Abstract A017: Cysteine-Engineered CAR-T Cells to Counter Antigen Escape in B Cell Lymphoma
Authors: Lühle Jost , Krost Simon , Goerdeler Felix , Seitz Christian , Seeberger Peter H. , Moscovitz Oren 
Published: February 2025

Abstract
The chimeric Antigen Receptor (CAR) T-cell approach has revolutionized the treatment of hematological malignancies, achieving remarkable success. However, despite its outstanding achievements, the technology still faces several challenges, including treatment-induced antigen escape. Thus, cancer cells often adapt to CAR-T therapy by suppressing target markers, leading to cancer relapse and treatment failure. This phenomenon renders CAR-T cells ineffective and poses a significant clinical challenge. Therefore, identifying alternative targets less prone to antigen escape is crucial. Abnormal expression of Protein Disulfide Isomerase (PDI) and other multifunctional redox enzymes in cancer cells alters the redox status of exofacial protein thiols or reactive disulfide. Our recent study identified abnormal redox states in B-cell lymphomas (BCL) as potential immunotherapy targets. In addition, we presented a single-domain antibody that specifically binds to BCL cells through a non-canonical cysteine in its complementarity-determining regions (CDRs). Building on these findings, we introduced a single-cysteine to a single-domain antibody fragment targeting the green fluorescent protein (GFP), inducing specific binding to BCL cells. We then expressed the cysteine-engineered single-domain antibodies as CARs on T cells, demonstrating their capacity to trigger cytotoxicity against BCL through their altered redox cell surface. Therefore, we generated bifunctional CAR-T cells by cysteine-engineering the clinically approved anti-CD19 single-chain variable fragment (scFv) CAR to overcome CD19 antigen escape. Our data demonstrate that T cells expressing the cysteine-engineered CD19-CAR effectively target various CD19-positive BCL subtypes. However, in contrast to their non-engineered counterparts, the inserted cysteine residue enables CAR-T cell activation and cytotoxicity against CD19-negative BCL cells in vitro and in a Mantle Cell Lymphoma (MCL) xenograft mouse model, extending survival in treated mice. Furthermore, we introduced cysteine mutations in different domains of the anti-CD19 scFv-CAR and meticulously assessed their activity and cytotoxicity against both CD19-positive and negative MCL cells. Based on the mutants varying cytotoxicity levels, we propose specific criteria for cysteine insertion to achieve optimal cytotoxicity of bi-specific CAR-T cells. Our findings present a novel class of bifunctional CAR-T cell therapy that targets both conventional antigens and altered redox states on the surface of BCLs, offering a promising strategy to mitigate the risk of antigen escape.
Citation Format: Jost Lühle, Simon Krost, Felix Goerdeler, Christian Seitz, Peter H. Seeberger, Oren Moscovitz. Cysteine-Engineered CAR-T Cells to Counter Antigen Escape in B Cell Lymphoma [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A017.