Abstract 2270: RG7769 (PD1-TIM3), a novel heterodimeric avidity-driven T cell specific PD-1/TIM-3 bispecific antibody lacking Fc-mediated effector functions for dual checkpoint inhibition to reactivate dysfunctional T cells

Based on the unprecedented clinical efficacy of PD-1/PD-L1 pathway checkpoint inhibitors (CPI), non-redundant immune checkpoints like TIM-3, LAG-3, TIGIT or BTLA are currently being targeted, by combinatorial approaches using monospecific or bispecific antibodies. Up-regulation of TIM-3 has been described as an adaptive CPI resistance mechanism, and internal prevalence data on archival samples of CPI-naïve and -experienced patients showed co-expression of PD-1 and TIM-3 in various tumor types, consistent with literature reports. Here, we describe RG7769 (PD1-TIM3), a novel avidity driven heterodimeric PD-1/TIM-3 1+1 bispecific CrossMabVH-VL intentionally designed as high affinity PD-1 (KD 250 pM, 37°C) and low affinity TIM-3 (KD 130 nM, 37°C) Fab-moieties to specifically target PD-1+ and PD-1+ TIM-3+ T cells through avidity gain, while bypassing PD-1- TIM3+ myeloid and NK cells. In contrast to IgG4-based PD-1 antibodies and conventional IgG1-based TIM-3 Fc-effector function competent antibodies, RG7769 harbors a PG LALA containing heterodimeric KiH IgG1 Fc-region rendering the BsAb refractory to drug shaving by FcgR-expressing macrophages in the TME, while retaining IgG-pharmacokinetics. RG7769 binds to PD-1 with higher affinity than pembrolizumab and nivolumab. X-ray crystallography demonstrated that the humanized PD-1 binding Fab recognizes a unique glycosylated epitope on PD-1, and potently blocks the PD-1/PD-L1 and PD-1/PD-L2 interactions in both biochemical and reporter cell line assays. The humanized TIM-3 binding arm was identified for maximal functional activity using mixed lymphocyte reaction (MLR) assays. Compared with bivalent TIM-3 antibodies, RG7769 shows reduced binding to TIM-3+ myeloid and NK cells, but binds preferentially to dysfunctional T cells expressing PD-1 or both PD-1 and TIM-3, like tumor infiltrating lymphocytes (TILs) in the tumor microenvironment. By virtue of its monovalency, RG7769 induced low antibody internalization on activated T cells when compared with bivalent TIM-3 antibodies, overcoming a major cellular sink for TIM-3 antibodies. In functional assays, RG7769 showed increased IFN-γ secretion by in vitro generated tumor-specific T-cells, increased ex vivo tumor-specific effector functions of T cells from PBMCs of melanoma patients, and enhanced the anti-tumor-activity of TILs from melanoma patients when compared to the monospecific parental PD-1 antibody. Finally, RG7769 showed superior efficacy in controlling s.c. MC38