Development of CLDN18.2-specific VHH CAR T cells for treatment of gastrointestinal cancers

Abstract

Solid tumors such as gastrointestinal and esophageal cancers continue to confer poor prognosis for patients, and treatment options remain limited. While hematological malignancies have benefitted from advances in targeted immunotherapies like Chimeric Antigen Receptor (CAR) T cell therapy, efficacy of CAR T cells in solid tumors lags due to a plethora of factors. Some of these issues could be overcome by using single-chain-only antibodies, nanobodies/VHHs, as opposed to single chain variable fragments (scFvs) for antigen recognition. In addition, a new gastric cancer biomarker, CLDN18.2, has recently emerged as a promising new tumor-specific CAR T cell therapy target. Therefore, this research aims to rapidly screen for and validate novel VHH sequences against promising CAR T cell targets such as CLDN18.2. Mice engineered to produce nanobodies were immunized with CLDN18.2-expressing dendritic cells, and the resulting VHH repertoires were isolated and amplified into VHH cDNA libraries. Libraries were cloned into a CAR backbone and the resulting VHH CAR libraries were transduced into a novel T cell reporter cell line. VHH CAR T cell libraries were screened for CLDN18.2-specific VHH sequences by co-culturing with CLDN18.2-expressing cells. Individual VHH CAR sequences were subsequently isolated and further validated for CLDN18.2-specificity. A subset of VHH CAR libraries contained CLDN18.2-specific and pan-CLDN18 VHH sequences. Moreover, a highly specific CLDN18.2 VHH was identified and shown to bind both overexpression and endogenously CLDN18.2-expressing cell lines, with no cross-reactivity for the isoform CLDN18.1. As the antigen-recognition moiety in primary human CAR T cells, this VHH was able to control CLDN18.2+ tumor cell growth. In addition, several pan-CLDN18-specific VHH sequences with varying affinities were identified and found to bind both isoforms, as well as control CLDN18+ tumor cell growth as a CAR moiety. Thus, we were able to isolate and characterize CLDN18.2- and pan-CLDN18 specific VHH sequences that can be functionally used in CAR T cells and demonstrate applicability of a nanobody-based screening pipeline.