Structure-guided engineering of the affinity and specificity of CARs against Tn-glycopeptides

Preeti Sharma, Venkata V.V.R. Marada, Qi Cai, Monika Kizerwetter, Yanran He, Steven P. Wolf, Karin Schreiber, Henrik Clausen, Hans Schreiber, David M. Kranz

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Article number201920662
Pages (from-to)15148-15159
Number of pages12
JournalProceedings of the National Academy of Sciences
Volume117
Issue number26
DOIs
StatePublished - Jun 30 2020

Keywords

  • CAR T cells
  • antibody engineering
  • neoantigen epitopes

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Structure-guided engineering of the affinity and specificity of CARs against Tn-glycopeptides'. Together they form a unique fingerprint.

Cite this