CB-1158 was synthesized at Calithera Biosciences  and dissolved in 100% DMSO for biochemical assays or in Milli-Q water (Millipore, Billerica, MA) for cell-based assays and in vivo studies. No endotoxin contamination of CB-1158 preparations was observed. All other chemicals were purchased from Sigma (St. Louis, MO) unless indicated otherwise.
Flow cytometry antibodies
The following anti-mouse antibodies were used for flow cytometry: CD45-V450 (30F11), CD45-BV510 (30F11), CD45-BV605 (30F11), CD8-BV510 (53–6.7), CD25-BV421 (PC61), CD25-BV605 (PC61) from BD Biosciences (San Jose, CA); CD3-PerCP-eFluor710 (17A2), CD45-PE-Cy7 (30F11), NKp46-eFluor660 (29A1.4), CD11b-PE-Cy7 (M1/70), CD68-PE-Cy7 (FA-11) from eBioscience (Thermo Fisher Scientific, Waltham, MA); CD3-PE (17A2); CD68-BV421 (FA-11), CD206-AlexaFluor488 (C068C2), CD11b-PerCP-Cy5.5 (M1/70), CD11b-BV605 (M1/70) from BioLegend (San Diego, CA); CD11b-PE (M1/70) from Stemcell Technologies (Vancouver, Canada); and Arg1-APC (polyclonal) from R&D Systems (Minneapolis, MN). The following anti-human antibodies were used for flow cytometry: CD66b-PE (G10F5), CD4-PerCP-Cy5.5 (SK3), CD8-APC (RPA-T8) from BD Biosciences; and CD15-eF450 (HI98) from eBioscience.
Recombinant arginase activity assays
Recombinant full-length human Arg1 was purchased from Enzo Life Sciences (Farmingdale, NY). Recombinant human arginase 2 (Arg2) comprising amino acids 23–254 was purchased from US Biological (Salem, MA). Activity assays using 2 nM Arg1 or 4 nM Arg2 were performed in reaction buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, 0.005% Triton X-100, 0.5 mM DTT, 0.5 mM MgCl2, 0.1 mM CaCl2, and 160 μM or 20 mM L-arginine, pH 7.4) at 37 °C for 30 min with a dose-titration of CB-1158. Activity was determined by a spectrophotometric assay using the QuantiChrom Urea Assay Kit (BioAssay Systems, Hayward, CA) or by quantification of the generation of 13C(5)-L-ornithine from 13C(6)-L-arginine using a SCIEX API4000 mass spectrometer (Applied Biosystems, Foster City, CA). Urea produced or 13C(5)-L-ornithine peak areas were plotted and fitted to a four-parameter equation using GraphPad Prism software (San Diego, CA) to determine IC50 values.
Native arginase activity in cell lysates
Human granulocytes or erythrocytes were purified from healthy donor peripheral blood using a pan-granulocyte negative selection kit (Stemcell Technologies) or centrifugation on a Ficoll layer, respectively. Frozen human hepatocytes were purchased from XenoTech (Kansas City, KS). Lysates were prepared by microtip sonication followed with clarification by centrifugation. Plasma samples from renal cell carcinoma (RCC) patients were obtained by Ficoll centrifugation of whole blood purchased from Conversant Biologics (Huntsville, AL). Granulocyte lysate was assayed at 0.094 mg/mL, as determined by bicinchoninic acid/BCA protein assay (ThermoFisher), in reaction buffer. Erythrocyte or hepatocyte lysates were assayed at concentrations empirically determined to consume 10–15% of 13C(6)-L-arginine in 30 min at 37 °C. Arginase activity was determined in lysates and plasma by quantification of the generation of 13C(5)-L-ornithine from 13C(6)-L-arginine in the presence of a dose-titration of CB-1158.
Native arginase activity in intact cells
Intracellular arginase activity was determined for the arginase-expressing HepG2 and K-562 cell lines as follows. HepG2 cells were seeded at 100,000 cells per well one day prior to treatment with CB-1158. K-562 cells were seeded at 200,000 cells per well on the day of CB-1158 treatment. Cells were treated with a dose-titration of CB-1158 in SILAC RPMI-1640 media (Life Technologies/Thermo Fisher Scientific) containing 5% heat-inactivated and dialyzed FBS, antibiotics/anti-mycotic, 10 mM L-arginine, 0.27 mM L-lysine, and 2 mM L-glutamine. The medium was harvested after 24 h and urea generated was determined with the QuantiChrom Urea Assay Kit. Wells containing media without cells were used as background controls. For assessing the effect of CB-1158 on Arg1 in primary hepatocytes, frozen human hepatocytes (XenoTech) were thawed, allowed to adhere onto collagen-coated wells for 4 h, and then incubated for 48 h in SILAC-RPMI containing 10 mM L-ornithine, no L-arginine, and a dose-titration of CB-1158, at which time the media were analyzed for urea.
Nitric oxide (NO) synthase (NOS) activity assays
Activity of 50 μM CB-1158 against 3 NOS isoforms, recombinant murine inducible NOS, recombinant bovine endothelial NOS, and native rat cerebellar neuronal NOS, was determined at Eurofins/Cerep Panlabs (Taipei, Taiwan) by either quantitation of radiolabeled L-citrulline or spectrophotometric measurement of nitrite.
All cell culture reagents were purchased from Corning (Corning, NY) unless indicated otherwise. The human cell lines, HepG2 and K-562, and the murine cell lines, LLC1 (LLC), B16-F10 (B16), CT26.WT (CT26), and 4T1 were obtained from American Type Culture Collection (ATCC, Manassas, VA). HepG2, K-562, CT26, and 4T1 were maintained in RPMI-1640 (Corning). B16 was maintained in DMEM (Corning). LLC was maintained in DMEM (ATCC). All media were supplemented with 10% fetal bovine serum (FBS), plus penicillin, streptomycin, and amphotericin. Cell lines were grown at 37 °C in a humidified 5% CO2 atmosphere.
Cells were seeded in fully-supplemented RPMI-1640 medium, treated with a dose-titration of CB-1158 in triplicate wells, and incubated for 72 h. Cytotoxicity was assayed by the addition of CellTiterGlo reagent according to the manufacturer’s instructions (Promega, Madison, WI) followed by fluorescence quantification on a Molecular Devices plate reader (Sunnyvale, CA).
T cell and NK cell proliferation assays
T cells or NK cells were purified from healthy donor human blood or from murine splenocytes using a negative selection kit for the appropriate cell type and species from Stemcell Technologies. Isolated T cells or NK cells were loaded with carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher) and stimulated for 72–96 h in complete growth medium containing a minimum of either 50 μM L-arginine (NK cells) or 100 μM L-arginine (T cells). For T cell stimulation, a solution of 10 μg/mL anti-CD3 (human clones UCHT1 or OKT3; murine clone 145-2C11) was used to coat the wells of a 96-well plate and then T cells were stimulated on immobilized anti-CD3 in the presence of 2 μg/mL soluble anti-CD28 (human clone CD28.2; murine clone 37.51). NK cells were stimulated with recombinant IL-2. Proliferation was quantified by analyzing CFSE dilution by flow cytometry (Guava flow cytometer, Millipore, Billerica, MA or Attune NxT flow cytometer, ThermoFisher).
T cell/myeloid cell co-culture assays
Granulocytes were purified from healthy donor peripheral blood using a pan-granulocyte negative selection kit (Stemcell Technologies) and incubated in SILAC-RPMI medium containing 10% charcoal-stripped FBS, antibiotics/anti-mycotic, 0.27 mM L-lysine, 20 μM MnCl2, 100 μM L-arginine, pH 7.4, and a dose-titration of CB-1158. Freshly isolated granulocytes were incubated for 48 h at 37 °C, during which time they spontaneously activate as determined by increased surface expression of CD66b and scatter properties. T cells isolated from the same healthy donor using a pan-T cell isolation kit (Stemcell Technologies) were loaded with CFSE and plated with immobilized anti-CD3 and soluble anti-CD28 in the presence of the aged granulocytes. The cells were co-cultured at several ratios of granulocytes to T cells as indicated in the figure or at a fixed ratio of 4 T cells to 1 granulocyte. Co-cultures were incubated for 3–4 days, at which time the medium was analyzed for L-arginine and L-ornithine by mass spectrometry and T cell proliferation was determined by flow cytometry. Granulocytic MDSC (G-MDSC) or granulocytes from cancer patients were isolated from whole blood purchased from Conversant Biologics. G-MDSCs were purified from the PBMC layer of a Ficoll gradient by positive selection for CD66b+ cells. Granulocytes were purified from the RBC layer of a Ficoll gradient using Hetasep (Stemcell Technologies). Granulocytes and G-MDSCs were characterized by flow cytometry for CD66b expression. Freshly isolated G-MDSC or granulocytes were incubated in co-culture medium containing 100 μM L-arginine for 48 h, at which time the cells were removed and the G-MDSC- or granulocyte-conditioned media were used for incubating healthy donor CFSE-loaded T cells on immobilized anti-CD3/soluble anti-CD28 for 3–4 days. Cytokines were quantified in the media from T cell co-culture assays using the Cytometric Bead Array kit according to the manufacturer’s instructions (BD Biosciences).
Murine tumor studies
Female wild-type C57BL/6 and Balb/c mice (5–6 weeks old) were purchased from Charles River Laboratories (Hollister, CA). Severe combined immune deficient (SCID, B6.CB17-PrkdcSCID/SzJ) and Pmel-1 TCR transgenic (B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J) mice (5–6 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed and treated in accordance with Institutional Animal Care and Use Committee guidelines. For the 4T1 tumor model, 105 cells were injected orthotopically into the mammary fat pad; for all other tumor models, 106 cells were injected subcutaneously (s.c.) in the right flank. For all studies, CB-1158 was administered by oral gavage twice per day at 100 mg/kg starting on study day 1 (1 day after tumor implant). Control groups received vehicle (water) twice daily by gavage. Tumor volume measured by digital caliper (length × width × width/2) and body weight were recorded three times per week. Animals were euthanized when tumors necrotized or volumes reached 2000 mm3. For the CT26 model, anti-PD-L1 antibody (5 mg/kg, clone 10F.9G2, BioXCell, West Lebanon, NH) was injected intraperitoneally (i.p.) on days 5, 7, 9, 11, 13, and 15. For the 4T1 model, anti-CTLA-4 antibody (5 mg/kg, clone 9H10, BioXCell) was injected i.p. on days 2, 5, and 8; anti-PD-1 antibody (5 mg/kg, clone RMP1–14, BioXCell) was injected i.p. on days 3, 6, and 9. 4T1 tumors were harvested on study day 25 into Fekete’s solution and tumor nodules were enumerated visually. Gemcitabine (Selleckchem, Houston, TX) was dosed 50 mg/kg i.p. on days 10 and 16 for the CT26 model, 60 mg/kg i.p. on days 6 and 10 for the LLC model, or 30 mg/kg i.p. on day 5 for the 4T1 model [17, 18]. With these regimens, gemcitabine modestly reduces tumor growth and spares most tumor-infiltrating immune cells, allowing for the evaluation of combination activity with CB-1158. For CD8+ cell depletion, mice were injected i.p. with anti-CD8 antibody (25 mg/kg, clone 2.43, BioXCell) on days −1, 0, + 5, and +10. For NK cell depletion, mice were injected i.p. with anti-NK1.1 antibody (25 mg/kg, clone PK136, BioXCell) in the LLC and B16 models or with anti-Asialo GM1 sera (20 μL, Wako Chemicals, Richmond, VA) in the CT26 model, per the same schedule as anti-CD8.
Conditional Arg1 deleted mice
Arg1 floxed mice were crossed to the Tie2-Cre deleter strain (The Jackson Laboratory) as previously described . Experimental mice were generated from crossing Arg1
+ males with Arg1
− females, with Cre negative littermates serving as wild-type controls. Mice were housed and treated in accordance with protocols approved by the Institutional Animal Care and Use Committee at St. Jude Children’s Research Hospital. LLC cells (106 per mouse) were injected s.c. in the flank region. Mice were orally gavaged with either 100 mg/kg of CB-1158 or an equivalent volume of vehicle control (water) every 12 h for 14 days. Mice were euthanized, and tumors excised and weights recorded. Myeloid deletion of Arg1 was confirmed via western blotting of IL-4-stimulated bone marrow-derived macrophages for all animals.
Adoptive T cell transfer studies
Activated gp100-specific CD8+ (Pmel-1) T cells were generated as described in Ya et al. . Briefly, splenocytes from Pmel-1 TCR transgenic mice were isolated, pulsed with 1 μM of murine gp10025–33 (Anaspec, Fremont, CA) and expanded for 1 week in the presence of 60 IU/mL recombinant human IL-2 (Peprotech, Rocky Hill, NJ). Cells were >90% CD8+ Vβ13+ T cells as determined by flow cytometry. C57BL/6 mice were inoculated s.c. with B16 tumor cells. CB-1158 was administered by oral gavage twice per day at 100 mg/kg starting 1 day after tumor implant. On day 7, lymphopenia was induced by a non-myeloablative chemotherapy regimen of 250 mg/kg cyclophosphamide and 50 mg/kg fludarabine administered i.p. The chemotherapy regimen was administered to all groups. On day 9, mice were administered 1 × 106 Pmel-1 T cells intravenously (i.v.). Mice receiving Pmel-1 T cells also received recombinant human IL-2 (200,000 IU/dose) administered i.p. twice daily for 3 days starting the day of T cell transfer.
Adoptive NK cell transfer studies
Balb/c mice were inoculated i.v. with 105 CT26 cells. On the same day as tumor inoculation, 106 NK cells (isolated from Balb/c spleens the day before injection and incubated with recombinant IL-2 and IL-15 for 18 h) were transferred to the mice. The injected NK cells were profiled by flow cytometry to be CD25+ and 80–90% pure with less than 0.4% T cells. Mice were treated with vehicle or CB-1158 for 14 days and then lungs were harvested into Fekete’s solution and tumor nodules enumerated visually.
Tumor dissociation and flow cytometry
Tumor-bearing mice treated with vehicle or CB-1158 (100 mg/kg BID) were sacrificed for flow cytometry analysis on study day 9 (B16), day 10 (4T1), or day 14 (CT26 and LLC). Excised tumors were placed on ice in RPMI-1640 medium containing 5% FBS, minced with a razor blade, and dissociated in RPMI-1640 supplemented with mouse tumor dissociation enzymes (Miltenyi Biotec, Bergisch Gladbach, Germany) on a GentleMACS Octo Dissociator With Heat (Miltenyi Biotec) according to the manufacturer’s instructions. Dissociated tumors were strained through 70 μm nylon mesh, washed with cold PBS containing 2% FBS, blocked with anti-CD16/CD32 (Fc block antibody, eBioscience), and stained for cell surface antigens. For B16 and 4T1 tumors, washed dissociated tumor cells were incubated with Dead Cell Removal MicroBeads (Miltenyi Biotec) and applied to a magnetic column prior to staining. For intracellular staining, cells were fixed and permeabilized using buffers purchased from R&D Systems or eBioscience for cytoplasmic or nuclear antigens, respectively. All tumor flow experiments were acquired on an Attune NxT flow cytometer and analyzed with FlowJo software version 10 (Ashland, OR), using fluorescence-minus-one controls for gating and single-stained OneComp eBeads (eBioscience) to set compensation matrices.
Gene expression analysis
LLC tumors from mice (N = 6 per group) treated with vehicle or CB-1158 (100 mg/kg twice daily) for 13 days were collected, placed into neutral buffered formalin overnight, transferred into 70% ethanol, and shipped to Core Diagnostics (Hayward, CA) for paraffin embedding. RNA was extracted for gene expression analysis and transcripts were quantified by NanoString Technologies (Seattle, WA).
LLC tumors from mice (N = 5 per group) treated with vehicle or CB-1158 (200 mg/kg twice daily) for 14 days were collected and flash frozen in liquid nitrogen. Tumors were homogenized in 50 mM Tris-HCl buffer containing 2 mM EDTA, pH 7.4 and protease inhibitors. The homogenate was centrifuged and the supernatant was collected and re-frozen. Cytokines in the supernatant were quantified by Myriad Rules Based Medicine (Austin, TX).
Automated IHC was performed by Indivumed (Hamburg, Germany) using the Discovery XT staining platform (Roche Diagnostics/Ventana Medical Systems, Mountain View, CA) on formalin-fixed and paraffin-embedded (FFPE) samples and tumor tissue microarrays (TMA). The rabbit anti-human Arg1 monoclonal antibody clone EPR6672(B) from Abcam/Epitomics (Burlingame, CA) was validated using 8 different cases of hepatocellular carcinoma (HCC) and one sample of normal liver tissue as positive control tissue; normal tonsil tissue and isotype control antibody were used as negative controls. IHC was performed on 11 different tumor histologies: non-small cell lung cancer (NSCLC, squamous and adenocarcinoma), breast cancer (triple negative and non-triple negative), gastric adenocarcinoma, colorectal cancer (CRC), prostate adenocarcinoma, pancreatic cancer, ovarian cancer, bladder cancer, and RCC. Arg1+ cells per mm2 were quantified by digital histopathology (OracleBio, Scotland, UK).
Tumor TMAs containing samples from patients with lung squamous cell carcinoma, CRC, RCC, esophageal carcinoma, and head and neck cancer were purchased from US Biomax and US Biolabs (Rockville, MD). Multiparameter immunofluorescence using the MultiOmyx platform for markers including Arg1, CD15, and CD68 was performed and analyzed by GE Clarient/NeoGenomics Laboratories (Aliso Viejo, CA).
Plasma Arg1 and L-arginine
The amount of Arg1 protein in plasma samples was determined by enzyme linked immunosorbent assay (ELISA, BioVendor, Asheville, NC) in specimens from healthy volunteers and patients with head and neck cancer (N = 5), HCC (N = 3), mesothelioma (N = 3), CRC (N = 3), T cell prolymphocytic leukemia (N = 2), melanoma (N = 2), bladder cancer (N = 4), NSCLC (N = 11), small cell lung cancer (N = 17), undefined lung cancer (N = 6), acute myeloid leukemia (N = 9), RCC (N = 9), and breast cancer (N = 2). Plasma L-arginine was determined by mass spectrometry in samples from patients with mesothelioma (N = 3), CRC (N = 3), NSCLC (N = 9), small cell lung cancer (N = 3), undefined lung cancer (N = 3), head and neck cancer (N = 3), and T cell prolymphocytic leukemia (N = 2). All cancer patient samples were purchased from Conversant Biologics.