In vitro activity: CB-1158 (previously known as INCB01158) is a potent and orally-bioavailable small-molecule inhibitor of the arginase. Myeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. CB-1158 has shown high anti-cancer activity in renal cell cancer, breast cancer, non-small cell lung cancer, acute myeloid leukemia, and other tumor types where arginase-secreting MDSCs are known to play an immunosuppressive role. Arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer, also the combination of CB-1158 with standard-of-care chemotherapy or other immunotherapies that target T-cell activation, such as CTLA-4 and PD-1 antibodies may yield improved clinical responses.

Kinase Assay: 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. 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.

Cell Assay: 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.