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Subtle Affinity-Enhancing Mutations in a Myelin Oligodendrocyte Glycoprotein-Specific TCR Alter Specificity and Generate New Self-Reactivity¹

Akshata Udyavar^2,*, Rajshekhar Alli^2,*, Phuong Nguyen^*, Lesley Baker^*, and Terrence L. Geiger^3,*

^* Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and Rhodes College, Memphis, TN 38112

We describe a simple iterative approach to augment TCR affinity, which we studied using a myelin oligodendrocyte glycoprotein-specific TCR. We hypothesized that single amino acid modifications in TCR CDR3 could enhance TCR sensitivity through focal interactions with antigenic peptide while minimizing the risk of cross-reactivity observed previously in TCR more broadly mutagenized using in vitro evolution techniques. We show that this iterative method can indeed generate TCR with Ag sensitivity 100-fold greater than the parental receptor and can endow TCR with coreceptor independence. However, we also find that single amino acid mutations in the CDR3 can alter TCR fine specificity, affecting recognition requirements for Ag residues over most of the length of the MHC binding groove. Furthermore, minimal changes in surface-exposed CDR3 amino acids, even the addition of a single hydroxyl group or conversion of a methyl or sulfhydryl moiety to a hydroxyl, can confer modified Ag-specific TCR with new self-reactivity. In vivo modeling of modified TCR through retroviral TCR gene transfer into Rag^–/– mice confirmed the biological significance of these altered reactivities, although it also demonstrated the feasibility of producing Ag-specific, positively selecting, coreceptor-independent receptors with markedly increased Ag sensitivity. These results affirm the possibility of readily generating affinity-enhanced TCR for therapeutic purposes but demonstrate that minimal changes in TCR CDR3 structure can promote self reactivity and thereby emphasize the importance of caution in validating receptors with even subtle alterations before clinical application.

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 National Institutes of Health Grant R01 AI056153 (to T.L.G.) and by the American Lebanese Syrian Associated Charities/St. Jude Children’s Research Hospital.

² A.U. and R.A. contributed equally to this paper.

³ Address correspondence and reprint requests to Dr. Terrence L. Geiger, Associate Member, Department of Pathology, St. Jude Children’s Research Hospital, 332 North Lauderdale, D-4047, Memphis, TN 38105. E-mail address: terrence.geiger{at}stjude.org

⁴ Abbreviations used in this paper: pMHC, peptide MHC; LN, lymph node; MOG, myelin oligodendrocyte glycoprotein; rhIL-2, recombinant human IL-2.

⁵ The online version of this article contains supplemental material.