Antibody and recombinant protein production and purification
The coding sequences of both human and murine PD1-Fc-OX40L, and the murine PD1(K78A)-Fc-OX40L mutant [18, 19], were codon-optimized for human cell line expression and directionally cloned into pcDNA3.4-hygro-mcs expression vector (Thermo). Vectors were then transiently transfected into mammalian cells and purified using affinity chromatography. DNA sequences for nivolumab, pembrolizumab, and tavolixizumab were synthesized and cloned into pcDNA3.4 vectors, transiently expressed in CHO cells and secreted protein isolated by affinity chromatography using Protein A (GeneArt).
Human (h) and mouse (m) PD1-Fc-OX40L protein were treated +/− the deglycosylase PNGase F (NEB, Cat #P0704) for 1 h at 37 °C according to manufacturer’s recommendations, and then +/− the reducing agent beta-mercaptoethanol. Samples were diluted in RIPA buffer prior to separation by SDS-PAGE. Primary antibodies used for probing hPD1-Fc-OX40L and mPD1-Fc-OX40L were obtained from Cell Signaling Technology, Jackson ImmunoResearch Laboratories, Inc. and R&D Systems.
For characterization of the h/m PD1-Fc-OX40L, high-binding ELISA plates (Corning) were coated overnight at 4 °C with 5 μg/mL of either recombinant hFc, hPD-L1-Fc, hOX40-His, mFc, mPD-L1-Fc or mOX40-His, in PBS (reagents were obtained from Jackson ImmunoResearch Laboratories, Sino Biological, Inc., Acro Biosystems and R&D Systems). Plates were then blocked with casein buffer for 1 h at room temperature (RT) and then probed with serial dilutions of the h/m PD1-Fc-OX40L, along with the appropriate standards (human and mouse; IgG, PD1-Fc, and OX40L-Fc) for 1 h at RT. Plates were washed and then detection antibody was added for 1 h at RT in the dark. Detection antibodies included anti-hIgG-HRP, anti-mIgG-HRP, anti-hOX40L and then anti-Goat-HRP, anti-mOX40L and then anti-Goat-HRP (all antibodies were obtained from Jackson ImmunoResearch Laboratories or R&D Systems). Plates were washed again and SureBlue TMB Microwell Peroxidase Substrate (KPL) was added to each well and allowed to incubate at RT for 20 min in the dark. To stop the reaction, 100 μL of 1 N sulfuric acid was added to each well and absorbance at 450 nm was read immediately on a BioTek plate reader. Samples were run at a minimum in triplicate and at multiple dilutions.
For the PD1-blocking ELISA, hPD-L1-Fc (Sino Biological, Inc.) was used to coat a high binding ELISA plate as described above. The following day, binding was detected using a recombinant hPD1-Biotin protein (Acro Biosystems) in combination with increasing concentrations of either hPD1-Fc-OX40L (to compete with PD1-Biotin binding for PD-L1) or an irrelevant fusion protein, which served as a negative control. Following this incubation, plates were washed as described above and probed with an avidin-HRP detection antibody (BioLegend) for 1 h at RT in the dark. Plates were then washed and analyzed as above. For IL2 (mouse and human) and TNFα functional ELISAs, cell culture supernatants were collected and assessed using commercial ELISA kits according to manufacturer recommendations (human IL2, human TNFα, and mouse IL2; R&D Systems).
Surface Plasmon resonance
Direct binding of h/m PD1-Fc-OX40L fusion protein to recombinant protein targets was performed using a BioRAD ProteOn™ XPR36 protein interaction array instrument. To determine the on-rates (Ka), off-rates (Kd) and binding affinities (KD) of hPD1-Fc-OX40L to its intended binding targets (referred to as ‘Ligands’), histidine-tagged versions of the human recombinant targets - PD-L1 (Acro Biosystems), PD-L2 (Acro Biosystems), OX40 (Acro Biosystems), FcγRIA (Sino Biological Inc.), FcγRIIB (Sino Biological Inc.), FcγRIIIB (Sino Biological Inc.) and FcRn (R&D Systems) were immobilized to an Ni-sulfate activated ProteOn™ HTG sensor chip (BioRAD). Increasing concentrations of the hPD1-Fc-OX40L fusion protein (referred to as ‘Analyte’) diluted in PBS/Tween (0.005%) buffer pH 7.0 was injected for 3 min followed by a dissociation phase of 5 min at a flow rate of ≥80 μl/min. hPD1-Fc and hOX40L-Fc (Acro Biosystems) recombinant proteins were used as positive control analytes for binding to their respective partners (PD-L1/L2 and OX40, respectively) and human IgG was used as a positive control for binding to Fcγ receptors and FcRn. To assess analyte binding to FcRn ligand, the pH of the buffer was reduced to pH 5.5. To minimize mass transport limitation, ligand concentrations were maintained at an optimized low concentration and flow rates over the chip were maintained at 80–100 μL/min.
Mouse PD1-Fc-OX40L fusion protein binding to mouse counterparts of the same targets was assessed as described above and the relevant recombinant proteins were obtained from Acro Biosystems, and R&D Systems.
Jurkat, CHO-K1, HeLa, HCC827, PC3, NCI-H2023, 4 T1, B16.F10, and CT26 cells were maintained in IMDM media supplemented with glutamine and 10% fetal bovine serum (FBS) at 37 °C in 5% CO2.
In vitro cell line generation
Stable cells lines were generated to assess in vitro binding of human and mouse PD1-Fc-OX40L ARC proteins (CHO-K1/hPD-L1, CHO-K1/hPD-L2, Jurkat/hOX40, HeLa/hOX40, CHO-K1/mPD-L1, CHO-K1/mPD-L2, and CHO-K1/mOX40). Briefly, cDNAs were amplified from CD3/CD28 activated human PBMCs or mouse splenocytes, and cloned into pcDNA3.1(−) (Life Technologies). Parental CHO-K1 and Jurkat cells were nucleofected with the 4D-Nucleofector and Cell Line Nucleofector Kit SE (Lonza) according to manufacturer’s directions. Cells were selected with antibiotics and single-cell cloned in order to generate stable, high-level expressing clones that were screened by flow cytometry.
Tumor model systems
For CT26 studies, BALB/C mice were subcutaneously implanted with 5 × 105 tumor cells into the rear flank on day 0. On treatment days, tumor bearing mice were either untreated or treated with 2 doses (on days 5 and 7) of the mPD1-Fc-OX40L ARC, anti-OX40 (clone OX86; BioXcell), anti-PD1 (clone RMP1–14; BioXcell), or anti-PD-L1 (clone 10F.9G2; BioXcell). Tumor area (mm2) and overall survival was assessed throughout the time-course. Survival criteria included total tumor area less than 175 mm2 with no sign of tumor ulceration. Complete responders, in which tumors established and were subsequently rejected are listed in the appropriate figures. A cohort of CT26 experimental mice was euthanized on day 13 for immune profiling in splenocytes and tumor tissue using flow cytometry. Serum was isolated from peripheral blood and cytokine levels were assessed using LEGENDplex (BioLegend) cytokine reagents. Tumors were excised from these mice and dissociated using a tumor dissociation kit (Milltenyi) and homogenized through a 100 μM strainer to isolate tumor cells and infiltrating immune cells.
For CD4/CD8 depletion experiments, mice were treated via IP injection of 100 μg of CD4 (clone GK1.5, BioXcell), CD8 (clone 2.43, BioXcell), or both CD4/CD8 depleting antibodies on days − 1, 1, and 10 of the experimental time-course. CT26 tumors were inoculated on day 0, as described above. Vehicle (PBS) or mPD1-Fc-OX40L (300 μg) treatments were given via IP injection on days 5 and 7 of the time-course. CD4 and CD8 populations in the peripheral blood were assessed at several time points to verify depletion.
LEGENDplex cytokine analysis
Experimental mice were euthanized through CO2 asphyxiation and cervical dislocation and whole blood was collected via cardiac puncture. Blood was allowed to coagulate for 30 min at room temperature and spun at 1000 g for 10 min to separate the serum. Serum was then transferred to a new 1.5 mL eppendorf tube. Cytokine analysis was performed using the LEGENDplex Cytokine Analysis kit (BioLegend) according to manufacturer recommendations and analyzed on the Sony SH800 or BD LSRII Fortessa. Flow cytometry results were converted to pg/mL secretion using BioLegend LEGENDplex software.
Briefly, isolated cells were stained with antibodies for 30 min on ice in the dark. Indicated antibodies were purchased from either Sony, BioLegend, or Abcam. Following this incubation period, stained cells were washed and resuspended in FACS buffer (1X PBS buffer containing 1% bovine serum albumin (BSA), 0.02% sodium azide and 2 mM EDTA). The AH1 tetramer (SPSYVYHQF) was purchased from MBL International. For intracellular FOXP3 staining, cells were fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set from Biolegend. Flow cytometry and cell sorting were performed on a Sony SH800 or BD LSRII Fortessa.
In vitro functional assays
Human co-culture assay
CD3+ T cells were isolated from PBMCs using a magnetic selection kit (Stemcell Technologies), and then stimulated for 2 days with a suboptimal quantity of bead bound CD3/CD28 (ThermoFisher) and soluble IL2 (BioXcell). During these 2 days, PC3 (PD-L1Low) and HCC827 (PD-L1High) cells were plated in multi-well tissue culture coated plates. After these 2 days, PC3 and HCC827 cells were treated with Mitomycin-C (Sigma) for 2 h 37 °C and 5% CO2 in order to prevent further cell division. Activated T cells were then plated on the Mitomycin-C treated tumor cells +/− PD1-Fc-OX40L for 5 additional days. On day 6 after initial T cell isolation, supernatants were collected from co-cultures and assessed using IL2 ELISA.An initial time-course was performed and determined that maximal proliferation and cytokine responses were observed between days 5 and 7 of the co-culture (data not shown). Thus, on days 5 and 7 after initial T cell isolation, the T cells in suspension in the co-culture systems were isolated and analyzed by flow cytometry for markers associated with proliferation (Ki-67) and T cell activation (IFNγ and TNFα).
The mouse functional co-culture assay depicted in Additional file 1: Figure S5H-I was performed identical to the human experiment performed above, however B16.F10 (PD-L1High) and 4 T1 (PD-L1Low) cells were used, as well as a mouse CD3 isolation kit (Stemcell Technologies), mouse CD3/CD28 beads (ThermoFisher), mouse IL2 (BioXcell), and a mouse IL2 detection ELISA.
Staphylococcus enterotoxin-B (SEB) assay
Primary PBMCs (or mouse splenocytes) were obtained from healthy donor leukopacs (Stemcell technologies) and incubated +/− increasing concentrations of the super-antigen SEB (List Biological Laboratories), in the presence or absence of the PD1-Fc-OX40L ARC, human/mouse IgG (Jackson Immunoresearch, Inc.), antibody controls, or single-sided fusion protein controls (Acro Biosystems and Sino Biologicals); as both monotherapies and combinations. All antibodies and ARC proteins were used at the concentrations indicated in the figures. After 3 days, culture supernatants were collected and assessed by ELISA for levels of IL-2 (R&D Systems). Additionally, the SEB assay was performed in human PBMCs depleted of CD4, CD8, or both CD4/CD8 cells, using magnetic positive selection kits (StemCell Technologies).
To account for variability between donors when directly comparing PD1-Fc-OX40L activity to antibody/fusion protein comparators, OD450 values were baseline-normalized by setting the first IgG control OD450 value to .1, and subtracting each treatment group by the numerical value needed to obtain the .1 control value. The concentration of secreted IL-2 was also quantitated in certain experiments, and ranged between ~ 500–2000 pg/mL.
Primary PBMCs were isolated and incubated as described above for the SEB assay. After 3 days, when the culture supernatant was removed for IL-2 ELISA analysis, proliferation of the remaining cells in culture was assessed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) (Promega) according to manufacturer’s instructions, and quantitated on a GloMax Navigator luminometer (Promega).
NFkB-luciferase reporter assay
The in-house engineered Jurkat/hOX40 cells, were stably transfected to generate a stable cell line that co-expressed an NFkB-luciferase reporter plasmid, pGL4.10[luc2] (Promega). Bright-Glo luciferase reagent and a luminometer were used to assess activation of NFkB signaling. The expression vector, luciferase reagent, and luminometer (Promega) were used according to manufacturer guidelines. Briefly, anti-CD3 (OKT3, at 1 μg/mL) was diluted in PBS and used to coat white 96-well plates (Costar) over night at 4 °C. The next morning, plates were washed to remove unbound anti-CD3 and 10,000 Jurkat/hOX40/NFkB-luciferase cells were plated in each well with 2 μg/mL functional grade CD28 (eBiosciences), +/− the ARC or other test agents. Plates were incubated at 37 °C/5% CO2 for 6 h, where after the Bright-Glo reagent was added and luminescence was assessed on a luminometer.
Human CD3+ cells were isolated and stimulated as described above, for 2 days. The day before co-culture, NCI-H2023 tumor cells were plated in 96-well 190 μM glass bottom plates (Greiner). The following day, tumor cells were treated for 30 min with Cell Tracker Orange (Invitrogen), HCS Nuclear Mask Blue (Invitrogen), and for 1 h with 150 μg of the human PD1-Fc-OX40L ARC previously labeled with Alex Fluor 647 (Invitrogen). Media was then removed and activated human T cells were seeded on the tumor cells at a ratio of 8:1 (T cell: tumor cell), along with CellEvent Caspase 3/7 Green Detection Reagent (Invitrogen). Images were then acquired over the course of 6 h at a 40X magnification on the GE IN Cell Analyze 2500HS System.
CD107a degranulation assay
C57Bl/6 mice were adoptively transferred with 5 × 105 OT-I (GFP) and 1 × 106 OT-II cells via tail vein injections. The following day, mice were vaccinated with Ova/Alum prepared as previously described . Three days after vaccination, CD3+ splenocytes were harvested and activated for 2 days according to the procedure described above. The day before co-culture, B16.F10-ova cells were plated in 24 well tissue culture plates. The following day, T cells were seeded on top of tumor cells at a ratio of 8:1 (T cell: tumor cell) +/− test agents. After 24 h, brefeldin A (BFA) and GolgiStop (BD Biosciences) were added to the cultures for 4 h and returned to the tissue culture incubator. After 4 h, suspension cells were removed and stained for surface expression of CD4, Vβ5.1/5.2, CD8, and CD107a, and intracellular expression of IFNγ (according to the flow cytometry procedure described above), and then analyzed by flow cytometry. All antibodies were purchased from either BioLegend or BD Biosciences.
Cleaved caspase 3/7 luciferase assay
The co-culture setup for this assay was identical to the CD107a assay above. Six hours after co-culture in white 96-well plates, apoptosis was assessed by reading out cleaved caspase 3/7, using the Caspase-Glo 3/7 Assay from Promega, according to manufacturer recommendations.
The co-culture setup for this assay was identical to the CD107a assay above. 1–3 days after co-culture in 96-well plates, DNA fragmentation was assessed using the TiterTACS Colorimetric Apoptosis Detection (TUNEL) Kit from Trevigen, according to manufacturer recommendations.
PBMCs were isolated from 50 healthy donor buffy coats and CD8+ T cells were depleted using CD8+ RosetteSep (StemCell Technologies). Cells were plated at a density of 4–6 million cells per mL in AIM-V medium (Gibco). On days 5, 6, and 7, cells were gently resuspended in 3 × 100 μL aliquots and transferred to each well of a round bottomed 96 well plate. Cultures were pulsed with 0.75 μCi [3H]-Thymidine (Perkin Elmer) in 100 μL AIM-V culture medium, and incubated for a further 18 h, before harvesting onto filter mats (Perkin Elmer), using a TomTec Mach III cell harvester. Counts per minute (Cpm) for each well were determined by Meltilex (Perkin Elmer) scintillation counting on a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer) in paralux, with low background counting.
PBMCs were isolated, CD8 cells depleted, and cultured in AIM-V as described above, and on day 8, cells were assessed. Briefly, ELISpot plates (Millipore) were pre-wetted and coated overnight with 100 μL/well IL-2 capture antibody (R&D Systems) in PBS. Cells were plated at a density of 4–6 million cells/mL in a volume of 100 μL per well, in sextuplicate. After 8 days, ELISpot plates were developed by sequential washing in dH2O and PBS (× 3), prior to the addition of 100 μL filtered biotinylated detection antibody (R&D Systems). Following incubation at 37 °C for 1.5 h, plates were further washed and incubated with 100 μL filtered streptavidin-AP (R&D Systems) for 1 h; and then washed again. 100 μL BCIP/NBT substrate (R&D Systems) was added to each well for 30 min at room temperature. Spot development was stopped by washing wells 3 times with dH2O. Dried plates were scanned on an Immunoscan Analyser and spots per well (SPW) were determined using Immunoscan Version 5 software.
Samples included human PD1-Fc-OX40L (.3 μM), the neoantigen KLH (Keyhole limpet haemocyanin; .3 μM) as a positive control, and Exenatide (Bydureon, 20 μM) as a clinical benchmark control; representing a non-immunogenic agent in this assay. For ELISpot, a mitogen positive control (PHA at 8 μg/mL) was included on each plate as an internal test for ELISpot function and cell viability.
An empirical threshold or Stimulation Index (SI), equal to or greater than 1.9 has been previously established (by the Contract Research Organization Abzena) whereby samples inducing responses above this threshold are deemed positive. SIs were calculated for each test article by dividing each value by the mean of media only control wells. One-way ANOVA was used to calculate significance versus the Exenatide control.
Experimental animal guidelines
All mouse protocols were designed based on IACUC guidelines and approved by a licensed veterinarian. Experimental mice were monitored daily and euthanized by CO2 asphyxiation and cervical dislocation prior to any signs of distress.
Experimental replicates (N) are shown in figures and figure legends. Unless noted otherwise, values plotted represent the mean from a minimum of 3 distinct experiments and error is SEM. Statistical significance (p-value) was determined using One-Way ANOVA with multiple comparisons. Significant p-values are labeled with one or more ‘*’, denoting *p < .05, **p < .01, ***p < .001 and ****p < .0001. Mantel-Cox statistical tests were used to determine the significance between the survival curves shown in Figs. 5 and 6. P-values are noted in the legends to these figures.