Using a deep mutational scan, Sharma et al. showed that the 237-antibody, which binds to Tn antigen – a cancer-specific, aberrantly O-glycosylated epitope of a murine protein (OTS8) – is affected by the peptide side chains. CAR T cells transduced with higher-affinity 237-scFv variants only slightly increased CAR T cell activation against Tn-OTS8-expressing cancer cells. Selection for 237-scFv variants against a different O-glycosylated epitope from human MUC-1 (Tn-MUC1) resulted in molecules reactive to Tn-MUC1, Tn-OTS8, and cancer cells lacking both antigens, suggesting a response against multiple Tn antigens.
Contributed by Anna Scherer
ABSTRACT: The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N-acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors.