Abstract 887: Killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2) immune checkpoint modulates CAR-T cell effector function
Authors: Fontela Miguel Gomez , Snedal Sebastian , Planes Elena Martinez , Abate-Daga Daniel 
Published: April 2023

Abstract
Significance: A modulatory role of killer immunoglobulin-like receptor 2DL2 (KIR2DL2) during T cell receptor (TCR) late signaling events has been proposed. However, its role in CAR-T cell effector function has not yet been studied. Our work aims to understand the differential modulatory effect of KIR2DL2 immune checkpoint on the effector function of therapeutic T cells used for the treatment of liquid and solid tumors.
Methods: We generated CAR-T cells, with or without KIR2DL2 overexpression, using a bicistronic retroviral vector based on the MSGV1 backbone. HLA-C1-deficient tumor cells (HLA-C1−/−) were generated by CRISPR/Cas-based knockout of the B2M gene. We designed gRNAs targeting KIR2DL2, validated their cleavage efficiency and proved the feasibility to abrogate its expression in primary T cells. We tested the inhibitory role of KIR2DL2 in vitro in HLA-C1+ or HLA-C1−/− tumor cells using impedance- and bioluminescence-based cytotoxic assays at different effector:target ratios. We evaluated cytokine production after coculture of CAR-T and target cells using the ELLA system. To assess the modulatory role of KIR2DL2 in vivo, we generated HLA-C1+ or HLA-C1− subcutaneous xenografts of human pancreatic adenocarcinoma (HPAC) cells, then treated the mice with KIR2DL2+ or KIR2DL2− PSCA-CAR-T cells. We assessed the effect of KIR2DL2 in a lymphoblastic leukemia (ALL) xenograft model (NALM6 cells), treated with either KIR2DL2+ or KIR2DL2-deficient CD19-CAR-T cells (i.v.).
Results: In vitro, KIR2DL2+ CAR-T cells were significantly less cytotoxic in an HLA-C1-dependent manner, regardless of their target antigen. Cytokine analysis after coculture showed that overexpression of KIR2DL2 in CAR-T cells impaired their ability to produce IFN-γ, IL-2 and TNF-α in presence of HLA-C1. In vivo, KIR2DL2-overexpressing PSCA-CAR-T cells injected in NSG mice harboring PSCA+/HLA-C1+ pancreatic tumors were not able to eliminate tumors. In contrast, KIR2DL2-overexpressing CAR-T cells transferred to mice harboring PSCA+/HLA-C1−/− tumor cells performed as well as their KIR2DL2− counterparts. Two gRNAs targeting KIR2DL2 were validated in primary T cells by means of an enzymatic cleavage assay and flow cytometry. Current efforts are focused on characterizing the impact of KIR2DL2 ablation on the anti-leukemia activity of CAR-T cells in vivo.
Conclusions: KIR2DL2 impairs CAR-T cell effector function and cytokine secretion in vitro in both pancreatic and ALL tumor models, and in vivo in the pancreatic model. We implemented abrogation of KIR2DL2 by CRISPR/Cas9 during CD19-CAR-T cell manufacturing. Ongoing efforts focus on testing the impact of KIR2DL2 ablation on the in vivo performance of CAR-T cells. These results strengthen the notion of KIR2DL2 as an immune checkpoint with a relevant role in T cell immunosurveillance and may constitute an actionable target to enhance adoptive cell therapies.
Citation Format: Miguel Gomez Fontela, Sebastian Snedal, Elena Martinez Planes, Daniel Abate-Daga. Killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2) immune checkpoint modulates CAR-T cell effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 887.