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.