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The inherent antigen cross-reactivity of the T cell receptor (TCR) is balanced by high specificity. Surprisingly, TCR specificity often manifests in ways not easily interpreted from static structures. Here we show that TCR discrimination between an HLA-A*03:01 (HLA-A3)-restricted public neoantigen and its wild-type (WT) counterpart emerges from distinct motions within the HLA-A3 peptide binding groove that vary with the identity of the peptide's first primary anchor. These motions create a dynamic gate that, in the presence of the WT peptide, impedes a large conformational change required for TCR binding. The neoantigen is insusceptible to this limiting dynamic, and, with the gate open, upon TCR binding the central tryptophan can transit underneath the peptide backbone to the opposing side of the HLA-A3 peptide binding groove. Our findings thus reveal a novel mechanism driving TCR specificity for a cancer neoantigen that is rooted in the dynamic and allosteric nature of peptide/MHC-I binding grooves, with implications for resolving long-standing and often confounding questions about T cell specificity., Competing Interests: Competing interests: C.A.K. and S.S.C. are inventors of patents related to the T cell receptor (TCR) sequences featured in this manuscript and are recipients of licensing revenue from Intima Bioscience shared according to Memorial Sloan Kettering Cancer Center (MSKCC) institutional policies. C.A.K. has consulted for or is on the scientific advisory boards for Achilles Therapeutics, Affini-T Therapeutics, Aleta BioTherapeutics, Bellicum Pharmaceuticals, Bristol Myers Squibb, Catamaran Bio, Cell Design Labs, Decheng Capital, G1 Therapeutics, Klus Pharma, Obsidian Therapeutics, PACT Pharma, Roche/Genentech, Royalty Pharma, and T-knife, and is a scientific co-founder and equity holder in Affini-T Therapeutics. S.S.C. is a scientific advisor and equity holder in Affini-T Therapeutics. B.M.B. is an inventor on patents relating to differences between mutant and self in identifying immunogenic neoantigens has consulted for or received funding from Merck, Pfizer, Eureka Therapeutics, and EnaraBio, and is on the scientific advisory board of T-cure Bioscience. The other authors declare no competing interests., (© 2025. The Author(s).)

SARS-CoV-2infection can induce multisystem inflammatory syndrome in children, which resembles superantigen-induced toxic shock syndrome. Recent work has suggested that the SARS-CoV-2 spike (S) protein could act as a superantigen by binding T cell receptors (TCRs) and inducing broad antigen-independent T cell responses. Structure-based computational modeling identified potential TCR-binding sites near the S receptor-binding domain, in addition to a site with homology to known neurotoxins. We experimentally examined the mechanism underpinning this theory-the direct interaction between the TCR and S protein. Surface plasmon resonance of recombinantly expressed S protein and TCR revealed no detectable binding. Orthogonally, we pseudotyped lentiviruses with SARS-CoV-2 S in both wild-type and prefusion-stabilized forms, demonstrated their functionality in a cell line assay, and observed no transduction, activation, or stimulation of proliferation of CD8 + T cells. We conclude that it is unlikely that the SARS-CoV-2 spike protein engages nonspecifically with TCRs or has superantigenic character., Competing Interests: Competing interests statement:M.E.B. is a founder and consultant of Kelonia Therapeutics and Abata Therapeutics. M.E.B. is an equity holder of Kelonia Therapeutics, Abata Therapeutics, and 3T Biosciences. The lentiviral targeting approach in this manuscript is the subject of US patent applications with M.E.B. and S.A.G. as coinventors.

The inherent cross-reactivity of the T cell receptor (TCR) is balanced by high specificity, which often manifests in confounding ways not easily interpretable from static structures. We show here that TCR discrimination between an HLA-A*03:01 (HLA-A3)-restricted public neoantigen derived from mutant PIK3CA and its wild-type (WT) counterpart emerges from motions within the HLA binding groove that vary with the identity of the peptide's first primary anchor. The motions form a dynamic gate that in the complex with the WT peptide impedes a large conformational change required for TCR binding. The more rigid neoantigen is insusceptible to this limiting dynamic, and with the gate open, is able to transit its central tryptophan residue underneath the peptide backbone to the contralateral side of the HLA-A3 peptide binding groove, facilitating TCR binding. Our findings reveal a novel mechanism driving TCR specificity for a cancer neoantigen that is rooted in the dynamic and allosteric nature of peptide/MHC-I complexes, with implications for resolving long-standing and often confounding questions about the determinants of T cell specificity., Competing Interests: Additional Declarations: Yes there is potential Competing Interest. C.A.K. and S.S.C. are inventors on patents related to the T cell receptor (TCR) sequences featured in this manuscript and are recipients of licensing revenue from Intima Bioscience shared according to Memorial Sloan Kettering Cancer Center (MSKCC) institutional policies. C.A.K. has consulted for or is on the scientific advisory boards for Achilles Therapeutics, Affini-T Therapeutics, Aleta BioTherapeutics, Bellicum Pharmaceuticals, Bristol Myers Squibb, Catamaran Bio, Cell Design Labs, Decheng Capital, G1 Therapeutics, Klus Pharma, Obsidian Therapeutics, PACT Pharma, Roche/Genentech, Royalty Pharma, and T-knife, and is a scientific co-founder and equity holder in Affini-T Therapeutics. S.S.C. is a scientific advisor and equity holder in Affini-T Therapeutics. B.M.B. is an inventor on patents relating to differences between mutant and self in identifying immunogenic neoantigens, and is on the scientitic advisory board or has consulted for Merck & Co., Pfizer, T-cure Bioscience, Eureka Therapeutics, and EnaraBio. Conflicts of interest statement C.A.K. and S.S.C. are inventors on patents related to the T cell receptor (TCR) sequences featured in this manuscript and are recipients of licensing revenue from Intima Bioscience shared according to Memorial Sloan Kettering Cancer Center (MSKCC) institutional policies. C.A.K. has consulted for or is on the scientific advisory boards for Achilles Therapeutics, Affini-T Therapeutics, Aleta BioTherapeutics, Bellicum Pharmaceuticals, Bristol Myers Squibb, Catamaran Bio, Cell Design Labs, Decheng Capital, G1 Therapeutics, Klus Pharma, Obsidian Therapeutics, PACT Pharma, Roche/Genentech, Royalty Pharma, and T-knife, and is a scientific co-founder and equity holder in Affini-T Therapeutics. S.S.C. is a scientific advisor and equity holder in Affini-T Therapeutics. B.M.B. is an inventor on patents relating to differences between mutant and self in identifying immunogenic neoantigens, and is on the scientific advisory board or has consulted for Merck & Co., Pfizer, T-cure Bioscience, Eureka Therapeutics, and EnaraBio.

Neoepitopes arising from amino acid substitutions due to single nucleotide polymorphisms are targets of T cell immune responses to cancer and are of significant interest in the development of cancer vaccines. However, understanding the characteristics of rare protective neoepitopes that truly control tumor growth has been a challenge, due to their scarcity as well as the challenge of verifying true, neoepitope-dependent tumor control in humans. Taking advantage of recent work in mouse models that circumvented these challenges, here, we compared the structural and physical properties of neoepitopes that range from fully protective to immunologically inactive. As neoepitopes are derived from self-peptides that can induce immune tolerance, we studied not only how the various neoepitopes differ from each other but also from their wild-type counterparts. We identified multiple features associated with protection, including features that describe how neoepitopes differ from self as well as features associated with recognition by diverse T cell receptor repertoires. We demonstrate both the promise and limitations of neoepitope structural analysis and predictive modeling and illustrate important aspects that can be incorporated into neoepitope prediction pipelines., Competing Interests: Competing interests statement:P.K.S. and B.M.B. are inventors on patent US11338026B2 which relates to neoepitope discovery and differences from self.