Discovery and characterization of GI-128: A PD-L1 x LILRB1/2/4 bispecific antibody as a macrophage engager

Immunosuppressive activity of myeloid cells within the tumor microenvironment (TME) contributes significantly to tumor immune evasion. Leukocyte immunoglobulin-like receptor subfamily B (LILRB) members are inhibitory receptors primarily expressed on myeloid cells. LILRB1, LILRB2 and LILRB4 are upregulated on tumor-infiltrating myeloid cells across many cancers. LILRB1 and LILRB2 suppress cytotoxic T cell and NK cell activity through interactions with their ligands such as HLA-G and HLA-G/beta2m. LILRB4 interacts with extracellular matrix proteins and subsequently promotes myeloid-mediated immunosuppression, leading to impairment of anti-tumor immunity. Although immune checkpoint inhibitors (ICIs) show broad clinical benefit, many patients are refractory or develop acquired resistance. ICI-resistant tumors often exhibit increased polarization of immunostimulatory M1 to immunosuppressive M2 tumor-associated macrophages. As macrophages are abundant in the TME across many cancer types, reprogramming of tumoricidal macrophages and their engagement with tumor cells could be a promising approach to elicit effective anti-tumor responses. Since high LILRBs expression correlate with poor prognosis and ICI non-responsiveness, and PD-L1 is highly expressed in multiple cancer types or positively correlates with LILRBs expression, we designed GI-128, a macrophage-engaging bispecific antibody composed of an anti-PD-L1 sdAb (single domain antibody) and an anti-LILRBs sdAb. Here, we showed that GI-128 candidates exhibited high affinity for both PD-L1 and LILRB1/2/4 and effectively suppressed their receptor-ligand interactions against each target in cell-based blockade assay or cell-based reporter assay. In functional assays using immune cells, GI-128 candidates enhanced macrophage-mediated phagocytosis of tumor cells. Furthermore, they promoted pro-inflammatory cytokine production in LPS-stimulated PBMC-derived monocytes and induced M1 polarization during macrophage differentiation. In addition, a trans-binding assay showed that GI-128 candidates bound simultaneously to PD-L1-expressing cells and to LILRB1-, LILRB2- or LILRB4-expressing cells, implying their functionality as macrophage-engaging molecules. These findings suggest that GI-128, a bispecific macrophage engager, has a promising potential to reinvigorate tumoricidal macrophage function in immunosuppressed PD-L1-positive tumors. In vivo anti-tumor efficacy of GI-128 is being investigated.