Background Immune checkpoint blockade (ICB) therapies including anti-CTLA4 and anti-PD1/L1 antibodies have revolutionized cancer treatment landscape by eliciting durable remission.¹ However, despite early response, most cancer patients eventually become resistant to the ICB therapies. The DNAM1 axis, an additional immunomodulatory pathway, is a potent regulator of innate and adaptive immunity.² The immunoglobulin receptors TIGIT and PVRIG, which interact with their ligands PVR and PVRL2 in DNAM1 axis respectively, are reported to contribute to primary or acquired resistance to PD-1/L1 blockade. Here, we report the internally discovered D3L-002, an anti-TIGIT×PVRIG bispecific antibody (bsAb), bound to TIGIT and PVRIG simultaneously and effectively blocked the interaction between TIGIT/PVR and PVRIG/PVRL2. D3L-002 could restore the function of exhausted T/NK cells and result in potent anti-tumor activity.

Methods The affinity of D3L-002 was examined by surface plasmon resonance (SPR) method. Cellular binding and in vitro blocking assays were measured by FACS or ELISA. T cell activation was detected via TCR/NFAT signaling and IFN-γ cytokine secretion. NK cell function was examined by degranulation and target cell lysis. Antibodies’ efficacy was studied in syngeneic mouse tumor models.

Results D3L-002, a IgG1 tetravalent bsAb with symmetric IgG-single-chain variable fragment (scFv) structure, showed high binding affinity to the extracellular domain of human TIGIT (K_D < 1 nM) and PVRIG (K_D < 10 nM) respectively. D3L-002 bound to human TIGIT and PVRIG expressing cells with sub-nanomolar EC₅₀ and was cross-reactive to respective cynomolgus homologs. Moreover, it demonstrated strong TIGIT/PVR and PVRIG/PVRL2 receptor-ligand interaction blocking activities in both cellular and protein level assays. This potent blocking translated well into enhanced functions in vitro. D3L-002 was able to activate TCR/NFAT signaling in Jurkat cells and showed better activity than anti-TIGIT and anti-PVRIG parental mAb alone, or their combination. D3L-002 could also enhance degranulation and cytotoxicity of primary NK cells and reinvigorate exhausted CD8+ T cells. In MC38 model, D3L-002 showed a trend of better anti-tumor efficacy than parental anti-TIGIT (P>0.05) and anti-PVRIG mAb (P<0.01). In addition, the combination of D3L-002 with anti-PDL1 mAb showed more potent efficacy than Atezolizumab monotherapy (P<0.01). MoA study indicated that D3L-002 monotherapy treatment depleted Treg, while combination with Atezolizumab induced CD8+ T cell proliferation in tumor microenvironment.

Conclusions D3L-002 is a novel TIGIT×PVRIG bsAb which demonstrated potent anti-tumor effect via TIGIT and PVRIG co-blocking in both in vitro and in vivo models. D3L-002 might provide a novel treatment approach for solid tumors and overcome resistance to current ICB therapies.

References

1. Sharma P, Allison JP. The future of immune checkpoint therapy. Science. 2015;348(6230):56–61.

2. Alteber Z, Kotturi MF, Whelan S, Ganguly S, Weyl E, Pardoll DM, Hunter J, Ophir E. Therapeutic targeting of checkpoint receptors within the DNAM1 axis. Cancer Discov. 2021;11:1–13.