Description:
<jats:title>Abstract</jats:title>
<jats:p>Chimeric antigen receptors (CARs) have shown remarkable promise in treating hematological malignancies, especially those expressing CD19 antigen. On the other hand, identification and efficacy of CARs against antigens in solid cancers has remained a significant challenge. Defects in glycosylation in solid cancers are common, and can result in formation of cell surface-expressed neoantigens that are aberrantly glycosylated. Here, we focused on antigens that result from defects in O-linked glycosylation, leading to formation of unique antigens that possess an N-acetyl-galactosamine linked to a serine or threonine residue on a surface-expressed protein (also called Tn antigen), instead of extended glycosylation patterns. 237 is a mouse monoclonal antibody that was generated by a mouse immunized with cells of a spontaneous murine cancer, and that binds to a Tn-antigen linked to the mouse protein, OTS8. Previously, we showed that a CAR generated from the 237 single chain variable fragment (scFv) recognized, with low affinity, the human cell line Jurkat. More recently, we demonstrated that the 237 CAR could eradicate established Jurkat leukemia in a mouse model. Here, we used structure-guided directed evolution to engineer the binding site of 237 to bind to multiple Tn linked antigens, including MUC1, with higher affinity. CARs containing these engineered scFv variants recognized a panel of glycosylation-defective mouse and human cancer cell lines more effectively than the wild-type 237 CAR, with no activity toward cell lines lacking these defects. Cancer cell lines lacking MUC1 were also effectively recognized by the engineered variants, indicating their broadened specificity toward multiple Tn-linked antigens. Consistent with this finding, the CAR variants also demonstrated greater sensitivity toward several Tn-linked human peptides. To extend these studies, we are currently developing additional mouse models to study the efficacy of the engineered 237 CARs in the control of solid tumors. In summary, we developed efficient chimeric antigen receptors that recognize multiple, cancer-associated Tn linked antigens, based on a single antibody scaffold. We believe that their specific recognition of Tn antigen, together with broadened peptide-backbone reactivity, holds promise for cancer-specific recognition yet minimal antigen escape against tumors with defects in O-linked glycosylation, because cancer-specific Tn-linked epitopes on several independent proteins can be simultaneously targeted.</jats:p>
<jats:p>Citation Format: Preeti Sharma, Venkata VVR Marada, Monika Kizerwetter, Claire P. Schane, Yanran He, Steven P. Wolf, Karin Schreiber, Edward J. Roy, Henrik Clausen, Hans Schreiber, David M. Kranz. Engineering chimeric antigen receptors for adoptive T cell therapy of cancers that express the Tn antigen [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3238.</jats:p>