757 LBL-042, a novel bispecific antibody, has the potential to improve immune evasion in cancer by targeting not only T but also myeloid suppressive cells

<h3>Background</h3> The myeloid-derived immune suppression in the tumor microenvironment contributes to tumor immune evasion. LILRB1 (also known as ILT2) and LILRB2 (also known as ILT4) are inhibitory receptors with intracellular tyrosine-based inhibitory motifs (ITIMs) that recognize common MHC-I ligands. Both LILRB1 and LILRB2 are highly expressed on tumor-infiltrating myeloid cells, and LILRB1 is also expressed on tumor-associated T cells, macrophages and NK cells. LILRB1 and LILRB2 receptors inhibit cytotoxic T cells’ function by interacting with human leukocyte antigen-G (HLA-G) and MHC-I. In patients who do not respond to immune checkpoint inhibitor therapies, the upregulation of LILRB1 and LILRB2 expression is observed, suggesting that these receptors may serve as potential mechanisms of resistance. Here we report a novel bispecific antibody LBL-042, targeting PD-1 and LILRB1/2 simultaneously, could block both PD-1 and LILRB1/LILRB2 immune suppression, may improve immune evasion of TME and possibly overcome resistance to IO therapy. <h3>Methods</h3> A panel of diverse VHH antibodies against LILRB1/2 were developed using alpaca immunization and phage library screening. The candidate molecules underwent rigorous <i>in vitro</i> evaluation, including binding to LILRB1 and LILRB2, MHC-I and HLA-G blockade, myeloid cell functional assays, etc. The anti-PD-1 and LILRB1/2 bispecific antibodies were developed using the Leads Biolabs’s multi-specific antibody platform. These candidates include different anti-LILRB1/2 locations, valencies and IgG isotypes. The binding and blocking capabilities of these bispecific antibodies were evaluated using ELISA and FACS. The <i>in vitro</i> activities on myeloid cells were studied in macrophage phagocytosis, MDSC reprogramming and DC maturation assays. T cell proliferation and cytokine release were assessed using a mixed lymphocyte reaction assay. Furthermore, <i>in vivo</i> anti-tumor efficacy was investigated in A375-HLA-G PD-1 resistant tumor model. <h3>Results</h3> The LBL-042 bispecific antibody exhibited high affinities to both PD-1 and LILRB1/2. It could block the binding of LILRB1/2 to MHC-I and HLA-G. Blockade of LILRB1/2 enhances macrophage phagocytosis, MDSC reprogramming and DC maturation. In MLR assay, it enhanced T cells’ activation. LBL-042 also demonstrated potent anti-tumor effects <i>in vivo</i>. <h3>Conclusions</h3> The novel bispecific antibody LBL-042, which targets PD-1 and LILRB1/2, unleash the suppression of T cells through anti-PD-1, while reprogramme or activate macrophages and DCs through LILRB1&amp;2 inhibition. These effects reprogramme the immune-suppressive microenvironment, ultimately leading to a synergistic immune effect with anti-PD-1. It may overcome the resistance to PD-1 monotherapy induced by myeloid cell immune-suppression.