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Visualization of p53_264–272/HLA-A*0201 Complexes Naturally Presented on Tumor Cell Surface by a Multimeric Soluble Single-Chain T Cell Receptor¹

Xiaoyun Zhu^*, Heather J. Belmont^*, Shari Price-Schiavi, Bai Liu^*, Hyung-il Lee^*, Marilyn Fernandez^*, Richard L. Wong, Janette Builes^*, Peter R. Rhode^* and Hing C. Wong^2,*

^* Altor BioScience Corp., Miramar, FL 33025; Pathology Associates Inc., Frederick, MD 21701; and University of Florida, Gainesville, FL 32612

Intracellular Ags are processed into small peptides that are presented on cell surfaces in the context of HLA class I molecules. These peptides are recognized by TCRs displayed by CD8⁺ T lymphocytes (T cells). To date, direct identification and quantitation of these peptides has relied primarily on mass spectrometry analysis, which is expensive and requires large quantities of diseased tissues to obtain useful results. Here we demonstrate that multimerization of a soluble single-chain TCR (scTCR), recognizing a peptide from p53 presented in the context of HLA-A2.1, could be used to directly visualize and quantitate peptide/MHC complexes on unmanipulated human tumor cells. Tumor cells displaying as few as 500 peptide/MHC complexes were readily detectable by flow cytometry. The scTCR/multimers exhibited exquisite recognition capability and could distinguish peptides differing in as little as a single amino acid. We also demonstrate that scTCR/multimers could specifically stain human tumors generated in mice, as well as tumors obtained from patient biopsies. Thus, scTCR/multimers represent a novel class of immunostaining reagents that could be used to validate, quantitate, or monitor epitope presentation by cancer cells.