Abstract 7532: NB005.1, a fully humanized anti-KLRG1 blocking mAb enhances effector function of human CD8 T-cells

Abstract

Abstract Introduction: Immune checkpoint blockers (ICBs) have revolutionized treatment, enhancing survival and benefiting many patients. However, many patients fail to respond to the treatment, necessitating research for new targets and improved approaches. The Killer cell Lectin-like Receptor G1 (KLRG1), expressed on a subset of NK and T-cells harbors an immune receptor tyrosine-based inhibitory motif (ITIM) within its cytoplasmic domain, indicating its inhibitory function. On CD8+ T-cells, KLRG1 is expressed on highly differentiated antigen-specific effector memory T-cells (TEMs) and CD45RA+ effector memory T cells (TEMRA). These cells are known to exhibit strong cytotoxicity potential and display developmental plasticity following KLRG1 blockade. And 16-48% of CD8+ T-cells that infiltrate the tumor microenvironment (TME) are KLRG1 positive. We hypothesize that the blockade of KLRG1 should enhance the effector function of KLRG1+ CD8 T-cells in the TME and provide therapeutic benefit as a standalone or in combination with existing ICBs. Methods: Monoclonal antibodies (mAbs) targeting the human KLRG1 extracellular domain (ECD) were generated through mouse immunization and hybridoma technology. Purified mAbs from positive clones were screened using CHO-K1 and Jurkat cells overexpressing human or cynomolgus KLRG1 and human primary CD8 T-cells (n=3) to determine EC50 values. Epitope binning was performed, and high-affinity clones from each bin with cyno cross-reactivity were humanized to IgG1-Fc LALA variant. The Abs were evaluated for ligand blocking and enhancement of CD8 T-cell effector function through multiple functional assays. The assay measured enhancement of IFNγ or TNFα by KLRG1+ human CD8+ T-cells co-cultured with CHO-K1 expressing OKT3 and E-cadherin as artificial antigen-presenting cells (aAPCs) or PBMCs stimulated in with anti-CD3 and anti-CD28 Abs in presence of soluble E-cadherin. A human IgG1-LALA antibody (Ab) served as a negative control. PD-1 and KLRG1 synergy was evaluated in hPBMC assays. Results: NB005.1 demonstrated single digit nanomolar (nM) binding potency on human primary CD8 T-cells and Jurkat cells expressing human KLRG1 as identified by its EC50 value. Blocking of KLRG1 with NB005.1 on purified human CD8 T-cells in co-culture assay with aAPC expressing E-cad and OKT3 led to CD8 T-cell activation and increase in IFNγ production confirming inhibitory function of KLRG1. Similar result was obtained when the Ab was tested on hPBMCs stimulated with anti-CD3 and anti-CD28 Abs in presence of soluble E-cadherin. Conclusion: NB005.1, a fully humanized mAb, binds to human KLRG1 in the single digit nM range and effectively blocks the interaction with its ligand. This blockade enhances T-cell effector function as measured by IFNγ production, supporting the advancement of NB005.1 for preclinical testing, either as a standalone therapeutic or in combination with existing ICBs. Citation Format: Bishnu P. Nayak, Meiguang Xiong, Yajuan Xue, Binbin Wang, Shahrzad Lighvani, Shilpi Sharma, Dong Wang, Haojai Wang, Baiyang Wang, Yu Zhang, Xin Dong. NB005.1, a fully humanized anti-KLRG1 blocking mAb enhances effector function of human CD8 T-cells [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 7532.