Abstract 6345: In vitro characterization of a dual antagonistic anti-LILRB2/LILRB4 monoclonal antibody
Authors: Daugherty Bruce L. , Ge Xiaomei , Hsieh Christine L. , Zabel Brian A. , Lederman Seth 
Published: March 2024

Abstract
Introduction: Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are a potential therapeutic target in immune checkpoint cancer therapy, but MDSC-targeted therapies have yet been shown to improve survival. The leukocyte immunoglobulin (Ig)-like receptor B2 is a member of the leukocyte Ig-like receptor (LILR) family and is predominantly expressed on the surface of cells of the myelomonocytic lineage (monocytes, macrophages and dendritic cells). LILRB2 is a negative regulator of myeloid cells and is an immune checkpoint receptor. We chose to generate mAbs against LILRB2 as a potential immunotherapeutic for cancer.
Methods: PentaMice® strains (5 WT mouse strains comprising 9 MHC class II haplotypes to maximize Ab diversity) were immunized with human LILRB2. Lymphocytes were fused with a myeloma partner to generate lead monoclonal hybridomas. 24 purified anti-LILRB2 mAbs were generated and evaluated by arrayed surface plasma resonance (SPR, Carterra® LSA® instrument) to assign epitope bins. mAb humanization was performed in silico to maximize T20 “humanness” scores (algorithm developed by Curia). Humanized mAbs were transiently expressed using Curia’s TunaCHO™ platform and tested in in vitro MDSC assays. Autologous co-cultures of magnetic bead enriched-human blood-derived MDSC (HLA-DR-CD14+) model cells and T cells (CD3+) were stimulated with plate-bound anti-CD3/CD28 to induce T cell activation. Cytokine secretion was assessed 4 days later.
Results: A total of 384 hybridoma supernatants were selected from the primary screen. 245 were confirmed by ELISA and 178 stained HEK293 cells expressing LILRB2 cells by flow cytometry (FACS). 24 hybridomas were further selected, cloned and sequenced. Five epitope bins were identified via Carterra® LSA® instrument. Two clones were chosen for humanization, designated 6-I11A and 4-O19A and were recombinantly expressed with the human IgG4 isotype. FACS analysis on HEK293 cells expressing either huLILRB2 or huLILRB4 revealed that clone hu4-O19A-IgG4 was specific for huLILRB2. Surprisingly, clone hu6-I11A-IgG4 stained both huLILRB2 and huLILRB4. FACS analysis with hu4-O19A-IgG4 on human MDSCs exhibited an EC50 of 11.9 nM and a KD of 1.5 nM in binding to recombinant huLILRB2 antigen, while hu6-I11A-IgG4 had an EC50 of 3.2 nM and a KD of 0.6 nM. In co-cultures with human MDSCs and T cells, both hu6-I11A-IgG4 and hu4-O19A-IgG4 reversed cytokine suppression by the MDSCs by increasing GM-CSF, IL-10, TNFα, and IL-6, while suppressing IL-13, and had little effect on IL-17A, MIP-3α, IL-4, IL-2 and IFNγ compared to treatment with the isotype control, with hu6-I11A-IgG4 having a greater effect as an antagonist than hu4-O19A-IgG4.
Conclusions: We have identified a dual anti-LILRB2/LILRB4 antagonist. Targeting MDSCs with this antibody offers great promise for immunotherapy for cancer as well as its immense potential for synergy with PD-1 blockade.
Citation Format: Bruce L. Daugherty, Xiaomei Ge, Christine L. Hsieh, Brian A. Zabel, Seth Lederman. In vitro characterization of a dual antagonistic anti-LILRB2/LILRB4 monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6345.