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High-Affinity TCRs Generated by Phage Display Provide CD4⁺ T Cells with the Ability to Recognize and Kill Tumor Cell Lines¹

Yangbing Zhao^*, Alan D. Bennett, Zhili Zheng^*, Qiong J. Wang^*, Paul F. Robbins^*, Lawrence Y. L. Yu^*, Yi Li, Peter E. Molloy, Steven M. Dunn, Bent K. Jakobsen, Steven A. Rosenberg^* and Richard A. Morgan^2,*

^* Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and Avidex Ltd., Abingdon, Oxon, United Kingdom

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 µM to 26 pM). CD8⁺ T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (K_D value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, K_D 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range—with K_D values of 450 nM and 4 µM—demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8⁺ T cells, introduction of these TCR dramatically increased the reactivity of CD4⁺ T cells to tumor cell lines.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health.

² Address correspondence and reprint requests to Dr. Richard A. Morgan, Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Drive, MSC 1201, Building 10, Room 3W5940, Bethesda, MD 20892-1201. E-mail address: rmorgan{at}mail.nih.gov

³ Abbreviations used in this paper: pMHC, peptide-MHC; wt, wild type; RCC, renal cell carcinoma cell line; IVT, in vitro transcribed; ₂m, ₂-microglobulin; IRES, internal ribosomal entry site; TIL, tumor-infiltrating lymphocyte; MSCV, murine stem cell virus; SCT, single chain trimer.

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