealed that NKTR-214 plus vaccine treatment induced CD8+ effector T cell responses and enhanced associated cytokines IFN-gamma and TNF-alpha that mediate specific depletion of intratumoral, but not peripheral, Tregs.

Methods

CT26 colon carcinoma tumor bearing mice were treated with NKTR-214 or CTLA-4 and/or PD-1 checkpoint blockade. Tumor size, survival and tumor-specific effector T cell response was analyzed. To monitor antigen-specific immune response, we adoptively transferred naïve gp100-specific pmel-1 CD8+ T cells into mice bearing established B16 tumors, followed by a single vaccination (gp100 peptide + anti-CD40 mAb + TLR-7 agonist) alone or in combination with NKTR-214 or Aldesleukin (IL-2) given every 8 days. Detailed analysis of CD8+ T cells and Tregs was done by flow cytometry. Chemokines/cytokines levels in tumor and spleen were measured by luminex- based assay.

Results

NKTR-214 efficiently synergized with checkpoint blockade and with vaccination, improving overall survival and cure of mice in models of colon carcinoma and melanoma. NKTR-214 promoted the CD8+ T cell survival, expansion and release of associated cytokines, IFN-gamma and TNF-alpha, which synergized to induce apoptosis and inhibited proliferation of Tregs specifically in tumors (Figure 1) while preserving Tregs in peripheral tissues. In vitro cytokine treatment also confirmed that IFN-gamma and TNF-alpha together are both sufficient and required to block Treg proliferation. Preliminary results confirm similar therapeutic effects with cancer patients receiving clinical doses of NKTR-214.

Conclusions

NKTR-214 synergizes with checkpoint blockade as well as with vaccination to improve the survival, proliferation and tumor infiltration of effector CD8+ T cells while promoting selective intratumoral depletion of Tregs to establish effective anti-tumor immunity.

Fig. 1 (abstract P424).

NKTR-214 mediated intratumoral Treg depletion

P425 Pharmacokinetics and pharmacodynamic effects of ALKS 4230, an investigational immunotherapeutic agent, in cynomolgus monkeys after intravenous and subcutaneous administration

Lei Sun, PhD, Jared Lopes, PhD, Heather Flick, MS, Erin Murphy, MS, Heather Losey, PhD

Alkermes, Inc., Waltham, MA, USA

Correspondence: Lei Sun (Lei.Sun@alkermes.com)

Background

ALKS 4230 is an engineered cytokine designed to selectively activate the intermediate-affinity interleukin-2 receptor (IL-2R), expressed predominantly on natural killer (NK) cells and CD8+ T cells, which play an important role in driving immune responses in cancer. A first-in-human study of intravenous administration of ALKS 4230 in patients with advanced solid tumors (NCT02799095) is currently ongoing. To compare the pharmacodynamic responses in response to the intravenous and subcutaneous administration of ALKS 4230, two studies were carried out in cynomolgus monkeys.

Methods

In the first study, a single dose of ALKS 4230 was administered intravenously or subcutaneously. In the second study, ALKS 4230 was administered intravenously once daily on Days 1-5 or subcutaneously on Days 1 and 4. Serial blood samples were collected from each animal for determination of serum concentrations of ALKS 4230 and multiple proinflammatory cytokines as well as for immunophenotyping by flow cytometry.

Results

Overall systemic exposure to ALKS 4230 as measured by area under the serum concentration vs. time (C-T) curve (AUC) after a single subcutaneous dose of 1 mg/kg was comparable to that after an intravenous dose of 0.3 mg/kg, suggesting a subcutaneous bioavailability of ~30%. With comparable AUC but lower Cmax, subcutaneous administration elicited greater expansion of CD8+ T cells and CD56+ NK cells as well as a superior ratio of CD8+ T cells to CD4+CD25+FoxP3+ Tregs compared to intravenous administration. In addition, expansion of CD8+ T cells and CD56+ NK cells was sustained up to 12 days after a single dose. Total systemic exposure to ALKS 4230 was comparable after 5 daily intravenous doses of 0.1 mg/kg and 2 subcutaneous doses of 0.5 mg/kg (on Days 1 and 4) and resulted in similar expansion of total CD8+ T cells, NK cells and Tregs between the two dosing regimens. The serum IL-6 C-T profile mirrored the ALKS 4230 C-T profile, with a higher peak IL-6 level and a higher Cmax of ALKS 4230 following the last intravenous dose of 0.1 mg/kg compared to the last subcutaneous dose of 0.5 mg/kg.

Conclusions

Subcutaneous administration of ALKS 4230 can achieve similar total systemic exposure to ALKS 4230 compared to intravenous administration with less frequent dosing and a lower Cmax, leading to similar expansion of total CD8+ T cell and NK cell populations. Therefore subcutaneous administration may be a practical alternative to intravenous dosing and merits clinical evaluation.

Emerging Models and Imaging

P426 Chaos-based fractal radiomic features of nodule vasculature predicts response to immunotherapy on non- contrast lung CT

Mehdi Alilou, PHD¹, Marjan Firouznia, PHD¹, Pradnya Patil², Kaustav Bera, MBBS¹, Robert Gilkeson³, Prabhakar Rajiah³, Vamsidhar Velcheti, MD FACP², Anant Madabhushi, PhD¹

¹Case Western Reserve University, Cleveland, OH, USA; ²Cleveland Clinic, Cleveland, OH, USA; ³University Hospital Case Medical Center, Cleveland, OH, USA

Correspondence: Mehdi Alilou (me.alilou@gmail.com)

Background

Immune-checkpoint blockade treatments, particularly drugs inhibiting programmed death-ligand 1 (PD-L1) with its receptor, programmed cell death protein-1 (PD-1) has demonstrated promising clinical efficacy in patients with advanced non-small cell lung cancer (NSCLC). In spite of recent regulatory approval of several immunotherapy (IO) drugs, the objective response rate of these drugs is modest (~20%) at best. The complex nature of the host immune response makes tissue based biomarker development for IO response assessment challenging. Consequently, there is an urgent and critical unmet need to develop accurate, validated biomarkers to predict which NSCLC patients will benefit from IO. Previous research has shown that the morphology of the tumor feeding vessels plays a role in cancer aggressiveness as well as therapeutic refractoriness. Post-treatment tumors show significant improvement in vessel tortuosity abnormalities when compared before therapy initiation. Hence, we sought to evaluate whether computer extracted measurements of fractal features of nodule associated vessel morphology on baseline CT scans in NSCLC patients treated with Nivolumab could distinguish between patients who did and did not respond to the PD-1 inhibitor.

Methods

Our study comprised non-contrast CT scans of 61 patients obtained retrospectively from the Cleveland Clinic, including 31 patients who responded to Nivolumab and 30 non-responders. Patients who did not receive Nivolumab after 2 cycles due to lack of response or progression as per RECIST were classified as ‘non-responders’, patients who had radiological response or stable disease as per RECIST were classified as ‘responders’. From nodule annotations provided by a trained radiologist, a region-growing algorithm was used to segment the surrounding vasculature (Figure1A). A set of 12 vessel fractal radiomic (VFR) measurements pertaining to the fractal analysis, the state space reconstruction and Lyapunov exponent were extracted from each nodule associated vasculature. A Naive Bayes classifier was then used, in a 3-fold cross-validation setting through 200 iterations, to construct a classifier to identify which patients respond to nivolumab therapy.

Results

VFR features (Figure1B) were found to distinguish responders from non-responders to Nivolumab with an AUC=0.73±0.08 . Statistically significant difference was observed for two VFR features between responders and non-responders (p<0.009).

Conclusions

VFR were able to distinguish responders from non-responders for patients with NSCLC and treated with Nivolumab. The VFR could potentially serve as a predictive tool for response assessment for immune checkpoint inhibitors and enable selection of NSCLC patients who will benefit from IO; paving the way for design of more rational clinical trials with combination of IO agents.

Ethics Approval

The study protocol was approved under University Hospitals (UH) IRB 02-13-42C.

Fig. 1 (abstract P426).

See text for description

P427 Novel immune competent murine glioblastoma models derived from Nestin-CreER^T2 Quaking^L/L; P53^L/L; PTEN^L/L mice

Chao-Hsien Chen, MD¹, Renee Chin, MS¹, Genevieve Hartley, PhD¹, Takashi Shingu, PhD², David Hong, MD¹, Jian Hu, PhD¹, Michael A. Curran, PhD¹

¹The University of Texas MD Anderson Cancer Center, Houston, TX, USA; ²University of Texas MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Michael A. Curran (mcurran@mdanderson.org)

Background

Despite the success of immunotherapy in several cancers, antibody blockade of the immune checkpoint receptor PD-1 failed to improve the survival of recurrent glioblastoma multiforme (GBM) patients [1]. In contrast to this clinical reality, the widely used immunocompetent mouse model of GBM, GL261, is highly immunogenic and readily cured by T-cell checkpoint blockade therapy [2]. The resulting inability to model the immunotherapeutic sensitivity of human GBM preclinically prevents effective translation of murine observations to clinical therapies. Quaking (QKI) is a GBM tumor suppressor gene which is deleted, mutated or downregulated in the majority of human GBM [3,4], the expression level of which strongly correlates with patient survival [5]. We describe novel murine immunocompetent glioblastoma stem cell (GSC) lines derived from Nestin-CreER^T2 Quaking (QKI)^L/L; P53^L/L; PTEN^L/L (QPP) mice [5] and determine their sensitivities to immunotherapies.

Methods

We selected four lines, namely QPP4, 5, 7 and 8, after validation of their engraftment in C57BL6/J mice. The immunotherapeutic sensitivities in response to systemic CTLA-4 and PD-1 blockade therapies were determined by tumor growth kinetics and survival. The tumor microenvironment (TME) was evaluated by flow cytometry analysis.

Results

All four QPP lines express GSC markers, such as CD171 and α2β5, but lack PD-L1 or PD-L2 expression in vitro and in vivo, excepting limited PD-L1 expression by QPP7 in vivo. This fits the observation that only a small proportion of human GBM expresses PD-L1 [6]. These QPPs have distinct sensitivities to systemic checkpoint blockade in different niches. Subcutaneously, QPP4, 5 and 8 are sensitive to CTLA-4 blockade, and QPP7 is sensitive to both PD-1 and CTLA-4 blockades. In the brain, QPP5 and 7 remain sensitive to CTLA-4 blockade (n= 8-15, p<0.05), while QPP4 and 8 resist both PD-1 and CTLA-4 blockades (n= 9-15, p>0.05) (Figure 1). Preliminary analysis of the orthotopic TME of the checkpoint-resistant QPP8 line reveals no significant change in CD8 T cells, regulatory CD4 T cells (Treg), myeloid-derived suppressor cells (MDSCs), tumor associated macrophages (TAMs) and microglia infiltration, or CD8/Treg and CD8/MDSCs ratios with either CTLA-4 or PD-1 blockade (n=3-5). PD- L1 expression on monocytic MDSCs, TAMs and microglia in the PD-1 or CTLA-4 blockade group are significantly increased (p<0.05) (Figure 2), however, which could reveal the origins of the prognostic value of the PD-1/PD-L1 axis in human GBM [7].

Conclusions

The distinct checkpoint blockade sensitivities of QPP lines could fill the critical need for preclinical GBM models suitable for evaluating immunotherapeutics.

References

1. Reardon DA, Omuro A, Brandes AA, Rieger J, Wick A, Sepulveda J, Phuphanich S, de Souza P, Ahluwalia MS, Lim M, Vlahovic G, Sampson J OS10.3 Randomized phase 3 study evaluating the efficacy and safety of nivolumab vs bevacizumab in patients with recurrent glioblastoma: CheckMate 143. Neuro-Oncology. 2017; 19: iii21-iii21.

2. Reardon DA, Gokhale PC, Klein SR, et al. Glioblastoma eradication following immune checkpoint blockade in an orthotopic, immunocompetent model. Cancer Immunol Res. 2016; 4(2):124-135.

3. Hu J, Ho AL, Yuan L, et al. From the cover: neutralization of terminal differentiation in gliomagenesis. Proc Natl Acad Sci U S A. 2013; 110(36):14520-14527.

4. Brennan CW, Verhaak RG, McKenna A, et al. The somatic genomic landscape of glioblastoma. Cell. 2013; 155(2):462-477.

5. Shingu T, Ho AL, Yuan L, et al. Qki deficiency maintains stemness of glioma stem cells in suboptimal environment by downregulating endolysosomal degradation. Nat Genet. 2017; 49(1):75-86.

6. Hodges TR, Ott M, Xiu J, et al. Mutational burden, immune checkpoint expression, and mismatch repair in glioma: implications for immune checkpoint immunotherapy. Neuro Oncol. 2017; 19(8):1047-1057.

7. Nduom EK, Wei J, Yaghi NK, et al. PD-L1 expression and prognostic impact in glioblastoma. Neuro Oncol. 2016; 18(2):195-205.

Fig. 1 (abstract P427).

Orthotopic survival and immune sensitivities

Fig. 2 (abstract P427).

PD-L1 expression on myeloid cells in QPP8 TME

P428 Spatially-resolved, high-plex digital profiling enables characterization of complex immune biology of the colorectal cancer microenvironment

Sarah Church, Chris Merritt, PhD, Andrew White, BSc, Douglas Hinerfeld, PhD, Dan Zollinger, Giang Ong, MS, Kristi Barker, MS, Sarah Warren, PhD, Joseph Beechem, PhD

NanoString Technologies, Seattle, WA, USA

Correspondence: Sarah Church (schurch@nanostring.com)

Background

Spatial characterization of the tumor microenvironment (TME) interface between cancer cells, stroma and immune cells is essential for understanding tumor progression and discovering prognostic and predictive biomarkers. However, it has proven difficult to perform such studies in a highly multiplexed manner using limited sample quantity. Digital Spatial Profiling (DSP) has been developed as a research use instrument, software and chemistry for hi-plex profiling of mRNA and protein using an optical-barcode read-out. In this study, microsatellite stable (MSS) or instable (MSI) characterized colorectal tumors were characterized using DSP with 40 proteins or 48 RNA probes to evaluate active and suppressive immune mechanisms in both immune dense regions and tumor versus stroma.

Methods

Sixteen FFPE colorectal tumors that were characterized for Microsatellite stability status were mounted on slides. Tissue sections were stained with a cocktail of pan-cytokeratin, CD45, CD3 and DNA fluorescent markers and 48 RNA probes or 40 antibodies, each conjugated to a UV-photocleavable DNA barcode. Regions of interest (ROI) were delineated using the immunofluorescence followed by UV excitation of the defined ROIs, which releases the DNA barcodes for downstream quantitation on the NanoString nCounter® platform. Two strategies were used for selecting ROIs, 1) Geometric profiling of CD45-enriched hotspots in the tumor center and invasive margin and 2) Segment profiling of cytokeratin-positive tumor regions compared to cytokeratin-negative regions.

Results

We show that deep profiling of CD45-enriched regions from the invasive margin and tumor center of MSS and MSI tumors have different immunosuppressive and activated immune phenotypes. Comparing colorectal tumors characterized as MSS, DSP was able to differentiate immune hot and cold tumors despite MSS status. Further evaluation using segment profiling of tumors versus stroma also identified specific immune proteins and RNA pathways that were distinctly related to each compartment and were different between MSI and MSS tumors.

Conclusions

Our results suggests DSP has the potential to be used to predict patients' response to PD-1 immune checkpoint blockade with greater sensitivity than standard MSS/MSI profiling, and furthermore DSP may allow identification of unique localized immune characteristics that would guide combination therapeutic approaches.

P429 Integrative spatially-resolved, high-plex digital profiling enables characterization of complex immune biology in the tumor microenvironment of mesothelioma

Carmen Ballesteros Merino, PhD¹, Moritz Widmaier, PhD², Sarah Church³, Thomas Herz, PhD², Alexei Budco, MSC², Dasa Medrikova, PhD², Ivan Kanchev, PhD², Andrew White, BSc³, Douglas Hinerfeld, PhD³, Shawn Jensen, PhD¹, John Handy, MD¹, Rachel Sanborn, MD¹, Carlo Bifulco, MD¹, Sarah Warren, PhD³, Joseph Beechem, PhD³, Bernard A. Fox, PhD¹

¹Providence Portland Cancer Center, Portland, OR, USA; ²Definiens, Munich, Germany; ³NanoString Technologies, Seattle, WA, USA

Correspondence: Bernard A. Fox (foxb@foxlab.org)

Background

Malignant mesothelioma is an aggressive cancer with poor prognosis and few effective therapies. Since mesothelioma is derived from the mesothelium of the lung, we hypothesize that immune cells in the tumor microenvironment (TME) may behave differently than other solid tumors. In our previous studies, utilizing multi- plexed immunofluorescence, we did not find immune phenotypes associated with improved patient survival. Here we describe a novel combination of two technologies to spatially characterize the interface between mesothelioma cells, stroma and immune cells in the TME in a high-plex capacity.

Methods

Ten FFPE mesothelioma tumors were characterized by Definiens’ Immune-Oncology Profiling (IOP) and NanoString Digital Spatial Profiling (DSP). Three alternating sequential sections were stained with Definiens’ IOP (CD8/PD-1/FOXP3, CD68/PD-L1/CD3, Granzyme B). Definiens analysis was combined to identify localization of each marker in the tumor center, invasive margin or stroma. Twelve regions-of-interest (ROIs) were then selected based on the Definiens analysis for high-plex analysis on DSP on the interleaving slide: 4 CD68-enriched, 6 CD8- enriched and 2 CD3-low. For DSP analysis, each slide was stained with a combination of fluorescent-labeled antibodies (pan-cytokeratin, CD3, CD68) and a panel of 38-antibodies each conjugated to a unique UV- photocleavable DNA barcode. ROIs from Definiens’ defined analysis were overlayed on DSP fluorescent scans, followed by UV excitation of the defined ROIs, which releases the DNA barcodes for downstream quantitation on the NanoString nCounter® platform.

Results

We found strong correlation between Definiens and NanoString analysis of T cell and macrophage markers in selected regions. Generally, patients with longer survival (>6 months) had increased density of immune infiltrates including higher density of T cells, T-cell activation markers (PD-1), higher cytokeratin levels and decreased Ki67 in the tumor center and increased tertiary lymphoid structure makers (B cells) in the invasive margin. Furthermore, STING and VISTA were highly expressed across all mesotheliomas. However, the patient with the longest survival (>31 months) expressed an immune-excluded phenotype. Co-localization analysis revealed that high CD68 density was tightly correlated to PD-L1 expression and in at least one case additional suppressive macrophage markers, including CD163 and B7-H3.

Conclusions

Already this small data set demonstrates that integration of two novel high-plex spatial analysis techniques separates distinct immune mechanisms in the TME. Our analysis suggests that macrophages are highly associated with expression of immune-inhibitory signals in mesothelioma. Therefore, we hypothesize that analysis of additional mesotheliomas may guide the development of combination immunotherapy trials that will be effective against this incurable disease.

P430 Radiomic texture features from MR perfusion images predicts pseudoprogression from true progression in glioblastoma patients: A multi-institutional study

Nabil Elshafeey¹, Aikaterini Kotrotsou¹, Srishti Abrol¹, Islam Hassan¹, Ahmed Hassan¹, Kamel El Salek, MD¹, Fanny Moron², Meng Law³, Pascal Zinn², Rivka Colen, MD⁴

¹MD Anderson Cancer Center, Houston, TX, USA; ²Baylor College of Medicine, Houston, TX, USA; ³University of Southern California, Los Angeles, CA, USA; ⁴The University of Texas, Houston, TX, USA

Correspondence: Rivka Colen (rcolen@mdanderson.org)

Background

Pseudoprogression (PsP) is an inflammatory response associated with radiation and necrotic induced changes reflective of treatment, appearing as areas of increased enhancement on postcontrast T1-weighted images. Response assessment criteria, such as RANO, struggle to distinguish between true progression and PsP. Advanced imaging techniques (MR perfusion, MR diffusion) have been proposed as an alternative way of distinguishing between PsP and progressive disease (PD). However, the outcome of such studies underscores the need for novel tools distinguish between these. In this study, we sought to evaluate the utility of radiomic analysis of MR perfusion [Dynamic contrast enhancement (DCE) and Dynamic susceptibility contrast (DSC)] maps in differentiating PsP from PD.

Methods

Patients: A total of 98 patients were included in this multi-institutional IRB-approved study. All had pathological confirmation; 78 patients with PD and 20 patients with PsP. Radiomic Analysis: All patients underwent DSC and DCE perfusion MRI as part of their routine clinical care. Images were analyzed using Nordic ICE 2.3 (NordicNeuroLab); rCBV and Ktrans maps were obtained. Subsequently, an experienced radiologist delineated the entire tumor on DCE and DSC maps using 3D slicer (http://www.slicer.org) (Figure 1). The extracted 3D region-of- interest (ROI) parametric maps were imported in the radiomic pipeline. A total of 475 features (10 histogram-based and 375 higher-order texture features) were calculated for each parametric map. Statistical Analysis: An advanced feature selection method based on Minimum Redundancy Maximum Relevance (MRMR) was used to analyze the featureset and extract core features. Selected features were used to build a Support Vector Machine (SVM) model for prediction of PD versus PsP. To evaluate the robustness of the estimates made with the SVM models, leave-one- out cross-validation (LOOCV) was conducted. Finally, box plots of the 10 most relevant features and probability maps were calculated.

Results

MRMR identified 50 radiomic features that were further used to build the SVM model. The prediction of progression by LOOCV was significant p-value=0.031. Area under the curve (AUC), sensitivity and specificity were 89.26%, 81.82% and 100% respectively and the most discriminating features were variance and sum entropy (Figure 2). Box plots of the 10 most relevant features are shown in Figure 3.

Conclusions

This study demonstrates that MR perfusion radiomic analysis can discriminate between PsP and PD. Further validation and a comparative study of radiomic analysis of MR perfusion maps and conventional MR images would be valuable to determine which approach is more effective, and the added value in combining the two approaches.

Fig. 1 (abstract P430).

See text for description

Fig. 2 (abstract P430).

See text for description

Fig. 3 (abstract P430).

See text for description

P431 Radiomic Analysis differentiates between True Progression and Pseudo-progression in Glioblastoma patients: A large scale multi-institutional study

Srishti Abrol¹, Aikaterini Kotrotsou¹, Nabil Elshafeey¹, Islam Hassan¹, Ahmed Hassan¹, Tagwa Idris, MD¹, Kamel El Salek, MD¹, Ahmed Elakkad, MD¹, Kristin Alfaro-Munoz¹, Shiao-Pei Weathers¹, Fanny Moron², John deGroot¹, Meng Law³, Rivka Colen, MD¹

¹MD Anderson Cancer Center, Houston, TX, USA; ²Baylor College of Medicine, Houston, TX, USA; ³University of Southern California, Los Angeles, CA, USA; ⁴The University of Texas, Houston, TX, USA

Correspondence: Rivka Colen (rcolen@mdanderson.org)

Background

Treatment-related changes can occur as a result of multiple factors; these changes are often difficult to distinguish from true progression (PD) of the tumor using conventional MRI. Treatment-related changes or pseudoprogression (PsP) subsequently subside or stabilize without any further treatment, whereas progressive tumor requires a more aggressive approach. PsP mimics PD radiographically and may potentially alter the physician’s judgement. Hence, it can predispose a patient to overtreatment or be categorized as a non-responder and exclude him from clinical trials. Radiomic analysis results in the quantification of grey tone spatial variation thereby providing textural features that characterize the underlying structure of the object under investigation. This study aims at assessing the potential of radiomics to discriminate PsP from PD in glioblastoma (GBM) patients.

Methods

In this multi-institutional study, we evaluated 304 GBM patients retrospectively. All patients showed radiographic worsening in MRI, with/without clinical deterioration, and were evaluated for PD our PSP. 149 patients had histopathological evidence of PD and 27 of PsP. Remaining 128 patients were categorized into PD or PsP based on RANO criteria . Conventional MR images were acquired using typical clinical acquisition parameters. Three tumor phenotypes (ROIs), namely edema/invasion, necrosis, and enhancing tumor, were delineated by an experienced radiologist. A total of 1800 radiomic features were obtained for each patient. Statistical Analysis: An advanced feature selection method based on Minimum Redundancy Maximum Relevance (MRMR) was used to analyze the featureset and extract core features. Selected features were used to build a Support Vector Machine (SVM) model for prediction of PD versus PsP status. To evaluate the robustness of the estimates made with the SVM models, leave-one-out-cross-validation (LOOCV) and a 70-30% split was performed.

Results

Using the MRMR feature selection method, we could identify 100 significant features that were further used to build a SVM model. On LOOCV, the area under curve (AUC) was 90%, with a sensitivity and specificity of 97% and 72% respectively (Figure 3). Using 70% of the patient data for training and 30% for validation an AUC of 94% was achieved, with sensitivity of 97% and specificity of 75%. Five texture features i.e. energy, cluster shade, sum average, maximum probability and cluster prominence were found to be most predictive of nature of disease progression.

Conclusions

The proposed tool has the potential to advance clinical management strategies. Apart from its non-invasive nature, our methodology doesn’t require additional imaging and may act as a complementary tool for the clinicians.

Fig. 1 (abstract P431).

See text for description

Fig. 2 (abstract P431).

See text for description

Fig. 3 (abstract P431).

See text for description

P432 High tumor mutation burden (Hypermutation) in gliomas exhibit a unique predictive radiomic signature

Islam Hassan¹, Aikaterini Kotrotsou¹, Carlos Kamiya Matsuoka¹, Kristin Alfaro-Munoz¹, Nabil Elshafeey¹, Nancy Elshafeey¹, Pascal Zinn², John deGroot¹, Rivka Colen, MD³

¹MD Anderson Cancer Center, Houston, TX, USA; ²Baylor College of Medicine, Houston, TX, USA; ³The University of Texas, Houston, TX, USA

Correspondence: Rivka Colen (rcolen@mdanderson.org)

Background

Increase in tumor mutation burden (TMB) or hypermutation is the excessive accumulation of DNA mutations in cancer cells. Hypermutation was reported in recurrent as well as primary gliomas. Hypermutated gliomas are mostly resistant to alkylating therapies and exhibit a more immunologically reactive microenvironment which makes them a good candidate for immune checkpoint inhibitors. Herein, we sought to use MRI radiomics for prediction of high TMB (hypermutation) in primary and recurrent gliomas.

Methods

In this IRB-approved retrospective study, we analyzed 101 patients with primary gliomas from the University of Texas MD Anderson Cancer Center. Next generation sequencing (NGS) platforms (T200 and Foundation 1) were used to determine the Mutation burden status in post-biopsy (stereotactic/excisional). Patients were dichotomized based on their mutation burden; 77 Non-hypermutated (<30 mutations) and 24 hypermutated (>=30 mutations or <30 with MMR gene or POLE/POLD gene mutations). Radiomic analysis was performed on the conventional MR images (FLAIR and T1 post-contrast) obtained prior to tumor tissue surgical sampling; and rotation-invariant radiomic features were extracted using: (i) the first-order histogram and (ii) grey level co-occurrence matrix. Then, we performed Logistic regression modelling using LASSO regularization method (Least Absolute Shrinkage and Selection Operator) to select best features from the overall features in the dataset. ROC analysis and a 50-50 split for training and testing, were used to assess the performance of logistic regression classifier and AUC, Sensitivity, Specificity, and p-value were obtained. (Figure 1)

Results

LASSO regularization (alpha = 1) was performed with all the 4880 features for feature selection and 40 most prominent features were selected for logistic regression modelling. Our entire dataset ROC analysis showed an accuracy of 100%, sensitivity of 100% and specificity of 100% with p-value of 1.256-12, while our 70-30 split ROC analysis showed an accuracy of 96.7%, sensitivity of 85% and specificity of 100% and a p-value of 0.003; Our 50- 50 split ROC analysis showed an accuracy of 94%, sensitivity of 75%, and specificity of 100% and a p-value of 0.0008. (Figure 2, 3, 4)

Conclusions

An MRI-radiomic phenotype is predictive of the increase in TMB (Hypermutation) in both primary and recurrent gliomas.

Fig. 1 (abstract P432).

See text for description

Fig. 2 (abstract P432).

See text for description

Fig. 3 (abstract P432).

See text for description

Fig. 4 (abstract P432).

See text for description

P433 Advances in multiplex fluorescence immunohistochemistry: 9 color imaging; whole slide multispectral

Carla Coltharp, PhD, Yi Zheng, PhDRachel Schaefer, Ryan Dilworth, PhD, Linying Liu, Chichung Wang, Kristin Roman, MS, Clifford Hoyt, MS, Peter Miller, MS

PerkinElmer, Inc., Hopkinton, MA, USA

Correspondence: Peter Miller (peter.miller@perkinelmer.com)

Background

We describe two advances in multispectral fluorescence immunohistochemistry, a powerful tool for quantifying interactions within the tumor microenvironment. First, a fully-automated 8-plex assay plus DAPI counterstain on the same tissue section. Second, a novel scanning method that produces a multispectral whole slide scan of 6 markers plus DAPI counterstain in ~6 minutes (1x1.5 cm tissue section).

Methods

FFPE primary tumors were immunostained using Opal™ reagents manually or on a Leica BOND RX™. Imagery was acquired on a Vectra Polaris® automated imaging system and analyzed with inForm® and MATLAB® software.

Results

Two new Opal™ reagents (Opal 480 and Opal 780) were combined with currently available Opal 7-color kits to stain and distinguish 8 markers plus DAPI when imaged on the Vectra Polaris®.Figure 1 shows a 9-color panel on lung cancer: CD20 (Opal 480), PD-L1 (Opal 520), CD8 (Opal 540), FoxP3 (Opal 570), CD68 (Opal 620), PD-1 (Opal 650), Ki67 (Opal 690), and PanCK (Opal 780). Colors assigned to each marker, and associated component planes, are shown in Figure 1B.These 8 markers combine to generate more than 20 phenotypes relevant to immuno- oncology that can be studied in relation to local PD-L1 expression and proliferation state (Ki67+/-). For example, while the density of CD8+ cells was 8-fold lower in tumor than stroma (150 vs 1200 cells/mm^2), those CD8+ cells were >4x more likely to be proliferating in tumor vs stroma (28% vs. 6%).To interrogate interactions across a whole section, we additionally developed a multispectral whole-slide scanning method, demonstrated on lung cancer using a subset of 7 stains from the 9-color panel above. Phenotype and expression-level assessments of the unmixed whole slide scan describe distribution patterns of immune cells across the entire section.In measurements of crosstalk and dynamic range, whole-slide multispectral scanning performed comparably to established field-based multispectral imaging, and outperformed conventional fluorescence scanning by reducing crosstalk from up to 8% to under 2% (typically <0.5%) and extending the dynamic range of some channels by more than 50-fold.

Conclusions

We introduce a 9-color fIHC assay that distinguishes 8 markers plus DAPI counterstain on the same tissue section, increasing the depth of cellular interactions that can be studied within the tumor microenvironment.Additionally, we introduce a whole slide multispectral imaging method that provides rich quantitation of interactions among 6 markers at length scales spanning from cell biology to tumor physiology.

Fig. 1 (abstract P433).

See text for description

P434 Mathematical modeling of CAR T cell therapy outcomes to develop design specifications for CAR T cell engineering

Amritava Das, PhD¹, Rachel Grosser, undergraduate², Ambar Velazquez Albino, BS Student³, Krishanu Saha², Christian M. Capitini, MD²

¹Morgridge Institutes for Research, Madison, WI, USA; ²University of Wisconsin - Madison, Madison, WI, USA; ³University of Puerto Rico - Mayaguez, Mayaguez, PR, USA; ⁴Morgridge Institute for Research, Madison, WI, USA

Correspondence: Christian M. Capitini (ccapitini@pediatrics.wisc.edu)

Background

Chimeric antigen receptor (CAR) T cell therapy has demonstrated success in clinical trials [1], and two such therapies have now been approved within the USA [2]. Due to the heterogeneity of apheresis products from heavily treated cancer patients, no algorithms exist to predict the efficacy of manufactured CAR T cell products. CAR T cells are living drugs, that are capable of division, anti-tumor cytotoxicity and cytokine secretion post infusion. Based on previous models of virus-T cell interaction [3], we developed new models to estimate post-infusion CAR T cell division and cytotoxicity. Simulation results reveal important characteristics when elite populations of CAR T cells are present in the pool of infused CAR T cells.

Methods

Models were implemented in COPASI [4], a biochemical network simulation platform. Patient CAR T cell performance data extracted from previously published studies using WebPlotDigitizer [5]. Fitting of model parameters to published patient data and model inference performed using ABC-SysBio [6], a python-based toolkit implementing Approximate Bayesian Computation. Post-processing of outputs from COPASI and ABC-SysBio was performed on MATLAB.

Results

Any of the models developed (selection shown in Figure 1) could be fit to patient data, and ABC-SysBio can be implemented to select between the models given patient data. Model presented in figure 1A was used to determine the effects of having a large population of CAR T cells which can only undergo one cell division and a smaller elite population (1/1000th of maximum at infusion) capable of unlimited expansion. Broadly, the rates of division of high performance clonal CAR T cells (at most 4 h doubling time), and the rates of memory formation of CAR T cells (at least 0.383/day) were found to most significantly impact tumor clearance, while the cytotoxicity of the CAR T cells (ranging from 2 – 16 /day/cell) did not significantly impact tumor clearance in the mathematical models (Figure 2).

Conclusions

Surprisingly memory formation is more associated with complete remission than cytotoxicity and mirrors previous findings that correlate therapeutic success with memory formation [7]. Estimation of the parameter values for number of CAR T cell divisions, rates of division, memory formation, memory reactivation, CAR T cell depletion (exhaustion and non-exhaustion induced death) and anti-tumor cytotoxicity can be useful in determining the design specifications of successful CAR T cell therapy administrations across various clinical trials. Extrapolation of this model in a prospective setting will be needed for further validation.

References

1. Gill S, Maus MV,Porter DL. Chimeric antigen receptor T cell therapy: 25years in the making. Blood Rev. 2016; 30(3): 157-67.

2. June, CH, Sadelain M. Chimeric Antigen Receptor Therapy. N Engl J Med. 2018; 379(1): 64-73.

3. Wodarz D,Thomsen AR. Effect of the CTL proliferation program on virus dynamics. International Immunology. 2018; 17(9): 1269-1276.

4. Hoops S, et al. COPASI--a COmplex PAthway SImulator. Bioinformatics. 2006; 22(24): 3067-74.

5. Burda, BU, et al. Estimating data from figures with a Web-based program: Considerations for a systematic review. Res Synth Methods. 2017; 8(3): 258-262.

6. Liepe J, et al. ABC-SysBio--approximate Bayesian computation in Python with GPU support. Bioinformatics. 2010; 26(14): 1797-9.

7. Fraietta JA, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med. 2018; 24(5): 563-571.

Fig. 1 (abstract P434).

Mathematical modeling frameworks developed

Fig. 2 (abstract P434).

Heatmaps indicating the number of cancer cells

P435 Image-based analysis of the myeloid cell landscape in the 3D co-culture with tumor cells

Gera Goverse, PhD¹, Kuan Yan, PhD¹, Lars Geulen², Paul Vink, BS², Leo Price¹, Lidia Daszkiewicz, PhD¹

¹OcellO B.V., Leiden, Netherlands; ²Aduro Biotech, Oss, Netherlands

Correspondence: Gera Goverse (gera.goverse@ocello.nl)

Background

The myeloid cell compartment plays an important role in anti-tumor immune responses and represents a heterogeneous population with both cancer-promoting and cancer-restraining actions. Unleashing the full potential of cancer immunotherapies requires an understanding of the cellular mechanisms that govern these opposite actions. To date, high throughput relevant preclinical models for dissecting the interactions between different cellular players in the tumor microenvironment are lacking. Previously we have shown that our 3D image-based co-culture system allows assessing efficacy of immune modulators to enhance PBMC infiltration and tumoroid killing. Our main goal was to improve this model by incorporating a more complete human immune system. To do that we first generated diverse myeloid populations in a 3D environment and then used our image-based platform to describe the different subsets. The image analysis software was trained on a set of features that reproducibly allowed discrimination between undifferentiated monocytes, M1 and M2 macrophages and dendritic cells. The different myeloid subsets were next co-cultured with tumor cells to analyze the complex cellular interplay of the TME.

Methods

Different myeloid populations were generated in 3D from monocytes derived from healthy donors PBMCs. Polarized M1 and M2 macrophages, DCs and undifferentiated monocytes were then co-cultured in 3D with SKBR3 tumor cells or 3D tumoroids derived from this cell line. The cellular interactions were visualized using high-content microscopy and quantified with multiparametric morphometric analysis with OMinerTM software.

Results

3D image analysis enabled the discrimination of immune-tumor cell interactions and revealed the effect of myeloid cells on tumor growth in co-culture. Our approach also enables the analysis of how tumor-driven mechanisms regulate myeloid cell differentiation and contribute to the immunosuppressive microenvironment. These results provide a means to elucidate the bi-directional interplay between tumor and immune cells and allows for analysis of functional reprograming of the suppressive population towards a M1 phenotype induced by drug candidates.

Conclusions

The 3D assay presented here enables visualization and measurement of effects of immunotherapies on cells that engage in a more physiologically relevant spatial setting than when culturing them in traditional 2D cultures. Using morphological measurements different myeloid cell subsets can be distinguished, which offers a very attractive alternative for complex and labor-intensive phenotyping based on markers expression and cytokine release profiling. The ultimate goal is to develop a highly sophisticated platform for testing cancer immunotherapies that combines the complexity of the TME and the robustness of a high throughput screening platform.

P436 Image analysis simulations of needle biopsy tumor specimens to investigate CD8+ TIL heterogeneity

Thomas Herz, PhD¹, Victor Matvienko¹, Tobias Wiestler, PhD¹, Rene Korn, PhD¹, Keith Steele, DVM, PhD²

¹Definiens AG, Munich, Germany; ²MedImmune, Gaithersburg, MD, USA

Correspondence: Thomas Herz (therz@definiens.com)

Background

Core needle biopsies are used to histologically assess tumors when surgical excision is impractical. Such small samples may not be representative given the known heterogeneity of immune cell distribution, including CD8+ tumor infiltrating lymphocytes (TILs)[1].

Methods

Initially, 20 immunolabeled slides from purchased non-squamous NSCLC tumor resections were scanned and tumor region was manually annotated[1]. CD8(+) TILs were detected using Definiens Developer XD™ software[1,2]. Needle biopsies were simulated using an elliptical shape, with multiple iterations applied by varying the size, angle and positioning of that ellipse across the full resection using Python programming language[3], totaling in 24,200 single needle simulations per case. CD8(+) TIL density was determined for the tumor region contained within each simulated portion. Using the statistical software R[4], individual cores were compared to other cores in each sample, to the full tumor region and across all 20 cases.

Results

The heterogeneity of the CD8(+) TIL distribution is very well reflected in the statistical analysis of the number of CD8(+) TILs actually found within the needle biopsy to the expected number, based on the size of the needle ellipse and full slide CD8(+) TIL density. Even in cases with generally high correlation, a single biopsy location with changing the needle size or the angular component of the needle direction only can already produce a set of non- representative CD8(+) TIL densities. In a about 15% of all simulated cores, no CD8(+) TIL was found in the tumor region, spanning all dimensions of variation used in the simulation equally as well as cases.

Conclusions

One needle biopsy insufficiently represents the CD8+ TIL density of resected non-squamous NSCLC. Determining a clinically-feasible number of cores to accurately assess CD8 requires further study. Systematic measurement of sampling error should be extended to other markers of the immune response to cancer whose expression is known to be heterogenous, such as PD-L1.

References

1. Steele K, Tan TH, Korn R. Measuring multiple parameters of CD8+ tumor-infiltrating lymphocytes in human cancers by image analysis. J Immunother Cancer 2018;6:202. Baatz M, Zimmermann J, Blackmore CG. Automated analysis and detailed quantification of biomedical images using Definiens Cognition Network Technology®. Combinatorial Chemistry & High Throughput Screening 2009;12:908–163. Python Software Foundation [https://www.python.org]4. R Core Team (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. [https://www.R-project.org]

P437 In vivo synergistic effect of checkpoint blockade and radiation therapy against chordomas in a humanized mouse model

Wataru Ishida, MD¹, Kyle McCormick, BA², Aayushi Mahajan, MS², Eric Feldstein, BS², Michael Lim, MD¹, Jeffrey Bruce², Peter Canoll, MD PhD², Sheng-fu L. Lo, M.D.¹

¹Johns Hopkins University, Baltimore, MD, USA; ²Columbia University Medical Center, New York, NY, USA

Correspondence: Sheng-fu L. Lo (larrylo@jhmi.edu)

Background

It has been a challenge to apply immunotherapy (IT) to patients with chordomas, due to lack of clinically-translatable in vivo models. Currently, there are no well-established murine chordoma cell lines that can be injected to syngeneic mice or no transgenic mouse models that develop chordomas spontaneously, which would allow us to study the interaction between murine chordomas and murine immune cells. Hence, we aimed to develop a humanized mouse model, where human immune cells are engrafted into immunodeficient mice,[1,2] to overcome this limitation by studying the interaction between human immune system and human chordomas. We also sought to utilize it to study synergistic effect between IT and radiation therapy (RT) against chordoma.

Methods

Fifteen 10-12-week-old NSG mice were sub-lethally (1.5Gy) irradiated and then implanted with fetal thymic tissue and CD34+ stem cells that had been harvested from a fetus, whose HLA-types were partially-matched with those of the U-CH1 chordoma cell line. Reconstitution of immune cells in NSG mice was confirmed 8 weeks post- transplantation and then each animal (15 humanized NSG mice and 12 naïve NSG mice) was injected with U-CH1 cell suspension bilaterally and subcutaneously. Next, they were treated for 4 weeks as follows: A) control, isotype antibodies (Abs) injection (n=3), B) anti-human-PD-1 Abs (n=4), C) RT + isotype Abs (n=3, unilaterally to the left- sided tumor, 8Gy x 4), D) anti-human-PD-1 Abs and RT (n=5), E) naïve NSG mice (n=6, without the engraftment of human immune cells) + isotype, and F) naïve NSG mice (n=6) + anti-human-PD-1 Abs. During and after the treatment, anti-tumor activities were monitored via tumor size, flow cytometry, qRT-PCR, and immunohistochemistry.

Results

One week after the treatment, on the irradiated side, (D) demonstrated lowest tumor volume (Figure 1), highest number of human PBMCs, highest % of CD8+ human T cells, highest % of CD45RO+CD4+ human (memory) T cells, and lowest % of PD-1+CD8+ human T cells in the tumors via flow cytometry (Figure 2), and highest IFN- gamma in the tumors via qRT-PCR, compared to the other five groups with statistical significance. On the non- irradiated side, similarly D) had the smallest tumor compared to the others (P=0.09).

Conclusions

We demonstrated that this humanized mouse model could be a revolutionary platform to investigate IT against rare cancers such as chordomas, where murine equivalent cell lines are currently unavailable. The direct synergistic effect between IT and RT against chordoma as well as the potential abscopal effect was observed.

Acknowledgements

We would like to thank all members of Herbert Irving Comprehensive Cancer Center at Columbia University Medical Center for generous support and its shared resource as well as CCTI, especially Drs. Hui Wang and Yong- Guang Yang at the CCTI humanized mouse core as well as Dr. Siu-Hong Ho, the director of the CCTI flow cytometry core and Assistant Professor of Medical Sciences. We also would like to thank The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University and its oncology shared resources, particularly Drs. Alan Meeker and Sujayita Roy. These studies used the resources of the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources funded in part through Center Grant P30CA013696. Research reported in this publication was performed also in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

References

1. Kalscheuer H, Danzl N, Onoe T, et al. A model for personalized in vivo analysis of human immune responsiveness. Sci Transl Med. 2012;4(125):125ra130.2. Zitvogel L, Pitt JM, Daillere R, Smyth MJ, Kroemer G. Mouse models in oncoimmunology. Nat Rev Cancer. 2016

Ethics Approval

This study was approved by Columbia IACUC, protocol number AAAQ8458.

Fig. 1 (abstract P437).

See text for description

Fig. 2 (abstract P437).

See text for description

P438 Effect of CD3 affinity and normal tissue expression on the biodistribution and tumor targeting of MUC16xCD3 bispecific antibodies in MUC16 and CD3 humanized mice

Marcus Kelly, PhD, Alison Crawford, PhD, Jason Giurleo, PhD, Richard Tavaré, PhD, Sosina Makonnen, Carlos Hickey, Makenzie Danton, Cody Arnold, Lauric Haber, Eric Smith, PhD, Dangshe Ma, William Olson, PhD, Gavin Thurston, PhD, Jessica Kirshner, PhD

Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA

Correspondence: Marcus Kelly (marcus.kelly@regeneron.com)

Background

The tumor associated glycoprotein MUC16 is highly expressed in ovarian cancer with limited normal tissue expression, making it a suitable target for the development of CD3 binding T-cell engaging bispecific antibodies. Here we used non-invasive immuno-PET imaging as a powerful tool to determine the impact of each antigen binding arm on bio-distribution of MUC16-CD3 bispecific antibodies in mice. To dissect the role of CD3 affinity on antibody distribution, we assessed two bispecifics with varying CD3 affinity; MUC16-CD3_low and MUC16-CD3_high^, alongside the bivalent parental MUC16 antibody.

Methods

Antibodies were radiolabeled with positron emitting radionuclide Zirconium-89 (⁸⁹Zr) using the chelator deferoxamine (DFO) and demonstrated high radiochemical purity and immunoreactivity. Initial imaging and biodistribution studies were performed in SCID mice bearing MUC16+ OVCAR3 ovarian tumor xenografts to validate the MUC16 binding arm of the antibodies. Localization of ⁸⁹Zr-MUC16-CD3_low and ⁸⁹Zr-MUC16-CD3_high was next measured in tumor-free MUC16 and CD3 double humanized immunocompetent mice. A subsequent study assessed blocking CD3-dependent localization of ⁸⁹Zr-MUC16-CD3_high by a control CD3 antibody. Parental ⁸⁹Zr- MUC16-MUC16 antibody was assessed in matched humanized and normal mice to determine localization to MUC16 expressing normal tissues. Lastly, the uptake of ⁸⁹Zr-MUC16-CD3_low or ⁸⁹Zr-MUC16-CD3_high to ID8/VEGF/hMUC16 tumors was assessed in the double humanized mice.

Results

Immuno-PET imaging of 89Zr-MUC16-CD3_low, ⁸⁹Zr-MUC16-CD3high and ⁸⁹Zr-MUC16-MUC16 all demonstrated high and specific targeting to OVCAR3 xenografts (~70%ID/g). In the MUC16 and CD3 humanized mice, very high localization of ⁸⁹Zr-MUC16-CD3_high to CD3+ lymphoid tissues (spleen and lymph nodes) was observed. Relative ⁸⁹Zr-MUC16-CD3_low uptake in lymphoid tissues was greatly reduced. Conversely, blood levels of ⁸⁹Zr- MUC16-CD3_high were lower than ⁸⁹Zr-MUC16-CD3_low, resulting in higher tissue:blood ratios by ⁸⁹Zr-MUC16- CD3_high. Blocking with control CD3 bispecific significantly reduced localization of ⁸⁹Zr- MUC16-CD3_high to lymphoid tissues. Specific uptake of ⁸⁹Zr-MUC16-MUC16 in normal tissues was not observed. ⁸⁹Zr-MUC16-CD3_low and 89Zr-MUC16-CD3_high both showed significant uptake (50-60%ID/g) in ID8/VEGF/hMUC16 tumors. Tumor uptake between the antibodies was generally not significantly different despite the high lymphoid uptake of ⁸⁹Zr- MUC16-CD3_high.

Conclusions

⁸⁹Zr-MUC16-CD3_low and ⁸⁹Zr-MUC16-CD3high demonstrated specific localization to MUC16+ tumors and CD3+ lymphoid tissues, with lymphoid distribution correlating to relative CD3 affinity. Both MUC16-CD3 bispecifics demonstrated clear tumor localization in the presence of CD3+ tissues. This work demonstrates that immuno-PET is an ideal technology to monitor bispecific localization in vivo. Further studies may investigate any correlation between antibody biodistribution as monitored by immuno-PET and toxicity or efficacy observed during the optimization of these promising therapeutics.

P439 A novel high-throughput, high-content real-time imaging platform to assess immunogenic cell killing activity of immunotherapeutic agents using patient-derived tumor samples

Jenny Kreahling, PhD, Melanie Mediavilla-Varela, PhD, Melba Marie Page, PhD, Soner Altiok, MD, PhD

Nilogen Oncosystems, Tampa, FL, USA

Correspondence: Jenny Kreahling (jenny@nilogen.com)

Background

Immuno-oncology has revolutionized cancer care for many cancer types, however, the development of novel immunotherapeutics still faces many challenges due to lack of drug screening platforms that represent the complexity of the tumor microenvironment. Conventional cytotoxicity assays, such as chromium 51 and LDH release are limited in providing clinically relevant data about immunogenic cell death. The goal of this study was to develop an integrated confocal-based high-throughput, high-content real-time imaging platform to assess immunogenic cell killing activity of novel immunotherapeutic agents and to develop rational drug combinations using patient-derived tumor samples.

Methods

All patient tumor samples were obtained with patient consent and relevant IRB approval. For the confocal imaging platform, unpropagated 3D tumoroids measuring 100-150 micron in size were prepared from fresh tumor samples of non-small cell lung cancer using a proprietary technology developed at Nilogen Oncosystems. Cell-match studies utilized autologous patient-derived cell lines that were isolated and propagated from each patient’s tumor.

Results

In Cell-match studies, tumor cells and tumor infiltrating lymphocytes (TILs) were labeled with different cell tracker fluorescent dyes to monitor cell movements and locations. For 3D tumoroid assays samples were pre-labeled with proprietary fluorogenic markers to identify live and dead tumor cells. After treatment with different immune- stimulatory agents, real-time confocal imaging analysis was performed to assess apoptotic tumor cell death which was evaluated via the detection of changes in the permeability of cell membranes and activation of caspase 3 pathway. Comprehensive flow cytometry analysis was performed to corroborate confocal imaging findings on immunogenic tumor cell death (LIVE/DEAD viability markers and cleaved caspase 3) and TIL activation (CD25, CD69, Ki-67 and granzyme expression in CD4 and CD8 positive lymphocytes). A custom image analysis algorithm was developed for the collection of data in a structurally relevant environment on quantification of marker-specific cell number, cell viability and apoptosis in addition to structural and functional analysis of cells in intact 3D tumoroids.

Conclusions

The confocal-based high-throughput and high-content real-time imaging platform described here is physiologically relevant and allows rapid screening of multiple drugs and drug combinations based on their immunogenic cell killing activity in a cost-effective manner to accelerate drug discovery.

P440 Open-source digital image analysis of whole-slide multiplex immunohistochemistry

Nikhil Lonberg, HSDG, Nikhil Lonberg, HSDG, Nikhil Lonberg, HSDG, Carmen Ballesteros Merino, PhD, Shawn Jensen, PhD, Bernard Fox, PhD

Robert W Franz Cancer Center, Earle A Chiles Research Institute, Portland, OR, USA

Correspondence: Bernard Fox (foxb@foxlab.org)

Background

Successful digital image analysis (DIA) of cancer tissue is accurate and reproducible. These points of emphasis have brought procedures like the tissue microarray (TMA) and hotspot regions of interest (ROI) under scrutiny. The nature in which a pathologist selects TMAs and ROIs is conducive to bias. Whole Slide Imaging (WSI) offers a solution in its unbiased region selection and consideration of a larger tissue sample. However, options for softwares that can handle such large throughput are scarce. Additionally, while multiplex immunohistochemistry (mIHC) is becoming popular [1], documentation of its digital analysis tools remains minimal [2]. The combination of these procedures potentiates a deeper understanding of the tumor microenvironment. This study presents the whole-slide mIHC analysis capabilities of QuPath, an open-source application developed at Queen’s University Belfast [3].

Methods

A multiplex fluorescent stain panel was performed on patient samples. The slides were imaged and cells were detected and segmented in QuPath. QuPath parallelizes its workload to manage whole-slide throughput efficiently. Custom scripts were written that exhibit machine-learning and thresholding techniques to aggregate cell phenotype totals. Additionally, cell detection numbers were generated for specific ROIs and compared to a commercial DIA software. All scripts and protocols in this study are made public for replication and improvement by the community.

Results

QuPath’s automated cell segmentation and classification were demonstrated as a proof-of-concept for whole-slide multiplex immunohistochemistry analysis. Across an entire slide, cells positive for multiple markers were effectively segmented and properly phenotyped.

Conclusions

Open-source applications have become a driving force for innovation and collaboration in the field of digital image analysis. In litigating the strengths and weaknesses of QuPath for whole-slide mIHC analysis, we aim to advance the field’s knowledge of available software tools and bring attention to necessary points of growth in this rapidly changing industry.

References

1. Feng Z, Jensen SM, Messenheimer DJ, Farhad M, Neuberger M, Bifulco CB, Fox BA. Multispectral imaging of T and B cells in murine spleen and tumor. J Immunol. 2016;196:3943-3950. 2. Blom S, Paavolainen L, Bychkov D, Turkki R, Mäki-Teeri P, Hemmes A, Välimäki K, Lundin J, Kallioniemi O, Pellinen T. Systems pathology by multiplexed immunohistochemistry and whole-slide digital image analysis. Sci Rep. 2017; 7:1-13. 3. Bankhead P, Loughrey MB, Fernández JA, Dombrowski Y, McArt DG, Dunne PD, McQuaid S, Gray RT, Murray LJ, Coleman HG, James JA, Salto-Tellez M, Hamilton PW. Qupath: open source software for digital pathology image analysis. Sci Rep. 2017; 7:1-7.

P441 Withdrawn

P442 Automated quantification of whole-slide multispectral immunofluorescence images to identify spatial expression patterns in the lung cancer microenvironment

Lorenz Rognoni, PhD¹, Vinay Pawar, PhD¹, Tze Heng Tan, MSc, PhD, DiplIng¹, Felix Segerer, PhD¹, Philip Wortmann, PhD¹, Sara Batelli, PhD¹, Pierre Bonneau¹, Andrew Fisher, PhD², Gayathri Mohankumar, MS², David Chain, PhD³, Michael Surace, PhD³, Keith Steele, DVM, PhD³, Jaime Rodriguez-Canales, MD³

¹Definiens AG, Munich, Germany; ²Definiens Inc., Cambridge, MA, USA; ³Medimmune, Gaithersburg, MD, USA

Correspondence: Jaime Rodriguez-Canales (rodriguezcanalesj@MedImmune.com)

Background

Advancement in cancer immunotherapy is associated with unraveling the complexities of immune suppressive mechanisms across different cancers. Quantification on multispectral multiplex-immunofluorescence (mIF) images allows detection of several biomarkers in a single section. In addition, new evidence using mIF techniques suggests that spatial analysis reveals novel insights in the tumor microenvironment. However, multispectral imaging is tile based due to long scanning periods, which leads to insufficient data acquisition for significant spatial analysis. In this study, our goal is to develop an automated workflow to study the spatial patterns of infiltrating cells in the tumor microenvironment based on multispectral mIF whole slide scans. This was used to study the relationship between tumor proliferation and immune-response in non-small cell lung cancer (NSCLC) resections.

Methods

45 formalin fixed, paraffin embedded NSCLC resection samples were stained with a custom-developed 7-plex mIF panel (CD68, CD8, Ki67, PD1, PD-L1, pancytokeratins-CK & DAPI) using the Opal method (PerkinElmer). Tiled scans were acquired with a Vectra Polaris (PerkinElmer) multispectral imaging system. Definiens Insights services with custom algorithms was used to analyze the unmixed multispectral data as whole slide images.

Results

The 7-plex Opal staining was optimized for an automated staining platform to ensure high throughput and consistent sample processing. We developed a workflow which composes the tiled unmixed multispectral data to a whole-slide image and optimizes the layers for screen display and automated image analysis. Furthermore, images were shared on Definiens collaboration platform along with a chromogenic-IHC pseudocolor of the IF CK/DAPI signals and co- registered H&E section for pathologist annotations. These annotations were used in defining tumor center and invasive margin. The image analysis includes single-cell detection on the complete slide along with classification of subpopulations based on multi-marker positivity of individual cells. Part of the analysis is a high-quality tumor stroma separation based on the CK signal. The single-cell readouts were used to construct spatial biomarker- expression patterns (Figure 1), which shows distinct immunological areas in the tumor region and a possible correlation between tumor proliferation (Ki67) with the immune activity in the invasive margin.

Conclusions

We developed an automated workflow for quantitative mIF image analysis on whole-tissue slides. Additionally, our image analysis permitted identification of spatial patterns for immunoprofiling, where we could overcome the limitation of small regions of interests and provide significant amount of data on the whole tumor region.

Ethics Approval

Commercially available samples were obtained according to the declaration of Helsinki for this study.

Fig. 1 (abstract P442).

See text for description

P443 Haplotype human immune system (HIS) modeling and co-engraftment of PDX: ImmunoGraft® platform for evaluation of pharmacodynamics of Immuno Oncology therapeutics

Bhavana Verma, PhD¹, Champions Oncology c/o Mancini, PhD¹, Angela Davies, MD¹, David Sidransky, MD², Amy Wesa, PhD¹, Neal Goodwin, PhD¹

¹Champions Oncology, Rockville, MD, USA; ²Johns Hopkins University, Baltimore, MD, USA

Correspondence: Amy Wesa (awesa@championsoncology.com)

Background

Recent success of several immunotherapeutic regimens, such as checkpoint modulators has boosted development of next generation IO agents underscoring the need for robust preclinical platform to evaluate IO-therapies. The Champions ImmunoGraft® model utilizing humanized NOG mice is an innovative pre-clinical model for assessing the efficacy of IO agents against solid tumors. Improved immunodeficient mouse strains, such as triple transgenic NOG-EXL mice expressing huIL-3 and huGM-CSF, allows for superior HIS development. In this study, we evaluated human immune lineage development, tumor infiltrating leukocytes, and tumor response to checkpoint inhibitor utilizing this humanized mouse platform.

Methods

Human immune system component development in peripheral blood was assessed by flow cytometry across 9 donors 8 weeks post intravenous transplantation of cord-blood (CB) C34+ hematopoietic cells (HSC) in NOG and NOG-EXL mice. Next, NOG-EXL mice were humanized with CB-HSC from 2 donors, monitored for engraftment then implanted with a patient-derived xenograft (PDX) tissue from a non-small cell lung carcinoma (NSCLC) patient. Immune cell populations (T cells, macrophages, myeloid-derived suppressor cells (MDSC) and dendritic cells (DC)) were evaluated by flow cytometry at 4 and 6 weeks post-tumor implantation in various tissues. For nivolumab (α-PD-1; 10mg/kg) treatment, dosing was initiated at a tumor volume of 80-150 mm3. Responses were determined as changes in tumor volume.

Results

NOG-EXL (100%) consistently engrafted more readily than NOG (80%), with greater than 25% huCD45+ cells in the periphery. Some donor to donor variability was observed in HIS engraftment in both mouse strains; both strains permitted T cell, B cell and some myeloid cell development. T cell lineage development was equivalent in both strains at 12-weeks post–HSC transplantation. Improved myeloid lineage (CD33+) development was found in NOG- EXL animals. Macrophage, MDSC, as well as T cells were found in tumor infiltrates. Evaluation of PD-1 blockade in NSCLC PDX ImmunoGraft® in NOG-EXL mice indicated HIS donor variability impacted treatment efficacy in vivo.

Conclusions

ConclusionsImproved mouse strains that allow for robust reconstitution of immune compartments enhances value of ImmunoGraft® platform for screening new IO therapies. We demonstrated that NOG-EXL mice allow better engraftment and HIS development compared to NOG. Evaluation of nivolumab efficacy in NSCLC PDX model in this enhanced ImmunoGraft® indicates that PD-1 blockade is feasible, and offers an opportunity to evaluate therapeutics targeting myeloid populations. The ImmunoGraft® has the potential to advance translational IO drug discovery and development.

P444 Autologous human immune system (HIS) ImmunoGraft®: Mobilized peripheral blood (MPB) derived CD34 engraftment and lineage development

Bhavana Verma, PhD¹, Georgia Chen, PhD², Edmund Waller, MD, PhD, FACP², Neal Goodwin, PhD¹, Angela Davies, MD¹ , Amy Wesa, PhD¹, Nabil Saba, MD²

¹Champions Oncology, Rockville, MD, USA; ²Winship Cancer Institute of Emory University, Atlanta, GA, USA

Correspondence: Amy Wesa (awesa@championsoncology.com)

Background

Humanized mice generated by hematopoietic stem cells (HSC) transplant and co-engrafted with a patient-derived xenograft (PDX) represents a promising pre-clinical platform for studying immunological response to cancer and evaluation of immunotherapeutic interventions. These models are limited by the fact that the immune system developed in these mice is allogeneic to the tumor. To address this, we have innovated a platform to reconstitute autologous HIS in immunodeficient NOG-EXL mice with mobilized peripheral blood (MPB)-CD34 cells derived from a head and neck cancer patients along with PDX generated from the same patient tumor tissue.

Methods

Patients with head and neck squamous cell carcinoma (HNSCC) were consented for tumor and IRB approved apheresis for stem cell collection at Winship Cancer Institute of Emory University. The HSC collection protocol included mobilization with G-CSF and Plerixafor, prior to apheresis, isolation and cryopreservation of MPB-CD34 cells. PDX were established from biopsies or surgical specimens by passaging in immunodeficient mice. In parallel, Irradiated NOG-EXL mice were humanized by intravenous transplantation of HSC. Engraftment of human immune components (T cells, B cells and myeloid cells) in peripheral blood was assessed by flow cytometry up to 25 weeks, with terminal collections and assessment of immune components in spleen and bone marrow at 30 weeks.

Results

Twenty-eight PDX models were generated from 43 patients with HNSCC and evaluated by next-generation sequencing. In parallel, HIS engraftment assessed in circulation was observed at 8 weeks post-transplant in 100% of NOG-EXL mice; with 5-20% hCD45+ cells present. B cell development was predominant at early timepoints and declined over time. T cell development was observed starting at 15 weeks, with both CD4 and CD8 T cell subsets observed. Strong myeloid lineage (CD33+) development was observed starting at 8 weeks and persisted throughout the study. At 30 weeks we plan to evaluate immune compartments in blood, spleen and bone marrow of the humanized mice.

Conclusions

HSC mobilized from an adult patient with HNSCC was used to engraft and generate HIS-mice with B cells, T cells, and myeloid cells. In parallel, a matched PDX model was established from the same patient. The co-engraftment of HIS mice with an autologous PDX is in progress. This data demonstrate that mobilization and apheresis of HNSCC patients is technically and clinically feasible, and may permit the establishment of autologous HIS-PDX mice. The advanced autologous CD34-ImmunoGraft® has the potential to advance translational ImmunoOncology drug discovery and development.

Ethics Approval

Apheresis was performed based on an IRB approved protocol implemented at Winship Cancer Institute of Emory University

P445 Human CD33+ myeloid cells support metastatic colonization in a humanized mouse model of melanoma

Chun Yu, PhD¹, Jan Martinek, PhD¹, Te-Chia Wu¹, Kyung Kim¹, Elaheh Ahmadzadeh, PhD¹, Rick Maser², Florentina Marches¹, Patrick Metang², Pierre Authie², Hannah Brookes¹, Joshy George, PhD¹, Jacques Banchereau¹, A. Karolina Palucka, MD, PhD²

¹The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA; ²The Jackson Laboratory for Mammalian Gen, Bar Harbor, ME, USA

Correspondence: A. Karolina Palucka (karolina.palucka@jax.org)

Background

Metastatic melanoma remains an incurable disease for some patients due to treatment resistance and metastatic dissemination. Here we show that metastatic melanoma tumor samples from patients are infiltrated with myeloid cells and display STAT3-driven transcriptional profiles.

Methods

To study the biology of myeloid cells and melanoma cells in vivo, we used NSG mice with transgenic expression of human hematopoietic cytokines SCF/GM-CSF/IL-3 (NSG-SGM3) engrafted with human CD34+ hematopoietic progenitor cells.

Results

Humanized NSG-SGM3 mice when implanted with Me275 human melanoma cell line subcutaneously, developed multi-organ distant melanoma tumors. This was linked with the presence of circulating tumor cells and elevated serum biomarker lactate dehydrogenase (LDH). Among six melanoma cell lines analyzed, potential to form distant tumors was correlated with G0/G1 cell cycle status and proliferative capacity. Treatment with VEGF inhibitor Avastin significantly decreased the number of melanoma tumors in the spleen but not in the liver. Adoptive transfer experiments confirmed the critical role of human CD33+ myeloid cells in metastatic colonization and these cells displayed STAT-3-driven transcriptional profiles.

Conclusions

Thus, our model enables mechanistic and pre-clinical studies for the development of novel treatment strategies targeting human-specific molecular pathways controlling melanoma dissemination.

P446 Using monocytes to instill brain tumors with TILs

Tomasz Zal, PhD, Meenakshi Shanmugasundaram, Anna Zal, Shouhao Zhou, Amy Heimberger, MD, Tomasz Zal, PhD, Felix Nwajei, PhD

University of Texas MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Tomasz Zal (tzal@mdanderson.org)

Background

Tumors that localize in brain, including primary cancers and brain metastases, typically contain infrequent lymphocytic infiltrates. The paucity of tumor infiltrating lymphocytes (TILs) in brain tumors (BTs) presents a challenge for current immunotherapies reliant on TIL reactivation or lymphocyte (T cell) delivery. In emergent view, TILs are hailed by various types of tumor-associated myeloid cells, which can originate either from the organ’s tissue-resident macrophages, or from bone marrow sources. In healthy brain, the myeloid compartment is dominated by the resident microglia, whereas extracranial-derived myeloid cell subsets are rare outside of brain inflammation. However, the basis of TIL recruitment to brain tumor (BT) sites, or lack thereof, remains unclear.

Methods

We investigated TIL dynamics in BTs using longitudinal skull window multiphoton microscopy in multi- reporter mouse strains. Cyan fluorescent brain metastases were induced by infusing, into the carotid artery, syngeneic MCA205 (sarcoma), LLC (lung adenocarcinoma) or B16F10 (melanoma) cells, and GL261 glioblastoma was deposited directly into the brain. Endogenous T cells (hCD2-DsRed) were co-imaged with the microglia and monocytes and/or dendritic cells (CX3CR1-GFP and CD11c-YFP). We further distinguished the microglia, monocytic and dendritic cell subsets using three-color myeloid reporter mice (CX3CR1-GFP/CD11c-YFP/CCR2- RFP).

Results

Present in various densities across BT models, TILs were accompanied by myeloid cells expressing the fractalkine receptor CX3CR1, which is expressed predominantly on the microglia and monocytes or monocyte-derived cells. Fractalkine (CX3CL1) was upregulated on BT-juxtaposed neurons and CX3CR1-expressing monocytes adhered to and scanned the local vasculature. TILs were decreased, and their motility aimless, in mice lacking CX3CR1, resulting in increased BT growth. However, the TIL’s spatial and temporal densities correlated with dendritic-form cells expressing high levels of CD11c (DCs), rather than CD11c-negative monocytes or microglia. TIL migration was confined around CD11c-high DCs and the confinement radius was tighter, in coincidence with cancer cell killing, in tumors undergoing T-cell mediated immune rejection, compared with BTs that were progressing. Depletion of CD11c cells from mice with established BTs led to a rapid abandonment of BTs by the TILs, whereas infusion of (CD11c-negative) monocytes into the blood gave rise to intratumoral CD11c-high dendritic cells, which could attract TILs.

Conclusions

Our results identify the patrolling monocytes and their development into intratumoral DCs as critical cellular mediators of the adaptive immune surveillance of tumors in brain. Moreover, our results establish a proof of principle for the use of monocyte adoptive cell transfer as a potential therapeutic strategy for instilling BTs with TILs.

Acknowledgements

Funding was provided by the NCI (5R01CA137059, 5P50CA127001/DP150072), the University of Texas MD Anderson Cancer Center (IRG 30026195, MRP1152014) and the Schissler Family Fellowship (FN).

Ethics Approval

The study was approved by the MD Anderson Institutional Animal Care and Use Committee, protocol number 00000878-RN02.

Immune Effects of Chemotherapy

P447 A case of checkpoint inhibitor-induced celiac disease

Dana Alsaadi, Neil Shah, MD, Aline Charabaty, MD, Michael Atkins, MD

Georgetown University, Washington, DC, USA

Correspondence: Neil Shah; Michael Atkins (mba41@gunet.georgetown.edu)

Background

Immune checkpoint inhibitors (ICIs) have now become standard of care treatment for many malignancies. ICIs are associated with unique immune mediated adverse events (irAEs) due to dysregulation of immune activation. As treatment with ICIs is becoming more common, rare irAEs are also being recognized. Here we report a case of ICI- induced celiac disease.

Methods

N/A

Results

A 74-year-old Caucasian female with metastatic renal carcinoma received second line nivolumab (anti-PD1 antibody) after initial disease progression on sunitinib. Ipilimumab was added after she failed to respond to six cycles of nivolumab monotherapy. One week after her first cycle of combo treatment, she presented with nausea, vomiting, grade 1 diarrhea, and weight loss. She underwent endoscopy, which showed bile stasis in the stomach, normal appearing stomach mucosa, and non-bleeding erythematous mucosa in the duodenal bulb. Stomach biopsy showed moderate active chronic gastritis. Duodenal biopsy showed moderate chronic active duodenitis with focal neutrophilic cryptitis, mucosal erosions, villous atrophy, mildly increased intraepithelial lymphocytes, and moderate chronic inflammation in the lamina propria pathognomonic of celiac disease. Symptoms improved with gluten-free diet, twice-daily omeprazole and anti-emetics and she was able to continue on treatment.

Conclusions

There has been only one published case reporting ICI-induced celiac disease.[1] Our case report highlights a rare irAE (celiac disease) associated with ICI treatment. It is unclear whether the patient had previously undiagnosed celiac disease or whether ICIs triggered her enteritis. Our patient was able to continue treatment with ICIs with dietary modifications, suggesting correct diagnosis is critical for optimal patient outcome.

References

• 1. Gentile NM, D’Souza A, Fujii LL, Wu TT, Murray JA. Association between ipilimumab and celiac disease. Mayo Clin Proc 2013; 88: 414-417.

Consent

Consent was received.

P448 Blockade of EphB4-ephrin-B2 interaction remodels the tumor immune microenvironment in head and neck cancers

Shilpa Bhatia¹, Sana Karam, MD, PhD²

¹University of Colorado, Anschutz Medical Campus, Aurora, CO, USA; ²University of Colorado Denver, Aurora, CO, USA

Correspondence: Shilpa Bhatia (Sana.karam@ucdenver.edu)

Background

Identifying targets in the tumor microenvironment (TME) that act as barriers to an effective anti-tumor immune response has become an area of intense investigation. In the current study, we established EphB4-ephrin-B2 signaling as a key pathway that regulates both innate and adaptive arms of the immune system. Eph receptor tyrosine kinases and their membrane-bound ephrin ligands have been implicated in human malignancies and in immune cell development, migration, and activation in inflammatory models. However, direct evidence that supports the role of Eph-ephrin interaction in cancer-related immune response is lacking. We hypothesized that EphB4-ephrin-B2 interaction regulates TME by sustaining immunosuppressive cells-Tregs and TAMs thus negatively impacting the functional ability of CD8 T cells.

Methods

We used orthotopic models of head and neck squamous cell carcinoma to determine the role of EphB4-ephrin-B2 interaction in tumor immune microenvironment. Mice were treated with control agent or an EphB4-ephrin-B2 blocker in the absence or presence of radiation (RT). Tumor immune cell infiltrates were analyzed using mass cytometry and flow cytometry applications. ELISA or multiplex cytokine array were utilized to determine circulating cytokine/chemokine levels in plasma.

Results

We observed that inhibition of EphB4-ephrin-B2 signaling in vivo significantly reduced tumor growth and decreased the infiltration of Tregs, TAMs, and increased infiltration and activation of Teffector cells, without affecting CD4 T cell numbers. This was correlated with decreased Treg proliferation and activation when EphB4- ephrin-B2 signaling is inhibited. Since RT remains the mainstay in treatment of head and neck squamous cell cancer (HNSCC) patients, we combined EphB4-ephrin-B2 inhibitor with RT in our tumor model and observed further increase in CD8 and CD4 T cell infiltrates and activation status, and a significant decline in circulating IL-10 and TGF-β1 levels compared to the control group. A significant reduction of TAMs, favoring a polarization towards an anti-tumoral M1 phenotype, was also observed in EphB4-ephrin-B2 inhibitor+RT group. We also compared the efficacy of combining EphB4-ephrin-B2 inhibitor with RT to anti-PDL1+RT in an in vivo model known to develop resistance to anti-PDL1+RT therapy. Our data demonstrated that combining EphB4-ephrin-B2 inhibitor with RT was equally effective to that of anti-PDL1+RT in terms of anti-tumor growth response.

Conclusions

Our study provides the first insight into a novel role for EphB4-ephrin-B2 interaction in modulating tumor immune microenvironment in HNSCC. Our findings present a potential alternative in the form of EphB4-ephrin-B2 targeted therapeutics that can be tested in clinical trials in combination with RT for HNSCC patients.

P449 Improving PDAC outcomes through targeting immune populations and fibrosis by EphB4-ephrinB2 or Treg inhibition combined with radiation

Sana Karam, MD, PhD², Shilpa Bhatia¹

¹University of Colorado, Anschutz Medical Campus, Aurora, CO, USA; ²University of Colorado Denver, Aurora, CO, USA

Correspondence: Sana Karam (Sana.karam@ucdenver.edu)

Background

A driving factor in pancreatic ductal adenocarcinoma (PDAC) treatment resistance is the tumor microenvironment, which is highly immunosuppressive. One potent immunological adjuvant is radiation therapy (RT). Radiation, however, has also been shown to induce immunosuppressive infiltration, which can contribute to tumor progression. Another negative effect is the potential contribution to formation of fibrotic tumor stroma. To capitalize upon the immunogenic effects of radiation and obtain a durable tumor response, radiation must be rationally combined with targeted therapies to mitigate the influx of immunosuppressive cells and fibrosis. One such target is ephrinB2, which is overexpressed in PDAC and correlates negatively with prognosis. Based upon previous studies of ephrinB2 ligand-EphB4 receptor signaling, we hypothesized that inhibition of ephrinB2-EphB4 combined with radiation would regulate the microenvironment response post radiation, leading to increased tumor control in PDAC.

Methods

Immunocompetent C57BL/6 and immune compromised athymic nude mice were injected subcutaneously with either a patient derived xenograft (PDX) tumor, PANC 272, or a mouse pancreata derived cell line (FC1242) and randomized into PBS, B11 (an inhibitor of ephrinB2-EphB4 interaction), RT and B11+RT groups. Depletion studies were conducted using anti-IgG or anti-CD35 antibodies. To determine tumor immune cell infiltration, tumors were subjected to flow cytometric analysis. Plasma samples were subjected to ELISA to determine circulating TGFβ1 levels in control and treatment groups. Fibrosis was quantified following Masson’s Trichrome staining and PicroSirius Red staining.

Results

Our data show that combining ephrin-B2-EphB4 inhibitor with RT significantly reduces regulatory T-cell and neutrophil infiltration, TGFβ1 secretion, and stromal fibrosis, enhancing effector T-cell activation and decreasing tumor growth. Further, our data demonstrate that depletion of regulatory T-cells in combination with radiation reduces tumor growth and fibrosis as demonstrated by Masson’s Trichrome staining and PicroSirius Red staining.

Conclusions

These are the first findings to suggest that in PDAC, ephrinB2-EphB4 interaction has a profibrotic, pro-tumorigenic role, presenting a novel and promising therapeutic target.

P450 The stapled peptide ALRN-6924, a dual inhibitor of MDMX and MDM2, displays immunomodulatory activity and enhances immune checkpoint blockade in syngeneic mouse models

Luis Carvajal, PhD, Narayana Narasimhan, PhD, Jian-Guo Ren, PhD, Solimar Santiago, MS, Manoj Samant, PhD, David Sutton, PhD, Vincent Guerlavais, PhD, D. Allen Annis, PhD, Manuel Aivado, MD, PhD

Aileron Therapeutics, Inc., Cambridge, MA, USA

Correspondence: Manuel Aivado (maivado@aileronrx.com)

Background

The tumor suppressor p53 is one of the most pursued targets in oncology, playing a central role inducing cell cycle arrest, apoptosis and senescence in response to cellular stress and oncogenic signals. In addition to its intrinsic anti- tumor activity in cells, p53 activation can induce anti-tumor immunity and plays an important role in the regulation of innate and adaptive immunity. Therefore, p53-reactivating agents in combination with immune checkpoint blockade (ICB) may represent a powerful approach to optimize the body’s immunological response against cancer. ALRN-6924 is an α-helical stapled p53 peptide currently in clinical testing that has demonstrated anticancer activity as monotherapy [1]. In this study, we investigated whether p53 reactivation with ALRN-6924 can be leveraged as new combination partner for ICB.

Methods

Peripheral blood mononuclear cells (PBMCs) were stimulated with ALRN-6924 ex vivo for 24 hr. Gene expression and cytokine levels were measured using a validated TaqMan assay (ThermoFisher) and the Human XL Cytokine Array Kit (R&D Systems). Immune profiles from pre- and post-treatment tumor biopsy samples were evaluated by NanoString PanCancer IO360 and Immune Profiling RNA gene expression panels. Immunophenotyping of PBMCs was done by flow cytometry. Efficacy and immune cell profile (determined by flow cytometry and IHC) were evaluated in CloudmanS91 and MC38 syngeneic murine tumor models following treatment with ALRN-6924 alone and in combination with anti-PD-1 or anti-PD-L1, including re-challenge studies to test for immunological memory.

Results

Ex vivo stimulation of PBMCs with ALRN-6924 promotes transcriptional activation of genes involved in innate and adaptive immunity, and the production of immune-stimulating cytokines including INF-γ, IL-6 and IL-12. mRNA analysis of pre- and post- treatment tumor biopsies from patients treated with ALRN-6924 revealed a differential gene expression pattern consistent with conversion to an inflamed tumor phenotype. In syngeneic mouse models, ALRN-6924 was sufficient to promote infiltration of CD8+ T cells, polarization of M1 macrophages in mouse tumors and immunological memory. Moreover, ALRN-6924 synergizes with anti-PD-1 and anti-PD-L1 to induce anti-tumor immunity resulting in an increased number of mice achieving complete regressions (CR), in both p53 wild-type and mutant tumors, compared to single agents.

Conclusions

Reactivation of p53 with ALRN-6924 enhanced the effects of ICB therapy in mice. Furthermore, the present study suggests that ALRN-6924 modulates anti-tumor immunity in p53 wild type, and p53 mutant tumors, possibly via tumor cell extrinsic effects in the tumor microenvironment.

References

1. Funda Meric-Bernstam, M. S. Saleh, J. R. Infante, S. Goel, G. S. Falchook, G. Shapiro,K. Y. Chung, R. M. Conry, D. S. Hong, J. S. Wang, U. Steidl, L. D. Walensky, V. Guerlavais, M. Payton, D. A. Annis, M. Aivado, M. R. Patel, Phase I trial of a novel stapled peptide ALRN-6924 disrupting MDMX- and MDM2- mediated inhibition of WT p53 in patients with solid tumors and lymphomas. J. Clin. Oncol. 35, 2505–2505 (2017).

Ethics Approval

All studies involving human material or animal (in vivo) studies were approved under Aileron's IND122392 protocol and by the Institutional Animal Care and Use Committee at Charles River Laboratories, Morrisville, N.C. ASP #: 990202, respectively.

P451 Gal9/Tim-3 expression level is higher in patient with failed chemotherapy in AML

Justin Kline, MD, Paola Dama, PhD, Hongtao Liu, MD, PhD

University of Chicago, Chicago, IL, USA

Correspondence: Hongtao Liu (hliu2@medicine.bsd.uchicago.edu)

Background

Activation of immune checkpoint pathways in Acute Myeloid Leukemia (AML) may interfere with effective T-cell anti-tumor immunity, and is associated with immune evasion in pre-clinical leukemia models as it has been demonstrated. [1,2] It was previously reported that overexpression of CTLA4 and PD-1 is associated with more aggressive leukemia and progression from MDS to AML or AML relapse. While PD-1/PD-L1 blockade therapy can be effective as cancer immunotherapy, interruption of PD-1/PD-L1 interactions alone does not completely restore T cell function in some patients indicating the involvement of additional negative regulatory pathways, such as Tim- 3/Gal-9, in T cell exhaustion. Immune checkpoint pathways active in Acute Myeloid Leukemia (AML) patients, especially during the course of remission induction chemotherapy, have not been well-studied. We characterized these pathways in newly diagnosed AML patients enrolled in a phase I dose escalation trial that combined Selinexor a Selective Inhibitor of Nuclear Export (SINE) with high-dose cytarabine (HiDAC) and mitoxantrone (Mito) (NCT02573363) as induction therapy.

Methods

Multi-parameter flow-cytometry was performed on bone marrow specimens at diagnosis and following remission induction therapy in 26 patients with AML enrolled to the study to monitor the changes in expression of immune checkpoint receptors. Expression of CD47, PD-L1, PD-L2 and Gal-9 was assessed on CD34+ AML blasts and CD34- cell populations. In parallel, expression of inhibitory (PD1, CTLA4, LAG3, TIM3) and stimulatory co- receptors (CD28, ICOS, CD137, OX40, CD40L, HLA-DR) on CD4+ and CD8+ T cell subsets were evaluated. The positivity and frequency of parent in percentage of each markers was gauged by comparing with their FMO controls. Samples were analyzed using LSR Fortessa or LSRII Cytometers. The Mann Whitney Test, Spearman’s rank correlation and Runs Test analysis were applied. For all analyses, P-values <0.05 were considered statistically significant.

Results

The percentage of CD34- Gal9+ cells was significantly higher and was positively correlated with higher numbers of TIM-3-expressing T cells at the time of diagnosis in patients who experienced treatment failure (TF) after chemotherapy, compared to those in complete remission (CR). When comparing TIM-3 expression on CD4+ and CD8+ T cells in pre-treatment (diagnosis) to post induction therapy samples, the magnitude of increase measured by median fluorescence intensity (MFI) inversely correlated to response to therapy with increase TIM-3 MFI of > 50% in patients with TF.

Conclusions

This study provides preliminary evidence to support a rationale for incorporating antibodies against the Gal9/TIM3 pathway during and/or following remission induction therapy for AML.

References

1. Zhang L, Gajewski TF, Kline J. PD-1 / PD-L1 interactions inhibit antitumor immune responses in a murine acute myeloid leukemia model. Blood. 2009; 114(8), 1545–1552.

2. Zhou Q, Munger ME, Blazar BR. Coexpression of Tim-3 and PD-1 identifies a CD8+T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia. Blood, 2011;117(17), 4501–4510.

Ethics Approval

The study was approved by the Institutional Review Board at The University of Chicago, approval number 150412.

P452 Cisplatin treatment induces anti-tumor immune response in NSCLC by activation of the innate immune response pathway

Triparna Sen, PhD², Lixia Diao², Kavya Ramkumar², Carl Gay², Pan Tong², You-Hong Fan², Robert Cardnell², Don Gibbons, MD², John Heymach², Jing Wang², Lauren Byers, MD², Carminia Della Corte, MD²

¹The University of Texas MD Anderson Cancer Center, Houston, TX, Houston, TX, USA; ²MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Carminia Della Corte (cmdella@mdanderson.org)

Background

Platinum-based doublet chemotherapy plus anti-PD1 immunotherapy is a new standard of care for the treatment of advanced NSCLC patients. It is known that DNA damage can activate antitumor immune responses in cancer through release of cytosolic DNA leading to Stimulator of Interferon Genes (STING) pathway activation, production of neo-antigens, and release of pro-inflammatory cytokines. Our group has previously demonstrated that mesenchymal tumors with high EMT scores have the highest expression of targetable immune markers (1). However, the underlying mechanism of how platinum-based chemotherapy modulate the immune microenvironment is far from being fully understood in NSCLC. The aim of this study is to elucidate the effect of platinum-based chemotherapy on anti-tumor immune response and identify novel biomarkers to aid patient selection for chemotherapy and immunotherapy combination clinical trials.

Methods

We analyzed transcriptomic and proteomic expression of immune markers in NSCLC samples from two clinical datasets (MDACC-PROSPECT, n= 209) and The Cancer Genome Atlas (TCGA, n=1016). We also treated NSCLC cell lines with cisplatin to investigate its effect on DNA damage and changes in immune markers expression by western blot and Reverse-Phase Protein Array (RPPA analysis).

Results

Treatment with cisplatin increased DNA damage (increased γH2AX), and significantly upregulated PD-L1 and STING pathway protein expression in a panel of NSCLC cell lines.In the TCGA cohort, immune checkpoints and inflammatory cytokines mRNA expression is highly coordinated and positively correlated with EMT genes. In the TCGA lung adenocarcinoma (LUAD, n=515) cohort, high expression of effector chemokines (CXCL10, CCL5) and mediators of STING pathway (TBK1, TMEM173) were associated with high levels of CD274 (PD-L1) and other targetable immune markers (LAG3, IDO1, PDCD1LG2, CTLA4). These findings were further validated both in lung squamous (LUSC) TCGA and the PROSPECT cohorts (LUAD and LUSC). Interestingly, in the LUAD TCGA cohort, smoking status (another source of DNA damage) was significantly correlated with higher expression of STING and other immuno-modulatory genes, and EMT signature.

Conclusions

Our results demonstrate that in treatment naïve-NSCLC tumors, expression of PDL1 and other targetable immune markers correlate with expression of STING, EMT, smoking status and DDR pathway genes, and that treatment with cisplatin further enhances the immunogenicity of tumors through activation of the STING pathway in NSCLC cells. Our findings identify a novel mechanism by which cisplatin activates an innate immune response pathway in NSCLC. Furthermore the results identify potential biomarkers (EMT, smoking status, DDR protein expression) for patient selection in clinical trials.

References

1. Mak MP, Tong P, Diao L, et al. A Patient-Derived, Pan-Cancer EMT Signature Identifies Global Molecular Alterations and Immune Target Enrichment Following Epithelial-to-Mesenchymal Transition. Clin Cancer Res. 2016;22:609-20.

P453 Enhancing the anti-tumor immunity elicited by alpha radiation-based brachytherapy using immunoadjuvants and blockade of suppressor cells

Vered Domankevich-Bachar, DR¹, Adi Cohen², Margalit Efrati², Michael Schmidt², Hans Georg Rammensee³, Itzhak Kelson², Yona Keisari²

¹Tel Aviv University, Tel Aviv, Israel; ²Tel Aviv Uiniversity, Tel-Aviv, Israel; ³University of Tübingen, Tübingen, Germany

Correspondence: Yona Keisari (ykeisari@tauex.tau.ac.il)

Background

Diffusing alpha emitters Radiation Therapy (DaRT) is a novel brachytherapy treatment for solid tumors. DaRT seeds disperse short-lived alpha-emitting atoms in a therapeutically-significant range, which diffuse inside the tumor and destroy a sizeable part of it. Thus, for the first time, an efficient and safe method for treating the entire solid tumor by highly destructive alpha radiation is used. In situ tumor ablation is known to release tumor antigens and damage associated molecular pattern molecules (DAMPs) that lead to the induction of systemic anti-tumor immunity. Indeed, we previously reported that in the breast cancer carcinoma mice model DA3, DaRT-treated mice showed increased survival rates, and reduced rates of lung metastases and of primary- or challenged- tumor development. Here we aimed to boost the anti-tumor immune response induced by DaRT, locally and systematically, and to investigate the specificity of the response.

Methods

Mice breast (4T1) and colon (CT26) tumors, implanted subcutaneously, were treated with DaRT seeds with/without immunomodulatory agents. Immunomodulatory agents studied are the immunoadjuvants polyIC, CpG, and XS15, the MDSC inhibitor sildenafil, and the Treg inhibitor cyclophosphamide. Non-radioactive seeds (inert) served as control. Local- and systemic- responses were determined by tumor progression, host survival, response to challenge and lung metastasis. The specificity of the immune response was studied by Winn Assay and tumor challenge to cured tumor-bearing mice.

Results

It was found that in the CT26 colon cancer mice model: (1) combining DaRT with polyIC, CpG or XS15 significantly reduced tumor progression and prolonged survival. (2) Complete response was achieved when using DaRT combined with CpG and immune suppressor cells inhibitors. (3) Cured mice became resistant to CT26 cells but not to DA3 (breast cancer) cells. (4). Splenocytes from CT26 bearing mice cured by DaRT specifically reduced CT26 but not DA3 tumor take in naïve mice. In the triple negative breast cancer model, 4T1, treating the primary tumor with polyIC, prior to DaRT treatment, reduced tumor progression and eliminated lung metastases.

Conclusions

DaRT is currently tested under clinical trials in squamous cell carcinoma patients showing effective tumor control without adverse effects. The current results provide strong evidence for the induction of a specific- and systemic- immune response against tumor antigens following DaRT treatment. We propose DaRT as a safe and efficient novel strategy, not only for tumor ablation, but also for in situ vaccination of cancer patients.

P454 Elucidating the functional role of type-1 interferon signaling following a medium-dose intermittent cyclophosphamide schedule in preclinical breast cancer models

Kshama Doshi, PhD, Cameron Vergato, Kshama Doshi, PhD, Darren Roblyer, PhD, David Waxman, PhD

Boston University, Allston, MA, USA

Correspondence: David Waxman (djw@bu.edu)

Background

Many cytotoxic chemotherapy drugs, including the breast cancer standard of care drug cyclophosphamide (CPA), can induce immunogenic cell death when administered at medium-dose and intermittent (MEDIC) schedule [1]. Adaptive and innate immune responses generated in this manner can greatly potentiate chemotherapy drug efficacy and generate tumor-specific long-term immune memory. Cancer cells have also been shown to up-regulate type-1 interferon (IFNα/β) signaling in response to many chemotherapy drugs. Here we set out to elucidate the effects and mechanisms of immune activation in a breast cancer preclinical model using a MEDIC schedule of CPA.

Methods

We used an in-vitro IFN-based biomarker strategy to identify breast cancer models that can induce immunogenic responses following treatment with 4-hydroperoxy cyclophosphamide (4HC), a chemically activated form of CPA. Sub-lethal concentrations of 4HC were established by MTS assay and used to study induction of interferon- stimulated genes by qPCR in five breast cancer cell lines: 4T1, E0771, Emt6, Py230 and MCF7. Anti-IFN receptor- 1 antibody was used to verify the role of IFNα/β in 4HC-induced interferon-stimulated gene induction. CPA- induced immune activation was also evaluated in a syngeneic mouse tumor model. Mice with orthotopic tumors implanted in the 4th mammary fat pad were treated with a MEDIC schedule of CPA and tumor progression was monitored. Effects of drug treatment on IFN signaling and immune cell infiltration into the tumor compartment was evaluated by marker gene expression.

Results

Screening results revealed sub-lethal concentrations of 4HC significantly induced multiple interferon-stimulated genes, including Cxcl10 (~8 fold), Igtp (~15 fold) and Mx1 (~20 fold) in 4T1 breast cancer cells. In contrast partial to minimum induction was seen in E0771, Emt6, Py230 and Mcf7 cells. Further, anti-IFN receptor-1 antibody blocked > 90% of 4HC-induced interferon-stimulated gene induction in 4T1 cells. Conditioned media from 4HC- treated 4T1 cells also stimulated IFNα/β signaling in drug-naive recipient 4T1 cells. Finally, in syngeneic mouse tumor models, MEDIC CPA scheduling significantly reduced tumor progression resulting in tumor stasis. Diminished tumor growth kinetics was accompanied by significant increases in expression of interferon-stimulated genes and cytolytic enzymes in the tumor compartment.

Conclusions

Breast cancer cell autonomous activation of type-1 IFN signaling and downstream gene induction is activated in a syngeneic 4T1 breast cancer model treated with a MEDIC CPA regimen. Mechanistic features of this pathway, involving autocrine and paracrine type-1 IFN signaling loop, were characterized in-vitro. Studies assessing key immune players and their role in anti-tumor responses are in progress, and will be presented.

Acknowledgements

Grant support: DOD grant # U.S. Department of Defense (DOD) (W81XWH-15-1-0070)

References

1. Wu J, Waxman DJ. Immunogenic chemotherapy: Dose and schedule dependence and combination with immunotherapy. Cancer Lett. 2018;419:210-221.

P455 Inducible T cell Co-stimulator (ICOS) is upregulated on lymphocytes following radiation of tumors and ICOS agonism in combination with radiation results in enhanced tumor control

Michael Gough, PhD¹, Shelly Bambina¹, Monica Gostissa, PhD², Christopher Harvey, PhD², David Friedman, PhD¹ Marka Crittenden, MD, PhD¹

¹Earle A. Chiles Research Institute, Portland, OR, USA; ²Jounce Therapeutics Inc., Cambridge, MA, USA

Correspondence: Marka Crittenden (marka.crittenden@providence.org)

Background

Radiation and co-stimulatory ligands or checkpoint inhibitors have demonstrated improved anti-tumor immunity and overall survival in preclinical animal studies. However, the results of human trials suggest we have not yet found the optimal combination. Here we demonstrate upregulation of ICOS expression on T cells following focal tumor radiation and test the hypothesis that ICOS agonism in combination with radiation will enhance the immunologic effect of radiation resulting in increased survival.

Methods

BALB/c mice bearing CT26 tumors or C57BL/6 mice bearing Panc02 tumors were treated at d14 with 20Gy CT guided radiation therapy and anti-ICOS antibody or isotype control antibody was administrated i.p. Mice were followed for overall survival to 100 days post implantation. Animals were euthanized when tumors reached 1.2cm in greatest diameter. Flow cytometry was performed using a T cell panel on fresh whole blood, PBMC, or tumor infiltrating immune cells.

Results

24 hours following 20Gy focal radiation to a CT26 tumor there was a significant increase in the percent of circulating CD4 Treg that express ICOS in the blood (27.42% vs 18.02%, p<0.0001, n=5/group). Similarly, 7 days following radiation there was an increase in non-Treg CD4 cells expressing ICOS in the blood (7.73% vs 3.68%, p<0.0001, n=5/group) and the tumor (62.16% vs 34.04%, p=0.004, n=5/group). ICOS expression was also increased on CD8 T cells in irradiated tumors (25.34% vs 14.02%, p=0.007). In mice bearing CT26 tumors, ICOS agonist antibody was administered prior to, concurrent with, or 7 days post radiation. Concurrent administration was associated with the most significant increase in survival (50%) when compared to isotype control (0%), ICOS agonist antibody alone (10%), or radiation plus isotype (0%). In the less immunogenic Panc02 tumor model, no survival benefit was seen with radiation and ICOS therapy. However in the same model, dual PD-1 antagonism and ICOS agonism plus radiation led to a significant increase in survival when compared to all other combinations, with an increase in median survival from 46 days to 68 days, p=0.01 compared to radiation alone and was associated with a 25% long term survival.

Conclusions

ICOS is upregulated on T cells following radiation and targeting ICOS in combination with radiation is associated with improved survival. Timing appears important as the benefit is optimal when ICOS agonism is delivered concurrent with radiation rather than preceding or 7 days post-radiation. In poorly immunogenic tumors, addition of PD-1 antagonism to the combination can lead to improved survival.

Ethics Approval

Animal protocols were approved by the Earle A. Chiles Research Institute IACUC (Animal Welfare Assurance No. A3913-01). All experiments were performed in accordance with relevant guidelines and regulations.

P456 Pressure-enabled delivery of CAR-T cells into the porcine pancreas results in highly-targeted pancreatic delivery with minimal systemic exposure and no pancreatitis or severe systemic cytokine release

John Hardaway, MD, PhD¹, James Chomas, PhD², David Jaroch, PhD², Prajna Guha, PhD¹, N. Joseph Espat, MD, FACS³, Steven Katz, MD³, Aravind Arepally, MD⁴

¹Roger Williams Medical Center, Providence, RI, USA; ²Surefire Medical, Inc., WESTMINSTER, CO, USA; ³Roger Williams Medical Center, Boston University, Providence, RI, USA; ⁴Surefire Medical, Inc., Piedmont Medical Center, Westminster, CO, USA

Correspondence: Aravind Arepally (aravind.arepally@surefiremedical.com)

Background

The purpose of this preclinical study is to determine whether highly preferential delivery of T cells into the pancreas can be achieved while minimizing systemic exposure and avoiding systemic and pancreatic inflammation using the Surefire® Retrograde Venous-Pressure Enabled Drug Delivery (RV-PEDD) method and device, as compared to systemic venous infusion (SVI).

Methods

Healthy human donor CAR-T cells (Sorrento Therapeutics) or unmodified activated T cells were transferred into 10 normal adult swine by either (a) SVI (n=5) or (b) RV-PEDD via trans-hepatic access into pancreatic veins (n=5). Samples of peripheral blood (PB) were obtained at 15, 30, and 120 minutes after infusion. Serum was analyzed for porcine tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay (ELISA) as indices of systemic inflammation, whereas circulating CAR-T were quantified using flow cytometry. Liver and pancreatic tissues were harvested for histology, immunofluorescence (IF) of human CD3, and determination of human CD3 mRNA expression via qPCR.

Results

After SVI, the donor CAR-T cell fraction among circulating mononuclear cells was 13.7% at 15 minutes, 31.7% at 30 minutes, and 20.5% at 120 minutes, versus RV-PEDD that yielded 1.8% detection at 15 minutes, and undetectable cells at 30 and 120 minutes. With SVI, IF found substantial accumulation of donor CAR-T cells in PB and minimal pancreatic staining, as opposed to RV-PEDD infusion where substantial pancreatic accumulation and minimal PB staining had occurred (Figure 1). qPCR analysis of pancreatic tissues from RV-PEDD specimens revealed a 147-fold increase in CAR-T penetration, as compared to SVI. Alternatively, analysis of PB following SVI revealed a 61-fold increase in systemic exposure with negligible detection in the pancreas. Histologically defined pancreatic inflammation was not evident in any animal after RV-PEDD (Figure 2). Systemic inflammation, based on TNF-α and IL-6 levels, was not evident within 2 hours after RV-PEDD infusion, whereas pronounced increases of TNF-α and IL-6 levels were observed with systemic infusion.

Conclusions

RV-PEDD was associated with efficient and specific delivery of donor T cells into porcine pancreas compared to systemic infusion. RV-PEDD did not result in significant systemic exposure nor induce pancreatic or systemic inflammation. These data support clinical testing of Surefire® RV-PEDD technology to improve the therapeutic index with selective delivery of cellular therapeutics into the pancreas.

Ethics Approval

The study was approved by the T3 Labs IACUC on 6/15/16, with amendments on 11/17/16, 2/24/17.

Fig. 1 (abstract P456).

See text for description

Fig. 2 (abstract P456).

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P457 TP53 gene therapy emanating from the investigational agent SGT-53 is capable of augmenting cancer immunotherapy in multiple murine syngeneic tumor models

Joe Harford, PhD¹, Sang-Soo Kim, PhD¹, Kathleen Pirollo, PhD², Antonina Rait, PhD², Manish Moghe², Esther Chang, PhD²

¹SynerGene Therapeutics, Inc., Potomac, MD, USA; ²Georgetown University Medical Center, Washington, DC, USA

Correspondence: Joe Harford (harfordj@synergeneus.com)

Background

Manipulation of immune checkpoints has emerged as an important form of cancer immunotherapy [1,2]. However, a large number of patients do not respond or develop resistance to checkpoint blockade, and treatment-related toxicities can be limiting. The tumor suppressor p53 exerts anti-tumor activity by inducing apoptosis, but also participates in the regulation of cellular immune responses [3.4]. We have investigated the potential of TP53 gene therapy to augment cancer immunotherapy by combining an anti-PD1 antibody with SGT-53, an investigational nanomedicine that targets tumors and carries a plasmid encoding human wild-type p53 (Figure 1). SGT-53 is now in Phase II clinical trials [5,6].

Methods

We utilized mouse syngeneic tumor models that are relatively resistant to immunotherapy including an aggressive metastatic breast cancer (4T1), a non-small cell lung carcinoma (LL2), and a glioblastoma (GL261). Anti-tumor efficacies of an anti-PD1 antibody alone, SGT-53 alone or the combination of these agents were compared [7]. A number of markers with relevance to immune responses or tumor-induced immunosuppression were assessed by FACS or gene expression profiling.

Results

In all syngeneic models, SGT-53 increased tumor apoptosis and rendered the tumors immunologically “hot”. SGT- 53 plus anti-PD1 inhibited growth more than either single agent (Figure 2). The combination therapy dramatically reduced lung metastases by 4T1 breast tumors, while the anti-PD1 alone was ineffective. Based on relevant markers, SGT-53 treatment increased tumor immunogenicity, enhanced both innate and adaptive immune responses, and reduced tumor-induced immunosuppression. In mice bearing 4T1 tumors, injections of the anti-PD1 antibody killed the mice before tumors were themselves fatal. Addition of SGT-53 to the treatment regimen alleviated this fatal xenogeneic hypersensitivity to the anti-PD1 and extended the lives of 4T1-bearing mice (see Figure 3). We have identified genes that potentially underlie these observations.

Conclusions

Collectively, our data indicate that restoring p53 function via SGT-53 is able to boost anti-tumor immunity to enhance anti-PD1 immunotherapy by sensitizing tumors while reducing immune-related adverse events. Our data suggest that SGT-53, representing tumor-targeted TP53 gene therapy, has potential to augment immune checkpoint blockade agents while minimizing toxicity to improve outcomes in a variety of malignancies. Our data provide a strong mechanistic rationale for combining the investigational agent SGT-53 with checkpoint blockade agents in a clinical trial setting. It is possible that the SGT-53 would not only improve outcomes for cancer patients who already respond to immunotherapy, but also increase the percentage responding while minimizing adverse events related to the immunotherapy.

References

1. Mellman I, Coukos G, Dranoff, G, Cancer immunotherapy comes of age. Nature 2011; 480, 480-489.

2. Pardoll DM, The blockade of immune checkpoints in cancer immunotherapy. Nature Reviews Cancer 2012; 12, 252-264.

3. Lowe J, Shatz M, Resnick MA, Menendez D, Modulation of immune responses by the tumor suppressor p53. BioDiscovery 2013; 8, 2.

4. Munoz-Fontela C, Mandinova A, Aaronson SA, Lee SW, Emerging roles of p53 and other tumour-suppressor genes in immune regulation. Nature Reviews Immunology 2016; 16, 741-750.

5. Senzer D, Nemunaitis J, Nemunaitis D, Bedell C, Edelman G, Barve M, Nunan R, Pirollo KF, Rait A, Chang EH, Phase I study of a systemically delivered p53 nanoparticle in advanced solid tumors. Molecular Therapeutics 2013; 21, 1096-1103.

6. Pirollo K F, Nemunaitis J, Leung PK, Nunan R, Adams J, Chang EH, Safety and efficacy in advanced solid tumors of a targeted nanocomplex carrying the p53 gene used in combination with docetaxel: Phase 1b study. Molecular Therapeutics 2016; 24, 1697-1706.

7. Kim SS, Harford JB, Moghe M, Rait A, Chang EH, Combination with SGT-53 overcomes tumor resistance to a checkpoint inhibitor, OncoImmunology 2018; DOI: 10.1080/2162402X.2018.148498

Ethics Approval

All animal experiments were performed in accordance with and under approved Georgetown University GUACUC protocols.

Fig. 1 (abstract P457).

See text for description

Fig. 2 (abstract P457).

See text for description

Fig. 3 (abstract P457).

See text for description

P458 Contribution of an immune system to RACPP mediated drug delivery

Dina Hingorani, PhD, Maria Camargo, Matthew Doan, BS, Joesph Aguilera, Stephen Adams, Sunil Advani

University of California San Diego, La Jolla, CA, USA

Correspondence: Dina Hingorani (dhingoraniucsd@gmail.com)

Background

The interaction of tumor and host derived stroma is a complex process involving enzymes including matrix metalloproteinases (MMPs), specifically MMP2 / 9 [1]. The lack of efficacy from small molecule inhibitors to MMPs [2,3] and new approaches of macrophage mediated drug delivery strategies [4], propelled us to test the contributions of intrinsic tumor cell vs host stroma in the recruitment of macrophages in-vivo. We tested the hypothesis that drug delivering peptides that target the tumor-immune interplay have improved therapeutic efficacy and specificity.

Methods

Cy5:Cy7 FRET based ratiometric activatable cell penetrating peptides (RACPPs) were used to image syngeneic WT and MMP2/9 KO Polyoma middle T (PyMT) orthotopic tumors in C57/Bl6 WT and KO mouse models. The excised tumors were stained F4/80 pan-macrophage antibody markers. ACPP carrying the tumor radiosensitiser monomethyl auristatin E (MMAE) along with Beta integrin targeting moiety cRGD was injected in syngeneic lung tumor cell line (LL2) implanted in immune compromised athymic Nu/Nu mice and immune competent C57/Bl6 mice. The amount of released MMAE drug was analyzed in tumor vs surrounding normal tissue by LC-MS.

Results

The normalized Cy5/Cy7 ratio for the WT tumors established in WT mice (1.87 ± 0.11) was significantly higher than the ratio for the DKO tumors in WT mice (1.34 ± 0.07; p <0.003) or the DKO tumors in DKO mice (1.20 ± 0.08; p < 0.0002) (Figure 1A, 1B). As expected, the majority of the immune infiltration was at the periphery of the tumor with WT tumor having a significantly greater ability to recruit host macrophages into the tumor than DKO tumor cells (Figure 1C-1F). At 24 h, biodistribution measurements of released MMAE drug, revealed a higher tumor/muscle ratio of delivered drug (14 fold) in immune competent mice compared to immune-deficient mice(Figure 1G, 1H).

Conclusions

Interestingly, genotype tumor cells was more important than the host stromal component in promoting MMP-2/-9 activity in the tumors in this model system. Importantly, exploiting drugs that inhibit macrophage recruitment into tumors [4] and harnessing macrophage mediated drug delivery [5,6] in the tumor extracellular matrix may prove superior in eradicating tumors. In summary, our novel RACPP-drug conjugates can selectively localize to tumors and where they can be cleaved both by tumor cells and tumor-associated macrophages to provide improve the therapeutic index of systemically administered drugs [6,7].

References

1. Noël A, Jost M, Maquoi E. Matrix metalloproteinases at cancer tumor–host interface. InSeminars in cell & developmental biology 2008 Feb 1 (Vol. 19, No. 1, pp. 52-60). Academic Press.

2. Gura, Trisha. "Systems for identifying new drugs are often faulty." (1997): 1041-1042.

3. Richmond, Ann, and Yingjun Su. "Mouse xenograft models vs GEM models for human cancer therapeutics." (2008): 78-82.

4. Panni, Roheena Z., David C. Linehan, and David G. DeNardo. "Targeting tumor-infiltrating macrophages to combat cancer." Immunotherapy 5, no. 10 (2013): 1075-1087.

5. Li, Fu, Michelle Ulrich, Mechthild Jonas, Ivan J. Stone, Germein Linares, Xinqun Zhang, Lori Westendorf, Dennis R. Benjamin, and Che-Leung Law. "Tumor-Associated Macrophages Can Contribute to Antitumor Activity through FcγR-Mediated Processing of Antibody–Drug Conjugates." Molecular cancer therapeutics 16, no. 7 (2017): 1347-1354.

6. Buckel, Lisa, Elamprakash N. Savariar, Jessica L. Crisp, Karra A. Jones, Angel M. Hicks, Daniel J. Scanderbeg, Quyen T. Nguyen et al. "Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery." Cancer research 75, no. 7 (2015): 1376-1387.

7. Crisp, Jessica L., Elamprakash N. Savariar, Heather L. Glasgow, Lesley G. Ellies, Michael A. Whitney, and Roger Y. Tsien. "Dual targeting of integrin αvβ3 and matrix metalloproteinase-2 for optical imaging of tumors and chemotherapeutic delivery." Molecular cancer therapeutics 13, no. 6 (2014): 1514-1525.

Ethics Approval

All animal were performed under approved IACUC protocol number S15290 at the University of California San Diego.

Fig. 1 (abstract P458).

See text for description

P459 Detection of tumor-specific antibodies and their binding regions in mice cured from B78 melanoma

Anna Hoefges, MS¹, Amy Erbe, PhD¹, Drew Melby¹, Alexander Rakhmilevich, MD, PhD¹, Jacquelyn Hank, PhD¹, Claire Baniel, BS, BA¹, Clinton Heinze, BS¹, Irene Ong, PhD¹, Sean Mcilwain, PhD¹, Hanying Li, PhD², Richard Pinapati, PhD², Bradley Garcia, PhD², Jigar Patel, PhD², Zachary Morris, MD, PhD¹, Paul Sondel, MD, PhD¹

¹University of Wisconsin Madison, Madison, WI, USA; ²Roche Sequencing Solutions, Inc., Madison, WI, USA

Correspondence: Anna Hoefges (hoefges@wisc.edu)

Background

Antibodies can play an important role in both innate and adaptive immune responses against cancer. We present a study that identifies possible new targets for antibody-based immunotherapy. We have developed a peptide array to assess potential protein-targets for antibodies that are activated in melanoma-cured mice through a combined immunotherapy regimen. By using Roche-Nimblegen’s unique technology, we were able to test antibody-reactivity to ~650 proteins, using 12 separate serum samples per array chip. This technology will enable us to accurately determine the linear peptide-binding sequences recognized by the anti-tumor antibodies produced in cured mice.

Methods

Mice bearing large GD2-expressing B78 melanoma tumors were treated with a triple-combination of immunotherapy capable of inducing an “in situ vaccine” effect, enabling mice to be cured of their tumors with long-term immune memory [1,2]. This triple combination therapy includes external beam radiation to the tumor, intratumoral injection of a tumor-specific immunocytokine (anti-GD2 mAb linked to IL2) and anti-CTLA-4. Serum was collected from mice when mice had macroscopic tumors, as well as after mice were cured of large tumors and rejected a re-challenge with the same tumor type. Using flow cytometry, mouse serum was tested for antibody- binding against B16 (parental cell line of B78). Afterwards, the serum was used on a Roche-Nimblegen peptide-array to determine specific antibody-protein binding sites and affinity towards the tumor.

Results

We analyzed sera from 4 mice that rejected established B78 tumors with this combination immunotherapy and compared their early-tumor and post-rejection serum antibody binding. We also included serum from mice bearing large tumors and analyzed the data generated by assessing differential expression in mice that rejected tumors vs mice that had large tumors or serum from naïve mice. Flow results showed increased signal after treatment. Multiple proteins of interest were selectively identified on the peptide array with sera from the 4 mice that rejected their tumors. We are continuing to investigate these proteins.

Conclusions

We were able to identify murine proteins that were selectively recognized by antibodies in mice that were cured of a tumor with immunotherapy but not by sera from to mice that were not cured of the same tumor or sera from naïve mice. The identified candidates may be new targets for antibody-based therapies, for adaptive recognition and could help in the development of new treatments.

References

1. Morris ZS, et al. Cancer Research, 76:3929-3941, 20162. Morris ZS, et al. Cancer Immunology Research, Published online, May 2018

P460 Chemotherapy induced immunogenic cell death and response to STING agonist in high-grade serous ovarian cancer

Sarah Nersesian, MSc, Nichole Peterson, MSc, Julie-Ann Francis, Madhuri Koti, DVM, MVSc, PhD

Queen's University, Kingston, ON, Canada

Correspondence: Sarah Nersesian; Madhuri Koti (kotim@queensu.ca)

Background

High Grade Serous Carcinoma of the ovary (HGSC) is mostly diagnosed at late stages and primarily treated with surgery followed by platinum/taxane-based chemotherapy. Unfortunately, majority of the patients exhibit resistance to chemotherapy and ultimately succumb to the disease. We previously demonstrated that chemotherapy naïve HGSC patient tumours with early recurrence show an immunosuppressed or immunologically cold pre-existing tumour immune microenvironment with decreased expression of genes involved in Type I Interferon (IFN1) and T helper type 1 response. We also reported the efficacy of a novel “Stimulator of Interferon Genes” agonist in combination with carboplatin chemotherapy and PD-1 immune checkpoint blockade using the ID8-Trp53-/- immunocompetent mouse model of HGSC. Based on previous reports on the distinct immunogenic cell death inducing potential of carboplatin and doxorubicin and that HGSC patients are treated with liposomal doxorubicin as a second line chemotherapy, the current study was performed to determine whether the effect of STING agonist can be further enhanced using a specific chemotherapy drug.

Methods

ID8-Trp53-/- and ID8-Trp53-/-;Brca1-/- cells were implanted in C57/BL6 immunocompetent mouse model of HGSC. At four-week time point established tumours were treated with carboplatin or doxorubicin chemotherapy followed by STING agonist treatment. Immune profiling was performed at early mid and late time points by measuring systemic responses in splenic immune cells, plasma cytokine profiles and tumour immune transcriptomic profiling. Overall survival was measured as per our previously established protocols.

Background

High Grade Serous Carcinoma of the ovary (HGSC) is mostly diagnosed at late stages and primarily treated with surgery followed by platinum/taxane-based chemotherapy. Majority of the patients exhibit resistance to chemotherapy and ultimately succumb to the disease. Contemporary immunotherapies targeting the PD-1/PD-L1 immune checkpoints have not proven be efficacious in HGSC patients. Based on our patient tumour based findings [1] that chemotherapy naïve HGSC patient tumours, with early recurrence and resistant to chemotherapy, show an immunologically cold pre-existing tumour immune microenvironment (TME), we conducted pre-clinical evaluation of a novel “Stimulator of Interferon Genes” (STING) agonist in combination with carboplatin chemotherapy and PD-1 immune checkpoint blockade using the ID8-Trp53-/- mouse model of HGSC [2]. This report demonstrated the potential of STING agonists in sensitization of ovarian tumours to PD-1 immune checkpoint blockade therapy, for ovarian cancer patients. Given the distinct immunogenic cell death (ICD) inducing potential of carboplatin and doxorubicin and that HGSC patients are treated with liposomal doxorubicin as a second line chemotherapy, the current study was performed to determine whether the effect of STING agonist can be further enhanced using a specific chemotherapy drug.

Methods

ID8-Trp53-/- cells were implanted in C57/BL6 immunocompetent mice. At four-week time point, established tumours were treated with carboplatin or doxorubicin chemotherapy followed by STING agonist treatment. A custom NanoString panel of 60 known ICD associated genes was used to measure the chemotherapy type related gene expression changes at early time point post single or combination treatments. Doxorubicin treated tumours showed significantly higher expression of Cxcl10, Cd274, Isg15, Psmb9 and Calr. Addition of STING agonist to each chemotherapy treatment showed significantly higher expression of Cxcl10 and IsG15 in the doxorubicin + STING agonist treated mice compared to carboplatin. Interestingly, Ccl5 gene expression was higher in the tumours from carboplatin treated mice compared to those treated with doxorubicin. Plasma cytokine profiles showed distinct profiles of interferon induced cytokines post treatment. Doxorubcin + STING agonist treated mice showed longer survival compared to carboplatin + STING agonist treated mice.

Results

Findings from our study demonstrate that efficacy of STING agonists can be further exemplified by selectively combining with potent ICD inducing chemotherapy.

Conclusions

Our study shows that clinical potential of STING agonists can be best achieved via combining with a potent ICD inducing chemotherapy and are key to the design of STING agonist based clinical trials.

Acknowledgements

This study was funded by the Canadian Institutes for Health Research and Early Research Award support to MK.

References

1. Au KK, Le Page C, Ren R, Meunier L, Clément I, Tryshkin K, Peterson N, Kendall-Dupont J, Childs T, Francis JA, Graham CH, Craig A, Squire JA, Mes-Masson AM, and Koti M. STAT1 induced intratumoural TH1 immunity predicts chemotherapy resistance in high‐grade serous ovarian cancer. Journal of Pathology: Clinical Research. 2016. Sep 19;2(4):259-270.

2. Haffari A, Peterson, Khalaj N NK, Robinson A, Francis JA and Koti M. STING agonist therapy in combination with PD-1 immune checkpoint blockade enhances response to carboplatin chemotherapy in high-grade serous ovarian cancer. British Journal of Cancer. 2018. Jul 26. doi: 10.1038/s41416-018-0188-5.

P461 Withdrawn

P462 Fractionated radiation with PD-1 blockade promotes anti-tumor activity in mouse head and neck cancer

Go Inokuchi, MD, PhD¹, Elizabeth McMichael, PhD², Masahiro Kikuchi, MD, PhD¹, David Clump, MD PhD¹ Robert Ferris¹

¹University of Pittsburgh, Pittsburgh, PA; ²University of Pittsburgh Hillman Cancer Center, Pittsburgh, PA, USA

Correspondence: Robert Ferris (ferrrl@upmc.edu)

Background

Resistance to RT could be explained by the increased myeloid cells and upregulation of PD-L1 on tumor and myeloid cells. As 2Gy fractionated radiation therapy (RT) is the standard of care in HNSCC, pre-clinical investigations suggest that the addition of PD-1 blockade to RT could be clinically beneficial. Here, we investigated the immune response in a murine model of HNSCC to fractionated irradiation with or without PD-1 blockade.

Methods

Mice were inoculated with 2x106 murine tonsil epithelium E6/E7/H-ras transformed head and neck cancer cells (MEER) s.c. into both the neck and flank. Ten days following implantation, the neck tumor was irradiated with 20 Gy in 10 fractions. Anti-PD-1 therapy began following the initial dose of RT and continued every 3-4 days thereafter. Tumor growth was monitored and tumor volume was determined. Splenic and tumors tissues were collected 4 days after the final radiation dose for flow cytometric analysis.

Results

The effects of conventional 2Gyx10 fractionated RT was found to be greatly enhanced by the addition of PD-1 blockade, reducing tumor volumes by 7.2-fold. No clear abscopal effect on the non-irradiated flank tumor was observed. 2Gyx10 RT was better able to recruit myeloid and CD8+ T cells to the tumor site, an increase of 1.5-fold, as compared to 2Gyx5 fractionation. RT was shown to upregulate PD-L1 both on CD45- tumor cells and CD45+CD11b+ myeloid cells (p<0.05). Fractionated RT was also shown to increase CD8+ T cells activation through the production of IFN-gamma and TNF-alpha (p<0.001).

Conclusions

Concurrent PD-1 blockade with fractionated 2Gyx10 RT could activate the anti-tumor response in mouse head and neck cancer and warrants further investigation.

P463 HfO2 nanoparticles exposed to radiotherapy generate abscopal effect through activation of CD8+ T cells

Audrey Darmon, BS¹, Ping Zhang, MD, PhD¹, Sébastien Paris, PhD¹

Nanobiotix, Paris, France

Correspondence: Sébastien Paris (sebastien.paris@nanobiotix.com)

Background

When exposed to radiotherapy (RT), nanoparticles of hafnium oxide (HfO2-NP) increase radiation dose deposition from within the cancer cells. HfO2-NP is intended for a single intratumor injection. Results of phase II/III in locally advanced Soft Tissue Sarcoma patients demonstrated a significant superiority and clinical benefits of HfO2-NP activated by radiotherapy compared to the standard of care, with a good local tolerance among this patient’s population, validating their first-in-class mode of action. HfO2-NP+RT is currently evaluated in six other clinical trials including head and neck, prostate, liver and rectum cancers. Moreover, preclinical studies have demonstrated that HfO2-NP+RT can generate the abscopal effect, where RT alone cannot. Here, we further explored the role of T cells infiltrates in the establishment of abscopal effect following HfO2-NP intratumor injection and activation with RT.

Methods

In a first experiment, CT26 (murine colorectal cancer cells) were subcutaneously injected in both flanks of BALB/c mice. Once the right tumors reached a mean tumor volume of 115±30 mm3, they were intratumorally injected with HfO2-NP (or vehicle) and irradiated 24 hours later with 4Gy per fraction for 3 consecutive days. Tumors from both flanks were collected 3 days after the last RT fraction and immune cell infiltrates were measured using immunohistochemistry (IHC) and digital pathology analyses.In order to investigate the specific role played by CD8+ T cells in the antitumor immune response and the abscopal effect, the experiment was subsequently repeated with CD8+ T cells depletion prior treatment with HfO2-NP+RT or RT alone (use of anti-CD8 antibody).

Results

In the first experiment, the abscopal effect was observed in the group treated with HfO2-NP+RT only. Correspondingly, IHC analyses showed a stark increase of CD8+ T cells infiltrates and other immune cells in both flanks of mice with HfO2-NP+RT, while RT alone had no significant effect.In the CD8+ T cells depletion experiment, no abscopal effect was observed. Besides, the control of the tumor treated with HfO2-NP + RT was less efficient than the control of the tumor treated with HfO2-NP+RT in absence of CD8+ T cells depletion.

Conclusions

These in vivo data suggest that the immunogenic conversion of the tumor microenvironment induced by HfO2-NP+RT triggers the abscopal effect through the activation of CD8+ T cells. HfO2-NP+RT may potentiate a pro- inflammatory environment suitable for immune enabling drugs: it may act as effective in-situ cancer vaccine and be combined with immunotherapeutic agents across oncology.

Ethics Approval

All experiments were approved by the Institutional Animal Care and Use Committee of Institut Gustave Roussy, approval number 2016_ 031_4340.

P464 Molecular targeted radiotherapy (MTRT) enhances the efficacy of immunotherapy increasing complete response rates of both local and distant disease in a “cold” tumor models

Ravi Patel, MD, PhD, Reinier Hernandez, PhD, Peter Carlson, Ryan Brown, Abigail Jaquish, Luke Zangl, Raghava Sriramaneni, PhD, Joseph Grudzinski, PhD, Bryan Bednarz, PhD, Jamey Weichert, PhD, Paul Sondel, MD, PhD, Zachary Morris, MD, PhD

University of Wisconsin, Madison, WI, USA

Correspondence: Zachary Morris (zmorris@humonc.wisc.edu)

Background

Studies have shown that in some immunologically “cold” tumor models, distant disease can suppress the effect of in situ vaccines (IS) even at the primary site[1]. This may be overcome by delivering low dose radiotherapy (RT) to all tumor sites; yet delivering large field RT to metastatic disease can cause systemic lymphopenia. We have developed a strategy using a molecular targeted RT (MTRT), Y90-NM600 (YN6), that has selective uptake in nearly any tumor type or location to deliver RT to all sites of disease in a functionally “cold” metastatic tumor model.

Methods

Large (~150-200 mm3) B78 melanoma primary tumors and occult secondary (non-palpable at treatment) as well as B16 melanoma lung metastases were established in syngeneic mice. Combinations of immune checkpoint inhibition (ICI; anti-CTLA-4 and anti PD-1), IS (12 Gy RT + IT anti-GD2-mAb + IL2), or MTRT (50 μCi) were given [Figure 1]. Tumor growth was tracked to day (D) 30, Survival to D60, and mice with complete response (CR) were re- challenged with injection of B78 cells (D90) and unrelated Panc02 cells(D120). Tumor growth and survival studies were replicated in syngeneic 4T1 breast and NXS2 neuroblastoma models. Mechanistic studies using T-cell depletion, whole body external beam RT (WBEBRT), histology, and gene expression profiling were conducted.

Results

Tumor response was significantly improved with the addition of MTRT to each group, with highest response rate in the triple combination treatment group which had a CR as well as tumor specific immune memory in 83% of mice (p < 0.05). Development of secondary tumors and distant metastatic disease was also reduced in the triple combination treatment group (ICI + IS + MTRT), while dual treatment groups had varying levels of efficacy in treating primary, occult secondary, or metastatic disease [Figure 2]. Similar response and survival was replicated in 4T1 and NXS2 models with addition of MTRT. T-cell depletion revealed a reversal of the enhanced response seen with MTRT. Unlike MTRT, delivering WBEBRT did not enhance efficacy of immunotherapy. QPCR of MTRT gene expression demonstrated upregulation of STING/IFN/apoptosis pathways (Mx1/Ifnb/PDL1/DR5/ICAM1) that were greater than that achieved with equivalent doses of EBRT. Histological analysis of tumor samples showed significantly increased CD8+ infiltrates in the combination treatment group (p < 0.05).

Conclusions

Our results demonstrate that MTRT can effectively stimulate and enhance the generation of an immune response to combination IS and ICI immunotherapy treatments, enabling tumor eradication at primary, occult secondary, and metastatic sites of disease.

Acknowledgements

RSNA Fellow Award, ASCO Young Investigator Award, UW 20/20 Award, UW Cancer Center Core Grant

References

1. Morris, ZS, et al. Cancer Immunol Res 2018 Jul;6(7):825-34

Ethics Approval

This study was approved by the UW Institutional Animal Care and Use Committee.

Fig. 1 (abstract P464).

Treating mice with 1 large, 1 small and disseminated COLD B78/B16 tumors

Fig. 2 (abstract P464).

See text for description

P465 Comparison of peripheral immune response during chemoradiotherapy (CRT) with and without PD-1 blockade in patients with head and neck squamous cell carcinoma (HNSCC)

Juan Callejas-Valera, PhD¹, Juan Callejas-Valera, PhD¹, Daniel Vermeer¹, Christopher Lucido², Caitlin Williamson¹, Marisela Killian¹, William Spanos, MD¹, Paola Vermeer, PhD¹, Steven F. Powell, MD³

¹Sanford Research, Sioux Falls, SD, USA; ²University of South Dakota, Sioux Falls, SD, USA; ³Sanford Cancer Center, Sioux Falls, SD, USA

Correspondence: Steven F. Powell (Steven.Powell@sanfordhealth.org)

Background

While inhibitors of the programmed death-1 and its ligands (PD-1 and PD-L1/2) are active in recurrent/metastatic (R/M) HNSCC, their effects during curative intent therapy are unknown. Previous translational data demonstrated that standard, high-dose CRT decreases circulating CD4+ and CD8+ T-cell populations while increasing PD-1 expression and myeloid derived suppressor cells (MDSCs) [1]. To overcome this suppressive immunophenotype, we developed a clinical trial exploring the combination of the PD-1 inhibitor, pembrolizumab, with CRT using a low- dose chemotherapy regimen. Here we present data comparing the peripheral blood immune response during this novel therapy to standard CRT.

Methods

We evaluated peripheral blood mononuclear cells (PBMCs) from HNSCC patients from two clinical trials (NCT02586207, NCT01386632) and healthy volunteers (controls) to compare the peripheral blood immune response during CRT. Trial 1 used low-dose cisplatin (40 mg/m2 weekly x 6 doses) with pembrolizumab and Trial 2 used standard high-dose cisplatin (100 mg/m2 every 3 weeks x 3 doses) without PD-1 inhibition. We compared circulating immunocytes, including CD4+ and CD8+ T-cells, regulatory T-cells (T-regs), and MDSCs, utilizing multi-color flow cytometry at baseline, during (mid-treatment) and after (3 months post-radiation) CRT. Immune checkpoint expression (PD-1, TIM3, LAG3) on CD4/CD8+ cells was also compared between the groups. Changes in memory T-cell populations (effector memory; EM, central memory; CM, and effector memory RA; EMRA) were also evaluated.

Results

18 patient samples from trial 1 and 15 samples from trial 2 were viable for evaluation. Comparing the two treatments, there was no significant difference in key immunocyte populations during therapy (Figure 1). However, there was a significant decline in PD-1 expressing CD4+ and CD8+ T-cell populations during treatment with PD-1 inhibition and low-dose chemotherapy compared to standard treatment (Figure 2). Expression of other markers of immune exhaustion (TIM3, LAG3) rise in both groups throughout treatment. Memory T-cell populations (Figure 3) show that the pembrolizumab-based treatment increased the percentage of both EM and EMRA helper T-cells in contrast with standard treatment.

Conclusions

Our data demonstrates that the addition of a PD-1 inhibitor to a low-dose cisplatin CRT regimen can reduce circulating PD-1+ T-cells during therapy. However, these populations, as well as other markers of T-cell exhaustion rise by the end of therapy and could play a role in immune escape. Further characterization of this immune response is needed to determine the best approach to add novel immunotherapy agents in this treatment setting.

Acknowledgements

Sanford Research Flow Cytometry CoreSupported by a Center of Cancer Biology Research Excellence (COBRE) grant (5P20GM103548-08) from the National Institutes of Health

References

1. Parikh F, Duluc D, Imai N, Clark A, Misiukiewicz K, Bonomi M, Gupta V, Patsias A, Parides M, Demicco EG, Zhang DY, Kim-Schulze S, Kao J, Gnjatic S, Oh S, Posner MR, Sikora AG. Chemoradiotherapy-induced upregulation of PD-1 antagonizes immunity to HPV-related oropharyngeal cancer. Cancer Res. 2014;74(24):7205- 16.

Ethics Approval

Trial 1 (NCT02586207) was approved by the Western Institutional Review Board (WIRB), approval number 20152167. Trial 2 (NCT01386632)was approved by the Sanford Research Institutional Review Board (IRB), approval number MODCR00000588.

Fig. 1 (abstract P465).

Peripheral Blood Immunocyte Response during CRT

Fig. 2 (abstract P465).

Changes in Immune Checkpoint Expression

Fig. 3 (abstract P465).

The Effect on Memory T-cell Response

P466 TGF beta blockade enhances radiotherapy abscopal efficacy effects in combination with anti-PD1 and anti- CD137 immunostimulatory monoclonal antibodies

Inmaculada Rodriguez², Lina Mayorga¹, Tania Labiano³, benigno Barbes¹, inaki etxeberria², Mariano Ponz-Sarvise¹, arantza Azpilicueta², elisabeth Bolanos², Miguel F.Sanmamed¹, Pedro Berraondo², Felipe Manuel Alfonso Calvo¹, Mary Helen Barcelos-Hoff⁴, Jose Luis Perez-Gracia¹, Ignacio Melero, MD, PhD³, Maria Rodriguez-Ruiz, MD, PhD¹

¹Clinica Universidad de Navarra, Pamplona, Spain; ²Centro de Investigacion Medica Aplicada, Pamplona, Spain; ³Complejo Hospitalario de Navarra, Pamplona, Spain; ⁴University of California, San Francisco, San Francisco, CA, USA

Correspondence: Ignacio Melero (imelero@unav.es)

Background

Radiotherapy can be synergistically combined with immunotherapy in mouse models, extending its efficacious effects outside of the irradiated field (abscopal effects) [1]. We previously reported that a regimen encompassing local radiotherapy in combination with anti-CD137 plus anti-PD-1 mAbs achieves potent abscopal effects against syngeneic transplanted murine tumors up to a certain tumor size. Knowing that TGFβ expression or activation increases in irradiated tissues, we tested whether TGFβ blockade may further enhance abscopal effects in conjunction with the anti-PD-1 plus anti-CD137 mAb combination [2].

Methods

Mice bearing bilateral MC38 or 4T1 tumors were randomly assigned to 6 groups receiving or not radiotherapy (8Gy/3fx), in combination or not with intraperitoneal antibodies (anti-PD1 plus anti-CD137 and/or anti-TGFβ). Tumor tissue was processed to obtain single-cell suspensions for flow cytometry analyses Levels of TGFβ-1 in mouse tumor tissue homogenates and IFNγ in mouse plasma samples were measured by commercial ELISAs.

Results

TGFβ blockade with 1D11, a TGFβ neutralizing monoclonal antibody, markedly enhanced abscopal effects and overall treatment efficacy against subcutaneous tumors of either 4T1 breast cancer cells or large MC38 colorectal tumors. Increases in CD8 T cells infiltrating the non-irradiated lesion were documented upon combined treatment, which intensely expressed Granzyme-B as an indicator of cytotoxic effector capability.

Conclusions

Radiotherapy-induced TGFβ hampers abscopal efficacy even upon combination with a potent immunotherapy combination. Therefore TGFβ blockade in combination with radioimmunotherapy regimens results in greater efficacy.

Acknowledgements

We are in debt to Dr. Alan Korman (BMS, San Francisco CA) for his kind gift of monoclonal antibodies. We acknowledge generous help from Drs. Martinez-Monge, Aristu, Castañon and Gil-Bazo from the department of oncology at CUN. Excellent dosimetry by Arantza Zubiria and dedicated animal care by Eneko Elizalde are also acknowledged.

Trial Registration

NA

References

1. Rodriguez-Ruiz ME, Rodriguez I, Barbes B. Abscopal effects of radiotherapy are enhanced by combined immunostimulatory mAbs and are dependent on CD8 T cells and crosspriming. Cancer Res. 2016 Oct 15;76(20):5994-6005.

2. Vanpouille-Box C, Diamond JM, Pilones KA. TGFβ is a master regulator of radiation therapy-induced antitumor immunity. Cancer Res. 2015 Jun 1;75(11):2232-42.

Ethics Approval

The study was approved by Navarra Institutution‘s Ethics Board, approval number 117/14

P467 Augmenting immunity with IAP antagonists in PDAC

Kevin Roehle, PhD¹, Michael Dougan, MD, PhD¹, Stephanie Dougan, PhD²

¹Dana-Farber Cancer Institute, Bosotn, MA, USA; ²Massachusetts General Hospital, Boston, MA, USA

Correspondence: Michael Dougan (mldougan@partners.org)

Background

Pancreatic ductal adenocarcinoma (PDAC) is responsible for about 7% of all cancer-related deaths in the US. PDACs are fibrotic and dense tumors with little vasculature, and are rapidly metastatic. Thus far, cancer- immunotherapy with immune checkpoint blocking antibodies have largely failed in PDAC. The Inhibitor of Apoptosis (IAP) protein family comprises a diverse group of proteins, many of which have immunoregulatory roles. IAP antagonists are small molecule drugs that primarily inhibit cellular (c-)IAP1 and c-IAP2 protein leading to TNFa mediated apoptosis in tumor cells through alternative NF-kB signaling. In immune cells IAP antagonism leads to increased alternate NF-kB signaling, leading to increased survival of B cells, activation of dendritic cells and supporting activation of T cells in a costimulatory manner.

Methods

We evaluated the effects of the IAP antagonist LCL-161 in multiple syngeneic models of pancreatic cancer.

Results

Although LCL-161 did not induce TNFa mediated apoptosis in any of our tumor cell lines in vitro, we were able to induce robust immune-mediated regressions in an orthotopic and subcutaneous tumor models.

Conclusions

These responses were dependent on CD8 and CD4 T cells, and we show evidence for a direct effect of LCL-161 in augmenting T cell priming in pancreatic cancer.

P468 Transcriptomic profiles conducive to immune-mediated tumor rejection in human breast cancer skin metastases treated with Imiquimod

Mariya Rozenblit, MD¹, Wouter Hendrickx, PhD², Adriana Heguy³, Luis Chiriboga³, Cynthia Loomis³, Karina Ray³, Farbod Darvishian, MD³, Mikala Egeblad, PhD⁴, Davide Bedognetti, MD, PhD⁵, Sylvia Adams, MD³

¹Yale University, Connecticut, CT, USA; ²Sidra Medicine, Doha, Qatar; ³NYU, New York, USA; ⁴Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA; ⁵SIDRA, Doha, Qatar

Correspondence: Mariya Rozenblit (Mariya.Rozenblit@yale.edu)

Background

Imiquimod is a topical toll-like-receptor-7 agonist currently used for treating basal cell carcinoma. Recently, imiquimod has demonstrated tumor regression in melanoma and breast cancer skin metastases. However, the molecular perturbations induced by imiquimod in breast cancer metastases have not yet been characterized. Here, we describe transcriptomic profiles associated with responsiveness to imiquimod in breast cancer skin metastases.

Methods

Baseline and post-treatment tumor samples from eight patients treated with imiquimod in a clinical trial were profiled using Nanostring nCounter ® Human v1.1 PanCancer Immune Profiling Panel. Two of the patients had stable disease during the initial study and were found to have a systemic complete clinical response after subsequent treatment with fulvestrant after study completion which continued for several years. On follow up, these two patients also had disease remission for two years. An additional patient had a local partial anti-tumor response after eight weeks of imiquimod treatment and was labeled as a partial responder (PR). Five of the eight patients did not have an anti-tumor response and were defined as non-responders (NR). An integrative analytic pipeline was used to analyze gene expression data including pathway analysis and deconvolution.

Results

We showed that tumors from patients who achieved a durable clinical response displayed a permissive microenviroment, substantiated by the upregulation of transcripts encoding for molecules involved in leukocyte adhesion and migration, cytotoxic functions, and antigen presentation (Figure 1AB). Imiquimod triggered a strong T-helper-1 (Th-1)/cytotoxic immune response, characterized by the coordinated upregulation of Th-1 chemokines, migration of Th-1 and cytotoxic T cells into the tumor, and activation of immune-effector functions, ultimately mediating tumor destruction (Figure 1C).

Conclusions

Topical imiquimod can induce a robust immune response in breast cancer metastases, and this response is more likely to occur in tumors with a pre-activated microenvironment. In this setting, imiquimod could be utilized in combination with other targeted immunotherapies to increase therapeutic efficacy.

Acknowledgements

The work was supported by the AMA Foundation Seed Grant

Ethics Approval

The clinical trial was approved by the New York University Institutional Review Board

Fig. 1 (abstract P468).

See text for description

P469 TCR repertoire correlates of response in tumor-bearing mice treated with radiotherapy and CTLA-4 blockade

Nils-Petter Rudqvist, PhD¹, Claire Lhuillier, PhD¹, Erik Wennerberg, PhD¹, Jennifer Sims, PhD² , Sandra Demaria, MD¹

¹Weill Cornell Medical College, New York, NY, USA; ²Memorial Sloan Kettering Cancer Center, New York, NY, USA

Correspondence: Sandra Demaria (szd3005@med.cornell.edu)

Background

Tumor-targeted radiation therapy (RT) in combination with immune checkpoint blockade can activate tumor-specific T-cells to reject tumors. Yet, predictive features of effectively primed T cell repertoires (TCR) remain poorly understood. Using the 4T1 mouse model of triple negative breast cancer, where RT+CTLA-4 blockade elicits an anti-tumor T cell response that controls both the irradiated tumor and non-irradiated lung metastases and extends survival, we previously reported increased intratumoral CD8/CD4 ratio and CD8+ T cell clonality following RT+anti-CTLA-4 treatment [1]. Here, we determined the longitudinal changes of the TCR repertoires in the 4T1 carcinoma and its correlates with treatment response.

Methods

To analyze longitudinally the TIL repertoire before and after treatment with RT+anti-CTLA-4, mice were inoculated in both flanks with 4T1 cells (n=8/group). One tumor was resected 2 days before treatment (pre-TX) and the other was treated with RT (3X8 Gy) or anti-CTLA-4 antibody (3x200 μg i.p.) monotherapy or in combination and resected 1 day after treatment when immune-mediated tumor rejection is occurring in tumors treated with RT+anti- CTLA-4 (post-TX). No local tumor recurrence was observed, but mice succumbed of lung metastasis with the largest increase in survival (vs. untreated) in mice given RT+anti-CTLA-4 (p=0.0041). To assess the TIL TCR repertoire, dual-stage PCR amplification and high-throughput sequencing of the TCRa and b CDR3 regions was performed using mRNA isolated from total tumor.

Results

In tumors treated with RT and RT+anti-CTLA-4, both the TCRa and b repertoires increased in clonality compared to pre-TX, whereas a smaller increase in TCRb clonality was found after anti-CTLA-4 monotherapy. We have previously characterized the TCRb repertoire of expanded and activated CD8+ T cells recognizing the AH1 epitope from gp70 antigen (a tumor antigen expressed by 4T1 cells) in tumors of mice treated with RT+anti-CTLA-4 [1]. Using GLIPH [2], we identified a major AH1-specific CDR3b motif and found it present in pre-TX tumors of all animals, and its frequency increased in mice treated with RT or RT+anti-CTLA-4, consistent with our previous findings. In contrast, the V-J Jensen-Shannon divergence, assessed excluding AH1-specific T cells, was higher between pre- and post-TX tumors in all mice treated with RT, (independent of anti-CTLA-4 treatment) and correlated strongly with survival (cox regression; p=0.00054), suggesting priming and expansion of T cells targeting antigens other than AH1.

Conclusions

Together these data support the dominant role of RT in priming emergent or low-abundance T cell clonotypes, rather than the driving of already-prevalent clonotypes.

References

1. Rudqvist NP, Pilones KA, Lhuillier C, Wennerberg E, Sidhom JW, Emerson RO, Robins HS, Schneck J, Formenti SC, Demaria S. Radiotherapy and CTLA-4 blockade shape the TCR repertoire of tumor-infiltrating T cells. Cancer Immunol Res. 2018; 6(2): 139-150.

2. Glanville J, H. Huang A, Nau O, Hatton LE, Wagar F, Rubelt X, Ji A, Han SM, Krams C, Pettus N, Haas CSL, Arlehamn A, Sette SD, Boyd TJ, Martinez S, Davis MM. Identifying specificity groups in the T cell receptor repertoire. Nature. 2017; 547(7661): 94-98.

Ethics Approval

All experiments were approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee, approval number 2015-0028.

P470 Targeting DNA damage response promotes anti-tumor immunity through STING-mediated T-cell activation in small cell lung cancer

Triparna Sen, PhD¹, Bertha Leticia Rodriguez¹, Limo Chen, PhD¹, Naoto Morikawa¹, Junya Fujimoto¹, Lixia Diao¹, Youhong Fan¹, Jing Wang¹, Bonnie Glisson¹, Ignacio Wistuba, MD¹, Julien Sage², John Heymach¹, Don Gibbons, MD¹, Lauren Byers, MD¹

¹UT MD Anderson Cancer Center, Houston, TX, USA; ²Stanford University, Houston, TX, USA

Correspondence: Lauren Byers (lbyers@mdanderson.org)

Background

Despite recent advances in the use of immunotherapy, only a minority of small cell lung cancer (SCLC) patients respond to immune checkpoint blockade (ICB) with programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) antibodies as monotherapy or combination. We have previously established that SCLC is vulnerable to agents targeting the DNA damage response (DDR) pathway, including inhibitors of PARP and CHK1, and these findings have led to several clinical trials of DDR inhibitors for SCLC. Our recent findings demonstrate that CHK1 inhibitor, prexasertib, potentiate the anti-tumor response of PD-L1 blockade in SCLC. In the present study we have established the efficacy of combined PARP (olaparib) and PD-L1 blockade and further elucidated the underlying mechanism of DDR-mediated anti-tumor immune response in SCLC models.

Methods

SCLC models were treated with small molecule inhibitors prexasertib or olaparib with or without anti-PD-L1. End point analyses were done by immunoblot, immunohistochemistry, qRT-PCR, flow cytometry and reverse phase protein array (RPPA).

Results

We treated the tumor bearing B6129F1 mice with either IgG, olaparib (100mg/kg,4/7), anti-PD-L1 or combination. By 10days, all mice treated with olaparib+anti-PD-L1 combination had a complete tumor regression with no tumor growth upto 80days. Co-targeting DDR+PD-L1 significantly increased the level of CD8+ cytotoxic-T cell infiltration and decreased PD-1+/TIM3+ exhausted T-cell population (p<0.0001). CD8 depletion was able to reverse the anti-tumor effect of the combination demonstrating the role of cytotoxic T-cell infiltration and function in DDR+PD-L1 response. Next, we established, by genetic knockdown studies, that DDR-mediated immune response was facilitated through activation of the STING/TBK1/IRF3 innate immune response pathway. STING pathway activation enhanced expression of Type-1 interferon gene IFNβ and downstream chemokines, CXCL10/CCL5 and resulted in T-cell recruitment.

Conclusions

Our results demonstrate,for the first time, the remarkable efficacy of the combination of PD-L1 blockade with PARP/CHK1 inhibition and provide a strong scientific rationale for combining these modalities in clinical trials for SCLC patients. Combining DDR inhibition with ICB leads to T-cell recruitment and enhanced effector cell function in SCLC tumors, mediated by the activation of the innate immune response pathway STING/TBK1/IRF3 and increased IFNβ. The immune profiling and correlative biomarker data from our study provide valuable mechanistic insight and indicate the subset of the patient population who are most likely to respond to these treatments. Because prexasertib, olaparib and other PARP inhibitors are already in clinical trials for SCLC, we expect that this hypothesis has the potential for rapid translation into the clinic.

P471 Mertk is a therapeutic target in combination with radiation to promote adaptive immune tumor responses

Garth Tormoen, MD, PhD¹, Jason Baird, PhD², Gwen Kramer, BS², Shelly Bambina², Marka Crittenden, MD, PhD², Michael Gough, PhD²

¹Oregon Health & Science University, Portland, OR, USA; ²Earl A. Chiles Research Institute, Portland, OR, USA

Correspondence: Garth Tormoen (tormoeng@ohsu.edu)

Background

Mertk is a member of the Tyro3-Axl-Mertk (TAM) family of receptors and regulates phagocytosis of dying cells by macrophages. Cancer cells killed by radiation therapy direct repolarization of macrophages into immune suppressive phenotypes. Mertk-/- mice grafted with immunogenic tumors have enhanced tumor control following ionizing radiation compared to Mertkwt mice. Gas6 is the endogenous ligand for Mertk and its ability to signal through Mertk requires a post-translational vitamin k-dependent modification that is inhibited by warfarin.

Methods

Mertk-/- and WT mice were injected subcutaneously in the flank with 5E4 CT26 cells (BALB/c) or 5E6 Panc02-SIY cells (C57BL/6) and allowed to grow to 5 mm before treatment with 250 μg anti-CD8α antibodies, warfarin (0.5 mg/L drinking water) and subjected to a single dose of ionizing radiation (16 Gy) followed by 250 μg of OX40 or PBS I.P. 1-day post-RT. Peripheral blood was collected 6 days after RT and evaluated by Flow Cytometry for SIY- pentamer+CD8+ T cells.

Results

Radiation therapy results in tumor control in BALB/c mice, but tumor cure in Mertk-/- BALB/c mice. Tumor cure in Mertk-/- BALB/c mice was abrogated by depletion of CD8 T cells indicating that ligation of Mertk in tumor macrophages suppresses endogenous anti-tumor immunity following radiation therapy. Similarly, warfarin-treated mice had higher rates of tumor cure following radiation that was also abrogated by CD8 depletion. In C57BL/6 mice, Mertk-/- alone does not affect responses to radiation therapy in the Panc02 tumor model, but the combination of radiation therapy with anti-OX40 costimulation of T cell responses resulted in a significant increase in peripheral blood SIY+ CD8 T cells 5 days after treatment, and significantly improved survival compared to radiation alone.

Conclusions

Mertk-/- mice, and Mertkwt mice treated with warfarin to inhibit Gas6 experience increased tumor control following ionizing radiation in an adaptive-immune mediated manner in CT26 tumor models. In less immunogenic tumors, loss of Mertk-/- permitted tumor cure following radiation therapy when combined with the T cell costimulatory molecule OX40. These data demonstrate that Mertk suppresses adaptive immunity in irradiated tumors. Mertk is an attractive therapeutic target in combination with ionizing radiation and immune therapy to promote adaptive immune anti-tumor responses.

Ethics Approval

All animal studies were approved by the Earl A. Chiles Research Institute IACUC, Assurance No. A3913-01.

P472 Immunogenic tumor antigen is required in antitumor effect of cisplatin monotherapy and its combination with anti-PD-L1

Daiko Wakita, PhD¹, Toshiki Iwai, BS¹, Masamichi Sugimoto, PhD¹, Osamu Kondoh¹

Chugai pharmaceutical CO., LTD., Kamakura, Japan

Correspondence: Osamu Kondoh (kondoosm@chugai-pharm.co.jp)

Background

Although anti-PD-L1/PD-1 immunotherapy has shown marked clinical effect in a broad range of cancer, a subset of patients respond to monotherapy, and tumor mutation burden have been identified as potential predictive marker for responders. To extend the clinical benefits, combination of anti-PD-L1 and chemotherapy has been actively investigated. However the association of immunogenicity of neoantigen with antitumor effects of the combination of immunotherapy and chemotherapy is remained unknown. Here we investigated tumor antigen-specific T cell responses and antitumor effect of the anti-PD-L1 plus cisplatin combination therapy in mouse tumor models.

Methods

E.G7-OVA cell, expressing ovalbumin (OVA) gene as an immunogenic model tumor antigen, and its parental less immunogenic EL4 cell were subcutaneously inoculated into C57BL/6 mouse. The tumor-bearing mice were intraperitoneally treated with anti-mouse PD-L1 mAb (anti-PD-L1; 10 mg/kg, three times a week) and cisplatin (CDDP; 1 mg/kg, once at Day1). For CD8+ cell depletion experiments, the tumor-bearing mice were treated with CDDP (4 mg/kg, once at Day1) and anti–mouse CD8 mAb (twice a week from one day before the treatment initiation). To evaluate cytolytic activity, CD8+ T cells isolated at Day7 were co-cultured with CFSE labeled-tumor cells and the frequency of dying-tumor cell was measured by flow cytometry.

Results

Anti-PD-L1 alone and CDDP alone exhibited significant antitumor effect in E.G7-OVA-bearing mice and the combination therapy resulted in further effect than the each monotherapy at Day15. In parallel with the therapeutic effect, CD8+ T cells from tumor-draining lymph node exhibited higher cytolytic activity against E.G7-OVA in monotherapy group than that in control group, and highest cytolytic activity was observed in combination group. In contrast, even after the combination therapy, cytolytic activity against parental EL4 cells was hardly detected. In addition, less immunogenic EL4 were insensitive to monotherapies with anti-PD-L1, CDDP and their combination. Moreover, in immunogenic E.G7-OVA-bearing model, the higher dose of CDDP (4 mg/kg) showed direct cytolytic activity as well as CD8+ T cell dependent antitumor effect, whereas only direct cytotoxic effect was observed in EL4-bearing model.

Conclusions

In our model, not only anti-PD-L1 alone but also CDDP alone enhanced T cell responses against immunogenic tumor antigen (OVA) but not neoantigens in EL4 cell, indicating the higher impact of immunogenic tumor antigen in antitumor effects during anti-PD-L1 therapy and chemotherapy. We are further exploring the contribution of subdominant epitopes of OVA or antigens other than OVA to antitumor effect.

P473 Enhancing abscopal responses to radiation therapy by manipulating autophagy

Takahiro Yamazaki, PhD, Marissa Rybstein, Aitziber Buqué, PhD, Ai Sato, Lorenzo Galluzzi, PhD

Weill Cornell Medicine, New York, NY, USA

Correspondence: Takahiro Yamazaki (tay2007@med.cornell.edu)

Background

Macroautophagy (autophagy) is an evolutionary conserved cellular mechanism culminating with the lysosomal degradation of dispensable, damaged or potentially toxic cytoplasmic structures (e.g., permeabilized mitochondria). Autophagy helps cancer cells to adapt to harsh environmental conditions and to resist therapy. However, autophagy is also key for multiple steps of the anticancer immune response. Thus, whether autophagy should be inhibited or activated in the context of cancer therapy remains debated [1]. Since autophagy has been shown to play a key role in the removal of cytosolic DNA, which is one mechanism leading to type I interferon (IFN) secretion, and since type I IFN is required for systemic immune responses activated by radiation therapy (RT), we asked the question as to whether selectively inhibiting autophagy in cancer cells may boost the ability of RT to initiate anticancer immunity.

Methods

CRISPR/Cas9 technology was used to render mouse mammary carcinoma TSA and EO771 cells autophagy deficient, and chemical inhibitors of autophagy were employed. Autophagy-competent versus –deficient cells were characterized for autophagic proficiency (by immunoblotting), growth (in vitro and in vivo), resistance to cell death induced by starvation, chemotherapy and RT (by multicolor flow cytometry and clonogenic assays) and production of type I IFN (by PCR and ELISA). Alongside, cancer cells were employed to generate synchronous tumors in immunocompetent syngeneic mice. Only one of these tumors (that was either autophagy-competent or-deficient) was irradiated in the context of CTLA4 inhibition, and the response of both the irradiated and non-irradiated (abscopal) tumor was monitored.

Results

Autophagy inhibition reduced the growth of mouse mammary carcinoma cells, in vitro and in vivo, limited their clonogenic potential (at baseline) and increased their sensitivity to multiple stressors. Moreover, pharmacological and genetic autophagy inhibition increased the capacity of mouse mammary carcinoma cells to secrete type I IFN in response to radiation. Finally, immunocompetent mice bearing syngeneic autophagy-deficient mouse mammary carcinoma cells mounted improved abscopal responses to RT (in the context of CTLA4 blockade) as compared to immunocompetent mice bearing syngeneic autophagy-competent cells, as determined by growth inhibition of a distant, non-irradiated, autophagy-competent lesion.

Conclusions

In conclusion, autophagy inhibits abscopal responses by limiting the release of type I IFN by irradiated cancer cells. We will test the innovative hypothesis that selective autophagy inhibition in cancer cells may synergize with autophagy activation at the whole-body level (by nutrient restriction or physical exercise), hence enabling superior therapeutic responses to radiation.

References

1. Rybstein MD, Bravo-San Pedro JM, Kroemer G, Galluzzi L. The autophagic network and cancer. Nat Cell Biol. 2018;20(3):243-251.

Ethics Approval

The study was approved by Weill Cornell Medicine‘s Ethics Board, approval number 2017-0007.

P474 Antibody targeting of WNT signaling modulator dickkopf1 (DKK1) enhances innate anti-tumor immunity and complements anti-PD-1 therapy

Mike Haas, Heidi Heather, Franziska Schürpf-Huber, Lane Newman, Walter Newman, PhD, Mike Kagey, Min Yang, PhD

Leap Therapeutics, Cambridge, MA, USA

Correspondence: Min Yang (myang@leaptx.com)

Background

DKK1, a secreted modulator of the Wnt and PI3K/AKT signaling pathways, may contribute to an immunosuppressive tumor microenvironment by influencing Tregs, MDSCs and NK cell functions [1] while also affecting tumor cell NK target expression [2]. DKK1 is expressed in a variety of tumor types and elevated levels frequently correlate with poor survival. DKN-01 is a neutralizing IgG4 monoclonal against DKK1, and mDKN-01 is a murine version of DKN-01.

Methods

mDKN-01 has been evaluated in B16 and 4T1 syngeneic mouse tumor models as monotherapy and in combination with anti-PD-1

Results

As a monotherapy mDKN-01 demonstrated a highly reproducible inhibition of tumor growth in a B16 melanoma syngeneic model, with concomitant infiltrates of CD45+ CD11b immune cells. Intratumoral MDSC are reduced and show elevated expression of PD-L1. mDKN-01 anti-tumor activity is lost in immunocompromised NSG mice and following depletion of NK cells in immune competent mice. In addition, mDKN-01 anti-tumor activity is retained in a RAG1-/- mouse, highlighting the T cell independence and NK dependence of the anti-tumor mechanism. Combination therapy with anti-PD-1 and mDKN-01 showed cooperative tumor growth inhibition in the B16 model, and reduction of lung metastasis in a 4T1 breast tumor model. These data indicate the potential for DKN-01 complementarity with checkpoint blockers.

Conclusions

In murine studies mDKN-01 is an innate-immune IO agent with effects on NK and MDSC cells. mDKN-01 plus anti-PD-1 are more active together than either agent alone in two murine syngeneic models. Preliminary clinical results demonstrate that DKN-01 in combination with pembrolizumab is well tolerated and is clinically active in esophagogastric cancer, including in patients previously treated with other checkpoint inhibitors or in immune resistant phenotypes not expected to respond to pembrolizumab alone. Clinical trial information: NCT02013154

Trial Registration

Clinical trial information: NCT02013154

References

1. Chae WJ, Ehrlich AK et al. The wnt antagonist dickkopf-1 promotes pathological type 2 cell-mediated inflammation immunity. 2016; 44:246-258.

2. Chae WJ, Park JH, et al. Membrane-bound dickkopf-1 in foxp3+ regulatory T cells suppresses T-cell-mediated autoimmune colitis. Immunology. 2017;265-275.

3. D'Amico L, Mahajan S. Dickkopf-related protein 1 (Dkk1) regulates the accumulation and function of myeloid derived suppressor cells in cancer. J Exp Med. 2016; 213:827-40.

4. Malladi S, Macalinao DG, et al. Metastatic latency and immune evasion through autocrine inhibition of WNT Cell. 2016;165:45-60.

P475 Avadomide in combination with nivolumab results in increased activated and memory T-cells and enhances CD8+ tumor infiltration in hepatocellular carcinoma patients

Patrick Hagner, PhD¹, Fadi Towfic, PhD¹, Alfredo Romano, MD¹, Julien Edeline, MD², Carlos Gomez-Martin, MD, PhD³, Antoine Hollebecque, MD⁴, Robin Kate Kelley, MD⁵, Armando Santoro, MD, PhD⁶, Michael Pourdehnad, MD¹, Anita Gandhi, PhD¹

¹Celgene Corporation, Summit, NJ; ²Centre Eugene Marquis, Rennes, France; ³Hospital 12 de Octubre, Madrid, Spain; ⁴Institut G. Roussy, Villejuif, France; ⁵University of California San Francisco, San Francisco, CA, USA; ⁶Humanitas University, Rozzano-Milan, Italy

Correspondence: Patrick Hagner (phagner@celgene.com)

Background

Avadomide (CC-122) binds E3 ubiquitin ligase CRL4^CRBN, resulting in degradation of transcription factors Aiolos and Ikaros, and activation of T cells. Preclinical and clinical data indicate that avadomide exerts strong immunomodulatory activity through enhanced ADCC and a shift in T-cell subsets from naïve to effector and memory subsets. Avadomide is in clinical development in multiple hematologic diseases and has been explored in solid tumors, including hepatocellular carcinoma (HCC), as single agent (NCT01421524) and in combination with nivolumab (NCT02859324). In preclinical models, avadomide plus nivolumab demonstrates synergistic activation of T cells and significantly enhanced immune-mediated cytotoxicity against HCC cells. Here, we report the effects of combining avadomide with nivolumab on peripheral blood T-cell subsets and activation status and on trafficking of immune cells to the tumor in patients with HCC.

Methods

Peripheral blood T-cell subsets were analyzed by flow cytometry. Tumor biopsies were analyzed by immunohistochemistry or RNA sequencing with deconvolution analyses to identify immune cell populations.

Results

Avadomide, as single agent and in combination with nivolumab, results in decreased absolute peripheral CD4+ and CD8+ naïve (CD45RA+/CD45RO–) T cells and increased memory (CD45RA–/CD45RO+) and activated (HLA- DR+) T cells, without significantly affecting total CD3+, CD4+ or CD8+ populations. Interestingly, the combination demonstrated a trend towards greater increase in activated (+182%) and memory (+257.9%) CD4+ T cells compared with avadomide alone (+123.2% and +12.2%). Increased levels of peripheral Treg populations were detected within 15 days of treatment initiation, and the CD8/Treg ratio declined from 7.8 at screening to 2.7 on C1D15. To understand the effects on tumor microenvironment, we performed RNA sequencing on paired tumor biopsies from patients receiving combination treatment collected at enrollment and six weeks after treatment initiation (n=9). Deconvolution analyses identified increased infiltration of T-cell populations, dendritic cells and macrophages, and decreased B-cell populations in on-treatment biopsies relative to pre-treatment. Immunohistochemistry confirmed significantly increased CD8+ T-cells in on-treatment biopsies relative to pre-treatment in patients receiving the combination (P=0.04), while no significant changes in CD8+ T-cell infiltration were observed in patients receiving single agent avadomide (P=0.65).

Conclusions

Avadomide is a potent immunomodulating agent with multiple immune activating properties. Avadomide plus nivolumab leads to significantly greater CD8+ T-cell tumor infiltration compared with single agent avadomide. These findings provide proof-of-concept for the combination of avadomide with checkpoint blockade in solid tumors and demonstrate potential for further clinical and biomarker studies to ascertain relative contribution of avadomide over nivolumab monotherapy and assess efficacy.

Trial Registration

ClinicalTrials.gov identifier NCT01421524 and NCT02859324.

Ethics Approval

This study was approved by the research ethics boards of all participating institutions.

Consent

Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Immunosuppressive Cells in the Tumor Microenvironment

P476 EWS-FLI1 expression level modulates T-cell mediated tumor apoptosis in Ewing sarcoma

Claire Julian, Ariel Klinghoffer, Hether Bernard, MD, Linda McAllister-Lucas, Kelly Bailey, MD, PhD

University of Pittsburgh, Pittsburgh, PA, USA

Correspondence: Kelly Bailey (kelly.bailey@chp.edu)

Background

Metastatic Ewing sarcoma is a deadly bone cancer most commonly diagnosed in children and is driven by the fusion oncoprotein EWS-FLI1. The level of EWS-FLI1 expression can change Ewing cell behavior. Interestingly, lower levels of EWS-FLI1 are associated with increased expression of ICAM-1, a surface protein reported in some cancers to influence tumor cell: T-cell interaction and promote T-cell activation. Very little is known about the immune response to Ewing sarcoma tumor cells and elucidating the mechanisms regulating the immune response to Ewing tumor cells may reveal much needed new treatment opportunities for patients with metastatic disease. In this study, we seek to determine the impact of tumor cell EWS-FLI1 expression level on T-cell mediated Ewing sarcoma tumor cell apoptosis.

Methods

We performed real-time monitoring of tumor cell caspase 3 activity in Ewing tumor cell/T-cell co-cultures. For this analysis, Ewing sarcoma tumor cell populations with ‘high’ or ‘low’ EWS-FLI1 expression were prepared by either: 1) using flow cytometry to isolate naturally occurring populations or 2) using EWS-FLI1 siRNA to generate EWS- FLI1 ‘low’ cells. Human T-cells were isolated from random donor buffy coat, and T-cells were activated using a CD2/3/28 antibody cocktail. Surface expression of ICAM-1, PD-L1 and PD-L2 was determined by flow cytometry analysis. Blocking antibodies were also utilized.

Results

EWS-FLI1 ‘low’ cells demonstrated a significant decrease in T-cell mediated tumor cell apoptosis upon introduction of ICAM-1 blocking antibody. Based on this, we questioned whether EWS-FLI1 ‘low’ cells would be more susceptible to T-cell mediated apoptosis that EWS-FLI1 ‘high’ cells (that lack surface ICAM-1). Notably, despite having higher ICAM-1, we found that EWS-FLI1 ‘low’ cells are actually less susceptible to T-cell mediated apoptosis that EWS-FLI1 ‘high’ cells, suggesting that EWS-FLI1 ‘low’ cells possess an ability to evade T-cell- mediated tumor killing. Further, in comparison to EWS-FLI1 ‘high’ cells, EWS-FLI1 ‘low’ cells respond to interferon-gamma treatment with dramatically greater transcriptional upregulation of PD-L1 and PD-L2. We then assessed the impact of PD-1 blocking antibody on T-cell mediated tumor cell apoptosis and found that treatment of EWS-FLI1 ‘low’ cell/T-cell co-cultures with blocking antibody significantly enhances T-cell-induced tumor cell apoptosis.

Conclusions

We have shown that Ewing cells with lower EWS-FLI1 are more resistant to T-cell mediated apoptosis than cells with higher EWS-FLI1. As such, EWS-FLI1 ‘low’ cells may serve as negative regulators of the immune response in Ewing tumors. These data highlight that Ewing tumor cell heterogeneity can influence the anti-tumor immune response.

Acknowledgements

KMB is supported by Alex’s Lemonade Stand Foundation Young Investigator Award, The Children’s Cancer Research Fund Emerging Scientist Award and the NIH 2K12HD052892-11A1

P477 HMBD-002-V4: A novel anti-VISTA antibody that uniquely binds murine and human VISTA and potently inhibits tumor growth by remodeling the immunosuppressive tumor microenvironment

Jerome Boyd-Kirkup, PhD, Dipti Thakkar, PhD, Vicente Sancenon, PhD, Siyu Guan, PhD, Konrad Paszkiewicz, PhD, Piers Ingram, PhD

Hummingbird Bioscience, South San Francisco, CA, USA

Correspondence: Piers Ingram (p.ingram@hummingbirdbio.com)

Background

Immune checkpoint therapies have shown unprecedented clinical activity in several types of cancer, however, less than 30% of patients respond. VISTA is a co-inhibitory immune checkpoint receptor of the B7 family and functions to suppress human T-cell activity. VISTA is highly expressed on tumor infiltrating myeloid cells including myeloid derived suppressive cells (MDSC), which have been associated with resistance to immunotherapy. Increases in VISTA+ cells have also been observed in response to PD1 and CTLA4 therapy. Targeting VISTA could represent a novel treatment axis in the non-responder population.Despite the promise of VISTA, limited structural information, lack of a definitive ligand, and incomplete data on expression in normal vs. disease contexts, have made development of drug candidates challenging. Further, previous anti-VISTA antibodies have only bound to rodent or human VISTA, making it impossible to translate pre-clinical efficacy and safety data to predict patient response.

Methods

HMBD-002-V4 is a humanized anti-VISTA antibody developed using Hummingbird Bioscience’s proprietary Rational Antibody Discovery platform to target a specific epitope predicted by structural modeling to block ligand binding and be conserved between human, cyno and murine VISTA.

Results

In vitro, HMBD-002-V4 showed dose-dependent inhibition of the interaction between VISTA and the putative ligand VSIG3 for both human and mouse orthologs, and further demonstrated release of VISTA inhibition on T-cell activity and increased secretion of pro-inflammatory cytokines in human ex vivo assays.In vivo, HMBD-002-V4 showed single agent tumor growth inhibition (TGI) of up to 40% in syngeneic murine CDX models, however, efficacy was significantly improved if combined with anti-PD(L)1 antibody where TGI above 94% was possible. Profiling of representative tumors by FACS revealed MDSC infiltration in these models that was significantly increased after treatment with anti-PD(L)1 antibody and associated with an increase in immunosuppressive serum cytokines. Conversely, HMBD-002-V4 efficacy was associated with decreased MDSC infiltration for both monotherapy and combination arms and a remodeling of the tumor microenvironment towards a pro-inflammatory phenotype. In models without MDSC infiltration, HMBD-002-V4 showed poor efficacy. HMBD-002-V4 was evaluated for pharmacokinetics and toxicology and demonstrated excellent serum half-life of 11 days, with no observable toxicity in multiple animal models. Further, HMBD-002-V4 has been optimized for manufacturability, including high expression titers and stability.

Conclusions

HMBD-002-V4 represents a promising therapeutic candidate for the treatment of VISTA-mediated suppression of anti-tumor immunity. Predictive biomarkers of response to HMBD-002-V4 are currently being explored in multiple indications and the first-in-human trial of HMBD-002-V4 is planned for 2019.

Ethics Approval

The study was approved by the SingHealth Institutional Animal Care and Use Committee, approval number 2016/SHS/1230.

P478 Platelets as immune suppressors in anti-cancer immune responses

Ana Micaela Carnaz Simões, Morten Holström, PhD, Mads Andersen, PhD, Per Thor Straten, PhD

Center for Cancer Immune Therapy, Herlev, Herlev, Denmark

Correspondence: Ana Micaela Carnaz Simões (ana.micaela.carnaz.simoes@regionh.dk)

Background

Platelets (PLTs) are well-known players during cancer progression. For several cancers, an increased number of circulating PLTs correlates with poor prognosis. PLTs help cancer cells by modulating angiogenesis and/or directly binding cancer cells, which facilitates the metastatic process [1,2]. These cells and their soluble factors can also protect cancer cells from immune attack by mechanisms that are poorly understood. Studies focused on autoimmune conditions, have shown that exhausted PLTs form aggregates with T cells, downregulating T cell activation, proliferation and interferon-ɣ production [3,4]. Nevertheless, no similar study has been conducted in the context of cancer.

Methods

Our study investigated the presence of circulating PLT-immune cell aggregates in myeloproliferative neoplasm (MPN) patients. To that purpose, cryopreserved peripheral blood mononuclear cells were analyzed by multicolor flow cytometry for PLT bound -T, -NK, -B and -CD3+/CD56+ cells, as well as CD4 and CD8 T cell subpopulations. Furthermore, to assess the effect PLT-binding has on T and NK cell anti-tumor reactivity, in vitro cytotoxic response was continuously monitored over 40 hours, using the xCELLigence technology.

Results

Our preliminary results show that, when compared to healthy donors, MPN patients have an increased number of PLT bound CD8+ T, NK and CD3+/CD56+ cells. Finally, our results indicate that platelets can modulate the T and NK cells tumor reactivity in distinct manners; the presence of PLTs impairs the killing capacity of T cell whereas it seems to enhance it on NK cells. However, further studies are needed to confirm our preliminary results.

Conclusions

N/A

References

1. Borsig L. The role of platelet activation in tumor metastasis. Expert Rev Anticancer Ther. 2008;8(8):1247-1255. doi:10.1586/14737140.8.8.1247.

2. Bambace NM, Holmes CE. The platelet contribution to cancer progression. J Thromb Haemost. 2011;9(2):237- 249. doi:10.1111/j.1538-7836.2010.04131.x.

3. Zamora C, Canto E, Nieto JC, et al. Functional consequences of platelet binding to T lymphocytes in inflammation. J Leukoc Biol. 2013;94(3):521-529. doi:10.1189/jlb.0213074.

4. Zamora C, Cantó E, Nieto JC, et al. Binding of platelets to lymphocytes: A potential anti-inflammatory therapy in rheumatoid arthritis. J Immunol. 2017;198(8):3099-3108. doi:10.4049/jimmunol.1601708.

P479 NG-641: an oncolytic T-SIGn virus targeting cancer-associated fibroblasts in the stromal microenvironment of human carcinomas

Matthieu Besneux¹, Brian Champion, PhD¹, Nalini Marino¹, Marilena Patsalidou¹, Gianfranco di Genova¹, Sam Illingworth¹, Stefania Fedele¹, Lorna Slater¹, Darren Plumb¹, Katy West¹, Joshua Freedman, BS², Len Seymour², Kerry Fisher, MD PhD¹, Alice Brown, PhD¹

¹PsiOxus Therapeutics Ltd, Abingdon, UK; ²Oxford University, Oxford, UK

Correspondence: Brian Champion (brian.champion@psioxus.com)

Background

NG-641 is a modified variant of enadenotucirev (EnAd), an Ad11p/Ad3 chimeric group B adenovirus, which retains all the functional properties of enadenotucirev, while also mediating the expression of transgenes designed to target the breakdown of the stromal barrier and reverse immune suppression within the tumor microenvironment (TME). As an approach to immunogene therapy targeting stromal rich tumors, we have created a transgene-modified variant of EnAd expressing a bi-specific T-cell activator molecule (FAP-TAC) recognizing human fibroblast activating protein (FAP) on cancer associated fibroblasts (CAFs) and CD3 on T-cells. To enhance the activity of the bispecific molecule, particularly in tumors with poor immune cell infiltration (“excluded phenotype”), NG-641 also encodes an immune-enhancer module (IM), consisting of the chemokines CXCL9 and CXCL10 and the type I interferon IFNa..

Methods

FAP-TAC constructs comprising linked ScFv antibodies specific for human FAP and CD3 were designed and used to generate EnAd viruses expressing the FAP-TAC transgene such that expression was under the virus major late promoter (MLP) to allow broad or tumor-selective expression, respectively. Tumor and fibroblast cell lines and freshly isolated malignant peritoneal ascites and surgically excised tumor samples from carcinoma patients were used to evaluate virus activities.

Results

Initial studies with different viruses expressing FAP-TAC alone or with different additional transgenes showed that FAP-TAC activity generated by NG-641 infection was essentially the same as that with viruses bearing only the FAP-TAC transgene. In tumor cell lines, NG-641 selectively secretes functional FAP-TAC molecules, as determined in cocultures of FAP+ fibroblasts and PBMC-derived T cells. Production of the IM transgenes CXCL9, CXCL10 and IFNa was confirmed by specific ELISA assays, with functionality evaluated by reporter cell, FACS and cell migration assays. T cell-activation mediated by FAP-TAC leads to cytokine secretion and cytotoxicity towards the fibroblasts. Studies with unseparated primary tumor samples (containing tumor cells, CAFs and infiltrated immune cells) also demonstrated potent activation of the endogenous T-cells, indicating that virus produced FAP-TAC is a potent T-cell activator despite suppressive influences of the TME.

Conclusions

In this study, we have shown that CAFs can be effectively targeted for T-cell mediated destruction by NG-641, a tumor stroma targeting transgene-bearing oncolytic virus. This is associated with strong activation of endogenous T- cells to kill CAFs even in the presence of an immunosuppressive microenvironment. Systemic dosing of such a virus to patients with stromal rich tumors may provide an effective approach for reversing immune suppression within the TME and driving effective anti-tumor immunity.

P480 Transcriptomic characterization of tumor vs. peripheral blood NK cells in head and neck cancer patients

Fernando Concha-Benavente, MD, PhD², Robert Ferris¹

¹University of Pittsburgh, Pittsburgh, PA, USA; ²UPMC Hillman Cancer Center, Pittsburgh, PA, USA

Correspondence: Robert Ferris (ferrrl@upmc.edu)

Background

NK cells play a crucial role in tumor immunosurveillance with a unique capacity of killing cancer cells via antibody dependent cellular cytotoxicity (ADCC), particularly in the setting of head and neck cancer (HNC) where cetuximab, an EGFR-specific mAb, has been used since 2006. Cetuximab-activated NK cells induce dendritic cell (DC) activation, tumor antigen cross-presentation and expansion of EGFR-specific T cells, linking innate and adaptive antitumor immunity. However, the benefit of cetuximab is only seen in 10-15% of patients [1]. Moreover, NK cell dysfunction has been associated with increased risk of cancer [2-5] and poor clinical prognosis [6-8]. Therefore, characterizing whether NK cells infiltrate HNC tumors and their expression profile is important in order to reverse NK cell dysfunction and improve cancer immunotherapy

Methods

NK cells were sorted by flow cytometry from freshly isolated HNC peripheral blood lymphocytes (PBL) and paired tumor infiltrating lymphocytes (TIL). Sequential RT reactions produced amplified dsDNA which was fragmented, end labeled with biotin and hybridized to the Human Clariom S array. First level data analysis was performed using Affymetrix Expression Console using RMA normalization algorithm

Results

Initial exploratory grouping analysis (EGA) showed that PBL (n=9) and TIL (n=6) NK cells had a divergent expression profile. Hierarchical clustering (HC) analysis of paired PBL and TIL NK cells (n=4) revealed that TIL NK cells had a total of 1345 differentially expressed genes, from which 713 were downregulated more than 2-fold when compared to PBL NK cells. We found that Th1 type transcription factors such as EOMES and BLIMP-1, activation/cytolytic markers such as CD38, NKG2F, CD247, granzyme A, granzyme H, perforin 1 and FCGR3A (CD16a) and FCGR3B (CD16b) which are key molecules for ADCC were significantly downregulated in TIL NK cells. Likewise, sphingosine-1-phosphate receptor 1 (S1PR1), a critical mediator of NK cell traffic and retention in inflamed tissues, Th1 type induced T/NK cell attracting chemokines CCL5 and CCL4 and innate immunity activator, toll-like receptor 3 (TLR3) were downregulated in TIL NK cells.

Conclusions

These findings suggest an exhausted phenotype of TIL NK cells characterized by downregulation of Th1 type activation markers compared to NK cells in the periphery. Reversing NK cell dysfunction is key in order to improve antitumor immunity of HNC patients.

References

1. Ferris R L, Jaffee E M, Ferrone S. Tumor antigen-targeted, monoclonal antibody-based immunotherapy: clinical response, cellular immunity, and immunoescape. J Clin Oncol 2010. 28: 4390-4399.

2. Hersey P, Edwards A, Honeyman M, McCarthy W H. Low natural-killer-cell activity in familial melanoma patients and their relatives. Br J Cancer 1979. 40: 113-122.

3. Fauriat C, Just-Landi S, Mallet F, Arnoulet C, Sainty D, Olive D, Costello R T. Deficient expression of NCR in NK cells from acute myeloid leukemia: Evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction. Blood 2007. 109: 323-330.

4. Saito H, Osaki T, Ikeguchi M. Decreased NKG2D expression on NK cells correlates with impaired NK cell function in patients with gastric cancer. Gastric Cancer 2012. 15: 27-33.

5. Schantz S P, Shillitoe E J, Brown B, Campbell B. Natural killer cell activity and head and neck cancer: a clinical assessment. J Natl Cancer Inst 1986. 77: 869-875.

6. Coca S, Perez-Piqueras J, Martinez D, Colmenarejo A, Saez M A, Vallejo C, Martos J A, Moreno M. The prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma. Cancer 1997. 79: 2320-2328.

7. Takeuchi H, Maehara Y, Tokunaga E, Koga T, Kakeji Y, Sugimachi K. Prognostic significance of natural killer cell activity in patients with gastric carcinoma: a multivariate analysis. Am J Gastroenterol 2001. 96: 574-578.

8. Ishigami S, Natsugoe S, Tokuda K, Nakajo A, Che X, Iwashige H, Aridome K, Hokita S, Aikou T. Prognostic value of intratumoral natural killer cells in gastric carcinoma. Cancer 2000. 88: 577-583.

P481 The epigenetic underpinnings of regulatory T cell fragility in the tumor microenvironment

Becky Dadey, BS¹, Abigail Overacre-Delgoffe, PhD², Ting Wang¹, Zhe Sun¹, Rong Zhang, MS², Wei Chen², Natalie Rittenhouse², Amanda Poholek², Tracy Tabib¹, Robert Lafyatis¹, Creg Workman, PhD¹, Dario Vignali, PhD¹

¹University of Pittsburgh, Pittsburgh, PA, USA; ²Children's Hopsital of Pittsburgh, Pittsburgh, PA, USA

Correspondence: Dario Vignali (dvignali@pitt.edu)

Background

Regulatory T cells (Tregs) are a suppressive cell population that limit the anti-tumor response. However, systemic ablation of Tregs cannot be utilized as a therapy due to massive autoimmune defects. Our lab has demonstrated that Treg-restricted deletion of cell surface protein Neuropilin (Nrp1, CD304) results in substantially reduced tumor growth with no autoimmune defects [1]. We have shown that Treg-restricted deletion of Nrp1 in the TME does not result in loss of Foxp3 expression and “ex-Treg” generation but rather causes them to exhibit an effector-like phenotype including loss of suppressive function and production of interferon gamma (IFNү), which we refer to as Treg fragility [2].

Methods

We sought to understand the epigenetic underpinnings between Nrp1-sufficient and -deficient Tregs from the tumor microenvironment that could lead to this ‘fragile’ state. To do so we performed bisulfite treatment from ZymoEZ Direct Kit followed by Sanger Sequencing to identify differences in DNA methylation. We utilized ATAC sequencing to identify discrepancies in chromatin accessibility following the Greenleaf protocol [3]. We also utilized TCR sequencing from Adaptive Biotechnologies per the manufacturer’s protocol. For single cell RNAseq, we loaded 3500 cells/sample using ChromiumTM Single Cell 3' Gel Bead Kit and Chromium Single Cell 3'v2 Library Kit. Samples were sequenced on a NextSeq500. Finally, CUT&RUN ChIP-seq was perform following the Henikoff protocol [4].

Results

We found that Tregs lacking Nrp1 in the TME have a differential methylation signature at the Conserved Non-coding Sequence 2 (CNS2) locus of the Foxp3 gene, albeit no difference in the chromatin accessibility at this locus, no change in single cell RNAseq, and maintenance of Foxp3 protein expression. We also found that Nrp1-deficient Tregs are not peripherally-induced Tregs but rather are thymically-derived.

Conclusions

We have identified an intriguing change in the DNA methylation status of the CNS2 locus of Foxp3 in the Nrp1-deficient Tregs from the tumor microenvironment but no loss in Foxp3 expression. This finding conflicts with current data suggesting that CNS2 hypermethylation shuts off Foxp3 expression. Additional experiments will be required to understand how this locus maintains Foxp3 protein despite DNA methylation. Future studies will also examine the epigenetic mediators that might cause this differential methylation or if extrinsic factors in the TME promote differential methylation.

References

1. Delgoffe GM, Woo SR, Tunis ME, Gravano DM, Guy C, Overacre AE, Bettini ML, Vogel P, Finkelstein D, Bonnevier J, Workman CJ, Vignali DA. Stability and function of regulatory T cells is maintained by a neuropilin-1- semaphorin-4a axis. Nature. 2013; 7466, 252-6

2. Overacre-Delgoffe AE, Chikina M, Dadey RE, Yano H, Brunazzi EA, Shayan G, Horne W, Moskovitz JM, Kolls JK, Sander C, Shuai Y, Normolle DP, Kirkwood JM, Ferris RL, Delgoffe GM, Bruno TC, Workman CJ, Vignali DAA. Interferon-γ derives treg fragility to promote anti-tumor immunity. Cell. 2017; 169, 1130-41

3. Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 2013; 10:1213-1218

4. Skene PJ, Henikoff S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 2017; 6:e21856

P482 Humanization and characterization of novel, best in class isoform-specific anti-TGFβ monoclonal antibodies

Matteo Brioschi¹, Pamela Cheou², Jacques Van Snick, PhD², Catherine Uyttenhove, PhD², George Coukos, MD, PhD³, Gerd Ritter⁴, Steven Dunn, PhD³, Steven Dunn, PhD³

¹Ludwig Institute for Cancer Research, Epalinges, Switzerland., Epalinges, Switzerland; ²Ludwig Institute for Cancer Research Ltd, Brussels, Belgium., Brussels, Belgium; ³Ludwig Institute for Cancer Research, Epalinges, Switzerland. CHUV, Lausanne, Switzerland., Epalinges, Switzerland; ⁴Ludwig Institute for Cancer Research, New York, NY., New York, NY, USA

Correspondence: Steven Dunn (steven.dunn@chuv.ch)

Background

TGFβ is a conserved, highly pleiotropic and potent cytokine that has been implicated in tumor escape and progression via its modulation and suppression of multiple immune-cell related pathways within the tumor microenvironment [1]. Three homo-dimeric isoforms have been identified, which individually have been shown to drive context-dependent physiological and phenotypic responses, including aspects of proliferation, migration, differentiation, angiogenesis, and immune responsiveness [2]. Although a compelling target for tumor immunotherapy, the use of anti-TGFβ antibodies that do not adequately distinguish between the individual isoforms could give rise to on-target off-tumor toxicity, undesirable inflammatory adverse events or a zero-sum anti-tumor activity. To address this unmet need for more specific reagents, we have selected two antibodies for humanization from a panel of recently generated high-affinity murine mAb hybridomas which have extraordinary mono-isoform selectivity for TGFβ1 or TGFβ3 and which neutralize TGFβ-driven signalling in a cell reporter system with high potency [3]. Isoform specific TGFβ blockade with these antibodies is effective at delaying in-vivo tumor growth in melanoma and breast cancer models [4].

Methods

Antibody humanization was performed using a molecular engineering approach combining framework grafting, competitive screening of antibody fragments and selective back mutation of individual residues guided by assaying for TGFβ neutralization potency of the antibody constructs using a transformed mink lung epithelial cell (tMLEC) TGFβ reporter line. Binding kinetics were assayed by surface plasmon resonance. Physicochemical properties of the engineered antibodies and suitability for down-stream development were characterized using several techniques including transient expression in singular cell system, size-exclusion chromatography and thermal differential scanning fluorimetry.

Results

We report the successful conversion of two TGFβ isoform-specific mouse mAbs into humanized recombinant IgG4 molecules targeting human TGFβ1 or TGFβ3 respectively. Engineered variants of both parental TGFβ antibodies express efficiently in a transient human cell system, display good thermal stability and retain their potent binding and neutralization activity with regard to their cognate TGFβ isoforms. We were able to generate several humanized LCR1901 anti-TGFβ3 variants with significantly improved neutralization potency in cellular TGFβ signalling assays relative to the parental mAb, and with essentially undetectable levels of binding to TGFβ1 and TGFβ2.

Conclusions

We have generated a panel of humanized antibodies that specifically neutralize TGFβ3 or TGFβ1 signalling. Recent studies assigning a dominant gatekeeper role for TGFβ3 in malignant glioblastoma progression [5], as well as specifically implicating TGFβ1 in breast and gastric cancers [6,7], support the continued pre-clinical and clinical investigation of these novel TGFβ antibodies.

References

1. Neuzillet C, Tijeras-Raballand A, Cohen R, Cros J, Faivre S, Raymond E, de Gramont A. Targeting the TGFβ pathway for cancer therapy. Pharmacol Ther. 2015 Mar;147:22-31

2. Poniatowski ŁA, Wojdasiewicz P, Gasik R, Szukiewicz D. Transforming growth factor beta family: Insight into the role of growth factors in regulation of fracture healing biology and potential clinical applications. Mediators Inflamm. 2015;2015:137823

3. Uyttenhove C, Marillier RG, Tacchini-Cottier F, Charmoy M, Caspi RR, Damsker JM, Goriely S, Su D, Van Damme J, Struyf S, Opdenakker G, Van Snick J. Amine-reactive OVA multimers for auto-vaccination against cytokines and other mediators: perspectives illustrated for GCP-2 in L. major infection. J Leukoc Biol. 2011 Jun;89(6):1001-7

4. Gupta A, Budhu S, Giese R, van Snick J, Uyttenhove C, Ritter G, Wolchok J, Merghoub T. Isoform specific TGF-β inhibition in combination with radiation therapy as a novel immune therapeutic approach to cancer therapy. Journal for ImmunoTherapy of Cancer 2017; 5 (suppl 2) :87 Abstract 326 AND Gupta A, Budhu S, Giese R, van Snick J, Uyttenhove C, Ritter G, Wolchok J, Merghoub T. Targeting specific TGF-β isoforms in combination with radiation therapy leads to differential antitumor effects in mouse models of cancer. Cancer Res. 2018; 78(13 suppl): Abstract 4716

5. Seystahl K, Papachristodoulou A, Burghardt I,Schneider H, Hasenbach K, Janicot M, Roth P, Weller M. Biological Role and Therapeutic Targeting of TGF-β3 in Glioblastoma. Mol Cancer Ther. 2017 Jun;16(6):1177- 1186

6. Rodriguez G, Abrahamsson A, Jensen L, Dabrosin C. Estradiol Promotes Breast Cancer Cell Migration via Recruitment and Activation of Neutrophils. Cancer Immunol Res; 5(3); 234–47

7. Peng LS, Zhang JY, Teng YS, Zhao YL, Wang TT, Mao FY, Lv YP, Cheng P, Li WH, Chen N, Duan M, Chen W, Guo G, Zou QM, Zhuang Y. Tumor-Associated Monocytes/Macrophages Impair NK-Cell Function via TGFb1 in Human Gastric Cancer. Cancer Immunol Res. 2017 Mar;5(3):248-256

P483 Iron and HLA-G: Neglected immunosuppressive molecules in the human microenvironment

Xian Jiang², Robert Elliott¹

¹Mastology, Baton Rouge, LA, USA; ²the Breast Foundation, Baton Rouge, LA, USA; ³EEB clinic, Baton Rouge, LA, USA

Correspondence: Robert Elliott (relliot@eehbreastca.com)

Background

There are many immunosuppressive molecules in the tumor microenvironment that need to be inhibited, if we are to improve immunotherapy in the Stage IV patients. There are many reports on the possible role of HLA-G in cancer immunosuppression, but very little on the role of iron. The immunosuppressive effect of iron and HLA-G in the tumor microenvironment has been neglected and ignored as therapeutic targets. This study was implemented to determine if iron and HLA-G, as neglected molecules enhance tumor immunosuppression, and could be cancer immunotherapy targets.

Methods

We examined HLA-G expression in normal mammary and breast cancer cell lines and human normal and breast cancer tissue. This examination was done by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Intracellular iron levels were manipulated in the human MCF-7 and MDA-MB-231 breast cancer cell lines. Cytolysis of these cell lines was measured after exposure to the natural killer cell line NK-92 MI (NK). The gene expression of ferritin heavy chain (FTH1) was determined as was the production of nitric oxide (NO) and tumor necrosis factor alpha (TNFa).

Results

RT-PCR confirmed HLA-G expression was absent in the normal epithelial MCF-12A cells showing no mRNA expression, however, the cell lines MCF-7, MDA-MB-231and T-47D had various levels of HLA-G mRNA expression. IHC was performed on 38 breast cancer specimens and on 12 normal breast specimens. Fifty-eight percent (22/38) of the cancer had medium to strong staining, but only 8.3% (1/12) of the normal specimens had medium staining. The difference was significant (p<0.05). When NK-92 MI cells were co-cultured with MCF-7 and MDA-MB-231 cells, NO and TNF-a were released into the media. The addition of iron inhibited the cytolysis of cancer cell lines. Deferoxamine (DFOM), an iron chelator, increased NK-92 MI cytolysis of MCF-7 and MDA-MB- 231 cells. The cytotoxicity of the breast cancer cells was reversed by the addition of iron. This cytotoxicity is induced by NO released from S-nitro-N-acetyl – penicillamine (NO donor). RT-PCR showed the iron chelator reduced FTH1 expression, while iron upregulated the expression of FTH1.

Conclusions

HLA-G antigen is expressed in trophoblastic placental cells as an immunotolerant molecule to protect the fetus from maternal alloreactivity. Its expression in cancer cells contributes to cancer immunosuppression. Increased iron in the tumor microenvironment and cancer cells inhibited cancer cells cytolysis by NK cells by antagonizing NO and TNFa cytotoxicity and the upregulation of ferritin expression. We hope this study will stimulate researchers to investigate the role of HLA-G and iron as therapeutic targets of the cancer microenvironment. Cancer immunotherapy in Stage IV patients will be improved by the inhibition of these neglected molecules.

P484 Pharmacokinetics, pharmacodynamics, and safety of FLX475, an orally-available, potent, and selective small- molecule antagonist of CCR4, in healthy volunteers

Sjoerd van Marle, MD², Ewoud-Jan van Hoogdalem, PhD, RPh², Daniel Johnson¹, Abood Okal, PhD¹, Paul Kassner, PhD¹, David Wustrow, PhD¹, William Ho, MD, PhD¹, Steven Smith¹

¹FLX Bio, South San Francisco, CA, USA; ²PRA Health Sciences, Groningen, Netherlands

Correspondence: William Ho (bill.ho@flxbio.com)

Background

Regulatory T cells (Treg) are essential for immune tolerance to self antigens, but can also dampen anti-tumor immune responses in the tumor microenvironment (TME). The predominant chemokine receptor on human Treg is CCR4, the receptor for the chemokines CCL17 and CCL22 [1], which are produced by tumor cells, tumor- associated macrophages and dendritic cells, as well as by effector T cells (Teff) in the setting of an inflammatory anti-tumor response. Preclinical studies with orally-available CCR4 antagonists have demonstrated potent inhibition of Treg migration into tumors, an increase in the intratumoral Teff/Treg ratio, and anti-tumor efficacy as a single agent and in combination with checkpoint inhibitors. [2]

Methods

A first-in-human, randomized, double-blind, placebo-controlled trial was conducted to examine the safety, pharmacokinetics (PK), and pharmacodynamics (PD) in healthy volunteers (HVs) of single and repeat dosing of FLX475, an orally-available, potent, and selective small-molecule antagonist of CCR4. Seven cohorts of 8 subjects each (6 drug, 2 placebo) were administered single doses ranging from 5 mg to 1000 mg. Six cohorts were administered daily doses of FLX475 for 14 days ranging from 25 mg to 150 mg, including two cohorts evaluating a loading dose administered on Day 1.

Results

FLX475 was well-tolerated, with no significant laboratory abnormalities or dose-limiting clinical adverse events. Dose-dependent increases in exposure were observed with low peak-to-trough ratios and a half-life of approximately 72 hours. Daily dosing without a loading dose demonstrated approximately 4-5x accumulation of FLX475 over 14 days. A receptor occupancy (RO) PD assay using study subject peripheral blood Treg [3] demonstrated a tight PK/PD relationship, suggesting that doses of approximately 75 mg PO QD and above are sufficient to maintain target drug exposure above the IC90 for human in vitro Treg migration.

Conclusions

In this first-in-human HV study, the oral CCR4 antagonist FLX475 was demonstrated to be well tolerated with outstanding PK properties. A robust PD assay measuring receptor occupancy on circulating Treg demonstrated the ability to safely achieve exposure levels predicted to maximally inhibit Treg recruitment into tumors via CCR4 signaling. These data have enabled the optimized design of an ongoing Phase 1/2 study of FLX475 both as monotherapy and in combination with checkpoint inhibitor in cancer patients.

Trial Registration

EudraCT 2017-003952-22

References

• 1. Talay O et al. Potent and selective C-C chemokine receptor (CCR4) antagonists potentiate anti-tumor immune responses by inhibiting regulatory T cells (Treg) [abstract]. In: Proceedings of the 110th Annual Meeting of the American Association for Cancer Research; 2017 Apr 1–5; Washington, DC. AACR; 2017. Abstract nr 4600.

• 2. Talay O et al. Potent and selective C-C chemokine receptor 4 (CCR4) antagonists inhibit regulatory T cell recruitment, increase effector T cell numbers, and potentiate anti-tumor responses in mice. J Immunother Cancer. 2017; 5(Suppl 2):467.

• 3. Okal A, Ho W, Wong B, Kassner P, Cutler G. Patient selection strategies and pharmacodynamic assays for CCR4 antagonists. J Immunother Cancer. 2017; 5(Suppl 2): 44.

Ethics Approval

Approved by the Independent Ethics Committee of the foundation “Stichting Beoordeling Ethiek Biomedisch Onderzoek” (Assen, The Netherlands), Study Code FLB307EC-173071, CCMO code NL63737.056.17.

P485 Testing of two bispecific SNIPER™ antibodies targeting mouse tumor-infiltrating Tregs

Anna Hoefges, MS¹, Bonnie Hammer², Amy Erbe, PhD¹, Alexander Rakhmilevich, MD, PhD¹, Jacquelyn Hank, PhD¹, Bryan Glaser, PhD², Lucas Bailey, PhD², Roland Green, PhD², Paul Sondel, MD, PhD¹

¹University of Wisconsin Madison, Madison, WI, USA; ²Invenra, Madison, WI, USA

Correspondence: Anna Hoefges (hoefges@wisc.edu)

Background

T regulatory cells (Tregs) are essential to help prevent autoimmune diseases. In the setting of cancer, however, Tregs can help cancer evade anti-tumor immunity by suppressing immunity. Murine studies have shown that if Tregs are selectively depleted, anti-tumor immunity can be enhanced and synergistic immunotherapy achieved, promoting tumor regression. However, currently available Treg-depletion agents can be non-specific and deplete/suppress other T cells, can fail to sufficiently deplete Tregs, or can potently deplete all Tregs, leading to toxic autoimmunity. We have developed and tested a way to selectively eliminate Tregs in the tumor microenvironment (TME) while leaving peripheral Tregs by using bispecific mAbs created using Invenra’s SNIPER™ technology. SNIPER™ bispecific antibodies have relatively weak affinity for two separate targets, limiting their binding and activity when only one target is present. However, when both targets are present, binding is much stronger due to the avidity effect. This allows specific subpopulations of cells to be more specifically selected for elimination by antibody drug conjugates or antibody dependent cellular cytotoxicity.

Methods

Two separate SNIPER™ bispecific mAbs, Inv-1 and Inv-2, were created. C57Bl/6 mice were injected with B78 melanoma tumors. Established tumors and spleens were harvested from mice and analyzed by flow cytometry to identify T cell populations and binding specificity of Inv-1 and Inv-2.

Results

We analyzed binding of the Inv-1 and Inv-2 to lymphocytes harvested from spleens and tumors from the B78 tumor-bearing mice. We used a standard Treg verification panel (CD4, CD25, Foxp3) to identify known Treg populations. Separate panels included the bispecific antibodies (Inv-1 or Inv-2). We found that Inv-1 binds to 59% of Foxp3+ cells extracted from the TME, but only to 18% of the splenic Foxp3+ cells. This shows a preferential binding for tumor-infiltrating-Tregs. Separately, Inv-2 bound to 81% of Foxp3+ cells extracted from the TME, but only to about 51% of the splenic Foxp3+ cells.

Conclusions

Both Inv-1 and Inv-2 selectively target Tregs, with a preference for Tregs present in the TME. In vivo administration of these antibodies may allow for selective depletion of tumor-associated Tregs. Selective depletion of TME-Tregs may result in a reduction in toxic autoimmune side effects associated with immune-activation in the setting of global Treg depletion. In turn, the removal of Tregs specifically from the TME, coupled with a reduction of potential toxic side effects, may enhance the efficacy and applicability of combining Treg depletion with other immune-activating immunotherapies.

P486 Antisense oligonucleotides targeting CD39 and PD-L1 modulate the immunosuppressive tumor microenvironment and have potent anti-tumor activity

Frank Jaschinski, PhD¹, Tamara Thelemann¹, Richard Klar, PhD¹, Monika Schell¹, Lisa Hinterwimmer¹, Sven Michel¹, Melanie Buchi², Abhishek Kashyap², Alfred Zippelius, MD²

¹Secarna Pharmaceuticals GmbH & Co. KG, Planegg-Martinsried, Germany; ²University of Basel, Basel, Switzerland

Correspondence: Frank Jaschinski (frank.jaschinski@secarna.com)

Background

Antisense oligonucleotides (ASOs) are a new therapeutic modality and have the potential to suppress expression of any RNA target. On the one hand they allow selective targeting of factors previously considered as undruggable, on the other hand -due to their different pharmacokinetic and pharmacodynamic properties- they can offer a complementary approach to more established modalities such as small molecule drugs or antibodies. In the present study, locked-nucleic-acid (LNA)-modified antisense oligonucleotides targeting PD-L1 and the ectonucleotidase CD39 were designed and their activity was tested in cell culture and syngeneic mouse models

Methods

In vitro activity of ASOs on target mRNA and protein expression was investigated in tumor cell lines and confirmed in isolated human T cells. Degradation of extracellular ATP and proliferation of immune cells were tested in isolated human T cells. In vivo, target activity and investigation of frequency of intratumoral Treg were investigated in the syngeneic MC38 mouse model. The MC38 model and the syngeneic EMT6 model were used to test effects on tumor growth or survival.

Results

In vitro, unformulated ASOs targeting PD-L1 and CD39 achieved potent target knockdown on mRNA and protein level in tumor cell lines and in isolated human T cells. CD39-specific ASOs potently reduced degradation of extracellular ATP in T cells. While treatment of T cells with ATP potently suppressed their proliferation, CD39- specific ASOs could reverse this effect. In syngeneic mouse tumor models, systemic treatment with CD39-specific ASO resulted in potent knockdown of CD39 expression e.g. in Treg, tumor-associated macrophages and myeloid- derived suppressor cells and in a reduction of the frequency of intratumoral Treg. Moreover, tumor growth was strongly reduced by CD39-specific ASO, as monotherapy. In combination with PD-1 antibodies, anti-tumor efficacy of antibodies was improved by ASO.Anti-tumor efficacy of-murine PD-L1 ASOs was demonstrated in syngeneic mouse models. In a breast cancer model, all tumor-bearing mice treated with the PD-L1 ASO rejected the tumor and remained tumor-free. Upon rechallenge, the vast majority of mice rejected the tumor cells demonstrating immunological memory formation. No signs of toxicity were observed.

Conclusions

We have shown, that ASOs targeting immunosuppressive factors are able to achieve potent target suppression in the relevant cell types in vivo and can induce potent anti-tumor effects as monotherapy and in combination therapy with antibody-based checkpoint inhibitors, thereby enhancing survival. Taken together, we developed innovative immunotherapeutic tools that will potentially improve treatment options for cancer patients in the future.

Ethics Approval

PBMC were obtained from leukapheresis products (Klinikum rechts der Isar, TU München, ethics commission reference: 329/16 S)

P487 The role of MultiOmyx in illustrating the pancreatic tumor microenvironment

Juncker-Jensen Juncker-Jensen, PhD, Jun Fang, Judy Kuo, Mate Nagy, Qingyan Au, Eric Leones, Flora Sahafi, RaghavKrishna Padmanabhan, Nicholas Hoe, Josette William, PhD, MD

NeoGenomics, Aliso Viejo, CA, USA

Correspondence: Juncker-Jensen Juncker-Jensen (anna.juncker-jensen@neogenomics.com)

Background

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an excessive amount of desmoplastic stroma seeded with inflammatory cells and it is one of the most aggressive forms of cancer with no current specific therapies. Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment (TME), and in most solid cancers increased TAM infiltration is associated with a poor prognosis. TAMs can be described as classically activated M1 types with pro-inflammatory antitumor functions, versus alternatively activated M2 types with immunosuppressive pro-tumor functions. The immunosuppressive functions of M2 TAMs can be exerted through release of cytokines and growth factors as well as via direct recruitment of T regulatory cells (Tregs), a subset of lymphocytes responsible for immune tolerance of the system to the tumor. While the differentiation from M1 to M2 in PDAC has been shown to be associated with a worse prognosis [1], not much is known about PDAC TAM polarization and its potential correlation to Treg recruitment.

Methods

We have used MultiOmyx, a proprietary, multiplexing assay with similar staining characteristics as standard IHC stains but with the significant advantage that 60 protein biomarkers can be interrogated from a single FFPE section. MultiOmyx protein immunofluorescence (IF) assays utilize a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining and deactivation.

Results

Using the pan macrophage marker CD68 in combination with either M1 marker HLA-DR or M2 marker CD163 we confirmed the presence of M1 (CD68+HLA-DR+) and M2 (CD68+CD163+) populations in 9 stage IIB non- metastatic PDAC FFPE samples, the vast majority being of the M2 subtype. Moreover, we found a positive significant correlation (Pearson’s correlation p<0.05) between the presence of M2 TAMs and Tregs (CD3+CD4+FoxP3+), but not between M1 TAMs and Tregs. Using our proprietary algorithms that takes into account the staining pattern for each specific biomarker, we will now examine the spatial relationship between the M1/M2 subtypes of TAMs and Tregs in the stromal and intratumoral areas and compare our findings to those found in samples from patients with stage IV metastatic PDAC.

Conclusions

We demonstrate a positive significant correlation between the presence of M2-TAMs and Tregs in the TME of PDAC, suggesting a possible pathway in which TAM-polarization plays an immunosuppressive function by recruiting Tregs.

References

1. Kurahara H et al. Significance of M2-polarized tumor-associated macrophages in pancreatic cancer. JSurgRes. 2011;167:e211-219

P488 Targeting hIDO1 with 3rd generation antisense oligonucleotides for modulation of the tumor microenvironment

Richard Klar, PhD¹, Sandra Kallert, PhD², Tamara Thelemann¹, Sven Michel¹, Monika Schell³, Lisa Hinterwimmer¹, Alfred Zippelius, MD², Frank Jaschinski, PhD³

¹Secarna pharmaceuticals GmbH & Co. KG, Planegg/Martinsried, Germany; ²University of Basel, Department of Biomedicine, Switzerland; ³Secarna pharmaceuticlas GmbH & Co. KG, Planegg-Martinsried, Germany

Correspondence: Frank Jaschinski (frank.jaschinski@secarna.com)

Background

Targeting the immunosuppressive microenvironment of tumors has emerged as a promising treatment option for oncologic indications in the last years. However, despite long lasting remissions in a small subset of patients the majority does not respond to the currently available immunotherapies, possibly caused by the existence of a plethora of immune suppressive factors. One of those factors is indoleamin-2,3-dioxygenase 1 (IDO1), an enzyme that degrades tryptophan to kynurenines which in turn can result in a suppression of immune effector cells.

Methods

As an alternative approach to small molecule IDO1-inhibitors, we designed antisense oligonucleotides (ASOs) with specificity for human IDO1 (hIDO1). ASOs were synthesized as GapmeRs with flanking locked nucleic acids to increase stability and affinity to the target RNA. The knockdown efficacy of ASOs on the mRNA and protein level was investigated in cancer cells and human immune cells without addition of a transfection reagent. The effect of hIDO1 knockdown in cancer cells on the production of L-Kynurenine and the proliferation of cocultured T cells was investigated. We furthermore developed ASOs with specificity for murine IDO1 (mIDO1) to investigate the efficacy of ASO-mediated IDO1 knockdown in syngeneic tumor models.

Results

We identified a subset of ASOs that resulted in a hIDO1 mRNA knockdown of >90% in cancer cell lines. Two highly potent ASOs with IC50 values in the low nanomolar range were selected for further experiments. The treatment of cancer cells as well as human immune cells resulted in reduction of IDO protein levels by >85%. Importantly, we observed a complete block in the production of immunosuppressive L-Kynurenine in ASO treated cells and IC50 values in the low nanomolar range. In line with those results, we observed a strongly increased proliferation of T cells when hIDO1 was knocked down in cocultured tumor cells. Preliminary in vivo experiments suggest that treatment of tumor-bearing mice with mIDO1-specific ASOs results in the knockdown of IDO1 in tumor cells as well as tumor infiltrating myeloid cells.

Conclusions

We selected highly potent hIDO1 ASOs that efficiently knock down hIDO1 mRNA and protein in cancer cells as well as primary human cells and potently reduce the immunosuppressive capacity of cancer cells. Potent mouse specific IDO1 ASOs have been identified and will be used for in vivo efficacy studies in tumor-bearing mice. Taken together, we developed an innovative immunotherapeutic tool to block the expression of hIDO1 that will potentially improve treatment options for cancer patients in the future.

Ethics Approval

PBMC were obtained from leukapheresis products (Klinikum rechts der Isar, TU München ethics commission reference: 329/16 S)

P489 Prognostic significance of tumor-associated macrophage content in head and neck squamous cell carcinoma: A meta-analysis

Ayan Kumar, BS, Alexander Knops, BA, Brian Swendseid, MD, Ubaldo Martinez-Outschoom, MD, Larry Harshyne, PhD, Nancy Philp, PhD, Ulrich Rodeck, MD PhD, Christopher Snyder, Adam Luginbuhl, MD, David Cognetti, MD, Jennifer Johnson, MD, Joseph Curry, MD

Thomas Jefferson University, Philadelphia, PA, USA

Correspondence: Joseph Curry (joseph.curry@jefferson.edu)

Background

Head and neck squamous cell carcinoma (HNSCC) develops within a complex cellular microenvironment that promotes tumor growth, thus providing multiple potential therapeutic targets. Among those are macrophages, which are abundant in and around tumor tissue, and have been implicated in the growth, development, and persistence of HNSCC [1]. However, the relationship between the density and composition of tumor-associated macrophages (TAMs) and clinicopathologic markers of disease is poorly defined [2,3]. Inconsistent findings may be a result of differences in approach to TAM detection. Some authors have measured total macrophage content in tumor tissue, while others have stained tumor samples for individual subtypes of TAMs, which include M1 (pro-inflammatory) and M2 (immunosuppressive). Here we review the published evidence concerning the relationship of the phenotypes of tumor-associated macrophages with HNSCC prognosis.

Methods

We conducted a meta-analysis of the existing publications investigating the relationship between TAM density (total and M2 subtype) and T stage, nodal involvement, vascular invasion, lymphatic invasion, and tumor differentiation (Figure 1). A total of thirteen studies were included (Table 1) [2-14]. Forest plots and risk ratios were generated to illustrate overall effects.

Results

Higher density of both total and M2 subtype of TAMs in the tumor microenvironment is associated with advanced T stage, increased rates of nodal positivity, presence of vascular invasion, and presence of lymphatic invasion (p < 0.0001; Figures 2-5). There is no significant association between TAM density, either total or M2 subtype, and tumor differentiation (Figure 6).

Conclusions

Increased TAM density, particularly those of the M2 phenotype, correlates with poor clinicopathologic markers in HNSCC and is associated with poor clinical prognosis. Yet, it is unknown whether and how TAMs contribute to poor prognosis in this tumor type. Additional investigation into the mechanisms behind TAM recruitment and polarization will help define the feasibility of TAM-targeted therapies.

References

• 1. Marcus B, Arenberg D, Lee J, et al. Prognostic factors in oral cavity and oropharyngeal squamous cell carcinoma. Cancer. 2004; 101(12):2779-2787.

• 2. Fang J, Li X, Ma D, et al. Prognostic significance of tumor infiltrating immune cells in oral squamous cell carcinoma. BMC Cancer. 2017; 17.

• 3. Shigeoka M, Urakawa N, Nakamura T, et al. Tumor associated macrophage expressing CD204 is associated with tumor aggressiveness of esophageal squamous cell carcinoma. Cancer Science. 2013; 104(8):1112-1119.

• 4. Balermpas P, Rödel F, Liberz R, et al. Head and neck cancer relapse after chemoradiotherapy correlates with CD163+ macrophages in primary tumour and CD11b+ myeloid cells in recurrences. Br J Cancer. 2014; 111(8):1509-1518

• 5. Fujii N, Shomori K, Shiomi T, et al. Cancer‐associated fibroblasts and CD163‐positive macrophages in oral squamous cell carcinoma: their clinicopathological and prognostic significance. Journal of Oral Pathology & Medicine. 2012; 41(6):444-451.

• 6. Hu Y, He M-Y, Zhu L-F, et al. Tumor-associated macrophages correlate with the clinicopathological features and poor outcomes via inducing epithelial to mesenchymal transition in oral squamous cell carcinoma. J Exp Clin Cancer Res. 2016; 35.

• 7. Huang H, Liu X, Zhao F, et al. M2-polarized tumour-associated macrophages in stroma correlate with poor prognosis and Epstein-Barr viral infection in nasopharyngeal carcinoma. Acta Otolaryngol. 2017; 137(8):888-894.

• 8. Liu S-Y, Chang L-C, Pan L-F, Hung Y-J, Lee C-H, Shieh Y-S. Clinicopathologic significance of tumor cell-lined vessel and microenvironment in oral squamous cell carcinoma. Oral Oncology. 2008; 44(3):277-285.

• 9. Lu C-F, Huang C-S, Tjiu J-W, Chiang C-P. Infiltrating macrophage count: a significant predictor for the progression and prognosis of oral squamous cell carcinomas in Taiwan. Head Neck. 2010; 32(1):18-25.

• 10. Matsuoka Y, Yoshida R, Nakayama H, et al. The tumour stromal features are associated with resistance to 5‐FU‐based chemoradiotherapy and a poor prognosis in patients with oral squamous cell carcinoma. APMIS. 2014;123(3):205-214.

• 11. Sugimura K, Miyata H, Tanaka K, et al. High infiltration of tumor‐associated macrophages is associated with a poor response to chemotherapy and poor prognosis of patients undergoing neoadjuvant chemotherapy for esophageal cancer. Journal of Surgical Oncology. 2015; 111(6):752-759.

• 12. Wang S, Sun M, Gu C, et al. Expression of CD163, interleukin-10, and interferon-gamma in oral squamous cell carcinoma: mutual relationships and prognostic implications. Eur J Oral Sci. 2014; 122(3):202-209.

• 13. Yamagata Y, Tomioka H, Sakamoto K, et al. CD163-positive macrophages within the tumor stroma are associated with lymphangiogenesis and lymph node metastasis in oral squamous cell carcinoma. Journal of Oral and Maxillofacial Surgery. 2017; 75(10):2144-2153.

• 14. Zhu Y, Li M, Bo C, et al. Prognostic significance of the lymphocyte-to-monocyte ratio and the tumor-infiltrating lymphocyte to tumor-associated macrophage ratio in patients with stage T3N0M0 esophageal squamous cell carcinoma. Cancer Immunol Immunother. 2017; 66(3):343-354.

Fig. 1 (abstract P489).

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Table 1 (abstract P489). See text for description

Fig. 2 (abstract P489).

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Fig. 3 (abstract P489).

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Fig. 4 (abstract P489).

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Fig. 5 (abstract P489).

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Fig. 6 (abstract P489).

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P490 Characterization of the tumor immune microenvironment in treatment-naïve EGFR-mutant NSCLC uncovers a suppressed immune phenotype

Xiuning Le¹, Alexandre Reuben¹, Marcelo Negrao, MD¹, Won-Chul Lee, PhD¹, Edwin Parra¹, Carmen Behrens, MD¹, Humam Kadara, PhD², Ignacio Wistuba, MD¹, Jianjun Zhang, MD, PhD¹, John Heymach¹

¹MD Anderson Cancer Center, Houston, TX, USA; ²American University of Beirut, Beirut, Lebanon

Correspondence: John Heymach (jheymach@mdanderson.org)

Background

Although immune checkpoint blockade (ICI) has been successfully utilized in treating patients with non-small cell lung cancer (NSCLC), the benefit of ICI for patients with advanced EGFR-mutant NSCLC has been limited. Intriguingly, recent data from IMpower150 subgroup analysis demonstrated that combination of VEGF blockade with anti-PD-L1 and chemotherapy induces response in patients with EGFR/ALK driven NSCLC, suggesting modulating the tumor microenvironment may enhance response to anti-PD-1 blockade in EGFR/ALK lung cancers. In order to develop effective immune therapy combinations, it is critical to understand tumor immune microenvironment (TME) and identify negative regulators in EGFR-mutant lung cancer for rational design of clinical trials. [1]

Methods

We leveraged a published set of stage I-III lung adenocarcinomas with immune profiling and sequencing data (PROSPECT cohort, Kadara et al Ann Oncol 2018). We selected a set of 94 adenocarcinomas including 14 cases with EGFR-sensitizing mutations with immune profiling by immunohistochemistry for ten immune markers (PD- L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, FoxP3 and Granzyme B). Gene microarray data were also available to evaluate the tumor microenvironment while CIBERSORT was used to infer immune cell subpopulations.

Results

PDL1 and GzmB were significantly lower in the EGFR-mutant cases, as expected. CD4 was higher in EGFR-mutant tumor center. Forty-four key immune regulators’ levels we evaluated to further evaluate the tumor microenvironment. IFNG was lower while TGFbeta was higher in EGFR-mutant cases, suggesting a suppressive TME. Other negative regulators, including CTLA4, LAG3, TIM3, TIGIT, IL6 and VEGFA were not differentially expressed. Lastly, CIBERSORT analysis revealed CD4+ memory T cells were decreased in the EGFR-mutant cases.

Conclusions

Results from this analysis are consistent with prior knowledge of EGFR-mutant NSCLC, demonstrating a PD-L1 low, GzmB low, IFNG low and TGFbeta high immune phenotype, suggesting a suppressive tumor microenvironment. CD4+ T cells composition needs to be further understood as subpopulation of CD4+ T cells (T regs) might be contributing to the suppressed TME. These results represent an initial step for rationale combination of immune therapy to modulate the suppressive TME, which might lead to enhanced treatment efficacy to benefit patients with EGFR-mutant lung cancers.

References

1. Kadara H, et al. Ann Oncol. 2018 29(4):1072

P491 The DNA methyltransferase inhibitor, guadecitabine, has beneficial immunomodulatory effects on myeloid derived suppressor cells, and augments adoptive immunotherapy for solid tumors

Andrea Luker, PhD candidate, Harry Bear, M.D., Ph.D., Daniel Conrad, Ph.D., Laura Graham

Virginia Commonwealth University, Richmond, VA, USA

Correspondence: Harry Bear (harry.bear@vcuhealth.org)

Background

Myeloid Derived Suppressor Cells (MDSC) are a regulatory population that accumulates in tumor microenvironments. They are a significant hurdle in treating cancer because they dampen anti-tumor responses as well as hinder the effects of immunotherapy. Here we show that the DNA methyltransferase inhibitor (DNMTi), Guadecitabine, has beneficial immunomodulatory effects on MDSCs and 4T1 tumor cells, both in vitro and in vivo.

Methods

Purified MDSCs or 4T1 cells were cultured for 48-72 hours with 1μM Guadecitabine and then analyzed for surface molecules by flow cytometry or further incubated with fluorescently-labeled Ovalbumin for 30 minutes to allow for antigen uptake. 4T1 tumors (50k, flank) were established in syngeneic Balb/c mice for 10 days before administering four daily doses of Guadecitabine (50μg, i.p.); mice were sacrificed at day 16 to collect tissues for analysis. Guadecitabine and AIT (50 million expanded draining lymph node cells) were administered concurrently starting at day 3, and tumor size was monitored until a humane endpoint. All experiments include n=3+ with at least two experimental repeats.

Results

MDSCs cultured with Guadecitabine had increased expression of MHC II, CD80, and CD86 exclusively in Ly6C+cells, with no effect on the Ly6G+ subpopulation. These Ly6C+ MDSCs also demonstrated significantly enhanced antigen uptake in vitro. Guadecitabine-treated 4T1 cells exhibited significantly increased MHC I and PD-L1 expression over vehicle-treated cells. Guadecitabine treatments in 4T1 tumor-bearing mice immediately reduced the tumor-induced MDSC accumulation in the spleen (Figure 1), bone marrow, and blood, thereby restoring normal cellularity. Similar to the in vitro findings, the remaining MDSCs in Guadecitabine-treated mice had increased levels of MHC II and co-stimulatory molecules. Subsequently, animals sacrificed at day 16 exhibited a significant reduction in tumor area (Figure 2). Finally, we tested the effectiveness of Guadecitabine in combination with T cell adoptive immunotherapy (AIT). Individually, Guadecitabine or AIT alone slowed the initial tumor progression over the first 2-3 weeks. When applied together, however, there was an additional synergistic effect that strongly suppressed tumor growth and prolonged overall survival for an additional 2+ weeks (Figure 3).

Conclusions

In vitro, the DNMTi Guadecitabine selectively alters the Ly6C+ MDSC subpopulation toward an immune-stimulatory phenotype, and induces expression of immunogenic surface molecules on 4T1 cells. In vivo, treatment with Guadecitabine reduces tumor burden by mainly affecting MDSC accumulation and phenotype. In addition, Guadecitabine enhances the effectiveness of AIT to suppress tumor progression and prolong overall survival.

Acknowledgements

We would like to thank Astex Pharmaceuticals for providing the Guadecitabine, as well as the Massey Cancer Center for pilot grant funding.

Ethics Approval

These studies were conducted with the permission and oversight of the VCU Institutional Animal Care and Use Committee.

Fig. 1 (abstract P491).

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Fig. 2 (abstract P491).

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Fig. 3 (abstract P491).

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P492 Myeloid-derived suppressor cells (MDSC) assessment using a fully automated sequential chromogenic multiplex assay

Anna Martirosyan, Dr, Assil Benchaaben, Aurélie Collignon, MS, Emilie Bonzom, Trainee, Matthieu Duval, Apprentice, Emmanuel Prestat, PhD, Christophe Haond, Jacques Fieschi, PhD

HalioDx, Marseille, France

Correspondence: Jacques Fieschi (Jacques.Fieschi@haliodx.com)

Background

Despite significant advances in the recent years, the response rate to immune checkpoint inhibitor therapies for non-small cell lung cancer (NSCLC) is only about 20%. There is a strong and urgent need to identify new diagnostic biomarkers to predict which patients can benefit from an immune checkpoint blocker treatment. Extensive animal data and several clinical trials indicate that immunosuppression is a limiting factor of effective anti-tumoral immunotherapy. In this context, the presence of immunosuppressive elements such as myeloid-derived suppressor cells (MDSC) in the tumoral microenvironment might be a major factor contributing to resistance to checkpoint inhibitors. In recent years, several studies have shown a correlation between the level of MDSC and stage, overall survival, and response to therapy in NSCLC patients. For instance circulating MDSC were negatively associated with the immune response to cancer vaccine. Furthermore, the accumulation of MDSC has also been reported to correlate with the progression-free survival and the response to chemotherapy, as well as metastatic burden in NSCLC patients. Last, but not least the intra-tumoral accumulation of MDSC is associated with unfavorable prognosis.

Methods

Here we assessed the presence and abundance of this major immunoregulatory population within the NSCLC microenvironment by using an automated sequential chromogenic multiplex assay.

Results

A unique combination of biomarkers (CD11b, CD15, HLA-DR, CD14, LOX1, and S100A9) was developed to detect and quantify different populations of MDSC on a single FFPE tumor tissue section. Briefly, a tissue section was sequentially stained, digitized, unstained and re-stained with antibodies targeting the six markers. Images of the whole slide were then analyzed by digital pathology: first, a newly developed software was used to co-register the 6 virtual slides and perform colors deconvolution. Then detection of positive cells was performed for each marker independently, using Indica Lab’s HALO software. The M-MDSC (monocytic MDSC) and PMN-MDSC (polymorphonuclear MDSC) populations were defined as being respectively CD11b+CD14+HLA- DRnegativeS100A9+cells and CD11b+CD15+HLA-DRnegativeLOX1+cells. In addition, tissue segmentation tools were used to assess MDSC densities in parenchyma, tumor stroma and invasive margin regions.

Conclusions

The detection and quantification of MDSC in NSCLC patients could be a key parameter to predict patient’s responses to anti-tumoral immunotherapy. This new tool will allow to evaluate the immunosuppressive landscape of NSCLC tumors.

P493 B cells in glioblastoma are associated with diminished survival

Ileana Mauldin, PhD, Jasmin Jo, MD, Nolan Wages, PhD, Samuel Young, BS, Loren Erickson, PhD, Maria- Beatriz Lopes, MD, PhD, Craig Slingluff, MD, Camilo Fadul, MD, FAAN

University of Virginia, Charlottesville, VA, USA

Correspondence: Camilo Fadul (fadul@virginia.edu)

Background

The demonstrated efficacy of immunotherapy, in the treatment of some cancers, has not been observed with glioblastoma (GBM). Characterization of the immune infiltrates in GBM may identify targets to drive the immune activation that improves the efficacy of immunotherapy. We have observed that GBM can harbor dense B cell infiltrates usually found cuffing blood vessels. B cells can act as antigen presenting cells driving local immune activation, or as regulatory B cells (Bregs) to suppress immune activation; they may also be localized in tertiary lymphoid structures (TLS) usually associated with better patient survival. The prevalence and role of B cell infiltrate in GBM is unknown.

Methods

Surgically-resected GBM from 48 patients were evaluated for immune infiltrate by multispectral Immunohistochemistry using the OPAL staining system, Vectra3 microscope, and Inform Software (PerkinElmer). Three multiplex panels were designed to enumerate CD20 infiltrates from three serial sections of tumors and to evaluate additional immune markers. RNA-seq gene expression data from the Cancer Genome Atlas (TCGA) was evaluated from GBM patients.

Results

In univariate assessments, low CD20+ cell densities were associated with better overall survival (OS) (median OS 31.7 months for 12 CD20low patients vs. 18.7 months for 36 CD20high patients, p=0.019). Multivariate analyses were done to assess key prognostic factors (CD20 density (low vs high), Karnofsky performance status (KPS; high vs. low), age, and MGMT methylation (methylated vs unmethylated). Decreased OS was observed from patients with high CD20+ infiltrate (Hazard ratio (HR)=2.84, p=0.03) and longer OS was associated with MGMT methylation (HR=0.49, p=0.04). Differences in age (HR=1.13, p=0.05) and KPS (HR=0.49, p=0.08) were trending in significance. We found that B cell infiltrates in GBM do not constitute TLS when examined for high endothelial venules. TCGA data also suggests that in GBM overexpression of MS4A1 (encoding CD20) is associated with decreased OS (p=0.01).

Conclusions

Our studies suggest a correlation between B cells and diminished patient survival, thus CD20 infiltrates may constitute a novel prognostic factor for GBM. We hypothesize that B cells in GBM may function as Bregs which would suppress immune activation. However, this hypothesis needs to be further evaluated by examining Breg function in tumors and evaluating the impact of GBM-infiltrating B cells on tumor-infiltrating T cells.

Ethics Approval

The study was approved by the University of Virginia's Ethics Board, approval number 20210.

P494 Targeting the CCR2/MCP-1 chemokine axis for cancer therapy

Payal Mittal¹, Tatiana Akimova², Craig Leach³, Jose Clemente³, Matthew Sender³, Liqing Wang², Joseph Marino³, Yao Chen³, Peiling Chen³, Brandon Turunen³, Wayne Hancock, MD, PhD², Payal Mittal, MD²

¹Children's hospital of Philadelphia/GSK, Philadelphia, PA, USA; ²Children's hospital of Philadelphia, Philadelphia, PA, USA; ³GlaxoSmithKline, Collegeville, PA, USA

Correspondence: Wayne Hancock (Whancock@pennmedicine.upenn.edu)

Background

Host anti-tumor responses, including the actions of cytotoxic T cells, play a key role in curtailing tumors, but can be hindered by multiple mechanisms active within the tumor microenvironment. One such mechanism involves the recruitment and expansion of immunosuppressive cells such as myeloid derived suppressor cells (MDSCs) in the tumor microenvironment. We are targeting the signaling pathways implicated in recruitment of MDSCs in tumor microenvironment.

Methods

We have utilized transplantable lung tumor cell line (TC1) to address the role of the CCR2/MCP-1 axis in MDSC associated tumor progression. Additionally, I utilized CCR2 knockout mice to demonstrate the dependence of progression of TC1 tumor on CCR2 signaling.I also used a thioglycolate-induced peritonitis model of inflammation to validate the ability of a CCR2 antagonist to inhibit trafficking of MDSCs to the site of inflammation and then tested the compound at this inhibitory concentration for its ability to impair CCR2 chemokine signaling within established tumors.

Results

Phenotypic profiling of TC1 tumors revealed maximal expression of CCR2 by tumor resident MDSCs, and MCP-1 by transplanted tumor cells, tumor associated macrophages (TAMs) and tumor associated neutrophils (TANs) respectively. Additionally, utilization of CCR2 knockout mice showed the dependence of progression of TC1 tumor on CCR2 signaling (tumors were significantly smaller in CCR2KO mice compared to WT mice). We used a thioglycolate-induced peritonitis model of inflammation to validate the ability of a CCR2 antagonist to inhibit trafficking of MDSCs to the site of inflammation in a dose-dependent manner with a maximal effect at a dose of 10 mg/kg, and then tested the compound at this inhibitory concentration for its ability to impair CCR2 chemokine signaling within established tumors. CCR2 antagonist promoted antitumor immunity in TC1 tumors.

Conclusions

In summary, genetic and pharmacologic data indicate that CCR2 targeting may be an important new component of immuno-oncology based therapies.

P495 Pegfilgrastim, but not Plinabulin, generates a blood myeloid cell (BMC) repertoire with a predominant immunosuppressive phenotype

Douglas Blayney, MD¹, Stephan Ogenstad², Yuankai Shi³, Lihua Du⁴, Lan Huang⁵, Ramon Mohanlal, MD, PhD, MBA⁵

¹Stanford Cancer Institute, Stanford, CA, USA; ²Statogen Consulting, LLC, Zebulon, NC, USA; ³Chinese Academy of Medical Sciences, Beijing, China; ⁴Wanchun Bulin Pharmaceuticals Limited, Dalian, China; ⁵BeyondSpring Pharmaceuticals, Inc., New York, NY, USA

Correspondence: Ramon Mohanlal (rmohanlal@beyondspringpharma.com)

Background

Tumors recruit BMC to the tumor microenvironment and modulate BMCs [immunosuppressive tumor-associated macrophages (TAM), neutrophils (TAN), and myeloid derived suppressor cells (MDSC)] in tumor microenvironment (Schupp, Cellular Immunology, 2017; Ginhoux, Nat Rev Immunology, 2014). Predominantly immature BMCs are associated with poor prognosis (Bergenfelz, PLoS One, 2015; Toor, Cancer Immunol Immunother, 2017). An elevated N-to-L Ratio (NLR) of NLR > 5, and reduced L-to-M ratio (LMR) of <3.2 are predictive of poor prognosis in cancer patients (pts) (Zhou; Nature, 2017; Sierzega; Ann Surg Onc, 2017). Chemotherapy (chemo) induced neutropenia (CIN) is mitigated with G-CSF such as pegfilgrastim (Peg). Plin is a novel non-G-CSF small molecule, with a different mechanism of action for CIN (LSK inhibition reversal; Lloyd AACR, 2017). Plin (by IV) and Peg (by SC) are given as a single dose-per-cycle. In contrast to Peg, Plin is given on the same day of chemo, 30 minutes after chemo, vs 24 hours after chemo with Peg. Plin does not cause bone pain, and has anti-cancer, immune-enhancing activity (Mohanlal, ASCO-SITC 2018). The Phase (Ph)2 portion of Study BPI-2358-105 (NCT03P171

*Corresponding author email: mbagarazzi@inovio.com6) in NSCLC pts, compared Plin (at different doses; n=55) with Peg for the prevention of Docetaxel (Doc) CIN. Plin (20 mg/m2) and Peg are equally effective for the prevention of Doc CIN, in respect to frequency and duration of severe neutropenia (Blayney, ASCO 2018). Since Plin and Peg both improve BMCs, we evaluated their respective immunosuppressive potential.

Methods

BMCs from cycle 1 of Ph2 study 105 was analyzed with either Plin (20 mg/m2; n=14) or Peg (6 mg; n=14). BMCs, including immature Ns ((pro)myelocytes and bands) were available through day (D) 15.

Results

In contrast to Peg, Plin did not show NLR>5 or LMR<3 (Table below). N bands were observed in 25% vs 0% of pts with Peg and Plin resp. (Pro)myelocytes were observed in 77% vs 14% of pts with Peg and Plin, resp (p<0.001).

Conclusions

Peg, but not Plin generates a BMC profile with a predominant immunosuppressive phenotype, while both are equally effective for the prevention of Doc CIN.

Table 1 (abstract P495). Blood Immune Cell Repertoire after Peg or Plin

P496 Innate and adaptive immune responses to metastatic colorectal cancer differ by sex and correlate with survival

Anita Ray, PhD¹, Robert Nofchissey, BS¹, Sarah Adams, MD², William Berry¹, Katherine Morris, MD, FACS³

¹OUHSC, Oklahoma City, OK, USA; ²UNM, Albuquerque, NM, USA; ³University of Oklahoma Stephenson Cancer Center, Oklahoma City, OK, USA

Correspondence: Katherine Morris (kmorris4@ouhsc.edu)

Background

Women with colorectal cancer (CRC) have a survival advantage over men. The mechanism behind this is unclear. CRC is strongly influenced by the tumor immune microenvironment (TME), with multiple immune cell types and signaling pathways implicated in its initiation, progression, and metastasis. Furthermore, murine models of sepsis have demonstrated increased numbers of peritoneal leukocytes and increased activation in females that correlate with improved survival [1,2]. Macrophages are vital participants in the CRC TME and can drive pro- and anti- inflammatory shifts. We hypothesized that the immune CRC TME is sex-dependent and contributes to improved survival in females.

Methods

Male and female C57/Bl6 mice were injected with 105 MC38 cells intraperitoneally. Age and sex-matched mice were sacrificed for normal controls (n=6 for all groups). Tumors and ascites developed in the peritoneal cavity in the tumor model. Mice were sacrificed when moribund or at Day 23 post-injection. Serum and tumor cytokine secretion were measured with Luminex™ 32-Plex Immunology assay and results compared by sex. Cells were stained with fluorescent antibodies and run on a flow cytometer alongside controls.

Results

Similar to human CRC patients, female mice survived longer than males (p=0.0354). While there were no significant differences in cytokine production in healthy mice, male and female mice bearing tumors had significant differences in both systemic and tumor-produced cytokines. In female TME, more G-CSF, IL-10, and GM-CSF were secreted. These cytokines influence macrophage polarization. Macrophage numbers at the tumor site were similar between the sexes, but tumors from female mice had increased IL-10 producing macrophages (p=0.0685), which is characteristic of M2-type macrophages. Mice with elevated IL-10+ macrophages survived longer (p=0.0005). M2 macrophages and G-CSF signaling cause differentiation of Th2 cells. Tumors from females showed increased total CD4+ T cells (p=0.0398), which also correlated with increased survival (p=0.0222). Animals with elevated IL-4 producing CD4 T cells survived longer (p=0.0195), suggesting that sex-specific innate and adaptive responses to CRC may contribute to the survival benefit seen in women.

Conclusions

Shifts within the TME can alter the trajectory of tumor progression and patient outcome. We report here marked differences in the TME of females and males in immune cell populations and behavior. These changes within the tumor are strongly correlated with survival, suggesting that they play a role in the survival gap between men and women with CRC. The origins of these sex-linked responses, and the potential effects on immune therapies warrant further study.

Acknowledgements

We would like to acknowledge the American Cancer Society, the Stephenson Cancer Center, and the University of Oklahoma Health Sciences Center Department of Surgery for funding, and the Laboratory for Molecular Biology and Cytometry Research at OUHSC for the use of the Flow Cytometry and Imaging facility which provided equipment and services.

References

• 1. Scotland R, Stables M, Madalli S, et al. Sex differences in resident immune cell phenotype underlie more efficient acute inflammatory responses in female mice. Blood 2011; 118:5918-5927.

• 2. Angele M, Schwacha M, Ayala A, et al. Effect of gender and sex hormones on immune responses following shock. Shock 2000; 14(2):81-90.

Ethics Approval

The study was approved by the OUHSC IACUC, protocol number 17-008-C.

P497 Study of anti-HER2 CAR T cells in the immunosuppressive ependymoma tumor microenvironment

Marissa Kane², Andrea Griesinger², Andrew Donson², Vladimir Amani², Nicholas Foreman², Davis Witt³, Jean Mulcahy Levy², Anandani Nellan, MD²

¹Children's Hospital Colorado, Aurora, CO, USA; ²University of Colorado Denver, Aurora, CO, USA; ³Thomas Jefferson University, Philadelphia, PA, USA

Correspondence: Anandani Nellan (anandani.nellan@ucdenver.edu)

Background

Ependymoma is the third most common childhood brain tumor and novel treatment methods are urgently needed. Complete surgical resection and radiation still results in a 10-year relapse rate of over 70%. Chemotherapy has failed to improve survival in patients with ependymoma. Chimeric antigen receptor (CAR) T cell therapy has been very effective in hematologic malignancies, but progress in solid tumors has lagged. The hostile tumor microenvironment of solid tumors has been implicated as a primary reason why CAR T cell therapy has only resulted in modest and temporary responses in patients. Previous research has shown that ependymoma tumor cells secrete cytokines that polarize surrounding monocytes into an immunosuppressive phenotype, which in turn renders tumor infiltrating T cells ineffective. Native T cells found in patients’ ependymoma tumor samples are incapacitated and this phenomenon may also affect engineered CAR T cells.

Methods

811 and 928 are two high-risk patient derived ependymoma cell lines that have confirmed HER2 surface expression and are used for in-vitro experiments. Human peripheral blood mononuclear cells are activated to promote T cell proliferation and transduced with retrovirus to express anti-HER2 CAR on the surface. Monocytes are cultured in 811 and 928 tumor conditioned media to polarize cells into an immunosuppressive phenotype (polarized monocyte media). T cells and monocytes from the same donor are used in each experiment. Flow cytometry is used to characterize exhaustion markers, as well as surface CAR expression of transduced T cells. Cytokine secretion will be analyzed with a Millliplex Human Cytokine Panel. T cell function will be assessed with an Incucyte live cell imager to quantify immune cell killing of tumor cells over time.

Results

Anti-HER2 CAR T cells have excellent pre-clinical efficacy against 811 and 928 cells as demonstrated by cytokine release after co-incubation and robust tumor cell killing. Anti-HER2 CAR T cells co-cultured in 811 and 928 polarized monocyte media exhibit higher numbers of surface inhibitory markers (PD-1, TIM-3, and LAG-3) compared to anti-HER2 CAR T cells cultured in AIMV media. Anti-HER2 CAR T cells co-cultured in 811 or 928 polarized monocyte media also have decreased CAR surface expression and a trend towards decreased tumor cell killing.

Conclusions

Anti-HER2 CAR T cells cultured in 811 and 928 polarized monocyte media have increased inhibitory markers and decreased CAR expression. Understanding the mechanism of exhaustion and downregulation of CAR expression may have therapeutic implications to improve the efficacy of CAR T cells against solid tumors.

P498 Characterization of the tumor microenvironment in a spontaneous mouse model of cholangiocarinoma: a robust model for evaluating therapeutic interventions for treating the disease

Luis Ruffolo, MD, Katherine Jackson, MD, Joseph Murphy, MSc, Nathania Figueroa, MD, Brian Belt, JD, David Linehan, MD, Peter Prieto, MD, MPH

University of Rochester Medical Center, Rochester, NY, USA

Correspondence: Peter Prieto (Apeter_prieto@urmc.rochester.edu)

Background

Cholangiocarcinoma is the second most common primary liver malignancy. Prognosis is dismal due to its resistance to conventional therapy and propensity to metastasize. Therefore, the development of effective strategies for treating cholangiocarcinoma represents a significant unmet clinical need, but models for evaluating more advanced approaches like targeted and immune based therapies are lacking. Cholangiocarcinoma is characterized by the presence of a dense fibro-inflammatory reaction and extensive tumor stroma that confers resistance to therapy. Although mice with targeted activation of Kras and loss of p53 (Kras-p53) in the liver spontaneously develop cholangiocarcinoma recapitulating the histological features of human disease, the composition of the tumor microenvironment (TME) remains largely unknown. Here, we examined the composition of the TME in Kras-p53 mice to identify additional targets for evaluating more advanced strategies for treating cholangiocarcinoma.

Methods

Histological staining and immunohistochemistry were performed on formalin-fixed, paraffin embedded and frozen tissue sections from human and mouse cholangiocarcinoma tumors for stromal and immune markers. Bone marrow (BM), peripheral blood, spleen, and single cell tumor and normal liver suspensions from Kras-p53 mice and littermate controls were processed for flow cytometry analysis. RNA was extracted from tumor and normal liver tissue and RNA-seq and qRT-PCR analysis were performed. Myeloid cells from BM, spleen, and tumor were isolated and functional assays were performed in-vitro.

Results

Human cholangiocarcinoma featured prominent immunosuppressive signatures including a dense inflammatory leukocyte infiltrate mainly comprised of myeloid cells of both monocytic and granulocytic origin including tumor associated macrophages (TAM) and neutrophils (TAN) respectively. Tumors from Kras-p53 mice featured a prominent fibro-inflammatory reaction (Figure 1) with a dense network of fibroblasts, collagen deposition and, hyaluronic acid. Flow cytometry demonstrated Kras-p53 tumors were infiltrated with significantly elevated levels of inflammatory leukocytes (Figure 2) including TAM and TAN. RNA-seq and qRT-PCR demonstrated Kras-p53 tumors expressed elevated levels of cytokines associated with myelopoiesis and mobilization of myeloid cells. In addition, tumors expressed increased levels of soluble factors and checkpoint markers associated with immune suppression including Tgf-β, Il10, Arg-1, Pd-l1, Pd-1, and Ctla-4. Myeloid cells isolated from Kras-p53 tumors were functionally trophic and suppressed T cell proliferation. Thus, these data suggest the TME of cholangiocarcinoma features additional targets for testing more advanced strategies including immune based therapies.

Conclusions

Cholangiocarcinoma tumors derived from Kras-p53 mice are highly desmoplastic and feature a prominent inflammatory immune infiltrate including highly immunosuppressive myeloid cells. Thus, Kras-p53 mice are a robust model to evaluate targeted and immune therapeutic interventions.

Ethics Approval

The study was approved by the University of Rochester UCAR Committee, approval number 2014-037E

Fig. 1 (abstract P498).

Tumors derived Kras-p53 mice highly desmoplastic

Fig. 2 (abstract P498).

Kras-P53 tumors increased inflammatory leukocytes

P499 Correlation of immune escape mediated defects in the HLA class I antigen-presenting machinery with the immune cell infiltration and prognosis in oral squamous cell carcinoma

Claudia Wickenhauser, MD, PhD, Barbara Seliger, MD, PhD

Luther University Halle-Wittenber, Halle, Germany

Correspondence: Barbara Seliger (Barbara.Seliger@uk-halle.de)

Background

Progression of oral squamous cell carcinoma (OSCC) is often associated with an evasion of tumor cells from the host immune surveillance, which is accompanied by a worse outcome of patients and might influence the efficacy of immunotherapies.

Methods

Since little information exist about the molecular mechanisms leading to tumor immune evasion and its correlation with the immune cell contexture the expression of HLA class I antigens and components of the antigen processing machinery (APM) was analyzed in three untreated and interferon (IFN)-γ treated OSCC cell lines as well as a panel of 160 human papilloma virus (HPV)-negative OSCC lesions and correlated with the composition of immune cell infiltration and clinical parameters.

Results

Immunohistochemical analyses of the OSCC lesions revealed that HLA class I heavy chain and β2-microglobulin (β2-m) as well as selected HLA class I APM components were significantly downregulated in OSCC lesions vs. non-neoplastic cells. This was in accordance to the predominantly low basal mRNA and protein expression of HLA class I APM components in OSCC cell lines, which was accompanied by low HLA class I surface expression. The impaired HLA class I APM component expression was enhanced by IFN-γ treatment suggesting a deregulation rather than structural alterations as a major mechanism of impaired expression. This was in line with a positive association of HLA class I APM expression levels with the frequency and composition of the immune cell infiltration of OSCC lesions and mediated by the T cell produced IFN-γ: Intra- as well as peri-tumoral density of CD8+ T lymphocytes and intra-tumoral density of FoxP3+ regulatory T cells correlated with membranous HLA class I heavy chain (HC) and β2-m expression and the trimeric HLA classI/β2-m/peptide complex, while membranous β2-m and cytoplasmic HLA class I HC expression positively correlated with the intra-tumoral density of CD4+ T lymphocytes. High cytoplasmic expression levels of HLA class I HC and β2-m, low cytoplasmic expression of the peptide transporter associated with antigen processing (TAP) subunit 1 and a high nuclear expression of LMP2 were significantly correlated with a poor overall survival (OS) of OSCC patients.

Conclusions

In conclusion, this study revealed marked differences between HLA class I-positive and negative tumors related to tissue structure, the composition of intra- and peri-tumoral leukocyte infiltration of the TME and OSCC patients’ prognosis. This knowledge might help to overcome immune escape and to improve the efficacy of immunotherapeutic strategies for OSCC patients in the future.

P500 Novel bispecific antibody preferentially and efficiently depletes tumor-associated immunosuppressive myeloid cells and enhances therapeutic responses to PD-L1 blockade in immune-excluded tumor model

Seng-Lai Tan, PhD, Sangeetha Palakurthi, PhD, Jacqueline Lee

Elstar Therapeutics, Cambridge, MA, USA

Correspondence: Seng-Lai Tan (thomas.tan@elstartherapeutics.com)

Background

Accumulation of tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) has been associated with tumor progression, poor prognosis, and inferior response to immune checkpoint therapy in many cancers. These cells render effector immune cells dysfunctional and promote angiogenesis and metastasis. Thus, TAMs and MDSCs are considered promising therapeutic targets in cancer immunotherapy.

Methods

We determined co-expression of CSF-1R and CCR2 on TAMs and MDSCs and generated a bispecific antibody (UniTI-01), which simultaneously binds CSF-1R and CCR2, to deplete these immunosuppressive cell populations, while sparing tissue-resident macrophages. In vitro and ex vivo assays using recombinant proteins, cell lines, and primary cells were conducted to determine UniTI-01 binding to both CSF-1R and CCR2 on the same cell and its effects on CSF-1 and CCL2-dependent functional assays. Plasma exposure, pharmacodynamic and therapeutic effects of UniTI-01 were assessed in murine syngeneic tumor models.

Results

We confirmed CSF-1R and CCR2 are co-expressed on TAMs and monocytic MDSCs (M-MDSCs) from ovarian and colorectal cancer patients as well as murine syngeneic tumors. Specific binding of the murine-reactive surrogate bispecific antibody UniTI-01 was demonstrated using murine CSF-1R and/or CCR2 in heterologous expression systems. Additionally, UniTI-01 effectively bound TAMs and M-MDSCs derived from several syngeneic tumor models. The monovalent anti-CSF-1R arm and anti-CCR2 arm of UniTI-01 exerted inhibitory activity in CSF-1- and CCL2-dependent functional assays in vitro, respectively. Importantly, UniTI-01 preferentially depleted TAMs and M-MDSCs over major organ tissue-resident macrophages, including Kupffer cells, in tumor-bearing mice. In contrast, an anti-CSF-1R monoclonal antibody induced significant depletion of tissue-resident macrophages in several organs. UniTI-01 treatment increased intratumoral T cells and CD8+ T cells:CD4+ Treg ratio across different syngeneic tumor models. In the immune-excluded model EMT6, a combination regimen of UniTI-01 and an anti-PD-L1 monoclonal antibody induced significant tumor regression compared to either agent alone. Finally, mice that cleared EMT6 tumor on the combination therapy developed specific and durable anti-tumor response demonstrated by their protection when re-implanted with EMT6 cells, but not with a different tumor cell line (CT- 26).

Conclusions

Dual targeting of CSF-1R and CCR2 using a bispecific antibody efficiently depletes TAMs and M-MDSCs without significantly affecting tissue-resident myeloid cells and may serve as a novel approach to enhance therapeutic efficacy of checkpoint blockade immunotherapy with a wider therapeutic window. Our data support development of a synonymous human UniTI-01 for clinical evaluation.

P501 Multiplex immunofluorescence evaluation of immune cell relationships within PDAC resection tissues using tailored analysis of multi-spectral image component data

Hannah Thomson¹, Alison Bigley, CSci, FIBMS², Lorcan Sherry, PhD², Mark Anderson, BSc², Mariana Beltran³, Dawn Lyster, MSc³, Mike Millar³

¹OracleBio Ltd., North Lanarkshire, Scotland, UK; ²OracleBio, Glasgow, UK; ³Aquila BioMedical, Edinburgh, UK

Correspondence: Lorcan Sherry (lorcan.sherry@oraclebio.com)

Background

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive exocrine tumour with an extremely poor prognosis where the application of checkpoint inhibitors has proven to be disappointing. One of the characteristics of PDAC is a desmoplastic process that is thought to create a barrier to potential responses of immune cells and reduce accessibility of therapeutic agents. PDAC phenotype is also known to be immune cell deficient. However, M2 macrophage aggregations have been identified within the tumour milieu that often co-express programmed cell death markers. The evaluation of PD-L1 expression and associated immune cell responses in Whipple’s resection tissues may be utilised to aid predicting patient outcome [1]. Here we use a 7-plex evaluation to exemplify the potential of multiplex immunofluorescence (mIF) combined with multispectral imaging and quantitative image analysis to examine relationships in immune, inflammatory and checkpoint expressing cell populations within PDAC surgical resection samples.

Methods

Exemplar PDAC resection sections were mIF labelled by Aquila BioMedical for 5 cell markers, including PD-L1, CD3, CD8, FoxP3, CD163, a pan cytokeratin epithelial marker and DAPI nuclear marker. The stained slides were digitised using the Vectra Polaris multispectral scanner (Perkin Elmer) and defined region of interest (ROI) images exported in multi-layered component data format. The mIF images were analysed by OracleBio using tailored applications developed in Visiopharm Oncotopix Software. These enabled the identification of tumour and stroma ROI, facilitated cell detection, classification and analysis and the determination of cell relationships within the tumour microenvironment.

Results

Across the n=5 resection samples, selected ROI displayed a range of tumour, stroma, lymphoid aggregates and connective tissue content. Analysis of cell populations indicated varying levels of CD3, CD8 and FoxP3 immune cell infiltrations. PD-L1 also showed a varied expression within tumour cells across samples while higher numbers of CD163 positive macrophage aggregations were identified within tumour.

Conclusions

Although knowledge of the underlying mechanisms of PDAC have advanced over the recent years, much still remains unclear. Multiplex IF data potentially enables a greater understanding of the complex mechanisms involved in PDAC, thereby furthering the development of drugs that target immune cells and may be indicative of response to treatment or predicting patient outcome.

References

1. Yamaki S, Yanagimoto H, Tsuta K, Ryota H, Kon M. PD-L1 expression in pancreatic ductal adenocarcinoma is a poor prognostic factor in patients with high CD8+ tumor-infiltrating lymphocytes: highly sensitive detection using phosphor-integrated dot staining. International Journal of Clinical Oncology. 2017 March 18. 22(4): 726–733.

P502 Novel approach of modulating immune cell metabolism in the tumor microenvironment to enhance efficacy of immunotherapy

Frank Boriello, MD/PhD², HongBum Lee³, Vincent O'Neil³, Ted Kim, PhD³, James Lederer, PhD⁴, Sanghee Yoo, PhD³

¹ImmunoMet Therapeutics Inc., Houston, TX, USA; ²Alloplex Biotherapeutics, Boston, MA, USA; ³ImmunoMet Therapeutics, HOUSTON, TX, USA; ⁴Brigham and Women’s Hospital/Harvard, Boston, MA, USA

Correspondence: James Lederer (jlederer@rics.bwh.harvard.edu)

Background

Cells adopt different metabolic strategies depending on their functional needs. Tumor cells deplete glucose by aerobic glycolysis, which can inhibit effector immune cells that may rely on aerobic glycolysis for effector activity [1]. It has been shown that immune cells that use mitochondrial oxidative phosphorylation (OXPHOS) for energy are able to co-exist with tumor cells in the TME. OXPHOS dependent immune cells include CD4+ regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSC), and tumor associated macrophages (TAM). These immune cell types are immune suppressive and metabolically compatible with tumor cells [2].

Methods

Human PBMC was used for immune suppressive cell differentiationCyTOF mass cytometry was used to characterize immune cellsSyngeneic in vivo animal studies using RENCA and CT26 were conducted for in vivo efficacy studies

Results

IM188 is an OXPHOS inhibitor drug with a biguanide core structure. Metformin is the canonical biguanide drug that has been safely used to control glucose levels in people with type II diabetes. The mechanisms for how biguanide drugs influence immune cells has not been well characterized. Since IM188 is an optimized biguanide targeting OXPHOS dependent immune cells, we studied the effects of IM188 on human blood immune cells (PBMCs) and on immune responses in mouse models of infection or cancer. PBMCs were differentiated under conditions to promote Treg or MDSC expansion in vitro in the absence or presence of IM188. Analysis of differentiated T cells by CyTOF mass cytometry showed reduced expression of multiple Treg markers such as Foxp3, CTLA4, and TGF-beta. In MDSC differentiation studies, we found that IM188 reduced MDSC expansion and their functional activity to suppress T cell proliferation. In mouse bacteria and virus infection studies, the most intriguing finding was the IM188 treatment caused increased CD8+ T cell expansion and increased IFN-gamma and TNF-alpha cytokine expression in CD8+ T cells. These observations suggest that IM188 can enhance T cell mediated immune responses. Finally, in syngeneic mouse tumor models, IM188 showed a good range of combination efficacy with anti-PD1 therapy. We measured increased T effector cells and decreased immune suppressive cell types at the tumor site in mice treated with IM188 or anti-PD-1 antibody.

Conclusions

In summary, IM188 shows metabolic reprogramming activity that may enhance immune functions by modulating immune cell differentiation and/or function by inhibiting OXPHOS-dependent cells and promoting aerobic glycolysis by effector immune cells.

References

• 1. Chang HA, Qiu J, O’Sullivan D, Buck MD, Noguchi T, Curtis JD, Chen Q, Gindin M, Gubin, MM, van der Wind GWJ, Tonc E, Schreiber,RD, Pearce EJ, and Pearce EL. Metabolic competition in the tumor microenvironment is a driver of cancer progression. 2015 ; 162:1229-1241.

• 2. Hossain F, Al-Khami AA, Wyczechowska D, Hermandez C, Zheng L, Reiss K, Valle LD, Trillo-Tinoco J, Maj T, Zou W, Rodriguez PC, Ochoa AC. Inhibition of fatty acid oxidation dodulates immunosuppressive functions of myeloid-derived suppressor cells and enhances cancer therapies. Cancer Immunol Res. 2015; 2:1236-127.

P503 Characterization of the immune desert in metastatic non-small cell lung cancer (NSCLC) and the use of cell proliferation to predict clinical response to immune checkpoint inhibitors (ICIs)

Jason Zhu, MD¹, Matthew Labriola, MD¹, Daniele Marin, MD¹, Shannon McCall, MD¹, Edwin Yau, MD, PhD², Grace Dy², Sarabjot Pabla, MSc, PhD, BS³, Sean Glenn, PhD³, Carl Morrison, MD, DVM³, Daniel George, MD¹, Tian Zhang, MD¹, Jeffrey Clarke, MD¹

¹Duke University, Durham, NC, USA; ²University of Buffalo, Buffalo, NY, USA; ³Omniseq Inc, Buffalo, NY, USA

Correspondence: Tian Zhang (tian.zhang2@duke.edu)

Background

Immune checkpoint inhibitors (ICIs) have profoundly reshaped the treatment landscape for advanced NSCLC. While patients who have an ICI response may have a deep and durable response, in unselected populations, the majority of patients will not respond, but will be exposed to potential immune toxicities. Current biomarkers such as PD-L1 are not sensitive nor specific for predicting response, particularly for non-inflamed (NI) or “immune desert” tumor microenvironments. Here, we describe the use of cell proliferation to evaluate response in immune desert NSCLC.

Methods

113 formalin-fixed, paraffin-embedded (FFPE) tumor samples of metastatic NSCLC were evaluated by RNA-sequencing to measure transcript levels of genes related to tumor infiltrating lymphocytes and cell proliferation, DNA-sequencing of 409 genes for tumor mutational burden (TMB), and PD-L1 status (Dako 22C3 antibody assay). Tumors were defined as inflamed or NI based upon RNA-sequencing analysis of CD8 compared to a reference population of more than 500 cases of multiple tumors. NI/immune desert tumors were defined as the lower 15th percentile of rank for CD8+ T-cells. Cell proliferation, defined as the mRNA expression of 10 genes (BUB1, CCNB2, CDK1, CDKN3, FOXM1, KIAA0101, MAD2L1, MELK, MKI67, TOP2A) was evaluated for association with PD-L1 IHC expression, TMB, and response to ICIs by RECIST 1.1 criteria.

Results

In our cohort of 113 cases, 12% (15/113) were classified as having an NI, immune desert tumor microenvironment. Of these 15 NI cases, 8 (53%) were proliferative and 7 (47%) were non-proliferative; in addition, 10 of 15 cases (67%) had high TMB (TMB-H). The majority (11/15, 73%) of NI cases were non-responders, with 4 cases having an objective response. For the 4 responders, 3 were non-proliferative; conversely, for the 11 nonresponders,7 were proliferative. For NI/TMB-H there were 9 were nonresponders, with 7 proliferative and 1 responder who was non- proliferative. For NI/TMB-L the ORR was 60% (3/5) with 2 of 3 responders being non-proliferative. Only 2 of these NI cases were PD-L1 positive (TPS>50%), one of which as a non-proliferative responder (50%). PD-L1 was negative in the other 13 cases, 3 of whom were responders (23%).

Conclusions

Biomarkers such as PD-L1 and TMB on their own may not be sufficient in predicting overall response in immune desert tumor microenvironments. In these cases, cell proliferation may be important in distinguishing patients who may have a clinical benefit to ICI. These results support that NI non-proliferative tumors are more likely to respond to ICI than NI proliferative tumors.

Ethics Approval

OmniSeq’s analysis utilized deidentified data that qualified as non-human subject research under IRB-approved protocols, approved by both Roswell Park Comprehensive Cancer Center (Buffalo, NY, BDR #080316) and Duke Cancer Institute (Durham, NC, PRO00088762).

Impact of Diet, Exercise, and/or Stress on Antitumor Immunity

P504 Nutritional measures to boost immunosurveillance of breast cancer by NK cells

Lorenzo Galluzzi, PhD¹, Aitziber Buqué, PhD¹, Maria Perez-Lanzon, MSc (Master of Science)², Takahiro Yamazaki, PhD¹, Guido Kroemer, MD, PhD²

¹Weill Cornell Medical College, New York, NY, USA; ²Centre de Recherche des Cordeliers, Paris, France

Correspondence: Guido Kroemer (kroemer@orange.fr)

Background

Hormone receptor (HR+) breast cancer (BC) is currently responsible for the majority of BC-related deaths in the US [1]. HR+ BC patients are usually managed by surgery, followed by adjuvant endocrine therapy ± chemotherapy. However, chemotherapy provides limited clinical benefits and is often associated with severe side effects[2], calling for the development of alternative treatment regimens. Over the past decade, immunotherapy with immune checkpoint blockers (ICBs) has achieved unprecedented clinical success in patients with a variety of tumors [3]. However, despite the fact that BC is also under immunosurveillance [4,5], BC patients are generally resistant to ICBs [6], especially in the case of HR+ disease [7]. Thus, there is an unmet need for improved therapeutic approaches to HR+ BC, at least in part reflecting the lack of adequate preclinical models to recapitulate the incidence, natural progression, metastatic dissemination and response to therapy of HR+ BC in immunologically competent hosts.

Methods

We extensively characterized endogenous BC driven in wild-type or genetically engineered C57BL/6 or BALB/c mice by slow-release medroxyprogesterone acetate (MPA) pellets and oral 7,12-dimethylbenz[a]anthracene (DMBA) for incidence, progression, histology, transcriptomic profile, and sensitivity to standard therapeutic agents as well as nutritional interventions.

Results

We demonstrate that MPA/DMBA-driven tumors resemble human HR+ BC in that (1) they display a similar morphology, (2) they express hormonal receptors, (3) they have a gene signature that largely overlaps with that of HR+/HER2- human BCs, (4) tumorigenesis depend on nuclear estrogen receptors, (5) tumor insurgence can initially be delayed by tamoxifen administration, but acquired resistance rapidly subsides, (6) they are under active immunosurveillance by the host immune system with a predominant role for NK cells, and once they form palpable nodules they exhibit limited immune infiltration, and (7) they develop according to rather heterogeneous kinetics. Moreover, MPA/DMBA-driven tumors resemble human HR+ BC because they respond to chemotherapy, PD-1 blockade and RT in a rather heterogeneous and poor fashion. We demonstrate that MPA/DMBA-driven carcinogenesis can be delayed by caloric restriction as well as administration of vitamin B6, vitamin D, and nicotinamide mononucleotide (NAM), in immunocompentent, but not immunodeficient, mice, an effect paralleled by increased amounts of NK cells in the spleen.

Conclusions

HR+ BC appears to evolve by evading NK cell-dependent immunosurveillance, suggesting that NK cell-activating strategies, including nutritional measures like NAM, as well as specific antibodies targeting NK cell receptors, may improve the efficacy of (immuno)therapeutic agents currently employed in the clinics for HR+ BC patients.

References

1. Munoz D, et al. Effects of screening and systemic adjuvant therapy on ER-specific US breast cancer mortality. J Natl Cancer Inst. 2014;106. 2. Jagsi R, et al. Impact of adjuvant chemotherapy on long-term employment of survivors of early-stage breast cancer. Cancer. 2014;120:1854-1862. 3. Sharma P, Allison JP. The future of immune checkpoint therapy. Science.2015;348:56-61. 4. Savas P, et al. Clinical relevance of host immunity in breast cancer: from TILs to the clinic. Nat Rev Clin Oncol. 2016;13:228-241. 5. Kroemer G, Senovilla L, Galluzzi L, Andre F, Zitvogel L. Natural and therapy-induced immunosurveillance in breast cancer. Nat Med. 2015;21:1128-1138. 6. Emens LA. Breast cancer immunotherapy: facts and hopes. Clin Cancer Res. 2018;24:511-520. 7. Rugo HS, et al. Safety and antitumor activity of pembrolizumab in patients with estrogen receptor-positive/human epidermal growth factor receptor 2-negative advanced breast cancer. Clin Cancer Res. 2018.

Ethics Approval

The study was approved by IACUC at the hosting institutions

P505 The gut microbiome of metastatic melanoma patients initiating systemic therapy is influenced by host factors including diet, probiotic and antibiotic use

Vancheswaran Gopalakrishnan, MPH, PhD¹, Christine Spencer, PhD², Jennifer McQuade, MD¹, Miles Andrews, MD, PhD¹, Beth Helmink, MD PhD¹, Alexandria Cogdill, MEng¹, Md Khan¹, Elizabeth Sirmans¹, Lauren Haydu, MS, BChe, MIPH¹, Eliza Posada¹, Elizabeth Burton¹, Isabella Glitza, MD, PhD¹, Rodabe Amaria, MD¹, Sapna Patel, MD¹, Adi Diab, MD¹, Michael Wong, MD PhD FRCPC¹, Hussein Tawbi, MD, PhD¹, Wen-Jen Hwu, MD, PhD¹, Michael Davies, MD, PhD¹, Patrick Hwu, MD¹, Robert Jenq, MD¹, Kelly Nelson, MD¹, Carrie Daniel- MacDougall, MPH, PhD¹, Lorenzo Cohen¹, Jennifer Wargo, MD, MMSc¹

¹UT MD Anderson Cancer Center, Houston, TX, USA; ²Parker Institute for Cancer Immunotherapy, New York, NY, USA

Correspondence: Jennifer Wargo (JWargo@mdanderson.org)

Background

The diversity and composition of the gut microbiome has been implicated in differential responses to immune checkpoint blockade in melanoma and other cancers [1-3]. However, little is known about the impact of diet and other lifestyle factors in this population.

Methods

We assembled a large cohort of early and late-stage melanoma patients (n=312) initiating systemic treatment at UT MD Anderson Cancer Center. In addition to biological specimens, we collected a comprehensive lifestyle survey, including the NCI dietary screener questionnaire, in a subset of patients (n=113). The fecal microbiome was characterized via sequencing of the V4 region of the 16S rRNA gene to determine diversity and compositional structure. Dietary components were dichotomized into high and low categories based on the median of estimated consumption. Differences in compositional structure between groups was determined using analysis of similarity (ANOSIM) for unweighted UniFrac beta diversity distances, and pairwise Mann-Whitney tests for taxonomic comparisons.

Results

The median age of melanoma patients in our cohort was 62 yrs (59% male; 86% Stage III/IV), and the most common treatment type was anti-PD1 based therapy (53.1%). There were no significant associations observed between alpha diversity and age, sex or body mass index among the melanoma patients. “Biotic” use, defined as self-reported use of either biotic was quite common (29% antibiotics, 42% probiotics), and was associated with lower alpha-diversity (p=0.01), with significant associations observed for both antibiotics alone (p=0.05) and for probiotics alone (p=0.02). Additionally, consumption of red meat (p=0.006), sugar-sweetened beverages (SSB) (p=0.048), and fruits and vegetables (FV) (p=.049) were also associated with differences in compositional structure with selective enrichment of Desulfovibrionales (high vs. low red meat =0.03), Mollicutes (in low vs. high SSB consumers, p=0.008), and Porphyromonadaceae (in high vs low FV consumers, p=0.001).

Conclusions

Prospective longitudinal studies are underway to assess the relationships between “biotic” use, dietary factors and the gut microbiome, and treatment response among patients, as well as functional studies in preclinical models. These data provide preliminary evidence that the gut microbiome of melanoma patients may be modifiable by host factors such as diet, use of antibiotics and probiotics, with potential therapeutic implications.

References

• 1. Gopalakrishnan V, et al. Gut microbiome modulates response to anti–PD-1 immunotherapy in melanoma patients. Science. 2018; 359(6371): 97-103.

• 2. Routy B, et al. Gut microbiome influences efficacy of PD-1–based immunotherapy against epithelial tumors. Science. 2018; 359(6371): 91-97.

• 3. Matson V, et al. The commensal microbiome is associated with anti–PD-1 efficacy in metastatic melanoma patients. Science. 2018; 359(6371): 104-108.

Ethics Approval

The study was approved by The University of Texas MD Anderson Center's Ethics Board, approval numbers LAB00-063, and PA15-0232

P506 Subcutaneous and intramuscular fat indices predict survival in advanced stage cancer patients treated with immunotherapy

Dylan Martini, BA¹, Julie Shabto, BA¹, Yuan Liu, PhD², Milton Williams¹, Amir Khan¹, Colleen Lewis³, Hannah Collins³, Mehmet Akce³, Haydn Kissick¹, Bradley Carthon, MD, PhD³, Walid Shaib, MD³, Olatunji Alese, MD³, Conor Steuer, MD³, Christina Wu, MD³, David Lawson, MD³, Ragini Kudchadkar, MD³, Bassel El-rayes, MD³, Suresh Ramalingam, MD³, Taofeek Owonikoko, MD, PhD³, R. Donald Harvey, PharmD¹, Viraj Master, MD, PhD¹, Mehmet Bilen, MD³

¹Emory University School of Medicine, Atlanta, GA, USA; ²Emory University, Atlanta, GA, USA; ³Winship Cancer Institute, Atlanta, GA, USA

Correspondence: Mehmet Bilen (mehmet.a.bilen@emory.edu)

Background

Obesity has been investigated as a prognostic indicator in patients with cancer [1]. In this study, we explored the association between different types of fat and clinical outcomes in advanced stage cancer patients treated with immunotherapy by developing a risk group classification.

Methods

We performed a retrospective analysis of 90 patients treated on immunotherapy-based phase 1 clinical trials at our center from 2009-2017. Baseline CT images at mid-L3 were obtained, and subcutaneous fat density, intramuscular fat density, and visceral fat density (cm2) were calculated using SliceOmatic (TomoVision, version 5.0) and converted to indices (SFI: subcutaneous fat index, IFI: intramuscular fat index, and VFI: visceral fat index) after dividing by height in meters-squared. Risk groups by PFS were created by a recursive partitioning and regression trees method for SFI and IFI, which were selected by a stepwise variable selection among all fat related variables (Figure 1). Cox proportional hazard model and Kaplan-Meier method were used for association with OS and PFS.

Results

Most patients (59%) were males and more than two-thirds (69%) had at least 2 prior lines of therapy. Melanoma (33%) and gastrointestinal (22%) tumors were the most common histologies. The medians for each of the indices were as follows: SFI = 62.78, IFI = 4.06, and VFI = 40.53. Low-risk patients (SFI 73) had significantly longer OS and PFS than intermediate-risk (SFI < 73 and IFI < 3.4) and poor-risk patients (SFI < 73 and IFI 3.4) (Table 1). Intermediate-risk patients also trended towards longer OS and PFS than poor-risk patients. Patients in the low-risk group had substantially longer median OS and PFS than intermediate and poor-risk patients per Kaplan-Meier estimation (Table 1, Figures 2-3).

Conclusions

Decreased subcutaneous fat and increased myosteatosis may decrease survival in advanced stage patients treated with immunotherapy. Future studies should investigate the interaction between different fat composition, the immune system, and the tumor microenvironment.

References

1. Demark-Wahnefried W, Platz EA, Ligibel JA, Blair, CK Courneya KS, Meyerhardt JA, et al. The Role of Obesity in Cancer Survival and Recurrence. Cancer Epidemiology, Biomarkers, and Prevention. 2012 Aug;21(8):1244-59.

Ethics Approval

The study was approved by the Emory University Institutional Review Board, approval number IRB00100973.

Fig. 1 (abstract P506).

See text for description

Table 1 (abstract P506). MVA† of fat risk with survival

Fig. 2 (abstract P506).

See text for description

Fig. 3 (abstract P506).

See text for description

P507 Body mass index as a predictor of survival in advanced stage cancer patients treated with immunotherapy

Julie Shabto, BA¹, Dylan Martini, BA¹, Yuan Liu, PhD², Milton Williams¹, Amir Khan¹, Colleen Lewis³, Hannah Collins³, Mehmet Akce², Haydn Kissick², Bradley Carthon, MD, PhD², Walid Shaib, MD², Olatunji Alese, MD², Conor Steuer, MD², Christina Wu, MD², David Lawson, MD², Ragini Kudchadkar, MD², Bassel El-rayes, MD², Suresh Ramalingam, MD², Viraj Master, MD, PhD², Taofeek Owonikoko, MD, PhD², R. Donald Harvey, PharmD², Mehmet Bilen, MD²

¹Emory University School of Medicine, Atlanta, GA, USA; ²Emory University, Atlanta, GA, USA; ³Winship Cancer Institute, Atlanta, GA, USA

Correspondence: Mehmet Bilen (mehmet.a.bilen@emory.edu)

Background

Body mass index (BMI) has been investigated as a prognostic factor for cancer patients [1], but the effect of BMI on clinical outcomes in patients on phase 1 clinical trials using immunotherapy-based treatment is not known. We investigated the association between BMI and survival in advanced staged cancer patients treated with immunotherapy.

Methods

We completed a retrospective analysis of 90 patients treated on phase 1 clinical trials using immunotherapy-based treatment regimens at Winship Cancer Institute of Emory University from 2009-2017. Baseline BMI was collected from the electronic medical records. Overall survival (OS) and progression-free survival (PFS) were measured from the first dose of immunotherapy to date of death or hospice referral and radiographic or clinical progression, respectively. Cox proportional hazard model was used for association with OS and PFS. BMI was analyzed as a continuous variable and as a categorical variable (normal or underweight: BMI < 25, overweight: 25 ≤ BMI < 30, obese: BMI ≥ 30).

Results

Most patients (59%) were males and the majority (81%) were Royal Marsden Hospital (RMH) good risk. Approximately two-thirds (69%) of patients received at least 2 lines of systemic therapy before being treated with immunotherapy on the clinical trial. Melanoma (33%), gastrointestinal (22%), and lung and head & neck (20%) were the most common tumor types. The median BMI was 27.4. When treated as a continuous variable in multi- variable analysis, a higher BMI was significantly associated with longer OS and PFS (Table 1). Patients with a normal or underweight BMI had significantly shorter OS (HR: 3.27, p-value: 0.005) and trended towards shorter PFS when compared to overweight and obese patients. The median OS and PFS of obese patients was 19.1 months and 4.7 months, respectively, while median OS and PFS of normal or underweight patients was 6.7 months and 1.9 months and median OS and PFS for overweight patients was 8.6 months and 2.3 months, respectively, per Kaplan- Meier estimation (Table 1, Figures 1-2).

Conclusions

Obesity may help prolong survival in advanced stage cancer patients treated with immunotherapy. Further studies are needed to elucidate the underlying biologic effect of adiposity on the tumor microenvironment and the immune system in patients treated with immunotherapy.

References

1. Azvolinsky A. Cancer Prognosis: Role of BMI and Fat Tissue. JNCI. 2014; Volume 106: page 6-7.

Ethics Approval

The study was approved by the Emory University Institutional Review Board, approval number IRB00100973.

Table 1 (abstract P507). MVA† of the association between BMI and survival

Fig. 1 (abstract P507).

See text for description

Fig. 2 (abstract P507).

See text for description

P508 Obesity promotes PD-1 mediated T cell dysfunction and tumor pro-gression but superior anti-tumor effects upon checkpoint blockade

Ziming Wang, MS, Jesus Luna, PhD, Cordelia Dunai, MS, Lam Khuat, Catherine Le, BS, Ethan Aguilar, Annie Mirsoian, Christine Minnar, PhD, Kevin Stoffel, MS, Ian Sturgill, Steven Grossenbacher, Robert Canter, MD, MAS, FACS, Arta Monjazeb, MD, PhD, William Murphy, PhD, Ziming Wang, MS

University of California, Davis, Sacramento, CA, USA

Correspondence: William Murphy (wmjmurphy@ucdavis.edu)

Background

PD-(L)1 signaling is central to T cell exhaustion which occurs with chronic antigen stimulation and results in T cell dysfunction. Blockade of the PD-(L)1 pathway augments T cell responses in a variety of viral and cancer models. Obesity, defined by high body mass index (BMI >30 kg/m2), is reaching pandemic proportions and is a major cancer risk factor. The impact of obesity on immune responses in general, and cancer immunotherapy in particular, is poorly understood.

Methods

Male B6 and female BALB/c mice were fed diets consisting of either 60% or 10% fat, respec-tively, starting from 6-week until 6-month old. DIO and control mice were injected with either B16-F0 (non-metastatic melanoma), B16- F10 (metastatic melanoma), 3LL (metastatic Lewis lung carcinoma), or 4T1 (metastatic breast carcinoma) cells. Tumor-bearing mice were treated intraperitoneally with aPD-1 mAb every other day at 250μg/mouse after an initial dose of 500μg/mouse for a total of 6 injections. Tumor progression was determined by caliber measure-ment, PET- CT, and quantification of metastases. Immune phenotypes and T cell function were measured by flow cytometry. Transcriptomes were analyzed by RNAseq.

Results

DIO mice were significantly heavier than control mice, with an average weight of 60g vs 42g in B6 mice, and 40g vs 20g in BALB/c mice. Tumors grew significantly faster in DIO mice com-pared to control counterparts as quantified by caliber measurement and PET-CT. T cells in the tumor microenvironment (TME) of DIO mice demonstrated features of exhaustion, including significantly increased expression of PD-1, Tim3 and Lag3, but decreased expression of Ki67. Transcriptomic analysis of sorted (>95% purity) CD8+ memory T cells from B16- bearing control and DIO mice also demonstrated the upregulation of exhaustion-related transcripts and down- regulation of effector-related transcripts in T cells from DIO mice. aPD-1 treatment led to signifi-cant reduction of tumor burden, inhibited development of metastases in DIO mice, and overall improved survival times. The enhanced checkpoint blockade responsiveness in DIO mice was associated with significantly increased CD8+ tumor- infiltrating lymphocytes (TILs), as well as increased TNFα and IFNγ-production by CD8+ T cells.

Conclusions

These data indicate a paradoxical impact of obesity on cancer in which immune dysfunction, and tumor progression are heightened, but checkpoint blockade efficacy is also enhanced. This study highlights obesity as a biomarker for cancer immunotherapy.

Innate Anti-Tumor Immunity

P509 Tumor spheroid model to dissect the interplay between myeloid cells and cancer cells

Elaheh Ahmadzadeh, PhD, Jan Martinek, PhD, Florentina Marches, Chun Yu, PhD, A. Karolina Palucka, MD

Jackson Laboratory, Farmington, CT, USA

Correspondence: A. Karolina Palucka (karolina.palucka@jax.org)

Background

The tumor microenvironment includes cancer cells as well as stromal cells, immune cells, epithelial cells, vasculature and extracellular matrix. Interactions between these components regulate tumor development, migration and metastasis. Current 2-dimensional (2D) in vitro culture methods fail to represent the multidimensional complexity of tumor microenvironment. The multicellular tumor spheroids support co-culture conditions and allow a 3D system that can be employed to investigate the integration of multiple cell types and gain insight into the interaction of cancer cells with other cells in their environment. We established such model to investigate the molecular mechanisms regulating the interactions between melanoma, myeloid cells and T cells.

Methods

Tumor Spheroids were developed using hanging drop technique with multiple melanoma cell lines, dermal fibroblasts and immune cells. Preliminary experiments were carried out to determine the optimum conditions for spheroid formation. Spheroids were cultured at density of 104 per cell type per drop and incubated for 3, 4, or 5 days.

Results

Spheroid formation occurred within 72 hours and the integrity of spheroids were maintained throughout the experiments. Cell viability and cell proliferation was monitored within spheroids for up to 5 days using Live and dead staining and CellTrace™ CFSE Cell Proliferation Kit, respectively. Immunofluorescence analysis of spheroid cryosections showed a homogeneous distribution of fibroblasts in spheroids. However, spheroid compaction and fibronectin expression varied between spheroids formed by different cell lines. We assessed the integration and localization of monocytes within the spheroids by adding purified blood CD14+ cells to the mixture of tumor cells and dermal fibroblasts at equal density of 104 cells per drop. Immunofluorescence analysis of serial sections of spheroid showed CD45+ cells scattered throughout spheroid. Furthermore, addition of CD14+ cells to tumor spheroids on day 3 resulted in infiltration of monocytes into spheroids in less than 24 hours.

Conclusions

Thus, our 3D model can be used to assess the distribution of immune infiltrates and the interaction of cancer cells with myeloid cells.

P510 Monoclonal antibodies targeting the MerTK receptor de-repress the innate immune response

Diego Alvarado, PhD, Mike Murphy, Laura Vitale, BS, Thomas O'Neill, BA, Andrew Proffitt, Jay Lillquist, Gwenda Ligon, Craig Polson, Anna Wasiuk, Jeffrey Weidlick, BS, Jenifer Widger, BA, Laura Mills-Chen, Andrea Crocker, BS, Colleen Patterson, Russell Hammond, Li-Zhen He, MD, Joel Goldstein, PhD, Lawrence Thomas, PhD, Henry Marsh, BS PhD, Tibor Keler, PhD, Richard Gedrich

Celldex Therapeutics, New Haven, CT, USA

Correspondence: Diego Alvarado (dalvarad.da@gmail.com)

Background

MerTK, a member of the TAM (Tyro3/Axl/MerTK) family of receptor tyrosine kinases (RTKs), is an important negative regulator of innate immunity. Activation of MerTK in myeloid cells by its ligands Gas6 or Protein S (PROS) promotes phosphatidylserine-dependent efferocytosis of apoptotic cells, inducing a tolerogenic state and mediating resolution of inflammation. MerTK deficient mice exhibit phenotypes consistent with systemic inflammation and auto-immunity. Importantly, MerTK ablation confers tumor immunity, increased pro- inflammatory cytokines and tumor lymphocyte infiltration. We hypothesize that pharmacological targeting of MerTK with monoclonal antibodies (mAbs) may lead to a similar pro-inflammatory response and recapitulate the anti-tumor effects observed in MerTK-/- mice.

Methods

Cultured human PBMCs, or monocyte-derived dendritic cells and macrophages were treated with a panel of MerTK-targeting mAbs for 24 hours alone, or in the presence of pro-inflammatory stimuli (LPS, CD40L or IFN-gamma). A panel of cytokines (eg. IL-1RA, TNF-a) were measured in supernatants by ELISA, or using a multiplex approach. Phospho-immunoreceptor (R&D) profiling from PBMC lysates was performed after treatment with mAbs or saline for 1 hour in full serum. The generation and characterization of MerTK knock-out and human MerTK transgenic mice will be presented.

Results

From a panel of human anti-MerTK mAbs derived from phage-display libraries or human IgG mice we identified several mAbs that enhanced cytokine and chemokine release from primary human immune cells, alone or in the presence of inflammatory stimuli. Surprisingly, mAb activity required Fc receptor binding as introduction of mutations in the Fc domain to abrogate FcR binding rendered the Abs ineffective, despite maintaining target binding. Surrogate mAbs targeting mouse MerTK elicited similar responses in vivo. Treatment of human PBMCs with a MerTK mAb resulted in reduced phosphorylation of ITIM-bearing immunoreceptors known to negatively regulate the immune response. In addition, human MerTK transgenic, and MerTK knockout mice have been generated and characterized with a view to establish in vivo proof-of-concept with human MerTK mAbs.

Conclusions

Pharmacological targeting of MerTK with monoclonal antibodies modulates cytokine production in human and murine model systems in a manner consistent with genetic ablation of the target. MerTK mAbs enhanced production of inflammatory cytokines and decreased the activity of inhibitory immunoreceptors. The anti-tumor activity of MerTK mAbs is planned using surrogate models and human MerTK transgenic mice.

P511 Co-expression of a chimeric NKG2D receptor with membrane bound IL-15 enhances natural killer cell function and long-term persistence in vitro and in vivo

Luxuan Buren (lburen@nkartatx.com)

Nkarta Therapeutics, South San Francisco, CA, USA

Background

Chimeric antigen receptors have been used successfully to retarget T cells in patients with hematologic malignancies. Natural killer (NK) cells offer an alternative to T cells for cellular immunotherapy, highly active and suitable for allogeneic use as they are not HLA-restricted and do not cause GVHD. A goal of NK cell engineering is to improve their in vivo persistence and recognition of cancer cells. Ligands of the natural killer group 2D (NKG2D) receptor are broadly expressed in solid tumor and hematological malignancies, making NKG2D an attractive target for NK cell engineering. This work was undertaken to demonstrate that NK activity and persistence can be elevated by simultaneous expression of chimeric constructs directing the expression of an activating NKG2D receptor (aNKr) and a membrane-bound form of IL-15 (mbIL-15).

Methods

NK cells were generated by co-culture of peripheral blood mononuclear cells (PBMC) with genetically modified irradiated K562 feeder cells. NK cells were transduced at an MOI of 1-2 with a bicistronic virus encoding an NKG2D aNKr and mbIL-15. NK expansion and NKG2D aNKr expression were evaluated by flow cytometry to detect the CD56+ CD3- cell population and the elevation of NKG2D expression over endogenous levels. In vitro cytotoxicity of transduced NK cells was measured using both flow cytometry and the IncuCyte S3 live cell analysis system. The in vivo activity of engineered NK cells was further assessed in a xenograft tumor model, using the osteosarcoma cell line U2OS engineered to express luciferase, with tumor growth measured using bioluminescence in NSG mice.

Results

NK cells were expanded for 7 days (40 fold to >100 fold) prior to transduction. Transduction increased of NKG2D expression in NK cells (>70%, N=8 donors) relative to control cells. NKG2D aNKr-mbIL15 NK cells could be maintained for up to 6 weeks in low IL-2 culture. aNKr-mbIL-15 expression significantly elevated cytotoxicity in NK cells against multiple tumor cell lines, inducing cell death of > 60% of target cells within 4 hours at 1:1 E:T ratio. One infusion of transduced NK cells tumor-bearing NSG mice resulted in long-term anti-tumor responses. Moreover, co-expression of mbIL-15 significantly delayed tumor growth relative to that observed in cells expressing only the NKG2D aNKr.

Conclusions

Transduction of NK cells with an NKG2D aNKr and mbIL-15 increases their cytotoxic activity and persistence. Based on these data, further development of NKG2D aNKr-mbIL-15 NK cells for clinical use will be pursued.

P512 AO-176, a next generation CD47 antibody, induces immunogenic cell death

Ben Capoccia, PhD, Ronald Hiebsch, Michael Donio, Alun Carter, Robyn Puro, Benjamin Capoccia, W. Casey Wilson, Daniel Pereira, Pamela Manning, Robert Karr

Arch Oncology, St. Louis, MO, USA

Correspondence: Daniel Pereira (dpereira@archoncology.com); Ben Capoccia

Background

Recent success in cancer immunotherapy has targeted immune checkpoints such as PD-1, PDL-1, and CTLA-4 to enhance the cytotoxic activity of the adaptive T cell immune response. While the clinical response to these therapies has been dramatic for some, many others have shown partial or even no response highlighting the need for alternative or synergistic approaches that activate innate immunity. Disruption of the interaction between SIRPα and CD47, an innate checkpoint inhibitor, using anti-CD47 antibodies, for example, is known to enhance innate immunity by increasing the phagocytosis of tumor cells by macrophages and dendritic cells (DCs) leading to processing and presentation of tumor antigens. Recently, we described AO-176, a next generation anti-CD47 antibody that blocks the CD47/SIRPα interaction, induces phagocytosis and causes a direct tumor cell-autonomous death while negligibly binding RBCs.Herein, we characterize the ability of our CD47 antibodies such as AO-176, to induce Immunogenic cell death (ICD) and Damage Associated Molecular Patterns (DAMPs) in tumor cells and to potentiate chemotherapy-induced ICD/DAMPs. ICD is a process whereby an agent induces cell surface exposure and release of DAMPs from dying cells which stimulates DCs and adaptive immune responses.

Methods

Tumor cells were treated in vitro with our CD47 antibodies either alone or in combination with chemotherapeutics followed by assessment of ICD/DAMPs using flow cytometry and biochemical assays. RNAseq was also performed on cells undergoing CD47 antibody mediated ICD/DAMP induction to better understand how CD47 inhibition may regulate ICD.

Results

AO-176 and other CD47 antibodies, developed by Arch Oncology, caused mitochondrial stress and loss of outer-membrane integrity, typically observed prior to cells undergoing apoptosis. In addition, CD47 antibody treatment induced a significant ER stress response at the genetic level resulting in the surface exposure of ER chaperone proteins calreticulin, Hsp90, and PDIA3. Concomitantly, our CD47 antibodies increased autophagy and JAK/STAT signaling which resulted in both ATP and HMGB1 release, respectively. Finally, we demonstrated that in combination, our antibodies potentiated the effects of ICD/DAMP-inducing chemotherapy (eg. Doxorubicin).

Conclusions

Here, we describe the unique ability of a specific subset of next generation CD47 antibodies, such as AO-176 to induce ICD/DAMPs. RNAseq analysis of treated cells also revealed alteration of several pathways, including those where DAMPs play a role. In summary, next generation CD47 antibodies such as AO-176 may provide a novel approach to enhancing the current landscape of checkpoint immunotherapy by enhancing both the innate and adaptive immune responses against tumors.

P513 Targeting adenosinergic immunometabolic suppression with engineered natural killer cells for immunotherapy of CD73+ solid tumors

Andrea Chambers, MS, Kyle B. Lupo, Jiao Wang, PhD

Purdue University, Lafayette, IN, USA

Correspondence: Andrea Chambers (sandro@purdue.edu)

Background

Genetically engineered natural killer (NK) cells have shown promise as immunotherapies for hematologic malignancies; however, clinical treatment of solid tumors is lagging. This setback is caused by many mechanisms, including accumulation of immunosuppressive adenosine (ADO) [1,2,3] generated from ectoenzymes CD39 and CD73 by cancer cells [4]. We have shown that ADO suppresses NK cell anti-tumor immunity, resulting in downregulation of activating receptor expression and impaired metabolic activity. To overcome immunometabolic suppression due to adenosinergic signaling, we are engineering NK cells directed against CD73 by imparting in situ ADCC-like activation upon NK cells using a novel genetic construct.

Methods

Peripheral blood-derived NK cells were isolated from healthy human donors. For ADO studies, NK cells were primed 24 hours with IL-2 (200 IU/ml or 400 IU/ml), IL-15 (100 ng/ml), or IL-12 (20 ng/ml) and IL-15 (100 ng/ml) with or without exogenous ADO (1 mM). Treatments were performed with adenosine A2 receptor inhibitor SCH58261, and EHNA, an ADO deaminase inhibitor. Cytotoxicity against CD73+ cells was measured using 7- AAD/CFSE staining, while IFNγ and activating marker expression were measured by flow cytometry. Differential gene expression due to ADO was determined by RNAseq. Engineering NK cells using a novel genetic construct was made by fusing CD73 scFv with CD16a-derived signaling domains before transcribing into mRNA. CAR NK cells were generated by electroporation of the mRNA, and tested for NK transfection efficiency and effector function against CD73+ solid tumors.

Results

Results show that ADO reprograms NK cells’ anti-tumor responses, and priming NK cells with IL-12 and IL-15 can partially mitigate ADO-induced immunosuppression. Using IL-12 and IL-15 was preferential to using IL-2, and IFNγ production in response to ADO was enhanced. Furthermore, ADO resulted in altered gene expression signatures that matched impaired IFNγ signaling and cellular metabolism in NK cells. To block adenosinergic signaling, a novel genetic construct that fuses CD73 scFv with in situ ADCC-like signaling was generated. Human NK cells were successfully electroporated with mRNA encoding the construct. These cells are currently being evaluated for effector function and ability to block CD73-induced immunosuppression on solid tumor targets.

Conclusions

The microenvironment of solid tumors is highly immunosuppressive via the activity of CD73 expressed on cancer cells. Our results demonstrate that ADO can act on specific NK cell pathways to cause NK cell inhibition. Harnessing these results, a novel CD73-targeting construct is currently being investigated to redirect NK cell function when targeting solid tumors.

References

1. Rezvani K, Rouce RH. The application of natural killer cell immunotherapy for the treatment of cancer. Front Immunol. 2015; 6. 2. Rezvani K, Rouce R, Liu E, Shpall E. Engineering natural killer cells for cancer immunotherapy. Molec. Therapy. 2017;25: 1769-1781 3. Ohta A. A metabolic immune checkpoint: adenosine in tumor microenvironment. Front Immunol. 2016; 6. 4. Antonioli L, Blandizzi C, et al. Anti-CD73 immunotherapy: A viable way to reprogram the tumor microenvironment. Oncoimmunology. 2016;5.

P514 High efficiency electroporation of primary human NK cells

Jian Chen, PhD, Xiaofeng Xia, PhD

Celetrix LLC, Manassas, VA, USA

Correspondence: Jian Chen (jchen@celetrix.com)

Background

(Looking to give a short talk as this technical breakthrough would help many labs in the field.) Natural killer (NK) cells have a great potential as a therapeutic agent against tumor cells. Genetic modifications of NK cells such as gene knock-out or exogenous protein expression are important for boosting NK cell expansion or NK cell killing specificity. Unfortunately, viral vectors that are commonly used for other types of cell immunotherapy have poor efficiency in NK cell transduction. As a physical method, electroporation theoretically should work well with NK cells but the special biology of NK cells have made it difficult to achieve high efficiency in NK cell electroporation and cell viability was also a major issue. For example, electroporation of plasmids used to have poor efficiency and high cell mortality in expanded NK cells.

Methods

Here we used a two-pronged approach to tackle the NK cell electroporation problem. First, a novel electroporation method was used involving a new device that has surpassed the performance of all other electroporation technologies on the market. Second, instead of using expanded NK cells, we used fresh un-expanded NK cells that were previously considered harder for electroporation.

Results

Using a relatively high cell concentration, we selected a high electric field strength and were able to quickly achieve a very high efficiency (40% to 50%) for fresh NK cells electroporated with plasmids. The viability of the NK cells after electroporation was between 85% and 95%. Electroporation of mRNA or Cas9/gRNA ribonucleoproteins (RNPs) is much easier than electroporation of plasmids and the new method would allow complex experimental designs such as co-transfection of RNP and plasmids for knock-in.

Conclusions

With the new high efficiency NK cell electroporation method, genetic modification for NK cells has become easily accessible, thereby allowing more possibilities of clinical anti-tumor applications of NK cells.

P515 Novel class of small molecule direct STING agonists as potential cancer immunotherapy

Monika Dobrzanska, Stefan Chmielewski, PhD, Magdalena Zawadzka, Adam Radzimierski, Karolina Gluza, Katarzyna Wojcik-Jaszczynska, Maciej Kujawa, Grzegorz Topolnicki, Grzegorz Cwiertnia, Aleksandra Poczkaj, Izabela Dolata, Jolanta Mazurek, Magdalena Mroczkowska, Agnieszka Gibas, Tushar Mahajan, Marcin Les, Wojciech Schonemann, Sylwia Sudol, Kinga Michalik, Magdalena Sieprawska-Lupa, Katarzyna Banaszak, Charles-Henry Fabritius, Karolina Wiatrowska, Agnieszka Adamus

S.A., Krakow, Poland

Correspondence: Monika Dobrzanska (monika.dobrzanska@selvita.com)

Background

Type I interferons are major players in mounting immune response to cancer cells. IFNβ release by antigen-presenting cells promotes spontaneous anti-tumor CD8+ T cell priming being largely dependent on activation of Stimulator of Interferon Genes (STING). In preclinical murine models intratumoral injection of cyclic dinucleotide (CDN) STING agonists induced regression of established tumors and generated long-term immunologic memory. Relative instability and chemical nature of cyclic dinucleotides limit their use as systemically available immunotherapeutics. Therefore, herein we report the discovery of potent and selective first-in-class non-nucleotide, non-macrocyclic, small molecule direct STING agonists, structurally unrelated to known chemotypes with potential for systemic administration routes.

Methods

Cytokine release has been determined by ELISA or AlphaLisa in human peripheral blood mononuclear cells (PBMC) obtained from healthy human subjects. Activation of STING pathway was monitored in THP-1 Dual reporter monocytic cell line. Human monocyte-derived macrophages (HMDM) and human monocyte-derived dendritic cells (HMDC) were differentiated from CD14+ cells (obtained from PBMC) in the presence of GM-CSF and GM-CSF with IL-4 for HMDM and HMDC, respectively. Mouse bone marrow-derived macrophages (BMDM) were obtained from C57BL/6 mice. Surface expression of the antigen-presenting cell maturation markers i.e. CD80, CD86, CD83 and HLA-DR was assessed by flow cytometry with corresponding isotype controls. The binding affinity was confirmed by Fluorescence Thermal Shift, Fluorescence Polarisation and Microscale Thermophoresis.

Results

STING agonists have confirmed direct binding to both mouse and human STING protein in three independent biophysical binding assays (FTS, MST and FP) and by additional crystallography studies. The compounds have fine-tunable ADME properties with particularly good solubility, permeability and human plasma stability. They selectively activated STING-dependent signaling in both THP-1 reporter assays and in primary cells of human and mouse origin. In vitro functional assays demonstrated their ability to induce cytokine responses (IFNβ, TNFα) in a panel of human peripheral blood mononuclear cell (PBMC), human monocyte derived macrophage (HMDM) and human dendritic cells samples with various STING haplotypes. Additionally, the compounds efficiently induced cytokine release in mouse bone marrow-derived macrophages. Pro-inflammatory cytokine profile was accompanied by up-regulation of the maturation markers, CD80, CD86, CD83 and HLA-DRon the surface of human antigen presenting cells.

Conclusions

These data demonstrate potent, novel, next-generation small molecule STING agonists activating STING-dependent signaling in both mouse and human immune cells to promote potential antitumor immunity. The compounds show good selectivity and in vitro ADME properties enabling further development for systemic administration as a single agent or in combinatory cancer immunotherapies.

P516 SIRPα blockade increases the activity of multiple myeloid lineage cells, enhances dendritic cell cross- presentation, and aids in remodeling the tumor microenvironment

Brian Francica, Jay Hyok Chung, Brandy Chavez, Erik Voets, PhD, Andrea van Elsas, Hans van Eenennaam, PhD, Meredith Leong

Biotech Europe, Oss, Netherlands

Correspondence: Brian Francica (BFrancica@aduro.com)

Background

Antagonizing the SIRPα-CD47 pathway is gaining traction as an effective and novel approach to immune manipulation as design of immunotherapies broadens to include blockade of innate immune checkpoints. Recently, the combination of tumor-targeting antibodies with SIRPα-CD47 blockade has provided promising clinical results, suggesting that increased phagocytosis of cancer cells is clinically relevant for treatment of hematologic cancers [1]. However, the ability for this combination to enhance phagocytosis in the context of solid tumors may be remarkably diminished for several reasons including reduced expression of “eat-me” signals like SLAMF7, increased immune suppression in the tumor microenvironment (TME), and the physical size of tumor cells when adhered in a complex heterogeneous environment. To achieve efficacy in solid tumor indications, it is important that therapies blocking the SIRPα-CD47 axis also potentiate adaptive immune mechanisms and not solely phagocytosis.

Methods

Subcutaneous mouse tumor models and a mouse bone marrow-derived dendritic cell (BMDC) cross-presentation assay were used to assess the efficacy of SIRPα blockade in solid tumors.

Results

Here we demonstrate that, in addition to increasing macrophage uptake of tumor cells in suspension, SIRPα blockade also functions to modify the myeloid compartment in the TME of solid tumors. In four independent subcutaneous mouse tumor models, we demonstrate that SIRPα blockade combines in a synergistic manner with PD-1 blockade to reduce tumor burden. In these models, anti-SIRPα therapy skews the DC population towards cross-presenting DC1 cells and increases the CD86 expression on myeloid cells in multiple immune tissues. In vivo and in vitro, SIRPα blockade correlates with lower levels of SIRPα present on the cell surface, and we hypothesize that a combination of downregulation and blockade may cause the skewing of myeloid lineages. Using a mouse BMDC cross-presentation assay, we also demonstrate that the blockade of SIRPα results in increased T cell expansion, supporting a role for SIRPα blockade in enhancing DC function.

Conclusions

Together, these data suggest that antagonizing SIRPα functions to skew myeloid cells. This results in enhanced T cell activation and that, when combined with PD-1 blockade, improves therapeutic efficacy in multiple mouse models. Based on these data in mouse models, an antibody with specificity for human SIRPα, ADU-1805, is being developed for use in clinical trials.

References

1. Liu X, Kwon H, Li Z, Fu Y-X. Is CD47 an innate immune checkpoint for tumor evasion? Journal of Hematology & Oncology. 2017Nov;10(1).

P517 Pan-allele anti-SIRPα antibodies that block the SIRPα–CD47 innate immune checkpoint

Erik Voets, PhD, Jay Hyok Chung, Paul Vink, BS, David Lutje Hulsik, Marc Paradé, Sanne Spijkers, Inge Reinieren-Beeren, Joost Rens, Wout Janssen, BS, Brian Francica, Meredith Leong, Andrea van Elsas, Hans van Eenennaam, PhD

Biotech Europe, Oss, Netherlands

Correspondence: Erik Voets (EVoets@aduro.com)

Background

SIRPα immunoregulatory activity on myeloid cells is activated by binding of its ligand CD47 [1,2], and blockade of the pathway may enhance anti-tumor immunity [3,4]. Hence the pathway is thought to represent a novel immune checkpoint. CD47, being ubiquitously expressed on normal cells and upregulated on many cancer cells, has been extensively studied in the context of “don’t-eat-me” [5,6]. Alternative strategies are focusing on directly targeting SIRPα because of its more restricted expression to myeloid-derived lineages [7].However, the identification of functional human SIRPα antagonistic antibodies has been hampered by the allelic variation in the SIRPα locus and its homology with the activating receptor SIRPβ and the decoy receptor SIRPγ.

Methods

Using Aduro Biotech’s B-select platform, we have identified and characterized ADU-1805: a highly selective pan-allele anti-SIRPα antibody (EC50 SIRPαV1/SIRPαV2 ≤ 3nM) that lacks appreciable SIRPβ binding (EC50 > 120nM) and cross-reacts with SIRPγ (EC50 ≤ 5nM).

Results

ADU-1805 potently blocks CD47 binding (IC50 ≤ 1.5nM) in all known human SIRPA genotypes (including homozygous and heterozygous genotypes) and antagonizes SIRPα–CD47 interaction on primary SIRPα+ myeloid cells (IC50 ≤ 4nM). In line with its antagonistic properties, ADU-1805 enhances tumor cell clearance by human granulocytes and macrophages. Furthermore, on the IgG2 subclass backbone selected during the humanization process, ADU-1805 exhibits improved activity relative to other IgG subclasses tested. Finally, unlike data with CD47-targeting antibodies, ADU-1805 does not trigger hemagglutination or platelet binding/aggregation in vitro, suggesting a reduced risk of red blood cell (RBC) and platelet depletion in vivo.

Conclusions

In summary, we have identified ADU-1805 as a potentially best-in-class antagonistic anti-SIRPα antibody with a unique binding profile as it binds all reported human SIRPα alleles but does not appreciably bind to the activating SIRPβ receptor. Blocking the SIRPα–CD47 innate immune checkpoint with ADU-1805 may modulate myeloid cells in the tumor microenvironment and promote antigen presentation and cross-priming of dendritic cells. We are currently advancing ADU-1805 through preclinical studies.

References

• 1. Oldenborg PA, Zheleznyak A, Fang YF, Lagenaur CF, Gresham HD, Lindberg FP. Role of CD47 as a marker of self on red blood cells. Science. 2000;288(5473):2051–4.

• 2. Oldenborg PA, Gresham HD, Lindberg FP. Cd47-signal regulatory protein α (Sirpα) regulates Fcγ and complement receptor–mediated phagocytosis. The Journal of Experimental Medicine. 2001Feb;193(7):855–62.

• 3. Tseng D, Volkmer J-P, Willingham SB, Contreras-Trujillo H, Fathman JW, Fernhoff NB, et al. Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response. Proceedings of the National Academy of Sciences. 2013;110(27):11103–8.

• 4. Liu X, Pu Y, Cron K, Deng L, Kline J, Frazier WA, et al. CD47 blockade triggers T cell–mediated destruction of immunogenic tumors. Nature Medicine. 2015;21(10):1209–15.

• 5. Jaiswal S, Jamieson CH, Pang WW, Park CY, Chao MP, Majeti R, et al. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell. 2009;138(2):271–85.

• 6. Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, Gibbs KD, et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell. 2009;138(2):286–99.

• 7. Yanagita T, Murata Y, Tanaka D, Motegi S-I, Arai E, Daniwijaya EW, et al. Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy. JCI Insight. 2017Dec;2(1).

P518 Non-canonical cross-presenting dendritic cells mediate anti-tumor immunity

Ellen Duong, ScB, Stefani Spranger, PhD

MIT, Cambridge, MA, USA

Correspondence: Stefani Spranger (spranger@mit.edu)

Background

Recent studies revealed a critical role for cross-presenting CD103⁺ dendritic cells (DC1) in both the induction and maintenance of CD8⁺ T cell immunity in the tumor. Exclusion of DC1 from the tumor microenvironment (TME) is a mechanism of immune evasion by the tumor and contributes to impaired responses to checkpoint blockade therapy. Elucidating the contributions of different DC subsets to the TME will be instrumental towards improving current immunotherapies.

Methods

We compared the myeloid infiltrate of acutely cleared regressor tumors versus progressively growing tumors, an approach that was previously used to phenotype dysfunctional T cells in the TME [1]. Murine syngeneic tumor lines expressing SIY were categorized as ‘progressor’ or ‘regressor,’ and implanted in wild-type, Rag2^-/-, Batf3^-/-, Clec9a^GFP/GFP, and CD11c-DTR bone marrow (BM) chimera mice. Flow immunophenotyping was used to profile the intratumoral myeloid compartment, and ELISpot was performed to determine the number of IFNγ-producing CD8⁺ T cells. To assess function, sorted myeloid cells were co-cultured with CD8⁺ T cells to evaluate their ability to induce T cell proliferation. Single cell-RNA-sequencing was performed to profile the cellular components of the TME in an unbiased fashion.

Results

In contrast to progressor tumors, regressor tumors were more infiltrated with DC1 than other DC types (non-DC1). A high DC1/non-DC1 ratio was correlated with increased intratumoral CD8⁺ T cell infiltration and was predictive of tumor control across different tumor types and mouse strains. Batf3^-/- and Clec9a^GFP/GFP mice, which lack functional DC1, were able to eliminate a subset of regressor tumors, suggesting that the regression of these tumors was independent of Clec9a-mediated cross-presentation by Batf3-driven DC1. By IFNγ-ELISpot, we found that while CD8⁺ T cell priming was completely ablated in Batf3^-/- and Clec9a^GFP/GFP mice implanted with progressor tumors, mice with regressor tumors retained ~50% CD8⁺ T cell priming. Ex vivo co-culture assays of 2C CD8⁺ T cells with sorted myeloid cells from SIY-expressing tumors in Batf3^-/- mice indicated the presence of CD11c⁺ cells capable of non-canonical cross-presentation. Ablation of the CD11c⁺ compartment using diphtheria toxin-treated CD11c-DTR BM chimeras resulted in loss of T cell priming and anti-tumor immunity in the regressor tumor. Single-cell sequencing of the regressor tumor indicated the presence of a novel DC subset capable of non-canonical cross- presentation in DC1-deficient mice.

Conclusions

Identifying the cell type(s) involved and the mechanism of non-canonical cross-presentation in regressor tumors can open new therapeutic avenues to stimulate the anti-tumor immune response when Batf3-driven DC1 are excluded from the tumor.

References

1. Williams et al. The EGR2 targets LAG-3 and 4-1BB describe and regulate dysfunctional antigen-specific CD8⁺ T cells in the tumor microenvironment. J Exp Med. 2017; 214(2):381-400.

P519 Agonist redirected checkpoint platform (ARC), engineering bi-functional fusion proteins (SIRP -Fc-CD40L), for cancer immunotherapy

George Fromm, PhD¹, Suresh de Silva, PhD², Taylor Schreiber, MD, PhD²

¹Shattuck Labs, Inc, Apex, NC, USA; ²Shattuck Labs, Inc., Durham, NC, USA

Correspondence: George Fromm (gfromm@shattucklabs.com)

Background

A majority of clinical responses to PD1/L1 blockade occur in patients with abundant intratumoral PD-L1 expression and lymphocyte infiltration, suggesting that additional efficacy may be found in combination therapies that increase either of these variables. Because PD1/L1 blockade augments the cytotoxic potential of T cells, synergistic pathways could include those that reduce immunosuppressive myeloid cells or enhance antigen presentation. Here we report the generation of a novel, two-sided human fusion protein (Agonist Redirected Checkpoint, ARC), incorporating the extra cellular domains of SIRPα and CD40L, adjoined by a central Fc domain; termed SIRPα-Fc-CD40L. The SIRPα-Fc-CD40L construct was designed to simultaneously enhance antigen uptake and cross-presentation (CD47 axis) and enhance antigen presenting cell maturation and function (CD40 axis), with a single compound.

Methods

Human and mouse SIRP -Fc-CD40L were produced and characterized using a range of biochemical assays to determine molecular weight, subunit composition & binding affinity; molecular assays to characterize in vitro/ex vivo binding, in vitro functional activity; and anti-tumor efficacy in multiple syngeneic tumor model systems. SIRP-Fc-CD40L has entered late stage manufacturing.

Results

The SIRPα end of the ARC bound immobilized CD47 at 3.59 nM affinity and also CD47 on the surface of human tumor cells both in vitro and in vivo, but importantly, did not bind human platelets, RBCs, nor induce hemolytic activity. The CD40L end of the ARC bound immobilized CD40 at 756 pM affinity and also CD40 on primary macrophages. The SIRP -Fc-CD40L ARC stimulated Fc receptor-independent NF B-luciferase signaling and also induced cytokine secretion from human PBMCs, both with and without TCR stimulation. Furthermore, when activated human dendritic cells or macrophages were co-cultured with CD47 positive human tumor cells, SIRPα - Fc-CD40L was shown to enhance phagocytosis of human tumor cells, and in vivo in mice, induced rapid activation of CD4+ and CD8+ dendritic cells. Finally, the therapeutic activity of SIRPα-Fc-CD40L in established murine MC38 and CT26 tumors was superior to CD47-blocking antibody, CD40-agonist antibody, and combination antibody therapy. Interestingly, anti-tumor response was heightened significantly when SIRPα-Fc-CD40L was combined with antibody blockade of CTLA4, PD1, or PDL1.

Conclusions

These data demonstrate feasibility of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule. Signal replacement of CD47 by CD40L may uniquely poise DCs/macrophages in the tumor microenvironment for activation and cross-presentation of tumor antigens following enhanced tumor cell phagocytosis.

P520 Natural killer (NK) cells orchestrate the antitumor activities of Listeria monocytogenes (Lm)-based immunotherapy

Rachelle Kosoff, PhD¹, Lauren Pettit, MS¹, Nithya Thambi, MS¹, Kimberly Ramos, Bachelors in Small Animal Science¹, Jeff Jones¹, Skye Kuseryk¹, Robert Petit, PhD¹, Michael Princiotta, MS, PhD¹, Kim Jaffe, PhD¹, Sandy Hayes, PhD²

¹ADVAXIS, INC, Princeton, NJ, USA; ²Advaxis Immunotherapies, Inc, Princeton, NJ, USA

Correspondence: Sandy Hayes (hayes@advaxis.com)

Background

Advaxis’ Lm-based immunotherapies are antigen-based immunotherapies that are designed to elicit tumor antigen- specific T cell effectors that recognize and kill tumor cells. However, because the tumor antigens are delivered by a bacterial vaccine vector, innate cytotoxic effectors, such as NK cells, may also be recruited to play a role in controlling tumor growth. The purpose of this study is to determine whether and how NK cells contribute to the antitumor activities of Lm-based immunotherapy.

Methods

Tumor growth inhibition was evaluated in C57BL/6 mice that were implanted with human papillomavirus (HPV)16+ TC-1 tumor cells and then immunized on days 8, 15 and 22 after tumor implantation with PBS or with axalimogene filolisbac (AXAL), an Lm-based immunotherapy expressing the HPV16 E7 protein. To in vivo deplete NK cells, anti-asialo GM1 antibody (Ab) was administered 1 day before tumor implantation and at 3-day intervals during the PBS or AXAL treatment regimen. For mechanistic studies, flow cytometric analysis and immune-related gene profiling of tumor infiltrating leukocytes (TILs) were performed at various time points after tumor implantation.

Results

We first compared intratumoral NK cell frequency and maturation in PBS- and AXAL-treated mice. Although the percentages of intratumoral NK cells in PBS- and AXAL-treated mice were equivalent, NK cells in tumors of AXAL-treated mice were more functionally mature, based on their high expression of CD11b and granzyme A, than NK cells in tumors of PBS-treated mice. To determine whether AXAL-induced NK cell activity was required for AXAL-mediated tumor control, we used anti-asialo GM1 Ab to in vivo deplete NK cells. In AXAL-treated mice, NK cell depletion resulted in a complete loss of tumor growth inhibition. Phenotypic and functional analyses of TILs revealed impaired dendritic cell (DC) maturation and significantly reduced infiltration of functional HPV- specific CD8+ T cells in NK cell-depleted AXAL-treated mice compared to AXAL-treated mice. Gene profiling and pathway analysis showed that the genes significantly downregulated in tumors of NK cell-depleted AXAL-treated mice versus tumors of AXAL-treated mice were involved in NK cell signaling, DC maturation, and interferon signaling.

Conclusions

Treatment of tumor-bearing mice with AXAL leads to NK cell activation, DC maturation and, by extension, an effective antitumor T cell response. These data suggest that NK-DC cross-talk, which leads to activation and maturation of both cell types, is a mechanism by which NK cells contribute to AXAL’s antitumor activities.

Ethics Approval

All mouse experiments were performed under approved IACUC protocols (0914A2016 and 0914B2016).

P521 T cell immunotherapies trigger innate immunity and aseptic inflammation leading to potent anti-tumor and off-targets effects

Daniel Hirschhorn-Cymerman, PhD¹, Jacob Ricca², Billel Gasmi, MD², Olivier De Henau, MD², Levi Mangarin, BS², Sadna Budhu, PhD², Yanyun Li, PhD MD², Czrina Cortez, BS², Cailian Liu, MD², Roberta Zappasodi, PhD², Sean Houghton³, Allison Betof², Katherine Panageas, PhD², Mario Lacuoture, MD², Tracvis Hollmann, MD PhD², Jean Albrengues, PhD³, Mikala Egeblad, PhD³, Taha Merghoub, PhD², Jedd Wolchok, MD, PhD²

¹Memorial Sloan Kettering Cancer Center, New York, NY, USA; ²MSKCC, New York, NY, USA; ³Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Correspondence: Jedd Wolchok (wolchokj@mskcc.org)

Background

Mobilizing the immune system to treat advanced cancers is now a clinical reality. Successful immune-based therapies that treat tumors are often accompanied by immune-related adverse events (irAE) that can occasionally present with severe and lethal symptoms. Currently, there are no well-defined preventative approaches to uncouple anti-tumor immunity from irAEs. The primary immunotherapies currently in clinical use include agents that activate T cell responses such as checkpoint blockade of inhibitory pathways and infusion of ex-vivo tumor-derived, or T cell receptor (TCR)-transgenic or chimeric antigen receptor-modified T cells. While the beneficial and toxic effects of T cell-based immunotherapies in the clinic are being extensively explored, the precise mechanisms underlying their activity remain the subject of intense investigation.

Methods

In the present study, we treated established tumors with melanoma-specific adoptive CD4+ T cell transfer and costimulation via OX40 or CTLA-4 blockade.

Results

We found that, in spite of adequate T cell stimulation, acute local inflammation plays a fundamental role in tumor elimination and related irAEs. While stimulated T cells are necessary for initiating a therapeutic response, activation of endogenous neutrophils constitute an important and necessary effector mechanism of tumor destruction and irAEs. Extensive neutrophil extracellular traps (NETs) were associated with irAEs. Furthermore, melanoma patients treated with checkpoint blockade who developed skin rashes equivalent to irAEs found in mice, showed increased survival and NETs were found in biopsies from rashes and tumors.

Conclusions

Our results bring forward a novel paradigm where T cells enact an anti-tumor immune response that is followed by an inflammatory effector mechanism provided by the innate immune system with curative as well as morbid effects in mice and patients.

Ethics Approval

All tissues were collected at MSKCC following consent to an institutional biospecimen collection study protocol approved by the MSKCC Institutional Review Board. Informed consent was obtained for all patients. The study was in strict compliance with all institutional ethical regulations

P522 Immune-profiling reveals DNAM-1 downregulation in tumor-infiltrating lymphocytes of renal cell carcinoma patients

Veronika Kremer, MSc¹, Christina Seitz, MSc¹, Ann-Helen Scherman Plogell, MD², Eugenia Colon, MD, PhD², Evren Alici, MD, PhD¹, Andreas Lundqvist, PhD¹

¹Karolinska Institutet, Stockholm, Sweden; ²Stockholm South General Hospital, Stockholm, Sweden

Correspondence: Christina Seitz (christina.seitz@ki.se)

Background

Natural killer (NK) cells infiltrate renal cell carcinoma (RCC) tumors and may play a key role in modulating tumor progression, as high NK cell frequencies have been correlated with improved patient survival. However, NK cells, as well as T cells, have been found be phenotypically and functionally suppressed in the tumor microenvironment.

Methods

We investigated immune cell phenotypes and secretion signatures in blood and primary tumors of RCC patients and applied the supervised multivariate analysis tool Orthogonal Projections to Latent Structures (OPLS) to correlate those with disease parameters.

Results

We found that DNAM-1 expression on intratumoral NK cells is associated with a lower Fuhrman grade, whereas PD-1 expression on peripheral blood lymphocytes correlates with a lower primary tumor stage. Furthermore, we identified differences in the immune profiles of blood and tumor of each patient using an OPLS approach. We showed that DNAM-1 is significantly downregulated on tumor-infiltrating T and NK cells compared with peripheral blood (p=0.0006). Indeed, our in vitro experiments suggested that this is likely triggered by contact with tumor cells.

Conclusions

Our results suggest that while T and NK cells can be activated by RCC tumors, they are also inhibited through DNAM-1 down-regulation, which seems to be a central mechanism of immune escape by RCC tumors.

Ethics Approval

The study was approved by the Regional Ethical Review Board in Stockholm, approval number #2013-570-31.

P523 Engineering responsive multi-functional natural killer cells derived from induced pluripotent stem cells capable of overcoming immunometabolic suppression for immunotherapy of solid tumors

Kyle B. Lupo, Andrea M. Chambers, Jiao Wang, PhD, Sandro Matosevic, PhD

Purdue University, Lafayette, IN, USA

Correspondence: Sandro Matosevic (sandro@purdue.edu)

Background

Targeted immunotherapy with engineered natural killer (NK) cells has proven to be a promising approach for the treatment of solid tumors. The pathogenesis of solid cancers, however, causes severe immunosuppression, due to mechanisms which include the generation of immunosuppressive adenosine by the cancer-associated enzyme CD73, as well as the expression of CD155, which causes immune cell dysfunction. Moreover, challenges in sourcing NK cells impair the development of immunotherapies for solid tumors, promoting interest in using induced pluripotent stem cells (iPSCs) as a source of allogeneic NK cells.

Methods

We have generated “off-the-shelf” NK cells differentiated from iPS cells using a novel feeder-free cell culture protocol. These cells can act as a source of allogeneic NK cells that can be genetically engineered for use in cancer immunotherapy. Additionally, we are genetically engineering NK cells with a responsive genetic construct which combines extracellular TIGIT, a ligand for CD155, with intracellular signaling elements that redirect inhibition induced by CD155/TIGIT interaction to trigger release of CD73 scFv. These multi-functional NK cells are capable of competitively binding to CD155+ cancer cells, displacing the inhibition induced by CD155/TIGIT interaction, and blocking CD73. Engineered NK cells are generated via transfection using mRNA electroporation and assessed for cytotoxic function against CD73+ targets.

Results

Initially, iPS cells were differentiated into hematopoietic progenitor cells. These cells were characterized via flow cytometry using cell surface markers expressed on hematopoietic progenitors – CD34, CD43, and CD45. Differentiation yielded CD34+/CD45+ and CD34+/CD43+ cell populations of approximately 20% and 13%, respectively, consistent with results described in literature using feeder-based protocols [1]. Following four weeks of NK cell culture, cells were further analyzed and yielded high expression of several NK cell maturation markers under optimal conditions (greater than 70%, 45%, and 55%, for CD3-/CD56+, CD94+, and NKp46+ cell populations, respectively). In parallel, we have generated a responsive construct targeting CD155 with the concomitant release of CD73 scFv and are characterizing its effect on NK cells’ anti-tumor immunity.

Conclusions

We have shown that NK cells can be generated from iPSCs using an efficient feeder-free protocol. We also synthesized a novel genetic construct which redirects TIGIT-induced inhibition and triggers release of therapeutic CD73 scFv. NK cells engineered with our construct are expected to inhibit immunometabolic suppression due to multi-functional CD73 and CD155 activity and enhance the killing ability of NK cells against solid tumor targets, allowing improved cancer targeting over traditional chimeric antigen receptor-NK therapies.

Acknowledgements

Jungil Moon, Ph. D., iPS Facility Coordinator, Purdue University, West Lafayette, I.N.

References

1. Bock A, Knorr D, and Kaufman D. Development, expansion, and in vivo monitoring of human NK Cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Journal of Visualized Experiments : JoVE. 2013;74: 50337.

P524 Preclinical characterization of BMS-986299, a first-in-class NLRP3 innate agonist with potent antitumor activity, alone and in combination with checkpoint blockade

James Burke², Michael Wichroski, PhD², Huilin Qi², Jie Fang², Adam Bata², Ramola Sane², Anwar Murtaza, PhD², Ashvinikumar Gavai, PhD², Ragini Vuppugalla², Frederic Reu², Dana Banas², Julie Carman, PhD², Damien Bertheloot³, Dennis Dean³, Luigi Franchi³, Shomir Ghosh³, Gary Glick³, Jonathan Graves³, Ana Kitanovic, PhD³, Eicke Latz³, Xiaokang Lu, BS³, Edward Olhava³, William Roush³, Brian Sanchez³, Andrea Stutz³, David Winkler, PhD³, John Hunt, PhD², Miguel Sanjuan, PhD², James Burke²

¹Chrysalis Medical, Hayward, CA, USA; ²Bristol-Myers Squibb, Lawrence Township, NJ, USA; ³IFM Therapeutics, Princeton, NJ, USA

Correspondence: Michael Wichroski (Michael.Wichroski@bms.com)

Background

Immune checkpoint inhibitors (CPI) targeting adaptive immunity have significantly improved patient outcomes in many tumor types, but other approaches are needed to extend clinical benefit to more patients. Targeting innate immunity to provide broader activation of the immune system may be one approach to complement CPI activity. The NLRP3 inflammasome pathway, a key mediator of innate immunity and immune homeostasis, promotes pro- inflammatory response through the maturation of cytokines interleukin-1β (IL-1β) and IL-18, which drive augmented adaptive immune and T-cell memory responses. Targeting innate immune activation through the NLRP3 inflammasome represents a novel and differentiated approach to activating the antitumor immune cycle. Here, we present the preclinical evaluation of a first-in-class NLRP3 agonist BMS-986299 ± CPI.

Methods

Cellular activation of the NLRP3 inflammasome was investigated using cell imaging, biochemical methods, and cytokine release assays. Selectivity of BMS-986299 was confirmed in NLRP3-deficient cells. To investigate whether local activation of NLRP3 could drive systemic antitumor immunity, resulting in abscopal antitumor effects, BMS-986299 (intratumorally 6 to 100 μg Q2D×3 or Q1W) ± anti-PD-1 or anti-CTLA-4 CPI (intraperitoneally) was evaluated in mouse syngeneic tumor models (eg, EG7 thymoma and MC38 colon adenocarcinoma); abscopal activity was measured in a noninjected distal tumor.

Results

BMS-986299 selectively activated the human and mouse NLRP3 inflammasome, resulting in inflammasome assembly (ASC speck formation), caspase-1 activation, and IL-1β and IL-18 cleavage/release. BMS-986299 induced IL-1β at EC₅₀ ≈ 0.5 μM in human PBMCs. In allogeneic mixed lymphocyte reaction assays, single-agent BMS-986299 enhanced T-cell activation; the addition of CPI resulted in further improvements. Pharmacodynamic studies revealed that intratumoral administration of BMS-986299 led to localized release of IL-1β and IL-18 within injected tumors. In the EG7 model, BMS-986299 was sufficient to induce complete regression in both injected and abscopal tumors; efficacy was abrogated in NLRP3 deficient mice, confirming that antitumor activity was NLRP3-dependent for this molecule. In the MC38 model, BMS-986299 in combination with PD-1 blockade resulted in reduced tumor growth in both injected and abscopal tumors (≥ 50% complete tumor regressions). In both models, all mice achieving complete tumor regression with BMS-986299 ± CPI rejected fresh tumor cells when rechallenged without further treatment, demonstrating long-term, durable antitumor immunity.

Conclusions

BMS-986299 is a first-in-class, selective NLRP3 innate agonist, with robust preclinical antitumor activity ± CPI. These data support the ongoing clinical evaluation of BMS-986299 as a novel therapeutic for the treatment of solid tumors in combination with CPI [NCT03444753].

Ethics Approval

This preclinical study was conducted in accordance with ethical principles and local laws/regulations. The use of samples were reviewed and approved by an institutional review board or independent ethics committee.

P525 Preclinical characterization of BMS-986301, a differentiated STING agonist with robust antitumor activity as monotherapy or in combination with anti–PD-1

Gary Schieven, PhD², Jennifer Brown², Jesse Swanson, BS², Caitlyn Stromko², Ching-Ping Ho, BS², Rosemary Zhang², Bifang Li-Wang², Hongchen Qiu², Huadong Sun², Brian Fink, PhD², Anwar Murtaza, PhD², John Hunt, PhD²

¹Chrysalis Medical, Hayward, CA, USA; ²Bristol-Myers Squibb, Princeton, NJ, USA

Correspondence: Gary Schieven (gary.schieven@bms.com)

Background

Immune checkpoint inhibitors (CPI) targeting adaptive immunity have improved patient outcomes in many tumors, but other approaches are needed to extend benefit to more patients. Targeting innate immunity to provide broader activation of the immune system may be one approach to complement CPI activity. Stimulator of interferon genes (STING) enhances antitumor immunity by inducing innate immune responses leading to T-cell priming and activation, resulting in a more effective antitumor response. Here we present the preclinical evaluation of the novel STING agonist BMS-986301 ± anti–PD-1.

Methods

BMS-986301 activity was studied in reporter cell lines and mouse/human peripheral blood mononuclear cells (PBMCs). T-cell proliferation and survival were evaluated in resting and activated T cells. Antitumor activity of BMS-986301 (intratumorally) ± anti–PD-1 (intraperitoneally) was evaluated in bilaterally implanted staged (100 mm³) CT26 or MC38 mouse tumor models; abscopal activity was measured in the noninjected distal tumor. Immune cell levels were measured by flow cytometry, with tetramer staining of tumor-reactive CD8⁺ T cells. The STING agonist ADU-S100 was used as a reference.

Results

BMS-986301 induced cytokine and Type I interferon response gene expression, with comparable potency in human and mouse PBMCs. In human PBMCs, comparable activity was observed across major STING variants. No responses were observed in STING-deficient cells or mice, demonstrating specificity. Because STING agonists can inhibit T-cell proliferation and survival, BMS-986301 was tested and showed low cytotoxicity toward CD8⁺ resting human T cells and decreased inhibition of proliferation of activated human T cells in vitro relative to ADU-S100. BMS-986301 monotherapy (250 ug every 4 days [Q4D]×3) achieved >90% complete regressions of injected and noninjected tumors in both tumor models, showing abscopal effects in a dose-dependent manner. Identical dosing with ADU-S100 (250 ug Q4D×3) provided 13% complete regressions in both tumor models. In the CT26 model, anti–PD-1 plus a single dose of BMS-986301 (100 ug) provided 80% complete regressions of injected/noninjected tumors; no complete regressions were observed with anti–PD-1 alone. BMS-986301 induced increased expression of genes associated with T-cell activation in tumors and draining lymph nodes, induced T-cell proliferation, and increased NK-cell infiltration into tumors. In the CT26 model, antitumor activity correlated with induction of circulating tumor-reactive T cells. All CT26 mice achieving complete regressions with BMS-986301 rejected fresh tumor cells without further treatment, demonstrating immunological memory.

Conclusions

BMS-986301 is a differentiated STING agonist, with promising preclinical antitumor activity alone and in combination with anti–PD-1, supporting its evaluation in future clinical studies.

Ethics Approval

This preclinical study was conducted in accordance with ethical principles and local laws/regulations. The use of samples were reviewed and approved by an institutional review board or independent ethics committee.

P526 The potential role of fibroblast activation protein as a natural killer cell immune checkpoint in pancreatic cancer

Louis Weiner, MD, Shangzi Wang, PhD, Allison O'Connell, MD/PhD Candidate

Georgetown University, Washington, DC, USA

Correspondence: Louis Weiner (weinerl@georgetown.edu)

Background

Immunotherapy has been largely ineffective in pancreatic cancer, partially due to the dense stromal fibrosis surrounding the tumor that creates an immunosuppressive microenvironment. The main cellular component of this fibrosis, activated pancreatic stellate cells (aPSCs), are marked by elevated expression of fibroblast activation protein (FAP). Here we investigate the relationship between FAP and the cytotoxic activity of natural killer (NK) cells.

Methods

To assess the relationship between aPSCs and NK cells we used a novel in vitro co-culturing system that utilizes primary donor-derived PSCs and a human NK cell line, NK92. We tested the ability of NK cells to kill aPSCs using CytotoxGlo and Annexin V assays. We monitored FAP expression and markers of activation in aPSC and NK cells using rt-qPCR, western blot and flow cytometry. To assess the effects of FAP inhibition we used a non-specific FAP inhibitor, talabostat, in vitro and in vivo. 1 μM of talabostat was added to coculturing conditions and NK lysis of aPSCs was determined. For in vivo studies forty female C57BL/6 mice were injected subcutaneously with 1X105 syngeneic MT3-2D cells (Kras+/G12D, p53+/-R172H derived from a PDAC KPC GEMM model [1]). Once tumors reached 40-50 mm3, ten mice per group were given either 30 ug of talabostat per mouse daily by oral gavage, 200 ug of anti-PD-1 per mouse twice a week by i.p., both, or neither. Control mice were treated with PBS. Treatment was terminated after 4 weeks and the mice were monitored, with tumor measurements occurring weekly.

Results

Here we demonstrate that the human NK cell line (NK92) is activated by and kills aPSCs, potentially via recognition of MICA/B on aPSCs by NK cell surface receptor NKG2D. Upon direct contact with PSCs, PSCs downregulate FAP expression and NK92 cells upregulate FAP. This is the first-time NK cells have been shown to produce FAP and that induction of FAP is mediated by cell-to-cell contact. Furthermore, FAP expression by NK92 cells is associated with an inactivate phenotype. FAP inhibition enhances NK92 killing of PSCs in vitro and enhances PDAC tumor clearance in vivo. The anti-tumor activity of FAP inhibition was enhanced upon addition of anti-PD-1 therapy. (Figures 1-5)

Conclusions

This suggests FAP functions as an NK cell immune checkpoint. FAP is expressed in NK cells after activation to attenuate cytotoxicity and can be inhibited to enhance anti-tumor immunity.

Acknowledgements

I'd like to acknowledge Dr. Stephen Byers and Dr. Ivana Peran for provided the PSCs and Dr. Kerry Campbell for providing the NK92 cells.

References

1. Boj SF, Hwang C-I, Baker LA, Chio IIC, Engle DD, Corbo V, Jager M, Ponz-Sarvise M, Tiriac H, Spector MS, et al. Organoid models of human and mouse ductal pancreatic cancer. Cell. 2015; 160:324–338.

Ethics Approval

This study was approved by Georgetown University’s IACUC, protocol #2016-1254

Fig. 1 (abstract P526).

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Fig. 2 (abstract P526).

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Fig. 3 (abstract P526).

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Fig. 4 (abstract P526).

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Fig. 5 (abstract P526).

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P527 Imprime PGG, a novel cancer immunotherapeutic, engages the complement system to prime innate immune effector functions

Xiaohong Qiu, BS, Ben Harrison, MS, Adria Jonas, MS, Anissa Chan, PhD, Nadine Ottoson, BS, Nandita Bose, PhD, Keith Gorden, BS

Biothera Pharmaceuticals, Eagan, MN, USA

Correspondence: Keith Gorden (kgorden@biothera.com)

Background

Imprime PGG (Imprime), an intravenously-administered soluble, yeast β-1,3/1,6-glucan, is currently in clinical development with tumor-targeting antibodies, anti-angiogenics, and checkpoint inhibitors. The fundamental mechanistic rationale for these therapeutic combinations is that Imprime, being a PAMP, primes innate immune effector functions to ultimately inspire an adaptive immune response-based anti-cancer immunity cycle. Imprime forms a tripartite immune complex (IC) comprising of Imprime, naturally occurring anti-β-glucan antibodies (ABA) and iC3b complement opsonin in subjects with sufficient ABA levels. Ex vivo human and in vivo mouse studies have shown that the innate immune receptor, FcgRIIA, and the pattern recognition receptors, complement receptor 3 (CR3) and Dectin-1, are critical for Imprime’s innate immune responses. However, the contributions of the complement system, a vital component of innate immunity, towards the functional activity of Imprime has not been thoroughly investigated. Imprime-ABA IC activates the classical complement pathway and releases C5a. As C5a is a well-known priming agent, and cross-talks with the other innate immune receptors, we hypothesized that Imprime- induced C5a will engage the C5a-C5a receptor (C5aR) signaling pathway to enhance Imprime binding and innate immune effector functionalities.

Methods

The role of C5a in Imprime-ABA binding to isolated neutrophils was evaluated by: a) adding exogenous C5a; b) using C5a-depleted serum, and c) using C5aR antagonist (C5aRA). Cytokine production in healthy subjects with sufficient ABA levels were measured 24hrs post-Imprime treatment in the presence or absence of C5aRA by multiplex luminex assays. The effect of C5a inhibitors was also evaluated in a chemiluminescence-based oxidative burst assay measuring reactive oxygen species (ROS) generated by Imprime-treated isolated neutrophils in response to Rituxan-bound B cell lymphoma cells. In order to test these endpoints in complement-depleted conditions, the whole blood was washed extensively to remove the plasma.

Results

Addition of exogenous C5a increased the percentage of neutrophils binding to Imprime in a dose-dependent manner. Furthermore, Imprime binding in the presence of C5aRA and C5a-depleted serum was significantly reduced. Functionally, C5aRA abrogated cytokine production ( IL-8, MCP-1, MIP-1alpha, and IL-6) in Imprime-treated blood. Likewise, Imprime-ABA induced ROS in high-ABA blood was greatly inhibited in C5a-depleted serum and could be rescued by replenishing complements. C5aRA also inhibited Imprime-induced ROS production. In a non- physiological, complement-depleted condition, Imprime bound predominantly via FcgRIIA, resulting in diminished cytokine and ROS responses.

Conclusions

These results collectively demonstrate that Imprime-induced C5a play a critical role in enhancing Imprime binding and functional responses, potentially by lowering the signaling threshold of the other innate immune receptors.

P528 Tumor-derived alpha fetoprotein suppression of mitochondrial metabolism via PGC1-α and SREBP-1 expression and activity in human dendritic cells

Patricia Santos, PhD, Ashley Menk, BS, Jian Shi, MD, Allan Tsung, MD, Greg Delgoffe, PhD, Lisa Butterfield, PhD

University of Pittsburgh, Pittsburgh, PA, USA

Correspondence: Lisa Butterfield (butterfieldl@upmc.edu)

Background

Alpha-fetoprotein (AFP) is an oncofetal antigen expressed during fetal development and by over 50% of hepatocellular carcinomas (HCC). AFP-L3 is the major isoform present in the serum of HCC patients and is associated with poor patient prognosis. While tumor-derived AFP (tAFP) contains >80% of AFP-L3, cord blood serum-derived AFP (nAFP) contains less than 5% of AFP-L3. We have previously shown that monocyte-derived dendritic cells (DC) cultured in the presence of AFP (in particular tAFP), retained a monocyte-like morphology, had decreased expression of DC maturation markers, and are poor stimulators of antigen-specific T cell responses. In this study, the effect of AFP on DC metabolism was examined.

Methods

PBMC were isolated from healthy donor (HD) or HCC patients using Ficoll-Paque density gradient centrifugation. HD monocytes were isolated from PBMC and cultured for 5 days with IL-4 and GM-CSF to generate DC in the presence of 10 μg/mL ovalbumin (OVA), nAFP or tAFP. DC were collected and tested for 1) mitochondria levels and function by flow cytometry, 2) metabolic function by seahorse extracellular flux analyzer, 3) expression of oxidative phosphorylation proteins, SREBP-1 and downstream gene targets via Western Blot, and 4) expression of PGC1-α via flow cytometry. PBMC from HCC patients were stained with surface markers to identify different circulating DC subsets prior to intracellular staining with PGC1-α.

Results

DC cultured in the presence of nAFP and tAFP show reduced expression of mitochondrial regulator PGC1-α. Furthermore, nAFP- and tAFP-DC had reduced mitochondrial mass and mitochondrial activity compared to OVA- DC. This was confirmed by a reduction in the basal oxygen consumption rate (OCR) in nAFP-DC and a more severe reduction in basal OCR in tAFP-DC, with changes in DC metabolism occurring within 24 hours of AFP exposure.

The decrease in oxygen consumption in DC exposed to nAFP and tAFP is attributed to downregulation of cytochrome c oxidase, responsible for the reduction of oxygen into water. Importantly, circulating myeloid DC from HCC patients have reduced PGC1-α expression compared to healthy donors. Lastly, there was a reduction in the expression of the transcription factor SREBP-1 and downstream targets FASN and ACLY in DC exposed to nAFP and tAFP, suggesting mechanistic inhibition of mTORC1 pathway in DC by AFP.

Conclusions

Collectively, these data show the profound negative effects of AFP on DC metabolism. These novel findings elucidate a key mechanism of immune suppression in HCC and may lead to new therapeutic approaches to reverse these effects.

Ethics Approval

The study was approved by the University of Pittsburgh's Institutional Review Board, approval number 0403105.

P529 Efficacy and safety profile of AU7R-104, a small molecule targeting CD47/SIRPα pathway

Murali Ramachandra, PhD², Pottayil Sasikumar, PhD¹, Chennakrishnareddy Gundala², Wesley Balasubramanian, PhD², Sudarshan Naremaddepalli, PhD², Archana Bhumireddy, MSc², Sandeep Patil, PhD², Amit Dhudashiya², Vijaysai Rayavarapu, MSc², Dodheri Samiulla, PhD², Sanjeev Giri, PhD², Rajesh Eswarappa, PhD, DABT, ERT², Kiran Aithal, PhD², Girish Daginakatte, PhD², Murali Ramachandra, PhD²

¹Aurigene Discovery Technologies, Bangalore, India; ²Aurigene Discovery Technologies Limited, Bangalore, India

Correspondence: Murali Ramachandra (murali_r@aurigene.com)

Background

CD47 is over expressed on many different human cancers and it is also known as a “don’t eat me” signal. Many studies have demonstrated that there is great potential for targeting the CD47-SIRPα pathway as therapy for cancer. Efforts have been made to develop therapies inhibiting the CD47-SIRPα pathway, through antibodies directed against CD47 and recombinant SIRPα proteins. We have developed a novel small molecule CD47 antagonist, AU7R-104, as therapeutic agent for solid and hematological cancers. AU7R-104 enhances phagocytosis of tumor cells and exhibits good drug-like properties with good anti-tumor activity. Here, we report the in vivo activity of AU7R-104 in different tumor models, biomarker characterization and safety profile of AU7R-104 in rodents and non-rodents.

Methods

We have identified preclinical candidate compound AU7R-104 with potent in vitro and in vivo activity. AU7R-104 was profiled extensively in different tumor models both as single agent and in combination with tumor specific antibodies and other anti-cancer agents. In the PK-PD and efficacy studies, efforts were made for biomarker characterization through multiplex and FACS analysis. Advanced profiling of AU7R-104 has been completed in DMPK and toxicological studies in rodents and non-rodents.

Results

AU7R-104 has potent anti-tumor activity both as a single agent and in combination with anti-cancer agents. In the PK-PD studies, AU7R-104 enhanced in vivo phagocytosis in both macrophages and dendritic cells. Multiplex analysis of serum samples indicated there was modulation of macrophage and T-cell mediated cytokines. In the advanced ADME assays, AU7R-104 demonstrated good drug-like properties without any significant alerts. AU7R- 104 combination treatments were well tolerated. Preliminary safety evaluation of AU7R-104 in both rodents and non-rodents indicated the lack of safety concerns typically associated with anti-CD47 antibodies or SIRPα-Fc protein therapeutics.

Conclusions

The above findings support further development of these orally bioavailable agents for use in the clinic

P530 Novel bispecific antibody targeting NKp30 receptor enhances NK-mediated killing activity against multiple myeloma cells and overcomes CD16A deficiency

Monia Draghi, PhD¹, Jennifer Watkins-Yoon¹, Jamie Schafer, PhD¹, Sara Haserlat¹, Sri Vadde¹, Xin Kai¹, Allison Nelson¹, Lucy Liu¹, Nora Zizlsperger, PhD¹, Amanda Oliphant¹, Michael Schmidt¹, Robert Tighe, BS²,

¹Compass Therapeutics, Cambridge, MA, USA; ²Compass Therapeutics LLC, Cambridge, MA, USA

Correspondence: Monia Draghi (monia.draghi@compasstherapeutics.com)

Background

Multiple myeloma (MM) is a malignant hematological disease characterized by a dysregulated growth of malignant plasma cells. Different therapeutic options are available for MM patients; however, the disease remains mostly incurable. B-cell maturation antigen (BCMA) is a promising target in MM because of its restricted expression in normal and malignant plasma cells [1]. NK cells have been implicated in the clinical efficacy of several therapies against MM and may contribute to the success of stem cell transplantation (SCT) by clearing residual cancer cells [2]. In patients with advanced MM, NK cell function is impaired by downregulation of activating receptors including NKG2D, 2B4, and CD16A (FcγRIIIA) [3,4]. Downregulation of CD16A is particularly problematic for conventional anti-BMCA antibodies seeking to elicit ADCC. In contrast, expression of NKp30 remains stable, providing a compelling rationale for the design of BCMA-targeted multispecific molecules that redirect NK cell killing by engaging NKp30 to overcome deficiencies in other activating NK receptors.

Methods

Our Stitchmabs™ and common light chain (LC) bispecific antibody platforms were employed to discover and engineer CTX-4419, a tetravalent, fully human, bispecific molecule consisting of a novel IgG1 antibody recognizing BCMA fused at the c-terminus to two anti-NKp30 Fab fragments with a common LC sequence. The in vitro activity of CTX-4419 was compared against a conventional anti-BCMA IgG1 antibody format for its capacity to induce killing of MM cells and cytokine and chemokine production by primary NK cells and NK cell lines.

Results

When tested with primary NK cells expressing both CD16A and NKp30, CTX-4419 induced potent NK cell cytotoxicity and cytokine production against tumor cells which was superior to the monoclonal IgG1 anti-BCMA control. In contrast to the anti-BCMA IgG1 control that activated only CD16A+ NK cells, CTX-4419 induced potent killing of MM cells and IFN-γ production by KHGY-1, a CD16A- NKp30+ cell line, showing that CTX-4419 can redirect NK cell subsets with low expression of CD16A to kill MM cells. Additionally, an Fc-silent version of CTX-4419 retained activity, further supporting a CD16A-independent function. Importantly, CTX-4419 did not activate NK cells in the absence of BCMA-expressing tumor cells, indicating no off-target effects.

Conclusions

We have engineered and characterized a first-in-class, differentiated bispecific NK cell engager that potently redirects NKp30+ NK cells to kill BCMA+ tumor cells. Unlike traditional anti-BCMA mAbs, CTX-4419 remains highly active in the absence of CD16A engagement. CTX-4419 in undergoing monotherapy assessment in pre- clinical models including experiments with patient derived samples.

References

• 1. Tai YT, Anderson KC. Targeting B-cell maturation antigen in multiple myeloma. Immunotherapy. 2015;7(11):1187-99.

• 2. Cooley S, Parham P, Miller JS. Strategies to activate NK cells to prevent relapse and induce remission following hematopoietic stem cell transplantation. Blood. 2018;131(10):1053-1062.

• 3. Fauriat C, Mallet F, Olive D, Costello RT. Impaired activating receptor expression pattern in natural killer cells from patients with multiple myeloma. Leukemia. 2006;20(4):732-3.

• 4. Costello RT, Boehrer A, Sanchez C, et al. Differential expression of natural killer cell activating receptors in blood versus bone marrow in patients with monoclonal gammopathy. Immunology. 2013;139(3):338-41.

P531 Redirecting immunometabolic suppression by targeting purinergic signaling enhances immunotherapy of solid tumors with CAR-NK cells

Jiao Wang, PhD, Sandro Matosevic, PhD, Kyle B. Lupo, Andrea M. Chambers

Purdue University, West Lafayette, IN, USA

Correspondence: Sandro Matosevic (sandro@purdue.edu)

Background

Engineering natural killer (NK) cells with chimeric antigen receptors (CARs) can enhance their targeting of solid tumor malignancies [1]. However, the CD73-mediated generation of adenosine (ADO) within the hypoxic microenvironment of many solid tumors induces severe immunometabolic suppression which impairs their activation and anti-tumor immunity (Figure 1A) [2–4].

Methods

In an effort to overcome purinergic immunometabolic suppression and improve NK cell-mediated anti-tumor function, we have developed an immunotherapeutic treatment approach which combines non-virally engineered NKG2D-retargeted CAR-NK-92 (NKG2D.CAR-NK-92) cells alongside neutralization of CD73 ectonucleotidase activity (Figure 1B). We have quantified the cytotoxicity of these engineered cells in terms of IFN-γ expression and degranulation activity, and characterized their anti-tumor effects and homing of on CD73+ solid tumors in vivo.

Results

CAR-NK-92 cells, expressing a chimeric immunoreceptor targeting NKG2D based on the piggyBac transposon system, showed significantly higher IFN-γ production, degranulation capacity, and lytic ability against solid tumor cells compared with wild-type NK cells. CD73 blockade was able to further enhance the killing ability of CAR- engineered NK cells against CD73+ solid tumor targets. In vivo, neutralization of CD73 activity promoted anti- tumor efficacy of the engineered NK cells against CD73+ human lung cancer xenografts, including a greater delay in tumor growth, no obvious toxicity, and increased tumor-infiltrating NK cells (Figure 1C-G). CD73 blockade could contribute to the delay in tumor growth in vivo independently of adaptive immune cells, innate immunity or NK cell-mediated ADCC, suggesting that CD73 might contribute to tumor metastasis via autocrine-like mechanisms outside of its ectonucleotidase activity.

Conclusions

Immunometabolism is emerging as a profound mediator of NK cell anti-tumor immunity. Immunotherapies targeting the adenosinergic signaling pathway, such as by neutralizing CD73 ectoenzymatic activity, had, however, not been evaluated on NK cells. Our studies demonstrate, for the first time, the potential of targeting CD73 to modulate purinergic signaling and enhance adoptive NK cell immunotherapy via mechanisms that could implicate autocrine tumor control as well as by mediating adenosinergic signaling. We also provided the basis for targeting the regulation of cancer metabolism as a promising strategy for enhancing the therapeutic efficacy of CAR-modified NK cells for immunotherapy of solid tumors. Based on these results, in order to achieve affective redirection of purinergic signaling while enhancing cancer targeting specificity, we are currently designing and characterizing a multi-functional CAR-NK cell which consists of a single chain antibody targeting CD73 alongside dual chimeric antigen receptor targeting, as a next-generation, single-agent immunometabolic therapy of solid tumors with NK cells.

References

• 1. Guillerey C, Huntington ND, Smyth MJ. Targeting natural killer cells in cancer immunotherapy. Nat Immunol. 2016;17(9):1025-36.

• 2. Zhang B. CD73: a novel target for cancer immunotherapy. Cancer Res. 2010;70(16):6407–11.

• 3. Antonioli L, Blandizzi C, Pacher P, Haskó G. Immunity, inflammation and cancer: a leading role for adenosine. Nat Rev Cancer. 2013;13(12):842-57.

• 4. Ohta A. A metabolic immune checkpoint: adenosine in tumor microenvironment. Front Immunol. 2016;7:109.

Fig. 1 (abstract P531).

See text for description

P532 Preclinical characterization of a first-in-class ILT4 antagonist, MK-4830

Luis Zuniga, PhD¹, Barbara Joyce-Shaikh, BS¹, Douglas Wilson¹, Holly Cherwinski¹, Yi Chen¹, Grein Jeff¹, Wendy Blumenschein, BA¹, Eric Muise, MS¹, Xiaoyan Du¹, Edward Hsieh¹, Sripriya Dhandapani¹, Gulesi Ayanoglu¹, Maribel Beaumont¹, Shuli Zhang¹, Michael Rosenzweig, DVM, PhD¹, Robert Kastelein, PhD¹, Robert Stein, MD PhD², Dennis Underwood, PhD², Milan Blanusa³, Rachel Altura, MD¹, Daniel Cua¹

¹Merck Research Laboratories, Palo Alto, CA, USA; ²Agenus Inc., Lexington, MA, USA; ³Pieris Pharmaceuticals GmbH, Freising, Germany

Correspondence: Luis Zuniga (luis.zuniga@merck.com)

Background

Myeloid-derived suppressor cells in the tumor microenvironment contribute to tumor immune evasion by suppressing local T cell activation, proliferation, and anti-tumor effector responses, identifying them as targets for therapeutic intervention. ILT4 is an inhibitory member of the immunoglobulin-like transcript (ILT) family of proteins. It is expressed primarily by myeloid cells, including those in the tumor microenvironment, and interacts with major histocompatibility (MHC) class I complexes and angiopoietin-like (ANGPTL) ligands. ILT4 signaling is associated with the induction of a tolerogenic phenotype in antigen presenting cells. We demonstrate the pre-clinical anti-tumor properties of a first-in-class anti-ILT4 monoclonal antibody which has now entered clinical trials in patients with advanced solid tumors.

Methods

The clinical candidate MK-4830 is a fully human monoclonal antibody that we selected for specificity, ligand blockade, and functional downstream signaling antagonism of ILT4. Primary human tumor tissue, blood, and serum were profiled for the expression of ILT4 and its ligands. Primary human PBMCs were used to discover MK-4830- dependent, myeloid-associated cytokine responses in vitro. A humanized mouse model implanted with a patient- derived melanoma cell line (SK-MEL-5) was used to evaluate the mechanism of action of ILT4 antagonism and its anti-tumor efficacy.

Results

MK-4830 is specific to ILT4 and does not bind other ILT-family receptors. MK-4830 blocks ILT4 ligand binding and reverses ILT4-mediated suppression of signal transduction. Blocking ILT4 in vitro enhances proinflammatory cytokine expression of GM-CSF and TNFα in LPS-stimulated human PBMC cultures. ILT4 is expressed in primary human tumor samples and ILT4+ myeloid cells are observed both in the periphery and in the tumor infiltrate within the humanized mouse tumor model. Administration of MK-4830 in the humanized mouse tumor model resulted in approximately 50% reduction in tumor growth, alterations in both splenic and tumor myeloid subset distributions, as well as changes in myeloid-centric chemokine and cytokine profiles.

Conclusions

MK-4830, a novel first-in-class antagonist ILT4 antibody, induces robust anti-tumor activity in a humanized mouse tumor model. The preclinical data presented here support the ongoing clinical evaluation of MK-4830 as an anti- cancer therapy and suggests its potential to target tumor-associated myeloid cells in combination with other immune checkpoint blockers.

Trial Registration

Study of MK-4830 as Monotherapy and in Combination With Pembrolizumab (MK-3475) in Participants With Advanced Solid Tumors (MK-4830-001). (2018). Retrieved from https://clinicaltrials.gov/ct2/show/results/NCT03564691?term=NCT03564691&rank=1 (Identification No. NCT03564691)

Ethics Approval

In vivo experiments used in this study were approved by Merck Research Laboratories’ Ethics Board, approval number P2021-400265-JAN.

P533 Gastrointestinal symptoms observed after chimeric antigen receptor T–cell therapy

Hamzah Abu-Sbeih, MD, Tenglong Tang, MD, Jason R. Westin, MD, David Richards, MD, Sattva S. Neelapu, MD, Yinghong Wang, MD, PhD

MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Yinghong Wang (ywang59@mdanderson.org)

Background

The new approach of chimeric antigen receptor T-cell therapy (CAR-T) has been proven to be a very effective treatment for hematological malignancies.[1, 2] The most notable drawbacks of CAR-T is cytokine release syndrome (CRS) and CAR-related encephalopathy syndrome (CRES).[3-6] Gastrointestinal adverse events (GI- AEs) associated with CAR-T have not been studied yet. Herein, we describe the incidence and features of GI-AEs observed after CAR-T.

Methods

This is a case series of patients with hematological malignancies who received CAR-T, as a clinical trial or standard of care where data publication was permitted by the primary investigators, and subsequently suffered from GI-AEs between 1/2012 and 5/2018. Other etiologies of diarrhea were excluded (Figure 1).

Results

Out of the 132 patients that received CAR-T, 21 (16%) experienced GI-AEs. The median age for the 21 patients was 59 years (range, 23-77; Table 1). Most patients had diffuse large B-cell lymphoma (67%). Ten patients experienced CRS, whereas, 7 experienced CRES. Interleukin-6 antagonist was required in 10 patients. Diarrhea was present in all 21 patients (Table 2); 62% grade 1, 33% grade 2 and 5% grade 3. Other associated gastrointestinal symptoms among these 21 were abdominal pain (38%), nausea and vomiting (38%), fever (38%), abdominal distension (10%), and bloody stool (5%). The median duration from CAR-T infusion to diarrhea onset was 5 days (range, 1-40). Eleven patients required treatment for GI-AEs with a median duration of 6 days. Sixteen patients had abdominal imaging evaluation; 3 (19%) of them had findings suggestive of gastrointestinal tract inflammation. Three (14%) patients experienced GI-AEs recurrence after improvement initially. Colitis was confirmed endoscopically in 1 patient; a 76 year old male who received 2 infusions of CAR-T. Five months later, he developed grade 3 diarrhea with abdominal cramps and 15 pounds weight loss. A stool infectious workup including PCR-based multiplex was negative. Colonoscopy demonstrated diffuse inflammation of the entire colon with histology showing glandular drop out, increased apoptosis, and focal erosions. He had no improvement with steroids and mesalamine, and was subsequently treated with oral immunoglobulin with partial improvement. However, his colitis relapsed and an additional trial of cholestyramine was unsuccessful. Lastly, a complete resolution of his gastrointestinal symptoms was achieved by an antibiotics course (vancomycin and piperacillin/tazobactam) for his new onset of pneumonia.

Conclusions

GI-AEs occur in 16% of patients receiving CAR-T. They are typically mild and self-limiting requiring only symptomatic treatment. Nevertheless, of a rare occurrence, it could lead to a refractory colitis.

References

• 1. Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013;3(4): 388-98.

• 2. Sadelain M. CAR therapy: the CD19 paradigm. J Clin Invest. 2015;125(9):3392-400.

• 3. Magee M.S., Snook A.E. Challenges to chimeric antigen receptor (CAR)-T cell therapy for cancer. Discov Med. 2014;18(100):265-71.

• 4. Grigor E.J.M., et al., Efficacy and safety of chimeric antigen receptor T-cell (CAR-T) therapy in patients with haematological and solid malignancies: protocol for a systematic review and meta-analysis. BMJ Open, 2017. 7(12): p. e019321.

• 5. Leick M.B., Maus M.V. Toxicities associated with immunotherapies for hematologic malignancies. Best Pract Res Clin Haematol. 2018;31(2):158-165.

• 6. Neelapu S.S., et al. Chimeric antigen receptor T-cell therapy - assessment and management of toxicities. Nat Rev Clin Oncol. 2018;15(1):47-62.

Ethics Approval

This case series was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center (IRB No.: PA18-0472).

Consent

This case series was granted waiver of consent.

Fig. 1 (abstract P533).

Included patients

Table 1 (abstract P533). Patient characteristics (Number of patients = 21)

Table 2 (abstract P533). Characteristics of gastrointestinal adverse events observed in our cohort

P534 Timely endoscopic and histological evaluation is critical to provide appropriate management for immune checkpoint inhibitor induced colitis

Hamzah Abu-Sbeih, MD, Faisal S. Ali, Wenyi Luo, MD, Wei Qiao, PhD, Gottumukkala S. Raju, MD, Yinghong Wang, MD, PhD

MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Yinghong Wang (ywang59@mdanderson.org)

Background

mmune checkpoint inhibitors (ICI) are efficacious treatments for advanced malignancies but can result in immune mediated diarrhea and colitis (IDC). Currently, the guidelines for the treatment of IDC depend only on clinical symptoms. Endoscopic and histologic features of such adverse events are not well studied in a manner that can help to gauge treatment plans. We aimed to characterize endoscopic and histologic features of IDC and to assess their association with clinical outcomes.

Methods

IOur study included patients who had undergone endoscopy for IDC (1/2010 to 4/2018). Patients with GI infection at time of onset were excluded. High-risk endoscopic features are ulcers deeper than 2mm, larger than 1cm, and extensive colonic involvement. Univariate and multivariate logistic regression were performed to assess the association of endoscopic and histological features with clinical outcomes.

Results

IA total of 182 patients was included; most were white (92%), males (65%) with a mean age of 60 years. Median time from ICI initiation to IDC was 7 weeks. Fifty-three percent had grade 3–4 diarrhea, and 32% grade 3–4 colitis. One-hundred forty-one patients received immunosuppressant therapy, and 41 received symptomatic therapy only (Table 1). Forty-nine patients had mucosal ulcerations, 66 non-ulcerative inflammation and 67 normal endoscopy. Calprotectin was higher in patients with ulceration (P=0.04). The sensitivity of lactoferrin to detect histologic and endoscopic inflammation was 90% and 70% respectively. Patients who underwent endoscopy >30 days of symptom onset required longer duration of steroids (P=0.02), had more recurrent symptoms (P<0.01) and received later infliximab/vedolizumab add-on therapy than did those who underwent endoscopy ≤30 days (P=0.03; Table 2). High-risk features were associated with more frequent (P=0.03) and longer duration (P=0.02) hospitalization and infliximab/vedolizumab requirement (P<0.01; Table 3). Patients with active histological inflammation had more recurrence (P<0.01) and repeat endoscopy (P<0.01; Table 4). Repeat endoscopy was required in 47 patients. A multivariate logistic regression revealed that longer ICPI treatment was associated with more frequent hospitalizations (OR 1.00; 95%CI 1.00-1.01; P<0.01; Table 5) and high-risk endoscopic features were associated with the requirement of infliximab/vedolizumab (OR 3.89; 95%CI 1.68-9.01; P<0.01).

Conclusions

IHigh risk endoscopic features and active histologic inflammation represent important markers of disease severity with clinical implications and should be used in a timely manner to devise IDC-focused treatment algorithms that incorporate a more intricate degree of specificity to improve upon the currently available guidelines.

Ethics Approval

This retrospective, single-center study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center (IRB No. PA18-0472).

Consent

This study was granted waiver for consent.

Table 1 (abstract P534). Association between patient characteristics and treatment group

Table 2 (abstract P534). Clinical outcomes of patients according to the timing of endoscopy from IDC onset

Table 3 (abstract P534). Patient with endoscopic inflammation involvement

Table 4 (abstract P534). Association between histological active inflammation and clinical characteristics

Table 5 (abstract P534). Multivariate logistic regression analysis of infliximab/vedolizumab use and hospital admission

P535 Upper gastrointestinal symptoms and associated endoscopic and histologic features in patients receiving immune checkpoint inhibitors

Hamzah Abu-Sbeih, MD, Tenglong Tang, MD, Wenyi Luo, MD, Wei Qiao, MD, David Richards, MD, Yinghong Wang, MD, PhD

MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Yinghong Wang (ywang59@mdanderson.org)

Background

Immune checkpoint inhibitors (ICPIs) have demonstrated high effectiveness in treating many types of malignancies. Gastrointestinal (GI) immune-related adverse events (irAE) are commonly reported, however, limited literature describes upper gastrointestinal tract toxicity. Therefore, we aimed to describe clinical, endoscopic and histological characteristics of upper GI tract injury related to ICPI treatment.

Methods

We studied consecutive patients who received ICPIs between April 2011 and March 2018 and developed upper GI symptoms that required esophagogastroduodenoscopy (EGD). Patients with Helicobacter pylori gastritis were excluded from our study. We performed descriptive statistical analysis using means and standard deviations for continuous variables and frequencies and percentages for categorical variables.

Results

Sixty patients developed upper GI symptoms between ICPI initiation and 6 months after the last infusion (Table1); majority were of white race with a mean age of 59 years. In our cohort, 42 patients had other risk factors of gastritis such as chemotherapy, radiotherapy, and non-steroidal anti-inflammatory drugs (Table2). Patients without these risk factors had isolated gastric involvement on endoscopy. Overall, histologic inflammation of the stomach was evident in 83% of patients, and inflammation of the duodenum was evident only in 38% of patients. The rate of ulceration was the same in the cohorts with and without other risk factors for gastritis (11% vs. 12%). Among patients who had both upper and lower endoscopic evaluation (n=38), 17 (45%) had histological inflammation involving upper GI tract only; these patients developed GI toxicity later than patients with GI toxicity involving both upper and lower (P=0.060; Table3). Isolated upper GI tract involvement was more frequent in patients undergoing anti-PD-1/L1 treatment (P=0.071). Likewise, isolated upper GI toxicity was associated with more frequent mucosal ulceration (P=0.02; Table4). Patients with concurrent upper and lower GI tract involvement received immunosuppressive therapy more often than did patients with isolated upper GI tract involvement. Majority of the isolated upper GI symptoms were treated with proton pump inhibitors and H2 blockers, with less immunosuppressant use.

Conclusions

Overall ICPI-related upper GI-toxicities had gastric involvement more often than duodenal involvement on endoscopic and histological level, which is also observed more in patients treated with PD-1/L1. Mucosal ulcerations were more frequently found in isolated upper GI toxicity than concurrent upper and lower GI toxicities. Patients without other risk factors for gastritis had isolated gastric involvement on endoscopy, with duodenal inflammation in 39% of patients histologically. Concurrent GI tract involvement required immunosuppressive therapy more often than isolated upper GI tract involvement.

Ethics Approval

This retrospective, single-center study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center (IRB No. PA18-0472).

Consent

This study was granted waiver for consent.

Table 1 (abstract P535). Patient baseline characteristics

Table 2 (abstract P535). Comparison of EGD characteristics between patient who received ICPI and had other risk factors and those who received ICPI and had no other risk factors

Table 3 (abstract P535). Clinical characteristics according to the endoscopic involvement of GI tract (n = 38)

Table 4 (abstract P535). Endoscopic characteristics of concurrent and issolated upper involvement

P536 The fate of immune–mediated diarrhea after the resumption of immune checkpoint inhibitor treatment

Hamzah Abu-Sbeih, MD, Faisal S. Ali, Jianjun Gao, MD, PhD, Yinghong Wang, MD, PhD

MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Yinghong Wang (ywang59@mdanderson.org)

Background

Immune–mediated diarrhea (IMD) is a leading cause for immune checkpoint inhibitor (ICPI) treatment discontinuation. Nonetheless, despite the occurrence of IMD initially, the remarkable efficacy of ICPIs encourages oncologists to resume ICPI treatment for cancer progression or as a maintenance. There is a paucity of evidence about the recurrence rate of IMD after ICPI resumption.[1] Hence, we assessed the risk and risk factors of IMD recurrence after ICPI resumption.

Methods

This is a cohort study of patients who had developed IMD and then resumed the same or different ICPI agent after improvement of IMD between 1/2010 and 4/2018. IMD was graded using CTCAE v4.03. A univariate followed by a multivariate logistic regression analyses were performed to assess the association of clinical covariates and IMD recurrence.

Results

Out of the 4864 patients who received ICPI treatment, 437 (8.9%) developed any grade IMD (Figure 1-2). Among them, 116 resumed ICPI treatment and were included in our analyses; 21 restarted anti-cytotoxic T-lymphocytes associated protein-4 (CTLA-4) and 95 anti-programmed death-1/ligand-1 (PD-1/L1). The median age was 60 years (Table 1). ICPI treatment discontinuation was due to IMD in 76 patients (66%). Seventy-nine patients (68%) required immunosuppressive therapy for the first event of IMD. The median duration from the last ICPI dose to the restart of ICPI treatment was 65 days (SD, 194). Overall, 37 (32%) patients experienced a recurrence of IMD (CTLA-4, 48%; PD-1/L1, 28%). Twenty-seven patients (73%) required immunosuppression for the recurrent IMD (Table 2); 15 of them discontinued ICPI treatment. The median duration from ICPI re-initiation to IMD recurrence was 63 days (range, 1–397). Severe IMD requiring immunosuppression initially was associated with higher grades (P<0.001) and more frequent immunosuppression requirement (P<0.001; Table 3) for the recurrent IMD. On multivariate logistic regression, patients who received anti-CTLA-4 based therapy initially had lower risk of IMD recurrence (odds ratio [OR], 0.20, 95% CI, 0.08-0.51; P=0.001; Table 4-5). The requirement for immunosuppression for IMD initially (OR, 3.04; 95% CI, 1.12-8.29; P=0.030) and the resumption of anti-CTLA-4 agents (OR, 3.89; 95% CI, 1.22-12.40; P=0.022) were associated with increased risk of IMD recurrence.

Conclusions

The resumption of anti-PD-1/L1 therapy has a lower IMD recurrence rate compared to anti-CTLA-4. Hence, ICPI therapy, especially anti-PD1-PD-L1, may be resumed in order to maximize the clinical benefit for patients who have limited alternative treatment options. Severe IMD requiring immunosuppression initially was a risk factor for the recurrence of severe IMD after ICPI resumption.

References

1. Pollack, MH, et al., Safety of resuming anti-PD-1 in patients with immune-related adverse events (irAEs) during combined anti-CTLA-4 and anti-PD1 in metastatic melanoma. Ann Oncol, 2018. 29(1): 250-255.

Ethics Approval

This retrospective, single-center study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center (IRB No. PA18-0472).

Consent

This study was granted waiver for consent.

Table 1 (abstract P536). General characteristics of the initial colitis event

Table 2 (abstract P536). Characteristics of the recurrent immune-mediated diarrhea based on the ICPI therapy resumed

Table 3 (abstract P536). Characteristics of the recurrent immune-mediated diarrhea for patients who needed immunosuppression for the initial immune-mediated diarrhea

Table 4 (abstract P536). Univariate logistic regression analysis of immune-mediated diarrhea recurrence

Table 5 (abstract P536). Multivariate logistic regression analysis of immune-mediated diarrhea recurrence

Fig. 1 (abstract P536).

The recurrence rate of immune-mediated diarrhea (IMD) after ICPI resumption according to the immunosuppression (IS) requirement for the initial immune-mediated diarrhea

Fig. 2 (abstract P536).

The reccurence immune-mediated diarrhea after ICPI resumption by the type of ICPI

P537 Immune checkpoint inhibitor–induced colitis as a predictor of survival in metastatic melanoma

Hamzah Abu-Sbeih, MD, Faisal S. Ali, Wei Qiao, PhD, Yang Lu, MD, Sapna Patel, MD, Adi Diab, MD, Yinghong Wang, MD, PhD

MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Yinghong Wang (ywang59@mdanderson.org)

Background

Gastrointestinal (GI) immune related adverse events (irAEs) commonly limit immune checkpoint inhibitors’ (ICPIs) treatment, which is very effective for metastatic melanoma. The impact of GI-irAEs and their immunosuppressive therapy on patients’ survival is not well studied. We aimed to assess the impact of GI-irAEs on overall survival (OS) and progression free survival (PFS) of patients with metastatic melanoma.

Methods

This is a retrospective study of patients with metastatic melanoma who received ICPI treatment and developed GI-irAEs from 1/2010 through 4/2018 with a mean follow-up duration of 1.7 years. A number of randomized patients who did not have GI-irAEs were included in our analysis. ICPI treatment response on CT and/or FDG PET/CT images was evaluated based on combined immune-modified Response Evaluation Criteria in Solid Tumors (RECIST) and immune-related RECIST 1.1. OS and PFS were defined as the time from ICPI initiation until death or last follow-up and until progression, death, or last staging, respectively. OS was redefined as the time from diarrhea onset to study the effect of immunosuppressive therapy. Kaplan-Meier curves were used to estimate unadjusted OS and PFS time distributions (Figure1-2). The Cox proportional hazards model was used to evaluate survival predictors. GI- and non–GI-irAE were included in the Cox model as time-dependent variables.

Results

A total of 243 patients were included in our analyses, majority were white (93%), males (64%) with a mean age of 58 years (Table 1). In our cohort, 173 patients (71%) had GI-irAEs; 124 (72%) received immunosuppression (Table 2). In multivariate Cox regression, ECOG 2-3 (HR 4.36, 95%CI 2.38-7.99; P<0.01), LDH ≥618 IU/L (HR 2.85, 95%CI 1.79-4.49; P<0.01), stage M1c (HR 4.66, 95%CI 1.69-12.78; P<0.01) were associated with worse OS rates (Table3). In contrast, longer duration of ICPI treatment (HR 0.86, 95%CI 0.81-0.92; P<0.01) and any grade GI- irAEs (HR 0.51, 95%CI 0.31-0.83; P<0.01) were associated with improved OS rates. Immunosuppressive treatment did not affect OS (HR 1.5, 95%CI 0.82-2.74; P=0.19). High-grade diarrhea was associated with improved OS (P=0.0492; Figure 3). Additionally, patients who developed GI-irAEs had longer PFS durations on multivariate Cox model (HR 0.44, 95%CI 0.29-0.64; P<0.01; Table 4).

Conclusions

GI-irAEs are associated with improved survival rates in patients with metastatic melanoma. Furthermore, higher grades of diarrhea are associated with improved patients’ OS, which could explain the finding that immunosuppressive therapy did not adversely affect OS. Therefore, the onset of GI-irAEs should be conveyed to patients as a favorable sign rather than an alarming one.

Ethics Approval

This retrospective, single-center study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center (IRB No. PA18-0472).

Consent

This study was granted waiver for consent.

Table 1 (abstract P537). Patient characteristics (n = 243)

Table 2 (abstract P537). Adverse events observed in our cohort

Table 3 (abstract P537). Multivariable Cox regression analysis for overall survival

Table 4 (abstract P537). Multivariable Cox regression analysis for progression free survival

Fig. 1 (abstract P537).

Kaplan-Meier overall survival curve stratified by immune checkpoint inhibitor (ICPI)-induced diarrhea/colitis status

Fig. 2 (abstract P537).

Kaplan-Meier progression-free survival curve stratified by immune checkpoint inhibitor (ICPI)-induced diarrhea/colitis status

Fig. 3 (abstract P537).

Kaplan-Meier overall survival curve stratified by diarrhea grade

P538 Precision medicine in immune checkpoint inhibitor–induced diarrhea and colitis treatment: the advent of organ targeted vedolizumab therapy

Hamzah Abu-Sbeih, MD¹, Faisal S. Ali¹, Dana Alsaadi², Joseph Jennings, MD², Wenyi Luo, MD¹, Zimu Gong, MD¹, David Richards, MD¹, Aline Charabaty, MD², Yinghong Wang, MD, PhD¹

¹MD Anderson Cancer Center, Houston, TX, USA; ²Georgetown University, Washington, DC, USA

Correspondence: Yinghong Wang (ywang59@mdanderson.org)

Background

Immune checkpoint inhibitor (ICPI), which is an efficacious treatment for advanced malignancies, is commonly limited by immune mediated diarrhea and colitis (IMDC). Steroids and infliximab are often used to treat severe IMDC given its immune mediated mechanism. These agents induce systemic immunosuppression with its associated morbidity. Additionally, systemic immunosuppression might hamper the effect of ICPI. Hence, we aimed to assess clinical outcomes of vedolizumab (a gut-targeted anti-integrin agent) as an alternative treatment for IMDC.

Methods

This is a retrospective multicenter case series of adult patients who had IMDC and received vedolizumab from 12/2016 through 4/2018 from MD Anderson Cancer Center and Medstar-Georgetown University. All patients had IMDC that is refractory to steroids and/or infliximab.

Results

Twenty-eight patients were included; 20 males (71%), 25 Caucasians (89%) with a mean age of 63 years (Table 1). The most common malignancy was melanoma in 7 patients (25%). Eight patients (29%) received anti-cytotoxic T- lymphocyte associated antigen-4 (CTLA-4), 12 (43%) programmed death protein-1 or its ligand (PD-1/L-1) and 8 (29%) combination therapy. Median time from ICPI to IMDC onset was 10 weeks (IQR 1-70). Fifteen patients (54%) had grade 2 and 13 (46%) had grade 3 or 4 IMDC. Diagnostic evaluations for IMDC are shown in (Table 2). The median reduction in fecal calprotectin values was 347 for vedolizumab initiation <14 days of IMDC onset and 197 for >14 days (Figure 1). Mucosal ulceration was present in 8 patients (29%), whereas non-ulcerative inflammation was present 13 (46%). All of our patients had features of active histological inflammation; 14 (50%) had concurrent features of chronicity, and 10 (36%) had features of microscopic colitis. The treatment and outcomes of IMDC are shown in (Table 3). Mean duration of steroid treatment was 96 days (SD 74). Seven patients received infliximab in addition to steroids and were refractory to it. Median number of vedolizumab infusions was 3 (IQR 1- 4). Mean duration of follow-up was 15 months. Twenty four patients (86%) achieved and sustained clinical remission. Repeat endoscopic evaluation was performed in 17 patients. Endoscopic remission was attained in 7 (54%) of the 13 patients who had abnormal endoscopic findings initially with 5/17 (29%) patients reaching histological remission as well. (Table 4) lists the characteristics of patients who had clinical remission. In our cohort, 1 patient developed skin rash and 1 had joint pain.

Conclusions

Vedolizumab could be an appropriate treatment for steroid refractory IMDC, with favorable outcomes and good safety profile.

Ethics Approval

This retrospective, single-center study was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center (IRB No. PA18-0472).

Consent

This study was granted waiver for consent.

Table 1 (abstract P538). Patient clinical characteristics (n = 28)

Table 2 (abstract P538). Patient diagnostic evaluation data (n = 28)

Table 3 (abstract P538). Treatment of colitis and outcomes (n = 28)

Table 4 (abstract P538). Vedolizumab therapy outcomes and clinical characteristics (n = 28)

Fig. 1 (abstract P538).

Decrease in calprotectin values after vedolizumab/infliximab therapy according to time from onset to treatment initiation

P539 Development of cutaneous squamous cell carcinoma in patients receiving anti PD-1/PD-L1 therapy

Amanda Herrmann, MD, PhD, Priyadharsini Nagarajan, MD, PhD, Vivek Subbiah, MD, Kelly Nelson, MD, Alexander J. Lazar, MD, PhD, Courteny Hudgens, MS, Khalida Wani, PhD, Michael T. Tetzlaff, MD, PhD, Jennifer A. Wargo, MD, MMSc, Anisha B. Patel, MD

UT MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Anisha B. Patel (apatel11@mdanderson.org)

Background

The utilization of immune checkpoint inhibitors (ICPI) has advanced in recent years. Antibodies targeting programmed cell death-1 (PD-1) and its ligand (PD-L1) are successfully used for treatment in a variety of cancers, including metastatic and inoperable cutaneous squamous cell carcinoma (cSCC). [1] Cutaneous adverse events are seen in approximately 40% of anti PD-1 treated melanoma patients [2], but few have described the development of new cSCC in the setting of anti PD-1 therapy [3].

Methods

We present a summary of 10 patients treated at MDACC with anti-PD-1/PD-L1 therapy for non-squamous cell cancers who developed cSCC. Seven patients presented to the dermatology clinic following development of skin lesions during the course of their anti-PD-1/PD-L1 therapy; three more were discovered with a limited retrospective chart review. Skin biopsies were interpreted from H&E stain as well as immunohistochemical antibody analysis for CD3, CD8, PD-1, and PD-L1.

Results

Two female and 8 male patients aged 51- 81 years, and developed biopsy-proven well-differentiated cSCC after a median of 4 months (range 3-16 months) following therapy initiation. Four patients developed multiple lesions over the course of several months, and in 70% of cases lesions developed during anti-PD-1/PD-L1 therapy. These lesions were treated conservatively with no complications or recurrence after 12 months follow up. Skin biopsies revealed well-differentiated cSCC, with full thickness keratinocyte atypia and at least superficial dermal invasion. Immunohistochemical analysis was performed using 8 biopsies from 4 patients. PD-L1 expression was present in 63% of samples [4]. The infiltrate was predominant and non-brisk at the periphery of the tumor with normal proportions of CD3:CD8 T lymphocytes. A median of 5% (range 1-13%) of the peripheral infiltrate had PD-1 expression.

Conclusions

There is a potential link between the development of cSCC lesions and anti-PD-1/PD-L1 therapy. While this link has yet to be solidified, we are actively investigating factors that may contribute to the development of these lesions in the setting of ICPI therapy, including whole exome sequencing, RNAseq, and TCR sequencing. We plan to compare cSCC samples resulting from various etiologies, including BRAF inhibitor- induced cSCC and ultraviolet light-induced cSCC, to determine if there are unique characteristics in the cSCC from our ICPI- associated tumor cohort of patients. Preliminary data shows increased PD-L1 expression in the drug-induced tumors. [4] These results will be important to clinicians moving forward, as the use of this class of therapeutics rapidly increases, and may soon include the treatment of advanced cSCC.

References

• 1. Migden MR, Rischin D, Schmults CD, et al. PD-1 Blockade with Cemiplimab in advanced cutaneous squamous-cell carcinoma. N Engl J Med. 2018 Jul 26; 379(4):341-351.

• 2. Villadolid J, Amin A. Immune checkpoint inhibitors in clinical practice: update on management of immune- related toxicities. Transl Lung Cancer Res. 2015 Oct; 4(5):560-75.

• 3. Freites-martinez A, Kwong BY, Rieger KE, Coit DG, Colevas AD, Lacouture ME. Eruptive keratoacanthomas associated with pembrolizumab therapy. JAMA Dermatol. 2017; 153(7):694-697.

• 4. Gambichler T, Gnielka M, Rüddel I, Stockfleth E, Stücker M, Schmitz L. Expression of PD-L1 in keratoacanthoma and different stages of progression in cutaneous squamous cell carcinoma. Cancer Immunol Immunother. 2017; 66(9):1199-1204.

Ethics Approval

The study was approved by UT MD Anderson Cancer Center's Institutional Review Board, protocol number PA17-1060.

Consent

Not applicable. Waiver of consent obtained through the IRB for PA17-1060.

P540 Safety of cancer patients with preexisting autoimmune diseases presenting in the emergency center following immune checkpoint blockade

Mohsin Shah, MD, Mazen N. Jizzini, MD, Imad El. Majzoub, MD, Aiham Qdaisat, MD, Cielito C. Reyes, DrPH, Sai-Ching J. Yeung, MD, PhD

The Univeristy of Texas MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Sai-Ching J. Yeung (syeung@mdanderson.org)

Background

Immune checkpoint inhibitors (ICI) have dramatically increased the survival of cancer patients, but their use can be limited by the occurrence of immune related adverse events (irAE) that may be serious and infrequently life threatening. Cancer patients with preexisting autoimmune diseases (PAD) were excluded from clinical trials. The safety of ICI in these patients is currently unknown. [1] The aim of this study was to evaluate the safety of ICI in patients with PAD that presented to the Emergency Center (EC) at a comprehensive cancer center.

Methods

Cancer patients with PAD who received ICI and presented to the EC between March 1, 2011 and February 29, 2016 at MD Anderson Cancer Center. We extracted data on patient’s characteristics (age, gender, preexisting autoimmune disease, treatment given for the underlying disease), type of checkpoint inhibitors used, reported irAE, how they were managed, whether immunotherapy was withheld or discontinued and their clinical outcome.

Results

Twenty two cancer patients with PAD were identified through institutional databases. Of these, 15 were males (68%). Mean age was 59.5 year (SD 11.6). Median Charlson Comorbidity Index was 9.5 (range 5-13). Most patients received anti PD-1 drugs (64%). Melanoma was the most common cancer (45%). Most frequent PAD were autoimmune thyroiditis and eczema. Eleven patients were actively receiving treatment for PAD at initiation of ICI therapy, of which 3 received immunosuppressive therapy (steroids in 2, disease-modifying antirheumatic drug plus rituximab in 1). Eighty six percent of cases experienced de novo irAE or PAD exacerbation. Fourteen percent were severe (Grade ≥3). Of these, 6 were managed with corticosteroids. Resolution of de novo irAE or exacerbation of PAD was achieved in 10 cases without the need to withhold or discontinue immunotherapy. Median time to last follow up or death from first infusion was 16.8 months [range 2-80]. Death was reported in 17 cases due to cancer progression.

Conclusions

Despite frequent de novo irAE or exacerbation of PAD, most patients with PAD who visited the ED tolerated ICI therapy well. Prospective studies are needed to establish the risk-benefit profile of ICI therapy in patients with PAD including those who did not need to visit EDs.

References

1. June CH, Warshauer JT, Bluestone JA. Is autoimmunity the Achilles׳ heel of cancer immunotherapy? Nat Med. 2017; 23: 540-547.

Ethics Approval

The study was conducted under a clinical research protocol approved by the institutional review board of The University of Texas MD Anderson Cancer Center.

Mechanisms of Resistance to Immunotherapy

P541 The immunosuppressive tumor microenvironment (TME) in Epstein-Barr virus (EBV)-positive and EBV- negative gastric cancers: implications for immunotherapy

Sepideh Besharati, MD, Tracee McMiller, MS, Mark Yarchoan, Qingfeng Zhu, PhD, Elizabeth Engle, MSc, Janis Taube, MD, MSC, Alan Berger, Robert Anders, MD, PhD, Suzanne Topalian, MD

Johns Hopkins University, Baltimore, MD, USA

Correspondence: Suzanne Topalian (stopali1@jhmi.edu)

Background

Chemotherapy-refractory gastric carcinomas (GC) are aggressive malignancies, and only ~15% respond to drugs targeting the PD-1/PD-L1 pathway. EBV+ GCs (10% of GCs) often contain chromosomal amplifications for PD-L1 and PD-L2. They have been reported to contain robust CD8+ T cell infiltrates and an interferon-gamma (IFNg) gene signature, suggesting immune stimulation by strongly immunogenic EBV proteins. The current study aimed to characterize immune cell subsets and checkpoint expression in EBV+ GC compared to EBV(-) GC.

Methods

After screening >1000 cases, 25 invasive primary GC specimens AJCC stage 1A–4 (11 EBV+, 14 EBV-, confirmed with EBER ISH) were identified from treatment-naïve patients. Immunohistochemistry (IHC) was conducted for CD3, CD4, CD8, CD20, CD68, FoxP3, PD-1, PD-L1, LAG-3, GITR, IDO1, CSF1R and COX-2. Immune cell densities were quantified. RNA was isolated from macrodissected areas of dense CD3+ T cell infiltrates juxtaposed to PD-L1+ stromal cells, and gene expression profiling (GEP) was performed using multiplex qRT-PCR for a panel of 61 candidate immune-related genes.

Results

IHC revealed that 17/25 GCs contained PD-L1+ stromal cells (range 5-75% positive cells) with no significant difference between EBV+/- specimens; however, only 3/25 specimens contained PD-L1+ tumor cells (all EBV+). There was a higher proportion of CD8+ vs. CD4+ T cells in EBV+ tumors (p=0.051). IHC analysis of EBV+/- GCs did not show significant differences in the proportions of other immune cell subsets or expression of immune modulators. However, GEP revealed that EBV+ tumors had higher expression of IDO1 (11-fold, p=0.02). In contrast, EBV(-) tumors overexpressed CD163, CSF1R and IL10 associated with suppressive M2 macrophages (p<0.10). In addition, EBV(-) tumors overexpressed the cancer-promoting genes CXCR4 (p=0.09), IL32 (p=0.03), and IL1A (p=0.02). Notably, PTGS2 (COX-2) and IL1B, involved in prostaglandin production supporting cancer progression and metastasis, were the most highly overexpressed genes in EBV(-) tumors (270-fold, p<0.001; and 24-fold, p=0.06, respectively). IHC showed COX-2 overexpression by EBV(-) tumors (p=0.068), consistent with GEP. IHC also indicated expression of COX-2 by normal gastric epithelium.

Conclusions

Gastric cancers are characterized by an immunosuppressive TME regardless of EBV status, with abundant expression of PD-L1 and other immune checkpoints. GEP revealed that EBV(-) GCs, which are much more common than EBV+ GCs, overexpress molecules such as COX-2, IL-1A, IL-1B, IL-10 and CSF1R. Our findings provide novel insights into the immune microenvironment of EBV+ and EBV(-) GC, and offer potential targets to overcome resistance to anti-PD-1/PD-L1 therapies in this disease.

Acknowledgements

Funded by the Bristol-Myers Squibb International Immuno-Oncology Network and NCI R01 CA142779.

P542 Exposure to anti-PD-1 causes Functional Differences in Tumor-Infiltrating Lymphocytes in Solid Tumors

Caitlin Creasy, MS¹, Cara Haymaker, PhD², Marie-Andrée Forget, PhD², Gopal Singh, PhD², Coya Tapia, MD, PhD², Chantale Bernatchez², Jeane Painter, PhD², Funda Meric-Bernstam, MD², Caitlin Creasy, MS¹

¹MD Anderson Cancer Center- UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA; ²UT MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Chantale Bernatchez (cbernatchez@mdanderson.org)

Background

The pervasive use of therapeutic antibodies targeting PD-1 puts it on target to become the standard of care for solid tumor malignancies. However, little is known as to how blockade of PD-1 may alter the function or phenotype of tumor-infiltrating lymphocytes (TIL). By investigating samples from pre-treatment and early on-treatment biopsies from patients with varying types of solid tumors treated with anti-PD1, we hope to elucidate drug-induced changes in TIL phenotype and function.

Methods

An ongoing Phase II clinical trial of anti-PD-1 in cohorts of patients with rare solid tumor types (NCT02721732) yielded mandatory core biopsies taken at baseline and day 15-21 after the first cycle of anti-PD-1 (Pembrolizumab, 200 mg). Upon receipt, half of the biopsy was mechanically disaggregated for TIL phenotyping, which we term “fresh” flow cytometry staining. The other half of the biopsy was used to propagate TIL ex vivo using the TIL 3.0 method, which includes IL-2, agonistic anti-4-1BB antibody (Urelumab, BMS), and anti-CD3 (clone OKT3). TIL phenotype and function were evaluated after 2 or 3 weeks of culture. Functionality was determined through sorting T cell subsets and measuring cytokine and chemokine secretion following anti-CD3 re-stimulation using MSD and Luminex platforms.

Results

Phenotypic analysis of the freshly stained and expanded TIL demonstrated an effector memory differentiation status before and after exposure to anti-PD-1. These TIL did not differ in their expansion of the CD4+ or CD8+ subsets. This is expected within the expanded TIL, given the predisposition to expand CD8+ TIL with the addition of anti-4- 1BB. Further, expanded TIL retained cytotoxic potential (perforin/granzyme B) after one dose of anti-PD-1. However, PD-1 expression on expanded CD8+ tended to be elevated after therapy (p=0.09). Further, TIL expanded after anti-PD-1 showed enriched CTLA-4 expression in CD4+ TIL (p=0.003). Functional analysis of 16 paired baseline and on-treatment expanded TIL show that CD4+ TIL with higher IL-4 secretion are accompanied by inhibited cellular growth (p<0.05). CD8+ TIL demonstrated an inverse relationship with growth and higher expression of PD-1, CTLA-4, and cytotoxic molecules, perforin/granzyme B (p<0.05). Further analysis in 12 of the 16 paired expanded CD8+ TIL samples for 65 soluble factors demonstrated an aberrant secretion profile post anti- PD1 treatment suggesting impaired function.

Conclusions

Our study assesses the ramifications of one dose of anti-PD-1 on TIL in rare solid tumors. We demonstrate that although phenotypically similar after undergoing checkpoint blockade, TIL tend to have a poorer functionality after anti-PD-1.

Ethics Approval

The study was approved by UT MD Anderson Cancer Center's IRB, approval number 2015-0948.

Consent

Written informed consent was obtained from the patient for publication of this abstract and accompanying images. A copy of the written consent is available for review by the Editor of this journal.

P543 Neoantigen heterogeneity as contributing factor for non-responders to neoantigen specific T-cell therapy in patients with metastatic gastrointestinal malignancies

Eric Groh, MD, Jared Gartner, MS, Todd Prickett, PhD, Yong Li, MS, Steven Rosenberg, MD, PhD, Paul Robbins, PhD

NCI, Bethesda, MD, USA

Correspondence: Paul Robbins (paul_robbins@nih.gov)

Background

In an initial pilot study, tumor infiltrating lymphocytes (TIL) that recognize neoepitopes were identified in 9 of 10 patients with metastatic gastrointestinal (GI) malignancies. These findings suggest that adoptive transfer of autologous neoepitope-reactive T cells may represent an attractive treatment strategy for patients with metastatic GI malignancies; however, objective response rates for this treatment strategy remain low. Factors that may influence therapies that target one or a relatively small number of mutations include intra- and inter-tumor mutational heterogeneity, which are evaluated in this study.

Methods

Whole exome sequencing (WES) was carried out on resected flash-frozen metastatic tumor samples and matched normal cellular DNA to identify somatically mutated gene products, and TIL cultures initiated from adjacent tumor regions. Co-culture of antigen presenting cells expressing individual tumor mutations and TIL allowed identification of neoepitope-reactive TIL which were then used for adoptive cell therapy. Additionally, FFPE tumor samples were obtained for each patient from which WES was performed to characterize tumor mutations.

Results

WES data was analyzed from 39 unique tumors from 12 patients with metastatic GI malignancy treated with neoepitope-reactive TIL. A mean of 3 tumors per patient were analyzed (range 2-6 tumors), and the mean number of mutations per sample was 101 (range 22-156 mutations). Inter-tumor heterogeneity was present in all 12 patients (Figure 1). The percent of mutations ubiquitously expressed in all samples from an individual patient ranged from 12.9% (Patient 4071) to 67.9% (Patient 3737). Mutation reactive TIL therapy resulted in an objective response in 2 of the 12 patients. The single neoantigen targeted by TIL administered to the 2 patients with objective responses was present in all additional FFPE samples studied. In contrast, for 7 of the 10 non-responders, targeted mutations that could not be detected 1 or more of the analyzed FFPE samples were identified (Table 1). These findings raised the concern that the lack of response seen in some patients receiving adoptive TIL transfer could be due to the absence of the targeted mutations in individual tumor lesions.

Conclusions

Inter-tumor heterogeneity of targeted mutations is presents in non-responders to neoepitope-reactive TIL therapy. This suggested one mechanism limiting the effectiveness of TIL therapy targeting a limited number of mutations. WES of primary and metastatic tumors from primary and metastatic tumors from individual patients may facilitate identification of ubiquitously expressed tumor mutations that, if successfully targeted by adoptively transferred TIL, may lead to improved object response rates.

Fig. 1 (abstract P543).

See text for description

Table 1 (abstract P543). See text for description

P544 Interferon-γ regulates the expression of PD-L1 and NKG2D ligand families in nasopharyngeal carcinoma

Lingling Guo¹, Yu Chen, MD, PhD², Chuaben Chen²

¹Fujian medical university affiliated cancer hospital, Fuzhou, Peoples Republic of China; ²Fujian Province Cancer Hospital, Fuzhou, Peoples Republic of China

Correspondence: Chuaben Chen (bencc@sina.com)

Background

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-related malignancy that is characterized by a large number of infiltrating lymphocytes in the tumor microenvironment. The CD8+ T-lymphocytes can release interferon γ (IFN-γ), a cytokine proven to be a strong stimulant of programmed death-ligand 1(PD-L1). Hence the high expression of PD-L1 is another characteristic of NPC. It is reported that PD-1/PD-L1 inhibitors have good therapeutic effects on solid tumors with high PD-L1 expression and extensive CD8+ T-lymphocyte infiltration. However, current clinical trials only show a limited effect of PD-1 inhibitors on recurrent or metastatic NPC. To find the mechanism of resistance to PD-1/PD-L1 inhibitors in NPC, we studied the co-expression status of the NKG2D ligand family, a group of activating molecules expressing on the surface of activated CD8+ T-lymphocytes, when IFN-γ is up-regulating the expression of PD-L1 in NPC.

Methods

We downloaded the RNA-Seq data of IFN-γ, PD-L1, MICA and ULBP2-4 in head and neck squamous cancer (HNSC, except NPC) and NPC respectively from the TCGA and the GEO databases, screened for the most related molecules to IFN-γ. We also administrated exogenous IFN-γ to three types of NPC cell lines, CNE-1, CNE-2 and 5- 8F, and did qPCR, flow cytometry (FC), Western-blot (WB) and RNA-Seq after 0, 4, 12, and 24 hours, to determine the expression level of above molecules at each time point.

Results

In 501 HNSC cases from the TCGA database, the expression of PD-L1 is significantly related to IFN-γ (R=0.65, P<0.001) while the correlation between MICA and IFN-γ is very weak (R=0.12, P=0.004). Among the ULBPs, ULBP3 has the most significant correlation with IFN-γ (R=-0.18, P<0.001). The 113 NPC cases from the GEO database show the similar results. All the cell experiments proved the positive correlation between PD-L1 and IFN-γ and the negative correlation between MICA and IFN-γ (P<0.05). All the ULBPs had a significant negative correlation with IFN-γ in WB and FC, whereas ULBP3 showed a best correlation in RNA-Seq and qPCR(P<0.05).

Conclusions

We confirmed that IFN-γ can up-regulate the expression of PD-L1 and change the expression of NKG2D ligands. ULPB3 has a most significant and time-dependent negative correlation with both IFN-γ. Therefore, we hypothesize the down-regulation of ULBP3 mediated by IFN-γ may be a unique mechanism of immune escape and primary resistance in NPC patients. Further studies are needed to explore the deeper molecular mechanism.

Fig. 1 (abstract P544).

See text for description

Fig. 2 (abstract P544).

See text for description

Fig. 3 (abstract P544).

See text for description

Fig. 4 (abstract P544).

See text for description

P545 The tumor inflammasome functions as a rheostat for regulating the efficacy of anti-PD-1 antibody immunotherapy by driving the recruitment of myeloid-derived suppressor cells

Brent Hanks, MD, PhD³, Bala Thievanthiran, PhD¹, Kathy Evans, BS¹, Nicholas DeVito, MD¹, Christine Xiao, BS¹, Yubin Kang, MD¹, David Hsu, MD, PhD¹, Alisha Holtzhausen, PhD²

¹Duke University, Durham, NC, USA; ²University of North Carolina, Chapel Hill, NC; ³Duke University Medical Center, Durham, NC, USA

Correspondence: Brent Hanks (brent.hanks@duke.edu)

Background

Despite the impact of anti-PD-1 antibody immunotherapy in oncology, many cancer patients do not benefit from this treatment modality. Mechanisms of resistance to checkpoint inhibitor immunotherapy in cancer remain poorly understood.

Methods

Various pre-clinical syngeneic and transgenic cancer models as well as an autologous humanized mouse model were utilized to investigate the genetic and cellular alterations of tumors escaping anti-PD-1 antibody immunotherapy based on immunohistochemistry, flow cytometry, RNAseq transcriptomic sequencing, and tandem mass spectrometry.

Results

Our studies have demonstrated that anti-PD-1 antibody immunotherapy promotes the recruitment of granulocytic myeloid-derived suppressor cells (Gr-MDSCs) to the tumor bed in a CXCR2-dependent manner. This process involves the upregulation of the CXCR2 chemokine ligand, CXCL5, which is induced by a Wnt5a-YAP non- canonical autocrine signaling pathway that, in turn, is stimulated by a HSP70-TLR4 signaling axis. Further investigation has demonstrated that this cascade of events is initiated by CD8+ T cell-dependent stimulation of the NLRP3 inflammasome in tumor cells which drives the release of HSP70 and serves to dampen the efficacy of anti- PD-1 antibody immunotherapy. This phenomenon has been observed in pre-clinical models of non-small cell lung cancer, melanoma, and pancreatic cancer, as well as an autologous humanized mouse model of renal cell carcinoma in response to anti-PD-1 antibody therapy but not anti-CTLA-4 antibody therapy or chemotherapy. Additional studies have revealed that this pathway also drives the accumulation of Gr-MDSCs in distant tissues such as the lung in a manner that is dependent upon the presence of the primary tumor. Genetic silencing of CXCL5 and pharmacological blockade of Wnt ligand signaling suppresses Gr-MDSC recruitment in response to anti-PD-1 antibody therapy and enhances the efficacy of checkpoint inhibitor therapy. CXCR2 and CXCR2 ligand expression correlates with Wnt5a and with cytolytic T cell markers in human melanoma tissues indicating that this pathway is clinically relevant. We have determined that levels of CXCL5 and other CXCR2 ligands are increased in the plasma of transgenic tumor models escaping anti-PD-1 antibody therapy. Clinical studies are ongoing to investigate relationships between anti-PD-1 antibody response and the plasma levels of CXCR2-dependent chemokines as well as genetic inflammasome polymorphisms in advanced melanoma patients undergoing checkpoint inhibitor therapy.

Conclusions

By promoting the recruitment and accumulation of Gr-MDSCs, the tumor inflammasome functions as a rheostat that modulates the T cell response generated by anti-PD-1 antibody therapy. The elucidation of this pathway provides novel therapeutic opportunities to expand on the efficacy of checkpoint inhibitor immunotherapy for cancer patients.

Ethics Approval

This study was approved by the Institutional Animal Care and Use Committee at Duke University, registry number A249-12-09.

P546 Targeted hypoxia reduction restores T cell infiltration and sensitivity to immunotherapy in prostate cancer

Priyamvada Jayaprakash, PhD, Midan Ai, PhD, Arthur Liu, Pratha Budhani, BS, MS, Todd Bartkowiak, Jie Sheng, Casey Ager, BS, Courtney Nicholas, PhD, Ashvin Jaiswal, Yanqiu Sun, Krishna Shah, Sadhana Balasubramanyam, Nan Li, Guocan Wang, PhD, Jing Ning, Anna Zal, Tomasz Zal, PhD, Michael Curran, PhD

The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Michael Curran (mcurran@mdanderson.org)

Background

Checkpoint blockade using anti CTLA-4 and anti PD-1 is effective in “hot” tumors like melanoma with pre-existing immune infiltrates (1); however, “cold” tumors like prostate cancer respond poorly. Within these tumors we find prevalent zones of hypoxia from which T cells are excluded. Hypoxia impedes T cell mediated antitumor immunity by creating a hostile microenvironment through acidification of the extracellular milieu, formation of abnormal vasculature lacking in adhesion receptors needed for T cell extravasation, and recruitment of immunosuppressive stromal populations such as myeloid derived suppressor cells (MDSC) and myofibroblasts (2-5) . We hypothesized that removing these hypoxic zones would restore T cell infiltration and effector function thus sensitizing prostate tumors to checkpoint blockade.

Methods

We utilized the hypoxia-activated prodrug, TH-302 (Evofosfamide) to ablate hypoxic zones of tumors and tested its efficacy alone and in combination with immune checkpoint blockade in reducing tumor burden and enhancing anti- tumor immunity using both transplantable and spontaneous models of prostate cancer. We investigated how this therapy impacted recruitment and function of tumor-infiltrating CD8+ T cells and immune suppressive MDSC.

Results

We found that TH-302 improved survival of prostate tumor bearing mice (OS 30%) and synergized with checkpoint blockade using αCTLA-4 and αPD-1 in curing 82% of mice compared to checkpoint blockade alone (OS 55%). This was concomitant with enhanced T cell and decreased MDSC infiltration into tumors. T cells infiltrating tumors treated with the combination of TH-302 and αCTLA-4/αPD-1 had higher proliferative capacity, improved effector cytokine production and greater mitochondrial biomass. In addition, MDSC from these tumors displayed reduced suppressive activity. Combination treatment caused a persistent defect in the ability of tumors to replenish their myeloid stroma. In addition, spontaneous prostate cancer in TRAMP transgenic mice, which is entirely checkpoint blockade resistant, responded dramatically to combination CTLA-4/PD-1 blockade when co-administered with the hypoxia-reducing agent TH-302.

Conclusions

We have shown that hypoxia reduction can sensitize otherwise resistant prostate tumors to immunotherapy. Hypoxic ablation is associated with the conversion of the immunosuppressive tumor microenvironment (TME) into an immune permissive one through improved survival, function and metabolic fitness of T cells and reduced infiltration and function of immunosuppressive MDSC. Our findings suggest that metabolic manipulation of the TME is a viable approach to enhance immunotherapy responses.

References

1. Curran MA, Montalvo W, Yagita H, and Allison JP. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci U S A. 2010;107(9):4275-80. 2. Corzo CA, Condamine T, Lu L, Cotter MJ, Youn JI, Cheng P, Cho HI, Celis E, Quiceno DG, Padhya T, et al. HIF-1alpha regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment. J Exp Med. 2010;207(11):2439-53. 3. Dang EV, Barbi J, Yang HY, Jinasena D, Yu H, Zheng Y, Bordman Z, Fu J, Kim Y, Yen HR, et al. Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1. Cell. 2011;146(5):772-84. 4. Ben-Shoshan J, Maysel-Auslender S, Mor A, Keren G, and George J. Hypoxia controls CD4+CD25+ regulatory T-cell homeostasis via hypoxia-inducible factor-1alpha. Eur J Immunol. 2008;38(9):2412-8. 5. Becker JC, Andersen MH, Schrama D, and Thor Straten P. Immune-suppressive properties of the tumor microenvironment. Cancer immunology, immunotherapy : CII. 2013;62(7):1137-48.

P547 Adoptive cell therapy and intratumoral nanoplexed poly I:C as an effective immunotherapy in interferon- signaling deficient melanoma

Anusha Kalbasi, MD², Kevin Hakimi, BS¹, Sarah Kremer¹, Christine Nguyen, BS¹, Davis Torrejon, MD¹, Giulia Parisi¹, Pedro Lopez-Casas, PhD³, Marisol Quintero, PhD, MBA³, Antoni Ribas, MD, PhD⁴

¹University of California Los Angeles, Los Angeles, CA, USA; ²UCLA, Los Angeles, CA, USA; ³Bioncotech, Madrid, Spain; ⁴University of California, Los Angeles, Los Angeles, CA

Correspondence: Antoni Ribas (aribas@mednet.ucla.edu)

Background

We examined the impact of defects in type I and/or II interferon signaling on the efficacy of adoptive cell therapy (ACT) with tumor-specific T cells in B16 murine melanoma.

Methods

Using CRISPR, we generated B16 tumor cell lines deficient in type I (Ifnar1-KO) or type II (Jak2-KO) interferon signaling, or both (Jak1-KO). ACT was performed using gp100-specific pmel T cells. Tumor cell were labeled with RFP to determine tumor-specific MHC-I expression in vivo. We also tested a nanoplexed formulation of poly I:C (BO-112) that activates TLR3/MDA5/RIG-I.

Results

Pmel ACT was effective against B16 tumors lacking type I (Ifnar1-KO) or type II (Jak2-KO) interferon signaling, but ineffective against tumors lacking both (Jak1-KO) (Figure 1). We observed a similar phenomenon in vitro, where growth of B16 tumor cells lacking type I or II interferon signaling (but not both) were inhibited by tumor- specific pmel T cells compared to non-specific T cells, provided the alternate interferon pathway was activated (p < 0.0001). We then examined interferon-dependence of MHC-I expression in vivo. We observed that basal MHC-I expression by B16 in vivo is dependent on type II, but not type I, interferon signaling. After pmel ACT, tumors lacking type II interferon (Jak2-KO) signaling were able to augment MHC-I expression compared to B2m-KO tumors (p = 0.068). Jak1-KO tumors did not express MHC-I even after pmel ACT (p = 0.5283). To overcome resistance of B16 Jak1-KO tumor cells to pmel ACT, we tested intratumoral delivery of BO-112, which has direct anti-tumor efficacy against B16 and augments anti-tumor efficacy of pmel ACT against B16. Notably, the direct anti-tumor effects of BO-112 are abrogated in the B16 Jak1-KO compared to wildtype B16 tumors both in vitro and in-vivo. Regardless, in combination with BO-112, pmel ACT was effective against B16 Jak1-KO tumors compared to non-specific T cells in combination with BO-112 (Figure 2). RNA sequencing of tumors 5 days after ACT revealed 209 genes enriched (fold change > 2, adjusted p-value < 0.05) in tumors treated with pmel ACT and BO- 112, which were not enriched in tumors treated with pmel ACT and vehicle or non-specific ACT and BO-112, including genes involved in T cell recruitment, antigen presentation, direct T cell cytotoxicity, and interferon signaling.

Conclusions

Our findings suggest ACT can be an effective immunotherapy in tumors lacking type I or II interferon signaling. For tumors lacking both type I and II interferon signaling, intratumoral BO-112 can resensitize tumors to ACT.

Fig. 1 (abstract P547).

See text for description

Fig. 2 (abstract P547).

See text for description

P548 Association between corticosteroid use at time of immune checkpoint inhibitor (CPI) therapy initiation and CNS disease control/survival in patients with brain metastases (BM)

William Kelly, MD¹, Neil Shah¹, Michael Serzan, MD¹, Barbara Ma¹, Matthew Blackburn¹, Sebastian Ochoa¹, Alice Knoedler¹, Jeevan Puthiamadathil¹, Ariana Santopietro¹, Subha Madhavan², Anas Belouali², Shruti Rao², Kellie Gardner, NP¹, Suthee Rapisuwon, MD¹, Stephen Liu, MD¹, Deepa Subramaniam¹, Michael Atkins, MD¹, Geoffrey Gibney, MD¹

¹Medstar Georgetown University Hospital, Washington, DC, USA; ²Georgetown University, Washington, DC, USA

Correspondence: William Kelly (08wkelly@gmail.com)

Background

CPI therapy targeting CTLA-4 and PD-1 has improved clinical outcomes in patients with BM [1], including objective responses in patients (pts) with active, untreated melanoma or non-small cell lung cancer (NSCLC) BM [2-4]. Pts with newly diagnosed BM often require corticosteroids at CPI initiation and the impact of such concomitant therapy on disease control and survival remains unclear. The clinical outcomes of pts with BM receiving CPI therapy and associations with corticosteroids and other prognostic factors were analyzed.

Methods

Pts with metastatic solid tumors and BM history treated with ipilimumab (anti-CTLA-4), nivolumab or pembrolizumab (anti-PD-1), and nivolumab/ipilimumab (nivo/ipi) at 3 Medstar Hospitals were identified by pharmacy records and chart review. Pts were excluded if initial BM occurred after CPI initiation or if baseline pretreatment/follow up brain MRI/CT imaging were not available. Data collected included demographics, baseline performance status, systemic corticosteroid use within 14 days of CPI initiation, provider-assessed best disease response and overall survival (OS).

Results

71 pts were identified (40 melanoma, 25 NSCLC, 3 renal cell carcinoma, 3 other). 55% were male, 86% had ECOG PS 0-1, and 66% had ≥2 brain metastases. 82% of pts had surgery and/or stereotactic radiosurgery for BM management prior to therapy. 22% of pts received anti-CTLA-4, 54% received anti-PD-1, and 24% received nivo/ipi. 52% had neurological symptoms and 24% received corticosteroids within 14 days of CPI initiation. The response rate (extracranial) was 23% and median OS was 13.8months for all pts. Survival was superior in pts with melanoma and those treated with nivo/ipi. BM control (no new BM or progression in treated BM) was seen in 38%. Extracranial disease control was associated with intracranial disease control (p=0.001). The use of corticosteroids was associated with BM progression (but not extracranial disease progression) and worse OS (median 5.8months vs 19.8months for no corticosteroid use, P=0.011). There was a trend for worse OS in patients with greater number of BM (p=0.053). The presence of baseline neurological symptoms was not associated with OS.

Conclusions

Pts with BM can have long-term survival with CPI therapy, particularly with nivo/ipi. There is general concordance between extracranial disease control and BM control, but discordance with BM progression can occur. The use of corticosteroids at time of CPI therapy in pts with BM is associated with worse BM control and survival. Pts initially requiring corticosteroids may benefit from alternative systemic therapy options.

References

• 1. Sloot S, et al. Improved survival of patients with melanoma brain metastases in the era of targeted BRAF and immune checkpoint therapies. Cancer, 2018;124(2):297-305.

• 2. Goldberg S, et al. Pembrolizumab for patients with melanoma or non-small-cell lung cancer and untreated brain metastases: early analysis of a non-randomised, open-label, phase 2 trial. Lancet Oncol, 2016;17(7):976-983.

• 3. Long G, et al. Combination nivolumab and ipilimumab or nivolumab alone in melanoma brain metastases: a multicentre randomised phase 2 study. Lancet Oncol, 2018.

• 4. Margolin K, et al. Ipilimumab in patients with melanoma and brain metastases: an open-label, phase 2 trial. The lancet oncology, 2012.

P549 Cell proliferation defines an additional mechanism of immune escape in non-small cell lung cancer

Carl Morrison, MD, DVM¹, Sarabjot Pabla, MSc, PhD, BS¹, Mary Nesline, MS¹, Jeffrey Conroy, BS¹, Sean Glenn, PhD¹, Antonios Papanicolau-Sengos, MD¹, Paul DePietro, PhD¹, Jacob Hagen¹, Blake Burgher, BS, RN¹, Vincent Giamo, BS, MS¹, Jonathan Andreas, MS¹, Felicia Lenzo¹, Maochun Qin, MD, MS¹, Yirong Wang, MS¹, Devin Dressman, PhD¹, Konstantin Dragnev, MD², Laura Tafe², Tian Zhang, MD³, Jeffrey Clarke, MD³, Matthew Zibelman, MD⁴, Luis de la Cruz-Merino⁵, Alexander MacKinnon⁶, Robin Jacob⁷, Oliver Binns⁸, Neel Shah⁹, Mark Gardner¹, Grace Dy¹⁰

¹OmniSeq, Inc., Buffalo, NY, USA; ²Dartmouth-Hitchcock Medical Center, Hanover, NH, USA; ³Duke University, Durham, NC, USA; ⁴Fox Chase Cancer Center, Philadelphia, PA, USA ⁵Hospital Universitario Virgen Macarena, Sevilla, Spain ⁶Medical College of Wisconsin, Milwaukee, WI, USA ⁷Meharry Medical College, Nashville, TN, USA; ⁸Mission Health System, Asheville, NC, USA; ⁹Northwest Oncology, Munster, IN, USA; ¹⁰Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA

Correspondence: Grace Dy (Grace.Dy@RoswellPark.org)

Background

Prior to this study, the most important mechanisms of immune escape in NSCLC include checkpoint blockade and low tumor mutational burden (TMB). In this study we have identified a third equally important mechanism of immune escape of cell proliferation in NSCLC.

Methods

169 NSCLC patients from ten institutions were evaluated for PD-L1 expression by IHC, TMB, and cell proliferation by RNA-seq of 10 immune and proliferation related genes including BUB1, CCNB2, CDK1, CDKN3, FOXM1, KIAA0101, MAD2L1, MELK, MKI67, and TOP2A, of which 46 represented historical controls and 113 had prior treatment with one more immune checkpoint inhibitors (ICIs) and for which response (RECIST v1.1) and survival were available.

Results

For all patients (n=169) the majority were non-proliferative (59.2%; 100/169), with a minority of proliferative results (40.8%; 69/169). Response rate in non-proliferative patients was 42.2% (27/64) versus 18.4% (9/49) in proliferative. Using a value of 10 mutations per megabase of DNA or greater as high the percent of TMB high cases in proliferative tumors (67%; 47/70) was higher than in non-proliferative tumors (47%; 47/99). The rate of PD-L1 IHC positive (TPS >50) was not different in proliferative (21.7%; 15/69) versus non-proliferative (26%; 26/100) supporting that PD-L1 expression is independent of proliferation status. Survival analysis using TMB high versus low did not show a survival advantage for all ICI-treated patients (p=0.7), proliferative cases (p=0.057), or non- proliferative cases (p=0.12). For 18 non-proliferative PD-L1 positive cases the response rate was 67% (12/18) and nearly double that in the corresponding proliferative group at 38% (5/14). For 46 non-proliferative PD-L1 negative cases the response rate was 35% (12/18) and more than three times that in the corresponding proliferative group at 11% (4/35). Survival for all combinations of PD-L1 and cell proliferation was greatest for PD-L1 positive non- proliferative cases, but perhaps equally if not more important was survival for PD-L1 negative non-proliferative cases was nearly equal to that for PD-L1 proliferative cases.

Conclusions

Cell proliferation, or more specifically high proliferation, is an important immune escape mechanism in NSCLC. Non-proliferation is an emerging biomarker for response to ICIs in NSCLC.

Ethics Approval

OmniSeq’s analysis utilized deidentified data that qualified as non-human subject research under IRB protocol (BDR #080316) approved by Roswell Park Comprehensive Cancer Center (Buffalo, NY).

P550 Defeating checkpoint resistance: Highly specific inhibition of latent TGFβ1 activation renders resistant solid tumors vulnerable to PD-1 blockade

Thomas Schürpf¹, Constance Martin, PhD¹, Christopher Littlefield, MSc¹, Christopher Chapron, MS¹, Stefan Wawersik, PhD¹, Ashish Kalra, PhD¹, Kevin Dagbay, PhD¹, Allison Simpson, BS¹, Francis Danehy, BS¹, Christopher Boston¹, Anastasia Nikiforov, MS¹, Susan Lin, BS¹, Justin Jackson, BS¹, Pichai Raman, PhD², Elizabeth Rainbolt, BS³, Laurie Comfort, BS³, David Harris³, Madelyn Cecil-Taylor³, Lorne Celentano³, Danielle Meadows³, gregory carven, PhD¹, Alan Buckler, PhD¹, Allan Capili, PhD¹, Abhishek Datta, PhD¹, Thomas Schürpf¹

¹Scholar Rock, Cambridge, MA, USA; ²Pichai Raman Consulting, Bryn Mawr, PA, USA; ³Charles River Discovery Services, Morrisville, NC, USA

Correspondence: Abhishek Datta (adatta@scholarrock.com)

Background

Despite the clinical breakthroughs achieved by cancer immunotherapy, a majority of patients fail to respond to PD-(L)1 inhibition due to primary or acquired resistance. Profiling of human urothelial cancer and melanoma tumors has recently implicated TGFβ activation as a potential mechanism of primary resistance to checkpoint therapies. However, therapeutic targeting of the TGFβ pathway has been hindered by dose-limiting cardiotoxicities, most likely due to inhibition of signaling from multiple TGFβ isoforms. Upon secretion, TGFβ growth factor is held in a latent complex with its non-covalently associated prodomain. TGFβ activation is induced by extracellular events that release the growth factor from this latent complex. We previously demonstrated that isoform-specific inhibition of TGFβ activation can be achieved by targeting the latent TGFβ complex. We hypothesized that the identification and inhibition of the predominant TGFβ isoform in tumors would enable a more targeted and potentially safer approach to TGFβ inhibition.

Methods

The Cancer Genome Atlas (TCGA) database was interrogated to assess mRNA levels of TGFβ isoforms. Antibody- mediated inhibition of TGFβ1 activation was tested using luciferase-based reporter cells. Efficacy of TGFβ1-selective inhibition in combination with anti-PD-1 was assessed in the MBT-2 bladder cancer and CloudmanS91 melanoma models.

Results

Bioinformatic analysis of TCGA data identified TGFβ1 as the predominant isoform in many human tumors. We generated high affinity, fully-human antibodies against latent TGFβ1. They inhibit the activation of latent TGFβ1 with no detectable binding to or inhibition of latent TGFβ2 or latent TGFβ3. Efficacy was tested in MBT-2 and CloudmanS91, two syngeneic mouse models that recapitulate key aspects of the primary PD-1 resistance phenotype of human disease. Inhibition of TGFβ1 activation is sufficient to completely block TGFβ signaling in MBT-2 tumors. Both models are largely resistant to anti-PD-1 or anti-TGFβ1 alone. However, the combination of anti-PD-1 with blockade of TGFβ1 activation leads to tumor growth delay, a substantial number of complete responses, and prolonged survival coupled with increased effector CD8+ T cell infiltration.

Conclusions

We show here that in many human tumor types, especially those for which checkpoint inhibitors are approved as therapies, TGFβ1 is the predominant isoform. Pharmacologic blockade of TGFβ1 activation is sufficient to sensitize TGFβ1-predominant tumors to PD-1 inhibition. These encouraging efficacy data and the potentially favorable safety profile of TGFβ1 isoform-selective inhibition establish a strong rationale for exploring therapeutic application of combining PD-(L)1 blockade with latent TGFβ1 inhibition in treatment of multiple cancer types.

Ethics Approval

Animal studies were conducted in compliance with CR Disovery Services IACUC ASAP # 980701 & #980702, and AAALAC Certification

P551 Suppression of immune response by tumor cell-induced XIAP-NFκB signaling and targeting strategies to overcome immunotherapy resistance in breast cancer

Michael Morse¹, Scott Sauer, PhD², Myron Evans³, Mohamed Ibrahim, MD², Xuhui Bao, MD², Pranalee Patel², Gayathri Devi, MSc, PhD²

¹Duke University Medical Center, Durham, NC, USA; ²Duke University School of Medicine, Durham, USA; ³St. Jude's, Memphis, USA

Correspondence: Gayathri Devi (gayathri.devi@duke.edu)

Background

Locally advanced breast cancers (LABC) that display lymphovascular invasion (LVI), such as inflammatory breast cancer (IBC), rapidly acquire therapeutic resistance and are highly lethal. A critical question is how, despite trafficking through lymphatics where they encounter immune effectors and inflammatory stress, do the tumor cells evade immune-mediated cell death. IBC expresses high levels of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein (XIAP). We previously identified that, in addition to its canonical function as a potent caspase inhibitor in both the extrinsic and intrinsic apoptosis pathways, XIAP activates nuclear transcription factor (NFκB) in suppression of two mechanisms of cell death caused by anti-tumor immune effectors (antibodies and T cells), granzyme-mediated cell death and accumulation of reactive oxygen species. Further, we identified a mechanism of stress-induced protein translational upregulation of XIAP in promoting tumor cell survival in models of IBC. Therefore, we hypothesized that stress-mediated XIAP-NFκB signaling can lead to a tumor cell-promoted immunosuppressive environment and targeting this signaling axis can enhance the efficacy of immunotherapy.

Methods

Using multiple cell models of aggressive breast cancer, we observed that increased XIAP expression caused decreased immune-mediated caspase activation, lower ROS induction/increased antioxidant protein, and NFκB target gene transcripts in the immune/inflammatory network. To directly test if increased XIAP-NFκB survival signaling can suppress efficacy of immunotherapy, we tested the FDA approved EGFR-specific monoclonal antibody (cetuximab), widely used in cancer therapy, in in vitro antibody-dependent cellular cytotoxicity (ADCC) assays and in in vivo tumor growth kinetic analysis of IBC cells with differential apoptotic capability when implanted orthotopically in the mammary fat pad of mice with functional NK activity.

Results

IBC cells with XIAP overexpression and resultant increases in NFκB target genes regulating antioxidant and immune factors were insensitive to cetuximab-mediated ADCC and resistant to cetuximab-mediated inhibition of in vivo tumor growth when compared to the ADCC-sensitive cell lines. In order to re-sensitize these XIAP overexpressing cells towards ADCC, we tested two strategies- 1. targeting XIAP-NFkB signaling using NRAGE peptide that blocks the XIAP-Tab1-Tak1 complex; 2. SMAC mimetics/birinapant, a synthetic small molecule and peptidomimetic of second mitochondrial-derived activator of caspases (SMAC) and inhibitor of IAP family proteins. Our results reveal enhanced immune-mediated cell death/sensitivity to immunotherapy.

Conclusions

Our in vitro and in vivo preclinical studies identify the cellular stress-mediated induction of the XIAP-NFκB signaling axis as a novel mechanism of immune evasion and reveal the potential of targeting this signaling pathway to improve breast cancer immunotherapy.

Acknowledgements

Supported in part by Department of Defense Partnership Idea grant awards [W81XWH-13-1-0046 (GRD); W81XWH-13-1-0046 (MAM)]; Duke School of Medicine Bridge Funds (GRD); Duke University Diversity Enhancement Fellowship (MKE) and the National Cancer Institute T32CA009111 (SJS).

Ethics Approval

The animal research was approved by the Duke University IACUC

P552 Analysis of TIL from human carcinoma combined with tissue imaging and in vitro models uncovers tumor- inflicted T cell deviations related to immune escape and strategies of intervention

Elfriede Noessner, PhD¹, Elfriede Noessner, PhD¹, Petra Prinz², Ilias Masouris³, Anna Mendler²

¹Helmholtz Zentrum Munchen, Munich, Germany; ²Helmholtz Zentrum München, Munich, Germany; ³Klinikum LMU München, München, Germany

Correspondence: Elfriede Noessner (noessner@helmholtz-muenchen.de)

Background

Many tumors are infiltrated with CD8 lymphocytes. Yet, tumors are not rejected suggesting that the tumor environment limits effector cell efficacy to control tumor growth.

Methods

Using human renal cell carcinoma, multiparameter fluorescence staining and confocal microscopy was performed to determine the status of lymphocytes in direct physical contact with malignant cells under the control of the local microenvironment. Ex vivo TIL analysis was used to identify TCR signaling alterations in CD8-TILs compared to CD8 T cells of non-tumor kidney.

Results

A special image analysis, modeled on the process of lytic granule exocytosis, was applied to identify CD8-TILs with active tumor recognition. The cytotoxic status of CD8-TILs, determined in relation to the TILs’ spatial distribution within the tumor, revealed a pivotal role of the tumor microenvironment in restraining lytic function of CD8-TILs. Some TILs appeared actively engaged in tumor recognition; however, there was no evidence that any CD8 cell was stimulated to produce IFNg. Application of in vitro models, which mimic conditions of solid tumors, identified tumor lactic acidosis as one potent factor abrogating TCR-stimulated IFNg production by inhibition of p38 and JNK/c-Jun activation. Ex vivo analyses of TILs identified TCR signaling alterations in CD8-TILs compared to CD8 T cells of non-tumor kidney, which were associated with failure to degranulate. These deviations were reversible concomitantly with gain in perforin and function.

Conclusions

The results reveal mechanisms of inhibition of CD8- and NK-TIL function imparted by the tumor environment which are related to immune escape in RCC. Based on identified alterations strategies to engineer T cells (i.e chimeric costimulatory proteins) and to modulate the tumor environment are designed to empower T cells for higher efficacy in adoptive therapy.

Acknowledgements

We acknowledge the patients and their families for donating tissue for analysis, and the clinicians and pathologists for the sample collection.

Ethics Approval

The ethics board of the Ludwig Maximilians University has approved the tissue collection

P553 Targeting mechanisms of immune suppression via CXCR2 inhibition to enhance checkpoint blockade

Elaine Pinheiro, PhD, Sarah Javaid, PhD, Ruban Mangadu, Marlene Hinton, Yun Wang, Sonia Feau, Amanda Watkins, PhD, Andrey Loboda, PhD, Xue Liang, Dario Gutierrez, Rebecca Ruck, Michael Rosenzweig, DVM, PhD, Vincent Giranda, MD, PhD

Merck Research Laboratories, Boston, MA, USA

Correspondence: Elaine Pinheiro (elaine.pinheiro@merck.com)

Background

Myeloid-derived suppressor cells (MDSCs) are an adverse cancer-wide prognostic population of immune infiltrating cells that contribute to tumor immune evasion by suppressing local T-cell activation and viability and influencing tumor progression by promoting tumor metastases, angiogenesis and tumor cell invasion [1]. PMN-MDSC cell signatures have emerged as a significant predictor of poor survival across solid tumors [2]. Here we explore the role of the CXCR2 pathway on PMN-MDSCs in the tumor microenvironment and the mechanisms of CXCR2-driven enhancement of checkpoint blockade.

Methods

Chemotaxis assays were performed. Anti-tumor activity was assessed in the B16-F10 syngeneic tumor model utilizing a small molecule antagonist of CXCR2, MK-7123 (Navarixin) and an anti-mouse PD-1 blocking antibody, muDX400. Molecular and cellular responses associated with anti-tumor activity were evaluated by RNA- Sequencing analysis, flow cytometry, and functional assays.

Results

Here we show that cancer patient PMN-MDSCs highly express the CXC G protein-coupled receptor, CXCR2, and that its chemokine ligand, IL-8, can influence the migration of this myeloid population. In addition, CXCR2 expression significantly correlates with poor survival across solid tumor types. In mouse tumor models, CXCR2 blockade disrupts the trafficking of CD11b(+)Ly6C(-)Ly6G(+) PMN-MDSCs/neutrophils in the tumor microenvironment and enhances the anti-tumor effect of checkpoint blockade. Preclinical studies to further explore the molecular and cellular mechanisms behind these combination effects will be presented, utilizing a small molecule antagonist of CXCR2, MK-7123 (Navarixin), and an anti-mouse PD-1 blocking antibody, muDX400. Tumor transcriptome and network analyses by RNA-sequencing reveal that MK-7123 + muDX400 combination treatment result in the enrichment of immune and tumor-related pathways. Moreover, changes to chemokine profiles favor enhanced T cell infiltration into the tumor. In addition, the effects of commensal microbiota on response to monotherapy and combination treatment will be explored.

Conclusions

Taken together, these preclinical oncology studies support the concept of targeting CXCR2 to increase the therapeutic efficacy of PD-1 blockade. Clinical investigation of Navarixin in combination with pembrolizumab/Keytruda is ongoing for the treatment of multiple cancers.

References

1. Veglia F, Perego M, Gabrilovich D. Myeloid-derived suppressor cells coming of age. Nat Immunol. 2018;19:108–119 2. Gentles AJ, Newman AM, Liu CL, Bratman SV, Feng W, Kim D, Nair VS, Xu Y, Khuong A, Hoang CD, Diehn M, West RB, Plevritis SK, Alizadeh AA. The prognostic landscape of genes and infiltrating immune cells across human cancers.Nat Med. 2015; 21(8):938-945

P554 Role of immune escape for resistance to cancer (immuno) therapy and its strategies targeting these mechanisms

Barbara Seliger, MD, PhD, Jurgen Bukur, PhD, MD

Martin Luther University Halle-Wittenber, Halle, Germany

Correspondence: Jurgen Bukur (juergen.bukur@uk-halle.de)

Background

Despite impressive durable clinical responses in tumor patients with distinct subtypes of cancer employing cancer immunotherapies, a high frequency of patients does not respond or develop resistances during treatment overtime. Therefore, identification of the underlying molecular mechanisms of these resistances as well as identification of novel therapeutic approaches to overcome them might significantly improve the clinical outcome and survival of patients.

Methods

A number of both tumor intrinsic as well as tumor extrinsic factors have been identified by us, which are involved in the escape of immune surveillance of different tumors.

Results

These include downregulation of MHC class I antigen processing and interferon signaling components, upregulation of co-inhibitory molecules, such as HLA-G and PD-L1, as well as downregulation of extracellular matrix proteins. These different alterations could occur either at the transcriptional, epigenetic or posttranscriptional level, while structural alterations leading to loss of expression of these immune modulatory molecules appear to be a rare event. Interestingly, impaired HLA class I APM component expression has been demonstrated to be directly associated with disease progression after adoptive T cell therapy. Next to these tumor intrinsic factors, the tumor microenvironment also plays an important role in immune escape. In particular, the immune cell compositions in peripheral blood as well as the spatial distribution of immune cells in the tumor microenvironment are key factors of immune suppression. This was directly associated with a worse prognosis, reduced survival and/ or lack of response to cancer (immuno)therapies. Furthermore, treatment of cells with cytokines, like interferons, as well as recombinant proteoglycans anti-oxidant substances, e.g. methyl selenic acid, and epigenetic drugs were able to enhance HLA class I surface expression thereby resulting in an enhanced immune response.

Conclusions

Thus, overcoming the acquired an intrinsic therapy resistance of tumors is an important tool for improving (immuno)therapeutic strategies.

P555 A role for mutant p53 in mediating T cell immune evasion in pancreatic adenocarcinoma and other solid tumors

Deborah Silverman, BS, Emily Ashkin, Simone Punt, PhD, Minying Zhang, Leila Williams, MSc, Anil Korkut, Jason Roszik, PhD, Anirban Maitra, MBBS, Patrick Hwu, MD

MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Deborah Silverman (DASilverman@mdanderson.org)

Background

Harnessing the immune system by altering the ability of T-cells to attack cancer has led to long-lasting cures in some tumors. Nonetheless, pancreatic ductal adenocarcinoma (PDAC), one of the most lethal malignancies, remains resistant to immunotherapy. To design effective therapies, it is critical to learn how PDAC evades the immune system.

Methods

We hypothesize that p53 mutations mediate tumor escape from T-cells in PDAC, and test whether subsets of p53 mutations in PDAC tumors impact T-cell migration and killing, using novel in vitro and inducible in vivo models, to characterize a new role for p53’s regulation in cancer.

Results

Here we present results suggesting that a subset of p53 missense mutants impact T-cell infiltration into and autologous T-cell killing in PDAC tumors, in a possible dominant gain-of-function mechanism.

Conclusions

These findings begin to elucidate the role of p53 activating mutations in tumorigenesis,and providing rationale to investigate mutant p53 immune-regulation in PDAC and other tumor types.

Acknowledgements

The authors thank and acknowledge Eran Kotler and Moshe Oren for generously providing H1299-p53-mutant lines; Gigi Lozano, Florencia McAllister, Russell Broaddus, Stephanie Watowich, and Katy Rezvani for their valuable advice and insight; Sara Leahey, Soraya Zorro Manrique, Elien Doordjuin, and Weiyi Peng for their advice and support; and the University of Texas MD Anderson Cancer Center Pancreatic Cancer Moon Shot Program for their generous financial support of this project.

References

• 1. Carter S L, Cibulskis K, Helman E, McKenna A, Shen H, Zack T, Beroukhim R. Absolute quantification of somatic DNA alterations in human cancer. Nat biotechnology. 2012; 30(5): 413-421.

• 2. Hingorani S R, Wang L, Multani A S, Combs C, Deramaudt T B, Hruban R H, Tuveson D A. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer cell. 2005; 7(5), 469-483.

• 3. Morton J P, Timpson P, Karim S A, Ridgway R A, Athineos D, Doyle B, Frame M C. Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer. PNAS. 2010; 107(1), 246-251.

• 4. Aschauer L, Muller P A. Novel targets and interaction partners of mutant p53 Gain-Of-Function. Biochem Soc Transactions. 2016; 44(2), 460-466.

• 5. Joerger,A C, Fersht A R. Structure–function–rescue: the diverse nature of common p53 cancer mutants. Oncogene. 2007; 26(15), 2226-2242.

• 6. McKenzie, J A, Mbofung, R M ., Malu, S, Zhang, M, Ashkin, E, Devi, S, & Xu, C. The Effect of Topoisomerase I Inhibitors on the Efficacy of T-Cell-Based Cancer Immunotherapy. J Natl Cancer Inst. 2018;110(7):777-786.

P556 STAT3-related cytokines drive IR-specific immune suppression of effector, memory and naïve, peripheral blood CD8+ T cells in cancer patients

Ashwin Somasundaram, MD, Dario A. Vignali, PhD, Anthony Cillo, PhD, James Herman, John Kirkwood, MD, Robert Ferris, MD, PhD, Tullia Bruno, PhD

University of Pittsburgh, Pittsburgh, PA, USA

Correspondence: Dario A. Vignali (dvignali@pitt.edu)

Background

Cancer patients that do not respond to PD1 blockade have increased inhibitory receptor (IR) expression in peripheral blood lymphocytes (PBL) and increased cytokine concentrations in the plasma. Cancer patients off therapy and with normal white blood cell counts are often at greater risk for infections, immune dysregulation, progressive disease or reactivation of viral infections. However, the exact mechanism of this systemic immunosuppression in cancer patients is not fully understood. We performed flow cytometric assays to assess both phenotype and function of peripheral CD8+ T cells in cancer patient samples and healthy donor controls. We hypothesize that cancer patients may have systemic immune suppression via cytokine-driven IR expression in all CD8+ T cells subsets, including naïve cells.

Methods

PBL were obtained from healthy donors and treatment-naïve NSCLC, HNSCC, and melanoma patients. IR (i.e. LAG3, PD1, CTLA4, etc) expression was assessed on CD8+ T cells, CD4+ T cells, and regulatory T cells. Cytokine concentrations were compared by Luminex between plasma from healthy donors and plasma from cancer patients with high and low IR expression on peripheral CD8+ T cells. Autologous micro-stimulation assays were performed on peripheral CD8+ or CD4+ T cells with antigen presenting cells plus or minus IR blockade.

Results

CD8+ T cells, including CD45RA+CCR7+CD62L+CD8+ T cells, from cancer patient PBL contain elevated total LAG3 expression which correlated with stage and elevated expression of other IRs. Further, CD8+ T cells from these patients had decreased proliferation, which was rescued with the addition of anti-LAG3 or anti-PD1. Plasma from these patients had significantly elevated levels of cytokines that can signal via STAT3 (i.e. IL-6, IL-8, IL-9), which were independently found to increase total IR expression in healthy donor, naïve CD8+ T cells.

Conclusions

The current understanding of PD1 blockade resistance has been limited to the tumor microenvironment (TME) and our findings support the growing body of literature that tumor-related systemic immune suppression is a potent mechanism of cancer progression. Patients with cancer have systemic elevations of cytokines that signal via STAT3 leading to increased IR expression in naïve, peripheral CD8+ T cells making them poised for exhaustion even before TCR binding. These findings suggest that IR blockade also plays a significant role in reversing immune tolerance outside of the TME and cytokine blockade may play a role in reversing PD1 blockade resistance.

Ethics Approval

The study was approved by the University of Pittsburgh's IRB and Ethics Board, approval number: PRO16070383.

P557 Overcoming genetically-based resistance mechanisms to PD-1 blockade

Davis Torrejon, MD¹, Gabriel Abril-Rodriguez, MS², Jennifer Tsoi², Ameya Champhekar², Giulia Parisi², Gardenia Cheung-Lau², Tom Wohlwender², Mykola Onyshchenko², Beata Berent-maoz², Catherine Grasso², Begoña Comin- Anduix, PhD², Siwen Hu-Lieskovan, MD, PhD², Antoni Ribas, MD, PhD²

¹UCLA Hematology-Oncology, Los Angeles, CA, USA; ²UCLA, Los Angeles, CA, USA

Correspondence: Antoni Ribas (aribas@mednet.ucla.edu)

Background

We studied loss of function (LOF) mutations within the interferon (IFN) pathway (JAK1 or JAK2) and in the antigen presentation pathway (beta-2-microglobulin-B2M) found in biopsies from patients who are resistance to anti-PD-1 therapy, and tested strategies to overcome the resistance.

Methods

Using CRISPR/Cas9 genome editing we generated JAK1, JAK2 and B2M knockout (KO) sublines of the murine MC38 carcinoma, a model of high mutational load cancer that responds well to anti-PD-1, as well as of human MART-1-positive melanoma cell lines, tested using in-vitro T cell co-culture systems. We analyzed signaling changes in human cell lines (parental and KOs) exposed to IFN-gamma using RNAseq. In addition, we performed in-vivo antitumor activity in the MC38 variants using mass cytometry (CyTOF) to characterize the tumor microenvironment. Finally, we tested strategies to overcome resistance mechanisms with SD-101 (TLR-9-agonist) and NKTR-214 (CD-122 biased agonist).

Results

The JAK1-KO sublines lost sensitivity to IFN-alpha, IFN-beta and IFN-gamma, while the JAK2-KO cell line was insensitive only to IFN-gamma induced signaling (PD-L1, MHC class I) and growth arrest (p<0.001 compared with IFN-alpha or beta). There was no difference in the in-vitro cytotoxicity by MART-1 specific T-cells against JAK1/2- KO-MART-1+ melanoma cells compared to the parental (94%, 95% vs 90% cytotoxicity at 10:1 E:T ratio, pNS). However, B2M-KO was resistant to killing by MART-1 specific T-cells (2% vs 90% cytotoxicity at 10:1 E:T ratio, p<0.0001). RNAseq differential gene expression analysis showed that the IFN-gamma-induced increased expression of antigen presenting machinery, IFN-gamma signaling and chemokines (CXCL9, CXCL10) were not expressed by JAK1/2-KOs. In the MC38 model, the significant antitumor activity of anti-PD-1 against the parental was lost in JAK1/2 and B2M KOs (Table1); in these KO sublines, CyTOF analysis revealed that anti-PD-1 therapy was unable to change tumor CD8 T-cell infiltration. Using JAK1/2-KOs cell lines we showed that intratumoral administration of the TLR-9 agonist SD-101 was able to overcome local resistance to anti-PD-1 even in abscopal sites, and the NKTR-214 overcame resistance to anti-PD-1 in the B2M-KO tumor growth and significantly increased survival.

Conclusions

JAK1/2 LOF mutant tumors result in loss of sensitivity to IFN induced antitumor effects but do not impair T cell recognition and cytotoxicity, while B2M LOF results in lack of antigen presentation to T cells and loss of antitumor activity. Both lead to in-vivo resistance to anti-PD-1 therapy, and JAK1/2 KO resistance can be overcome by a TLR9 agonist, and B2M-KO resistance can be overcome by a new generation IL-2.

Table 1 (abstract P557). Overcome resistance to PD-1 blockade

P558 Secondary resistance to immunotherapy associated with β-catenin pathway activation or genetic loss of phosphatase and tensin homolog (PTEN) in metastatic melanoma

Jonathan Trujillo, MD, PhD², Jason Luke, MD, FACP¹, Stefani Spranger, PhD³, Yuanyuan Zha, PhD¹, Karen Matijevich, RN, BSN², Thomas Gajewski, MD, PhD¹

¹University of Chicago, Chicago, IL, USA; ²Univeristy of Chicago, Chicago, IL, USA; ³MIT, Cambridge, MA, USA

Correspondence: Thomas Gajewski (tgajewsk@medicine.bsd.uchicago.edu)

Background

While immune checkpoint blockade therapy and even some vaccines have given rise to durable responses in many cases of advanced melanoma, a large fraction of patients subsequently develop secondary resistance to therapy. Mechanisms of immune-resistant cancer progression in this context are incompletely understood. Our lab previously showed tumor-intrinsic WNT/β-catenin activation can mediate T cell exclusion from tumor and primary resistance to anti-CTLA-4 + ant-PD-L1 therapy [1,2]. In addition, genetic loss of the tumor suppressor PTEN has been associated with defective T cell infiltration and primary resistance to PD-1 blockade [3]. However, whether secondary resistance might occur upon acquired oncogenic pathway alterations following initial response to immunotherapy is not known. We describe two metastatic melanoma patients who had a durable response to immunotherapy, but subsequently developed secondary resistance characterized by a phenotypic shift from a T cell- inflamed to non-T cell-inflamed tumor microenvironment, associated with oncogenic pathway alterations.

Methods

Case 1: A patient with metastatic melanoma was treated on a peptide/interleukin-12 vaccine protocol every 3 weeks for one year and achieved a partial response. Three years later a new metastatic lesion developed. Tumor gene expression profiling and histologic analysis for CD8 T cell infiltration and β-catenin expression were performed at baseline and at recurrence. Case 2: A patient with metastatic melanoma was treated with anti-CTLA-4 + PD-1 therapy and achieved a major partial response for a total of nineteen months. Baseline and treatment-resistant tumors underwent next-generation sequencing comprising a panel of commonly altered cancer genes for mutational and copy number analysis. Tumor biopsies were examined for CD8 T cell infiltration.

Results

Case 1: The baseline tumor prior to peptide vaccination demonstrated a T cell-inflamed gene signature and a robust intratumoral CD8 T cell infiltrate. In contrast, the recurrent treatment-resistant metastasis had a non-T cell inflamed phenotype and no infiltrating CD8 T cells. The new metastasis also acquired extensive expression of β-catenin, which was undetectable in the baseline lesion. Target antigens and circulating tumor-reactive T cells were detectable at the time of progression. Case 2: The on-treatment biopsy during anti-CTLA-4 + PD-1 therapy showed intratumoral CD8 T cells, while the recurrent metastasis lacked infiltrating CD8 T cells. The treatment-resistant metastasis uniquely harbored biallelic PTEN loss with no detectable PTEN protein present.

Conclusions

Our findings suggest that secondary resistance to immunotherapy may arise when tumor up-regulates β-catenin expression or undergoes genetic loss of PTEN, oncogenic events capable of driving T cell exclusion from the tumor microenvironment.

References

• 1. Spranger S, Bao R, Gajewski TF. Melanoma-intrinsic beta-catenin signalling prevents anti-tumour immunity. Nature 2015; 523:231-5.

• 2. Spranger S, Dai D, Horton B, Gajewski TF. Tumor-residing Batf3 dendritic cells are required for effector T cell trafficking and adoptive T cell therapy. Cancer Cell. 31:711-23.e4.

• 3. Peng W, Chen JQ, Liu C, et al. Loss of PTEN promotes resistance to T cell-mediated immunotherapy. Cancer Discovery. 2016; 6:202-16.

Ethics Approval

The study was approved by University of Chicago's Ethics board.

Consent

Consent was received

P559 Co-expression of TNF receptor 1 and 2 on human BRAF V600E+ melanomas is required for TNF-induced resistance to MAPK pathway inhibitors

Lazar Vujanovic, PhD, Cindy Sander, BS, Jian Shi, MD, John Kirkwood, MD, Lisa Butterfield, PhD

University of Pittsburgh, Pittsburgh, PA, USA

Correspondence: Lazar Vujanovic (vujanovicl@upmc.edu)

Background

The effectiveness of MAPK cascade-targeting therapies to treat patients with BRAF-V600E-mutant melanomas has been limited by a range of resistance mechanisms that may be driven by the tumor necrosis factor (TNF). TNF signaling is mediated through TNF receptor type-1 (TNFR1) and TNF receptor type-2 (TNFR2). TNFR1 signaling mediates apoptosis or cell survival/cytokine secretion, while TNFR2 selectively mediates cell survival/cytokine secretion. Although TNFR1 and TNFR2 are preferentially activated by soluble (sol)TNF and transmembrane (tm)TNF, respectively, they can crosstalk via shared signaling molecules. While TNF receptor 1 (TNFR1) is ubiquitously expressed, little is known about the expression patterns and functional roles of TNFR2 on melanomas.

The primary goals of this study are to evaluate whether TNFR2 is expressed on melanoma, to determine which TNFR mediates TNF- mediated resistance reprogramming to MAPK inhibitors (MAPKi) and to decipher whether INB03, a dominant-negative TNF biologic and specific antagonist of solTNF, can antagonize this therapeutic resistance pathway.

Methods

TNFR1/2 expression patterns on BRAF-mutant melanomas were evaluated by multi-color flow cytometry. Recombinant TNF was used to induce MAPKi resistance in melanomas. Activated human macrophages were used in transwell co-culture systems to induce MAPKi resistance in melanomas. The effectiveness of INB03 to antagonize this therapeutic resistance pathway was compared to an anti-TNF antibody and a selective NF-kB inhibitor. CRISPR/Cas9 system was utilized to edit out TNFR1 and TNFR2 on a melanoma cell line, and these knockout variants were used to test the intrinsic roles of these receptors in TNF-induced resistance to MAPKi. MTT viability assay was used as the readout for melanoma sensitivity to MAPKi.

Results

TNFR1 and TNFR2 were co-expressed by 48% of BRAF-V600E-mutant melanoma cell lines and primary melanomas. Interestingly, only cell lines that co-expressed TNFR1 and TNFR2 could acquire MAPKi resistance in response to recombinant and macrophage-derived TNF. Functional studies of TNFR1 and TNFR2 knockout cell lines indicated that both TNFR1 and TNFR2 signaling were necessary for the TNF-mediated induction of resistance to MAPKi. Finally, selective sequestration of both recombinant and macrophage-derived TNF using INB03 effectively prevented acquisition of resistance to MAPKi by BRAF-V600E mutant melanoma cell lines in vitro.

Conclusions

solTNF-mediated induction of MAPKi resistance in BRAF-V600E-mutant melanomas is predicated on the co-expression of TNFR1 and TNFR2. Our data indicate that almost half of BRAF-V600E-mutant melanomas express TNFR2. These results indicate that TNFR2 could be a biomarker that could be used to select for melanoma patients that could benefit from TNF-targeting therapies.

Acknowledgements

The authors thank David E. Szymkowski, Ph.D. of Xencor Inc. for providing the dominant-negative TNF biologic. This study was supported by the UPCI SPORE in melanoma and skin cancer (P50 CA121973) Developmental Research Project (DRP) award.We thank the following UPCI shared resources (supported in part by NIH P30CA047904): Flow Cytometry Facility and the Immunologic Monitoring Laboratory (Luminex).

Ethics Approval

Specimens collection was performed under IRB-approved protcol UPCI-96-099

P560 Resistance of CD44+ subpopulation to CTL though high production a protease inhibitor in colorectal cancer

Tomonori Yaguchi, MD, PhD, Tsubasa Miyauchi, Kenji Morii, MS, Yutaka Kawakami, MD PhD

Keio University School of Medicine, Tokyo, Japan

Correspondence: Yutaka Kawakami (yutakawa@keio.jp)

Background

Colorectal carcinoma (CRC) is generally resistant to immunotherapies, suggesting possible CRC-specific immunosuppressive mechanisms. In this study, we have identified markers which could define particular subpopulation harboring immuno-resistant properties and investigated the underlying mechanisms of the immunosuppression.

Methods

We analyzed the expression pattern of 30 CD (cluster of differentiation) antigens on 10 human CRC cell lines. We sorted a CRC cell line into the CD44-positive fraction and the CD44-negative fraction, and evaluated their sensitivity to CTL lysis. Gene expression profiles of the CD44-positive CRC fraction which showed resistance to CTL lysis were compared with those of the CD44-negative CRC fraction using the cDNA micro array. For the functional analysis of protease inhibitor X (PI-X) which was preferentially expressed in CD44-positive fraction, we evaluated the effect of PI-X knockdown by siRNA or PI-X overexpression in CRC cell lines on the sensitivity to CTL lysis in vitro and the effect of PI-X overexpression in murine CRC cells on the therapeutic efficacy of anti PD- 1 therapies in vivo. We also evaluated the correlation of the PI-X expression in human CRC and T cell infiltration and the patients’ prognoses by the analyses of immunohistochemistry and TCGA RNA-seq data.

Results

10 out of 30 tested CD antigens were heterogeneously expressed on the human CRC cell lines. Among these 10 CD antigens, we found that the CD44-positive fraction in human CRC cell lines were more resistant to tumor specific CTL-mediated killing compared to the CD44-negative fraction. cDNA microarray analysis revealed the CD44- positive fractions more highly expressed protease inhibitor X (PI-X) than the CD44-negative fractions. The expression level of PI-X was also positively correlated with that of CD44 in TCGA RNA-seq database. PI-X showed the highest expression in CRC among 17 human cancer tissues in meta-analysis using open-access gene expression data. The experiments of PI-X overexpression or PI-X knockdown in CRC cell liens showed PI-X could suppressed CTL-mediated killing. TCGA RNA-seq data showed the negative correlation between PI-X expression and CD8 expression. Moreover, analyses of immunohistochemistry and TCGA RNA-seq data revealed the best prognosis of the patients with low PI-X expression and high CD8+ T cell infiltration. In tumor-bearing mouse models, over expression of PI-X in a murine cancer cell line induced the resistance to anti PD-1 Ab therapies.

Conclusions

CD44 and PI-X may be potential biomarkers for prognosis and responses of CRC patients to cancer therapies including PD-1 blockade, and also be attractive therapeutic targets for combination immunotherapies.

Ethics Approval

The study was approved by Keio Univ. school of medicine Institutution‘s Ethics Board, approval number 20P16

P561 A new mechanism of ADCC resistance

David Zahavi, MS, BS¹, Dalal Aldeghaither¹, Louis Weiner, MD¹, Joseph Murray, MD, PhD², Elana Fertig², Garrett Graham¹, Yong-Wei Zhang¹, Allison O'Connell, MD/PhD Candidate¹, Junfeng Ma¹, Sandra Jablonski, PhD¹

¹Georgetown University, Washington, DC, USA; ²Johns Hopkins University, Ellicott City, MD, USA

Correspondence: Louis Weiner (weinerl@georgetown.edu)

Background

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism underlying targeted monoclonal antibody (mAb) therapy in cancer. The majority of patients develop resistance to mAb therapy; however the resistance mechanisms are not well characterized. In vitro modeling of ADCC provides an experimental system for uncovering tumor cell immune resistance mechanisms.

Methods

We continuously exposed epidermal growth factor receptor (EGFR) positive A431 cells to KIR-deficient NK92-CD16V effector cells and the anti-EGFR mAb Cetuximab.

Results

Persistent ADCC exposure yielded ADCC-resistant cells, that when compared with control ADCC-sensitive cells, exhibited reduced EGFR expression, overexpression of histone- and interferon-related genes, failure to activate NK cells, and no evidence of epithelial to mesenchymal transition. These properties gradually reversed following withdrawal of ADCC selection pressure. Remarkably, ADCC-resistant cells possessed lower expression of multiple cell surface molecules that contribute to intercellular interactions and immune synapse formation. Classic immune checkpoints did not modulate ADCC in this unique model system of immune resistance.

Conclusions

We show that the induction of ADCC resistance involves genetic and epigenetic changes that lead to a general loss of target cell adhesion properties required for the establishment of an immune synapse, killer cell activation, and target cell cytotoxicity.

Acknowledgements

We would like to thank Dr. Kerry Campbell for providing the NK92-CD16V cells.

Mechanisms of Toxicity

P562 Delayed immune-related events after discontinuation of immunotherapy – DIRE syndrome?

Marcus Couey, MD, DDS¹, Bell, MD, DDS, FACS¹, Ashish Patel, MD², Marka Crittenden, MD, PhD¹, Brendan Curti, MD¹, Rom Leidner, MD¹

¹Earle A. Chiles Research Institute, Portland, OR, USA; ²Providence Cancer Center, Portland, OR, USA

Correspondence: Marcus Couey (marcus.couey@providence.org)

Background

Although the temporality of immune-related adverse events (irAE) is well-recognized during immunotherapy to be highly variable and often delayed,[1] post-immunotherapy irAE are rarely described and potentially under- recognized. In 2013, two cases were reported in abstract form in Deutschen Dermatologischen Gesellschaft.[2] In July 2018 a case of autoimmune hepatitis eight months post-immunotherapy was reported in The Oncologist[3] and a dermatologic series appeared online in JAMA Dermatology.[4] With expanding indications for IO and an increasing number of clinical trials in the curative-neoadjuvant setting, larger numbers of patients are being treated in earlier stages of disease and often for short courses. Given this trend, under-recognition of delayed immune- related events (DIRE) after completion of immunotherapy could pose a growing clinical hazard.

Methods

We performed a literature review in PubMed and Google Scholar (search terms included in Table 1); DIRE syndrome was defined as immune-related events post-immunotherapy, newly incident beyond two elimination half- lives (t 1/2) of drug.

Results

We identified 10 cases, 6 by literature review (5 melanoma, 1 cutaneous SCC) and an additional 4 cases at our institution (4 HNSCC). Median cumulative immunotherapy exposure was 4 doses (range: 2 to 22 doses). Median interval from last immunotherapy dose to DIRE onset was 5 months (range: 2 to 28 months). All literature cases were in the recurrent/metastatic context; we report four cases in the curative-neoadjuvant context (italicized) with one recurrence.

Conclusions

An influx of neoadjuvant clinical trial design over the last 2-3 years, incorporating brief IO exposure (typically checkpoint blockade) followed by surgical resection and/or adjuvant therapy, is attracting interest in multiple tumor types in the curative setting.[5–9] In this context, it will be necessary to recognize an emerging phenomenon, which we have termed DIRE syndrome (delayed immune-related events). Clinical vigilance has the potential to reduce morbidity from delayed diagnosis, as these conditions are generally manageable with prompt initiation of treatment; or from misdiagnosis, to avert unnecessary/harmful interventions (in the autoimmune meningitis case we report, an Omaya reservoir was placed at an out-of-state hospital based on erroneous diagnosis of leptomeningeal carcinomatosis). Several factors confound diagnosis in the neoadjuvant-IO context: 1) intervening treatments with potentially overlapping toxicities; 2) brief and remote IO exposure; 3) reduced vigilance during NED surveillance, in contrast to active disease follow-up; 4) protracted process of diagnosis-by-exclusion. DIRE syndrome should therefore figure prominently in the differential diagnosis of patients presenting with diseases of unclear etiology, irrespective of elapsed post-immunotherapy interval.

References

• 1. Champiat S, et al. Management of immune checkpoint blockade dysimmune toxicities: a collaborative position paper. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol. 2016;27:559–574.

• 2. Abstracts of the 8th World Congress of Melanoma, the 9th Congress of the European Association of Dermatology (EADO), the 7th Interdisciplinary Melanoma/Skin Cancer Meeting, and the 3rd European Post-Chicago Melanoma Meeting. July 17-20, 2013. Hamburg, Germany. J. Dtsch. Dermatol. Ges. J. Ger. Soc. Dermatol. 2013;JDDG 11 Suppl 7:1–119.

• 3. Parakh S, Cebon J, Klein O. Delayed autoimmune toxicity occurring several months after cessation of anti-PD-1 therapy. The Oncologist. 2018;23:849–851.

• 4. Wang L, et al. Timing of onset of adverse cutaneous reactions associated with programmed cell death protein 1 inhibitor therapy. JAMA Dermatol. 2018; doi:10.1001/jamadermatol.2018.1912

• 5. Forde P, et al. Neoadjuvant PD-1 blockade in resectable lung cancer. N. Engl. J. Med. 2018;378:1976–1986.

• 6. Uppaluri R, et al. Neoadjuvant pembrolizumab in surgically resectable, locally advanced HPV negative head and neck squamous cell carcinoma (HNSCC). J. Clin. Oncol. 2017;35:6012–6012.

• 7. Ferris R, et al. LBA46An open-label, multicohort, phase 1/2 study in patients with virus-associated cancers (CheckMate 358): Safety and efficacy of neoadjuvant nivolumab in squamous cell carcinoma of the head and neck (SCCHN). Ann. Oncol. 2017;28.

• 8. Necchi, A. et al. Interim results from PURE-01: A phase 2, open-label study of neoadjuvant pembrolizumab (pembro) before radical cystectomy for muscle-invasive urothelial bladder carcinoma (MIUC). J. Clin. Oncol. 2018;36:TPS533-TPS533.

• 9. Powles, T. et al. A phase II study investigating the safety and efficacy of neoadjuvant atezolizumab in muscle invasive bladder cancer (ABACUS). J. Clin. Oncol. 2018;36: 4506–4506.

Table 1 (abstract P562). See text for description

P563 Characterizing immune-mediated adverse events of anti-PD-1 and anti-CTLA-4 monotherapies and combinations using a quantitative model based meta-analysis

Gabriel Helmlinger, PhD¹, Boris Shulgin, PhD², Yuri Kosinsky, PhD², Andrey Omelchenko, PhD², Lulu Chu, PhD¹, Ganesh Mugundu, PhD¹, Garrett DeYulia, PhD¹, Rodrigo Pimentel, MD¹, Kirill Peskov, PhD²

¹AstraZeneca, Waltham, MA, USA; ²M&S-Decisions LLC, Moscow, Russian Federation

Correspondence: Gabriel Helmlinger (gabriel.helmlinger@astrazeneca.com)

Background

Immune checkpoint inhibitors (ICIs) may be associated with treatment-mediated and immune-mediated adverse events (trAEs and imAEs, respectively), in particular when used in combination. The objective of this model-based meta-analysis (MBMA was to characterize immune mediated AEs of anti-PD-1 and anti-CTLA-4 monotherapies and combinations, normalized by drug exposure and drug potency.

Methods

We performed an exhaustive search of clinical ICI safety data in PubMed-Medline, the ASCO Abstracts, TrialTrove, and other sources. To quantitatively compare safety across monotherapy and combination studies, we normalized the data by drug exposure, as derived from pharmacokinetic models for anti-PD-1 and anti-CTLA-4 agents, and by drug potency. A cross-study model-based meta-analysis (MBMA) of safety was then performed for Grade 3&4 trAEs and imAEs, for five organ classes: gastrointestinal (GI), skin, pulmonary, hepatic, and endocrine. Exposure vs. safety meta-regression and subgroup analyses were implemented with the R software metafor package.

Results

Safety data from 133 cohorts out of 82 publications on 65 clinical trials were built into the study-level database for ICI trAEs and imAEs. Performing the corresponding MBMA, we determined: (a) no significant dependence on dose, for both Grade 3&4 total AEs and imAEs, under anti-PD-1 monotherapies, except for rare GI imAEs; (b) significant dose dependence, for both Grade 3&4 total AEs (increase from 23% [low] to 40% [high dose]) and hepatic AEs (increase 1% to 7%), under anti-CTLA-4 monotherapies, (c) significant anti-CTLA-4 dose dependence, for both Grade 3&4 total AEs (increase from 35% to 52%) and hepatic (increase 6.5% to 19%), GI, skin and endocrine AEs, under anti-CTLA-4 and anti-PD-1 combination treatments. AE rates for anti-PD-1 and anti-CTLA-4 combinations were supra additive vs. AE rates in the respective monotherapy, with an increase of ~10% for Grade 3&4 AES and ~15% for Hepatic AEs. Furthermore, under combination treatments, AE rates were from 5% (monotherapy) up to 20% (combination) higher in 1L patients (vs. other lines of treatment) and also about 15% higher in patients with positive PD-L1 status (vs. PD-L1 negative). Higher AE rates, generally, were also associated with higher efficacy responses to ICI therapies (Figure 1).

Conclusions

A comprehensive database combined with an exposure/potency-normalized MBMA of ICI-related imAEs enabled a quantitative comparison of AEs across anti-PD-1 / CTLA-4 mono- and combination therapies, and in relation to key patient characteristics (PD-L1 status, line of treatment) and efficacy measures. This analysis may support rational dose selection and can be applied to other ICI agents, in mono- and combination treatment settings.

Fig. 1 (abstract P563).

Dependence of Grade 3&4 AEs upon ICI drug exposure

P564 Interleukin-6 gene expression is highly upregulated in immune checkpoint mediated enterocolitis

Daniel Johnson, MD¹, Cara Haymaker, PhD¹, Khalida Wani, PhD¹, Wai Chin Foo, MD¹, Salah Eddine Bentebibel¹, Yinghong Wang, MD, PhD¹, Jonathan Curry, MD¹, Adi Diab, MD¹, Jennifer Wargo, MD, MMSc², Alexandre Reuben², Elizabeth Burton²

¹MD Anderson Cancer Center, Houston, TX, USA; ²MD Anderson, Houston, TX, USA

Correspondence: Adi Diab (adiab@mdanderson.org)

Background

A deep understanding of the immunobiology of checkpoint inhibitor (CPI) induced immune related toxicities (irAEs) could lead to development of strategies that uncouple autoimmunity from anti-tumor immunity. Immune- related enterocolitis (irEC) is the most common serious complication from CPIs. Interleukin-6 (IL-6) is a key cytokine in autoimmunity (rheumatoid-arthritis, inflammatory-bowel disease) contributing to acute and chronic inflammation and is an essential differentiating cytokine committing naïve CD4+T-cells into T-helper17 (Th17) lineage. The role of Th17 cells in irAEs is not fully explored, and their tumor immunity function is controversial. Through RNA gene-expression profiling, we sought to identify the critical immune pathways in irEC and how these compare to the immune signatures in CPI-responding tumor samples.

Methods

Total RNA from patient-matched irEC and normal colon FFPE tissue from patients [n=12] receiving CPIs (aPD-1 = 3, aCTLA-4= 7, aCTLA-4 + aPD-1 = 2) were profiled with the NanoString nCounter PanCancer Immune Profiling Panel (NanoPCIP). Of the 770 NanoPCIP-panel genes, fold change in gene expression were compared between the normal and inflamed colonic tissue using two-sample T tests. P-values were corrected using Benjamin-Yekutieli adjusted false discovery rate, and an adjusted p-value < 0.05 were considered significant. We also summarized fold- changes in gene-expression in CPI-responding melanoma tumors from a longitudinal NanoPCIP panel immune signature analysis previously performed at our institution.

Results

Significantly upregulated differentially expressed genes (DEGs) in the inflamed irEC tissue was observed in 52 genes compared to the normal colon control (adjusted p<0.05; figure 1). The highest up-regulated DEG encoded for IL6 (Fold change of +24.1). Other genes highly upregulated included IL-11 (a member of the IL-6-type cytokine- family) and genes that encode chemotactic molecules (Table-1).In our melanoma historical control, 173 DEGs significantly upregulated with a-CTLA-4 treatment (pre vs. on-treatment tumors) had a significant interaction with treatment-response. Only one gene was concordantly in our highest up-regulated DEGs in colitis (CXCL2). Most of our 10 highest colitis DEGs were not significantly up-regulated in tumors of CPI-responders compared to non- responders (Table-1). None of the 10 genes significantly and highest up-regulated in responding tumors were significantly upregulated in irEC (Table-2).

Conclusions

IL6 was the most significantly upregulated gene within inflamed-irEC samples compared to matched controls; the majority were not upregulated in association with tumor-response to CPIs. Our data suggest that IL-6 is important in irEC. IL-6-mediated inflammation may be more prevalent in irEC than in the responding tumors; targeting IL-6 may ameliorate irEC without hindering anti-tumor immunity.

Table 1 (abstract P564). See text for description

Table 2 (abstract P564). See text for description

Fig. 1 (abstract P564).

See text for description

P565 Characterization of lymphoid cells in synovial fluid from cancer patients with immunotherapy-associated arthritis

Sang Kim, MD, PhD, Roza Nurieva, PhD, Jean Tayar, MD, Huifang Lu, MD, PhD, Jennifer Wang, MD, Don Gibbons, MD, Guillermo Garcia-Manero, MD, Maria Suarez-Almazor, MD, PhD, Patrick Hwu, MD, Adi Diab, _MD

MD Anderson Cancer Center, Houston, TX, USA

Correspondence: Adi Diab (adiab@mdanderson.org)

Background

Immune related arthritis (ir-arthritis) is well documented Checkpoint inhibitors (ICIs) toxicity [1]. Ir AR occur in ~2% of cancer patients who receive ICI [2,3,4]. Although it is not life-threatening toxicity, it is clinically symptomatic toxicity and can severely impact the patients’ quality of life and can lead to disconsolation of ICI treatment. Usually, ir-arthritis requires a substantially prolonged period of immune suppression compared to other irAEs, which we may negatively impact and compromise the clinical anti-cancer benefit. A deeper understanding of the immunobiology of ir-arthritis and detailed immune-characterization of the inflamed tissues will possibly allow us to develop treatment strategies that lead to uncoupling autoimmunity from anti-tumor immunity. Here, we characterize synovial immune cells isolated from five patients who developed ir-arthritis post-ICI.

Methods

We analyzed synovial fluid from five symptomatic patients, who developed ir-arthritis (joint pain and swelling) after ICI treatment. Using flow cytometry, we stained lymphoid immune cells with lineage-specific markers and measured effector cytokines in the CD4+ T cell populations.

Results

Median time of joint aspiration from the first ICI-infusion was 34 weeks (Range [4,166]). Arthritis was initially treated with systemic or local injection of prednisone. Two patients achieved complete resolution of arthritis (“steroid responders”) while three patients remained refractory with partial relief/response to prednisone requiring additional/alternative treatment (“steroid refractory”). Immune analysis demonstrated that CD4+ T cells (53.1±13.2%) most abundant lymphoid immune-cells followed by CD8+ T cells, NK cells, NK T cells, and B cells. Most CD4+ and CD8+ T cells were effector memory phenotype. Several relevant CD4+ T-cell subsets were identified in the synovial fluid, including regulatory T cells (Treg), naïve T cells, Th1 (CXCR3hi CCR6lo), Th17.1 (CXCR3hi CCR6hi), Th17 (CXCR3lo CCR6hi), and follicular helper T cells (CXCR5+). Effector CD4+ T cell cytokines, including interferon gamma, IL-4, IL-17, and IL-21, were produced by both Tregs and non-Tregs. Interestingly, IL-17 producing non-Tregs were expanded in synovial fluid from steroid –refractory patients compared to synovial fluid from steroid-responders, suggesting that Th17 cells might play a role in persistent ir- arthritis and steroid resistance.

Conclusions

Although our preliminary data is very limited and descriptive in nature, we find the observed expansion of IL-17 producing non-Tregs in synovial fluid from steroid- refractory very intriguing and consistent with our prior recent publication of a successfully treated patients with steroid resistant ir-arthritis with IL-6 blockade [5]. Larger and prospective studies with longitudinal collection of tissue and blood are planned.

References

• 1. Suarez-Almazor ME, Kim ST, Abdel-Wahab N, et al. Review: Immune-Related Adverse Events With Use of Checkpoint Inhibitors for Immunotherapy of Cancer. Arthritis Rheumatol 2017;69(4):687-99.

• 2. Cappelli LC, Gutierrez AK, Baer AN, et al. Inflammatory arthritis and sicca syndrome induced by nivolumab and ipilimumab. Annals of the rheumatic diseases 2017;76(1):43-50.

• 3. Robert C, Schachter J, Long GV, et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. The New England journal of medicine 2015;372(26):2521-32.

• 4. Prieto PA, Yang JC, Sherry RM, et al. CTLA-4 blockade with ipilimumab: long-term follow-up of 177 patients with metastatic melanoma. Clin Cancer Res 2012;18(7):2039-47.

• 5. Kim ST, Tayar J, Trinh VA, et al. Successful treatment of arthritis induced by checkpoint inhibitors with tocilizumab: a case series. Annals of the rheumatic diseases 2017;76(12):2061-64.

Ethics Approval

The study was approved by MD Anderson Cancer Center's ethic board (Protocol number: PA16-0935)

P566 Hypoalbuminemia as a predictor factor for immune related adverse events (irAEs) in advanced melanoma patients treated with immune checkpoint inhibitors (ICIs)

E Rahma, MD², Steven Blum, MD², Jeffrey Ishizuka, MD,DPhil², Taha Qazi, MD³, Rawad Elias, MD⁴, Kruti Vora, BA⁵, Alex Ruan⁵, Anita Giobbie-Hurder, MS², Shilpa Grover, MD, MPH³, Rizwan Haq, MD, PhD², Meredith Davis², Maria Gargano, PA-C², Elizabeth Buchbinder, MD², Patrick Ott, MD, PhD², F. Stephen Hodi, MD²

¹Dana-Farber Cancer Institute, Boston, MA, USA; ²Dana Farber Cancer Institute, Boston, MA, USA; ³Brigham and Women Hospital, Boston, MA, USA; ⁴Boston Medical Center, Hartford, CT, USA; ⁵Harvard Medical School, Boston, MA, USA

Correspondence: E Rahma (osamae_rahma@dfci.harvard.edu)

Background

Despite the expansion of Immune checkpoint inhibitors (ICIs) indications there is limited data to date on predictors of ICIs’ immune related adverse events (irAEs).

Methods

Melanoma patients (pts) who received anti-CTLA-4 (ipilimumab), anti-PD-1 (pembrolizumab or nivolumab), or the combination at the Center for Immuno-Oncology and Melanoma at DFCI in the past 5 years (up to December 31- 2016) were included. Patients with irAEs were divided into 2 groups based on the severity of AEs: Grade (G) 1-2 and ≥ G 3. We analyzed the following patient characteristics and their prediction for irAE grade: Gender, age, BMI, ECOG, smoking and alcohol history, Flu or pneumonia vaccine administered within 6 months of starting ICIs, infections while on ICIs, history of autoimmune disease, asthma, and seasonal allergies. The following lab values were collected prior to starting ICIs: Albumin, LDH, neutrophil/lymphocyte ratio, and eosinophil count. We also analyzed the following: Prior chemo, targeted or radiation therapy, the presence of Kit, BRAF, or NRAS mutation, the number of metastatic sites, and concomitant medications (ACE and ARB inhibitors, NSAID, PPI, statins, antibiotics and vitamin D). Multivariable logistic regression of grade 3-4 vs. grade 1-2 irAEs was fit using the preceding characteristics as candidate predictors.

Results

We identified 213 patients who received a total of 246 ICIs (44 pts had 2 and 5 pts had 3 ICIs). The maximum grade irAEs reported were: G1 or 2 (92 pts, 43%), G 3 or 4 (121 pts, 57%). Table 1 summarizes the type of ICI the patient was taking at the time of the worst grade irAE. Patients who received combination of ICIs had significantly increased risk of grade 3-4 irAEs compared with patients who received single ICI. Patients with albumin levels above 4.2 had significantly reduced risks of G 3-4 irAEs compared with patients who had lower albumin level (table 2).

Conclusions

This is the first report to identify hypoalbuminemia as a predicting factor for the development of grade 3-4 irAEs while on ICIs. Hypoalbuminemia could represent poor nutritional status that may predispose patients to irAEs. We are in the process of performing correlative analyses using cytokine Luminex to identify inflammatory markers that could predict toxicity, and this will be correlated with the observation of an association between hypoalbuminemia and higher incidence of grade 3-4 irAE.

Acknowledgements

Parker Institute for Cancer Immunotherapy for providing funding for this project

Ethics Approval

The study was approved by Dana-Farber Institutional Review Board (IRB)

Table 1 (abstract P566). Type of ICI received at time of worst irAEs grade

Table 2 (abstract P566). Prediction Model for irAEs

P567 A meta-analysis of immune checkpoint inhibitors tumor type and dose-toxicity correlation

E Rahma, MD¹, E Rahma, MD¹, E Rahma, MD¹, E Rahma, MD¹, Joshua Reuss, MD², Ghazaleh Shoja E Razavi, MD³, Rawad Elias, MD⁴, Anita Giobbie-Hurder, MS¹, Samir Khleif, MD⁵

¹Dana-Farber Cancer Institute, Boston, MA, USA; ²Sidney Kimmel Cancer Center, Baltimore, MD, USA; ³Tom Baker Cancer Centre, Alberta, Canada; ⁴Boston Medical Center, Hartford, CT, USA; ⁵Georgetown University, Washington, DC, USA

Correspondence: Samir Khleif (snk48@georgetown.edu)

Background

The relationship between ICIs dose-escalation and toxicity has not been established. We performed a meta-analysis of clinical trials investigating ICIs to understand whether there is a correlation between dose or disease type and toxicity.

Methods

We searched PubMed and abstracts presented at national and international meetings for trials (T) using FDA-approved ICIs including Ipilimumab, Atezolizumab, Nivolumab, and Pembrolizumab. The rates of treatment-related grade 3-5 adverse events (G3/4 AEs) were collected and the overall incidence rates for each dose cohort (DC) were estimated using exact binomial methods. Generalized linear models with GEE were fit to assess important predictors of G3/4 AEs.

Results

A total of 52 T published between January 2010 and December 2017 were reviewed. The overall incidence rate of G3/4 AEs was 34% in melanoma T using Ipilimumab. Patients (Pts) treated at 3 mg/kg q3w (3 T) had 27% reduced risk of G3/4 AEs compared to 10 mg/kg q3w (3 T) (Figure 1, Table 1). There was no difference in the incidence of G3/4 AEs for urothelial cancer (2 T) vs. NSCLC (3 T) using Atezolizumab (1200mg q3w) (Figure 2, Table 2). The investigation of Nivolumab included 39 DC within 24 different T. We compared the following DC: 2mg/kg q3w (2 DC), 3 mg/kg q2w (20), ≤ 1mg/kg q2w (8), ≤ 1mg/kg q3w (2), 10 mg/kg q2w (4), 10 mg/kg q3w (3). The overall incidence rate of G3/4 AEs was 22% which was significantly lower for pts with NSCLC than any of the other tumor types (26-40% reductions). No relationships between dose and incidence of AEs were noted (Figure 3, Table 3). The Pembrolizumab analysis consisted of 23 DC from 17 reported T. Frequencies of DC: 2 mg/kg q3w (3), 200 mg q3w (8), 10 mg/kg q3w (5), 10 mg/kg q2w (7). The incidence of G3/4 AEs was significantly lower in melanoma compared to any of the other tumor types (20% risk reduction). Pts receiving flat dose of 200mg had significantly lower odds of G3/4 AEs compared to 2 mg/kg q3 (P= 0.04) or 10 mg/kg q2 or 3w (P= 0.01) (Figure 4, Table 4).

Conclusions

This is the largest meta-analysis to date investigating dose-toxicity relationship of ICIs. There is a clear correlation between increased dose and toxicity using CTLA-4 antibodies (Ipilimumab). However, there is no evidence of dose-toxicity correlation using Nivolumab, while a flat dose of Pembrolizumab was associated with lower toxicity compared to weight-based dose.

Fig. 1 (abstract P567).

Incidence of Grade 3-5 Adverse Events Ipilimumab

Fig. 2 (abstract P567).

Incidence of Grade 3-5 Adverse Events Atezolizumab

Fig. 3 (abstract P567).

Incidence of Grade 3-5 Adverse Events Nivolumab

Fig. 4 (abstract P567).

Incidence of Grade 3-5 Adverse Events Pembrolizumab

Table 1 (abstract P567). Dose-Toxicity Correlation of Ipilimumab

Table 2 (abstract P567). Tumor Type-Toxicity Correlation of Atezolizumab

Table 3A (abstract P567). Tumor Type-Toxicity Correlation of Nivolumab

Table 3B (abstract P567). Dose-Toxicity Correlation of Nivolumab

Table 4A (abstract P567). Tumor Type-Toxicity Correlation of Pembrolizumab

Table 4B (abstract P567). Dose-Toxicity Correlation of Pembrolizumab

P568 Pembrolizumab induced multiple immune related adverse events including myasthenia gravis, hepatitis and thyroiditis in a patient with thymoma

Kyunghoon Rhee, MD¹, Taeyeong ko, MD¹, Sangmin Chang, MD¹, Ji Hyun Rhee, MD², Lee Chun Park, MD¹, Young Kwang Chae, MD¹

¹Northwestern Univ, Feinberg School Med, Towson, MD, USA; ²Greater Baltimore Medical Center, Towson, MD, USA

Correspondence: Young Kwang Chae (young.chae@northwestern.edu)

Background

The rapidly developing field of cancer immunotherapy has shown remarkable progress in its utilization as a treatment option of various tumors in recent years. Although it is currently not recommended as a first line treatment for thymoma, pembrolizumab, which blocks the PD-1 pathway thereby enhancing the immune system, is used as an alternative option in treatment. However, it was shown to also induce rare immune-related adverse events (irAEs) in multiple organs [1, 2], which have been reported in limited cases.

Methods

Here we report a case of possible pembrolizumab induced myasthenia gravis (MG), hepatitis and thyroiditis.

Results

A 60-year-old female with metastatic thymoma on her second cycle of pembrolizumab presented with worsening SOB for two weeks, left ptosis, limited extra-ocular movement, lower bifacial, upper and lower extremity weakness. She was thought to have either pembrolizumab induced MG or unmasking of occult thymoma related MG, supported by elevated acetylcholine receptor binding antibodies. She was treated with pyridostigmine, IVIG, and plasmapheresis. On labs, TSH was found to be increased and free T4 decreased. Considering her normal thyroid functions before immunotherapy and the rapid development of hyperthyroidism within 2 weeks after the second cycle, pembrolizumab induced thyroiditis was suspected.In addition, she had gradually increasing Alk-P, AST, ALT and total bilirubin. Liver biopsy demonstrated marked portal and lobular T-cell infiltration with bile duct injury, consistent with immune modulator drug effect. She was treated with steroids and Cellcept with improvement in her LFTs. However, she developed septic shock and died.

Conclusions

This is a patient with stage IV thymoma on pembrolizumab who developed multiple irAEs. It is important to have a high clinical suspicion of such irAEs, and not only to discontinue the culprit PD-1 inhibitor but also to start early treatment for each involved organ. Since pembrolizumab is not a standard treatment of stage IV thymoma, there are only few reports of irAEs in thymoma. We do not know if pembrolizumab induced a new onset MG or exacerbated underlying MG. It is also unclear if simultaneous development of MG, hepatitis and thyroiditis is only unique in thymoma. Further investigation of irAEs in thymoma patients on pembrolizumab is therefore warranted.

References

• 1. Hofmann L et al. Cutaneous, gastrointestinal, hepatic, endocrine, and renal side-effects of anti-PD-1 therapy. Eur J Cancer. 2016 Jun;60:190-209

• 2. Zimmer L et al. Neurological, respiratory, musculoskeletal, cardiac and ocular side-effects of anti-PD-1 therapy. Eur J Cancer. 2016 Jun;60:210-25.

Consent

Consent top publish was received.

Microbiome and Anti-Tumor Immunity or I-O Agent Toxicity

P569 Novel Pharmacobiotic approach to enhance the tamoxifen efficacy using bacterial extracellular vesicles as the immunotherapy in breast cancer

Jeongshin An, MD,PhD¹, Yeun-yeoul Yang¹, Won-Hee Lee², Jinho Yang², Jong-kyu Kim¹, HyunGoo Kim¹, Se Hyun Paek¹, Jun Woo Lee¹, Joohyun Woo¹, Jong Bin Kim¹, Hyungju Kwon¹, Woosung Lim¹, Nam Sun Paik¹, Yoon-Keun Kim²

¹Ewha Womans University, Seoul, Korea, Republic of; ²MD healthcare company, Seoul, Korea, Republic of

Correspondence: Jeongshin An (rulru81@hanmail.net)

Background

The anti-cancer effect of bacteria has a long history. According to Bierman et al., spontaneous remission of cancer has been observed in patients with severe bacteremia[1]. The reason was not revealed at that time, but we studied that in breast cancer. There are four main ways in which microbiota affects cancer: probiotics, prebiotics, drugs that target microbial enzymes and microbial products that have anticancer properties[2]. Among them, bacterial extracellular vesicles(EVs) are one of microbial products. In this study, we investigated the effects of bacterial EVs on the growth of breast cancer cells and tamoxifen efficacy.

Methods

Here, we analized microbiota of urine samples by NGS to select the target EVs that were expected to affect the growth of breast cancer cells. A total of 347 female urine samples – from 127 breast cancer patients (cancer group) and 220 normal individuals (control group) – were collected and analyzed by NGS using a universal bacterial primer of 16S rDNA. Human breast cancer cells were cultured, and the cells were treated with EVs of S. aureus and K.pneumoniae for 72 h. Real-time polymerase chain reaction (PCR) and Western blotting for signalling molecule analysis were performed after treatment of EVs in each breast cancer cell.

Results

There was a significant difference in the distribution of bacterial EVs between the urine samples from breast cancer patients and from normal controls. Especially, S.aureus EVs were predominant in the normal group, and K.pneumoniae was abundant in the breast cancer group. Therefore, we selected these two bacterial EVs that may have an effect on breast cancer cell growth. We found that S.aureus and K.pneumoniae EVs down-regulated cell growth in MDA-MB-231 cells. We also found that S.aureus or K.pneumoniae EVs had a synergic effect on growth inhibition of while co-treated with tamoxifen. S.aureus EVs down-regulated mRNA expression of cyclin E2 and up- regulated that of TNF-alpha which was related ERK pathway while co-treated with tamoxifen.

Conclusions

The anti-cancer effect of S.aureus and K.pneumoniae was initiated by its bacterial EVs and consequently inhibited the growth of breast cancer cells in triple negative breast cancer cells and improved the efficacy of tamoxifen in ER-positive cells. In the near future, we plan to conduct animal studies which are expected to further clarify the effect of bacterial EV on breast cancer.

References

1 Bierman, H. R. et al. Remissions in leukemia of childhood following acute infectious disease. Staphylococcus and streptococcus, varicella, and feline panleukopenias. Cancer 6, 591-605 (1953).2 Zitvogel, L., Daillère, R., Roberti, M. P., Routy, B. & Kroemer, G. Anticancer effects of the microbiome and its products. Nature Reviews Microbiology 15, 465 (2017).

Ethics Approval

The study was approved by Ewha Womans University Medical Center‘s Ethics Board.

Consent

Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Fig. 1 (abstract P569).