Abstract 3433: Novel dual antagonist anti-LILRB1/LILRB2 antibodies reprogram myeloid cells and potentiate T cells

Background: Genome-wide association studies have identified HLA-G and LILRB1/2 loci associated with cancer risk. LILRB1 and LILRB2 are distinct inhibitory immune checkpoint receptors that recognize MHC-I ligands, particularly immunosuppressive HLA-G. LILRB1-mediated inhibition can lead to impairment of T-cell cytotoxicity and preventing the efficient engulfment of tumor cells by macrophages as a “don’t eat me” signal. LILRB2 is expressed primarily by myeloid cells, especially in the tumor microenvironment, as an important immunosuppressive signal to inhibit the stimulation of an immune response. A dual antagonist antibody targeting both LILRB1 and LILRB2 could be more effective for cancer immunotherapy than targeting either LILRB1 or LILRB2 individually. Methods: Mice were immunized with recombinant D1-D2 domain of LILRB1 and LILRB2 proteins. Resulted heavy chain and light chain variable regions of IgG were PCR amplified from splenocytes and bone marrow plasma cells. PCR amplicons were built into yeast libraries. Yeast displayed scFv were screened for LILRB1 and LILRB2 binding. The dual binders were converted to effector silent human IgG1 and evaluated for ability to block HLA-G binding to LILRB1/RB2. The dual binder and dual blocker were further assessed in series of assays using human T cells and various primary myeloid cell subsets, including human monocyte-derived macrophages (hMDMs), tumor-associated macrophages (TAMs), dendritic cells (DCs) and monocytes. Results: We have discovered a panel of anti-LILRB1/2 antibodies that bind to both LILRB1 and LILRB2 with high affinity and block the interactions to HLA-G efficiently. The anti-LILRB1/2 antibodies inhibit both LILRB1 and LILRB2 signaling and modulate the function of immune cells. Anti-LILRB1/LILRB2-mediatted LILRB1 blockade: 1) enhances phagocytosis of tumor cells by macrophages; 2) releases HLA-G mediated inhibition on TCR activation in Jurkat T cells over-expressing LILRB1; 3) promotes the activation of human primary T cells stimulated with allogenic DCs. Anti-LILRB1/LILRB2-mediatted LILRB2 blockad:1) enhances TNFα expression and inhibits IL10 expression in LPS-stimulated human PBMC; 2) reprograms both hMDMs and TAMs induced by tumor cells to polarize toward a more inflammatory phenotype, including enhanced T cell responses to stimulation by DCs and macrophages, increased expression of M1 markers and secreted proinflammatory cytokine TNFα, decreased expression of M2 markers and secreted anti-infla