TSR-033, a Novel Therapeutic Antibody Targeting LAG-3, Enhances T-Cell Function and the Activity of PD-1 Blockade In Vitro and In Vivo

TSR-033 binds with high affinity to human and cyno LAG-3. A, TSR-033 binds human and cyno LAG-3 expressed on the surface of CHO-S cells, but not mouse LAG-3, as assessed by flow cytometry. B, TSR-033 binds LAG-3 on resting CD4 and CD8 T cells in health human donors (n = 3); total LAG-3 was detected using a noncompeting antihuman LAG-3 antibody (Materials and Methods). C, TSR-033 inhibits binding of labeled LAG-3 Fc fusion protein to MHC Class II expressing Daudi cells. D, TSR-033 relieves LAG-3–mediated repression of an NFAT-responsive element, downstream of TCR signaling. Data are representative of at least two separate occasions.

TSR-033 amplifies T-cell effector function, particularly in combination with anti-PD-1. The functional activity of TSR-033 was evaluated in an MLR assay, in which primary human CD4 T cells were mixed with monocyte-derived dendritic cells from a different donor. In these studies, dendritic cells and allogeneic CD4 T cells were incubated (A) in the presence of TSR-033 or isotype control or (B) TSR-033, TSR-042, and isotype control at indicated concentrations for 48 hours, and activation of T cells was determined by measuring the level of IL2 secretion. C, The ability of TSR-033 and TSR-042 to induce IL2 from human PBMCs (n = 5 donors) stimulated with 100 ng/mL SEB for 3 days was assessed. Data are representative of at least two separate occasions.

Dual blockade of LAG-3 and PD-1 using TSR-033 and TSR-042 improves therapeutic efficacy and T-cell stimulation in a humanized NSCLC tumor mouse model. A, The combination of TSR-033 with TSR-042 has an additive effect (CDI = 1.001) on restricting tumor growth in HuNOG-EXL mice inoculated with A549 cells. Mice were randomized at tumor volumes of 80 to 120 mm³, followed by administration of the indicated regimens (Materials and Methods). Tumor growth inhibition at termination for each treatment arm is indicated in parentheses. B, Relative to TSR-042 monotherapy, the combination group had increased intratumoral T cells and proliferating CD8 T cells; *, P ≤ 0.05, unpaired Student T test. C, The combination of TSR-033 with TSR-042, compared with TSR-042 monotherapy, resulted in significant reduction in TAMs, M2 TAMs and increased M1/M2 ratios. TAMs were identified as CD45⁺CD3⁻CD20⁻CD68⁺, M2 TAMS, CD45⁺CD3⁻CD20⁻CD68⁺HLA-DR^loCD209⁺, and M1 TAMs, CD45⁺CD3⁻CD20⁻CD68⁺HLA-DR^hiCD209⁻. Data represent two independent experiments (n = 10 per treatment arm) and have been normalized to fold change over isotype control for each treatment arm in B; *, P ≤ 0.05, unpaired Student T test.

Increased effector-memory T cells and ex vivo cytokine production by splenic T cells from the combination group, relative to TSR-042 alone in a humanized NSCLC tumor mouse model. A, Increase in proliferating and effector-memory (CD45RA⁻CCR7⁻) CD4 and CD8 T cells in the combination group compared with TSR-042 alone. B, Significant increase in IFNγ and TNFα production by CD4 T cells in the combination group over TSR-042 alone, upon ex vivo stimulation of mouse splenocytes (Materials and Methods); *, P ≤ 0.05, unpaired Student T test.