{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,5]],"date-time":"2026-01-05T20:49:39Z","timestamp":1767646179168,"version":"3.48.0"},"posted":{"date-parts":[[2026,1,2]]},"group-title":"Bioengineering","reference-count":65,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,1,2]],"date-time":"2026-01-02T00:00:00Z","timestamp":1767312000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["R35GM131923"],"award-info":[{"award-number":["R35GM131923"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["T32GM086330"],"award-info":[{"award-number":["T32GM086330"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,1,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>T-cell receptors (TCRs) recognize peptides presented by MHC, enabling access to intracellular targets that are largely inaccessible to antibodies and difficult to target with small molecules.<\/jats:p>\n                <jats:p>Despite this potential, their inherent cross-reactivity limits tumor specificity, while single-antigen targeting provides limited coverage of intratumoral heterogeneity. Dual-TCR therapeutics comprising two distinct TCRs could enhance tumor specificity via combinatorial recognition while broadening coverage across heterogeneous antigens. However, practical development of dual-TCR therapeutics has been limited by \u03b1\/\u03b2 subunit mispairing that prevents efficient production and creates undesired binding properties. Here, we develop orthogonal TCR \u03b1\/\u03b2 interfaces that prevent subunit mispairing. Using computational multistate design and second-site suppressor strategies implemented in Rosetta, we identified over 250 TCR variants for experimental screening to assess protein stability and pairing fidelity. The top-performing designs achieved approximately 95% correct pairing, as validated by mass spectrometry and X-ray crystallography. Focusing mutations on constant domains and conserved framework regions of variable domains enabled broad applicability across diverse TCRs while preserving antigen recognition. Using these orthogonal interfaces, we developed trispecific T-cell engagers (TriTEs) that target two cancer-testis antigens and CD3 on T cells, demonstrating enhanced potency under dual-antigen engagement (EC50 of 380 fM) while maintaining high activity when targeting cells displaying a single antigen (EC50s of 48 pM and 20 pM). This orthogonal TCR interface technology establishes a generalizable platform for engineering multi-specific immune therapeutics targeting diverse cancer antigens.<\/jats:p>","DOI":"10.64898\/2026.01.02.697397","type":"posted-content","created":{"date-parts":[[2026,1,3]],"date-time":"2026-01-03T06:30:13Z","timestamp":1767421813000},"source":"Crossref","is-referenced-by-count":0,"title":["Computational design of orthogonal TCR \u03b1\/\u03b2 interfaces for dual-TCR therapeutics"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0939-7756","authenticated-orcid":false,"given":"Tomoaki","family":"Kinjo","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"},{"name":"Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1951-2949","authenticated-orcid":false,"given":"Shawn","family":"Yu","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"},{"name":"Division of Medical Oncology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0025-2377","authenticated-orcid":false,"given":"Nathan","family":"Nicely","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4609-4340","authenticated-orcid":false,"given":"Andrew","family":"Leaver-Fay","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7922-2156","authenticated-orcid":false,"given":"William Y.","family":"Kim","sequence":"additional","affiliation":[{"name":"Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"},{"name":"Division of Medical Oncology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4907-9699","authenticated-orcid":false,"given":"Brian","family":"Kuhlman","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"},{"name":"Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026010510301585000_2026.01.02.697397v1.1","doi-asserted-by":"crossref","first-page":"683","DOI":"10.3390\/cancers12030683","article-title":"Adoptive Cell Therapy\u2014Harnessing Antigen-Specific T Cells to Target Solid Tumours","volume":"12","year":"2020","journal-title":"Cancers"},{"key":"2026010510301585000_2026.01.02.697397v1.2","doi-asserted-by":"publisher","DOI":"10.1186\/s13045-019-0788-4"},{"key":"2026010510301585000_2026.01.02.697397v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2010.05.011"},{"key":"2026010510301585000_2026.01.02.697397v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/nrc3236"},{"key":"2026010510301585000_2026.01.02.697397v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/nrc.2017.36"},{"key":"2026010510301585000_2026.01.02.697397v1.6","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2007-02-075010"},{"key":"2026010510301585000_2026.01.02.697397v1.7","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1005802107"},{"key":"2026010510301585000_2026.01.02.697397v1.8","first-page":"188ra174","article-title":"Dual receptor T cells mediate pathologic alloreactivity in patients with acute graft-versus-host disease","volume":"5","year":"2013","journal-title":"Science translational medicine"},{"key":"2026010510301585000_2026.01.02.697397v1.9","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/gzg087"},{"key":"2026010510301585000_2026.01.02.697397v1.10","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.180.1.391"},{"key":"2026010510301585000_2026.01.02.697397v1.11","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-06-1450"},{"key":"2026010510301585000_2026.01.02.697397v1.12","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.0902055"},{"key":"2026010510301585000_2026.01.02.697397v1.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.molmed.2009.12.004"},{"key":"2026010510301585000_2026.01.02.697397v1.14","doi-asserted-by":"publisher","DOI":"10.1101\/2025.05.28.656745"},{"key":"2026010510301585000_2026.01.02.697397v1.15","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.19095"},{"key":"2026010510301585000_2026.01.02.697397v1.16","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-09-1450"},{"key":"2026010510301585000_2026.01.02.697397v1.17","doi-asserted-by":"publisher","DOI":"10.1038\/nm.2700"},{"key":"2026010510301585000_2026.01.02.697397v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(04)83004-0"},{"key":"2026010510301585000_2026.01.02.697397v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-381270-4.00019-6"},{"key":"2026010510301585000_2026.01.02.697397v1.20","doi-asserted-by":"crossref","first-page":"3031","DOI":"10.1021\/acs.jctc.7b00125","article-title":"The Rosetta all-atom energy function for macromolecular modeling and design","volume":"13","year":"2017","journal-title":"Journal of chemical theory and computation"},{"key":"2026010510301585000_2026.01.02.697397v1.21","doi-asserted-by":"publisher","DOI":"10.1002\/pro.3588"},{"key":"2026010510301585000_2026.01.02.697397v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-019-0163-x"},{"key":"2026010510301585000_2026.01.02.697397v1.23","doi-asserted-by":"publisher","DOI":"10.1080\/19420862.2015.1022694"},{"key":"2026010510301585000_2026.01.02.697397v1.24","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2016.02.013"},{"key":"2026010510301585000_2026.01.02.697397v1.25","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.2797"},{"key":"2026010510301585000_2026.01.02.697397v1.26","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0020937"},{"key":"2026010510301585000_2026.01.02.697397v1.27","doi-asserted-by":"crossref","first-page":"1055","DOI":"10.1002\/prot.22631","article-title":"Computational design of second-site suppressor mutations at protein\u2013protein interfaces","volume":"78","year":"2010","journal-title":"Proteins: Structure, Function, and Bioinformatics"},{"key":"2026010510301585000_2026.01.02.697397v1.28","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pcbi.1007714"},{"key":"2026010510301585000_2026.01.02.697397v1.29","doi-asserted-by":"publisher","DOI":"10.1021\/ja054718w"},{"key":"2026010510301585000_2026.01.02.697397v1.30","first-page":"1","article-title":"Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics","volume":"11","year":"2020","journal-title":"Nature communications"},{"key":"2026010510301585000_2026.01.02.697397v1.31","doi-asserted-by":"publisher","DOI":"10.1110\/ps.051936406"},{"key":"2026010510301585000_2026.01.02.697397v1.32","doi-asserted-by":"publisher","DOI":"10.1002\/pro.4428"},{"key":"2026010510301585000_2026.01.02.697397v1.33","doi-asserted-by":"publisher","DOI":"10.1002\/pro.2187"},{"key":"2026010510301585000_2026.01.02.697397v1.34","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1002\/prot.26030","article-title":"Perturbing the energy landscape for improved packing during computational protein design","volume":"89","year":"2021","journal-title":"Proteins: Structure, Function, and Bioinformatics"},{"key":"2026010510301585000_2026.01.02.697397v1.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.jim.2009.08.008"},{"key":"2026010510301585000_2026.01.02.697397v1.36","first-page":"189","volume-title":"Methods Enzymol","volume":"503","year":"2012"},{"key":"2026010510301585000_2026.01.02.697397v1.37","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkn838"},{"key":"2026010510301585000_2026.01.02.697397v1.38","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M115.707414"},{"key":"2026010510301585000_2026.01.02.697397v1.39","doi-asserted-by":"crossref","first-page":"2453","DOI":"10.4049\/jimmunol.1101268","article-title":"TCRs used in cancer gene therapy cross-react with MART-1\/Melan-A tumor antigens via distinct mechanisms","volume":"187","year":"2011","journal-title":"The Journal of Immunology"},{"key":"2026010510301585000_2026.01.02.697397v1.40","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-09-0547"},{"key":"2026010510301585000_2026.01.02.697397v1.41","doi-asserted-by":"publisher","DOI":"10.1038\/icb.2014.93"},{"key":"2026010510301585000_2026.01.02.697397v1.42","doi-asserted-by":"publisher","DOI":"10.1038\/nm.2764"},{"key":"2026010510301585000_2026.01.02.697397v1.43","doi-asserted-by":"publisher","DOI":"10.1016\/j.ctrv.2019.06.001"},{"key":"2026010510301585000_2026.01.02.697397v1.44","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2304753"},{"key":"2026010510301585000_2026.01.02.697397v1.45","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.87.18.7220"},{"key":"2026010510301585000_2026.01.02.697397v1.46","first-page":"45","article-title":"Engineering a humanized bispecific F (ab\u2019) 2 fragment for improved binding to T cells","volume":"7","year":"1992","journal-title":"International journal of cancer. Supplement= Journal international du cancer. Supplement"},{"key":"2026010510301585000_2026.01.02.697397v1.47","unstructured":"Hayes, C. , Hibbert, L. , Liddy, N. , Mahon, T. & Raman, M. T CELL RECEPTORS. WO patent WO 2017\/175006 A1 (2017)."},{"key":"2026010510301585000_2026.01.02.697397v1.48","unstructured":"Chester, F. et al. T CELL RECEPTORS SPECIFIC FOR THE NY-ESO-1 TUMOR ANTIGEN-HLA-A*02 COMPLEX. WO patent WO 2017\/109496 A1 (2017)."},{"key":"2026010510301585000_2026.01.02.697397v1.49","unstructured":"Addis Philip, W. et al. T CELL RECEPTORS. WO patent WO 2018\/234319 A1 (2018)."},{"key":"2026010510301585000_2026.01.02.697397v1.50","doi-asserted-by":"publisher","DOI":"10.1007\/BF00375376"},{"key":"2026010510301585000_2026.01.02.697397v1.51","doi-asserted-by":"publisher","DOI":"10.1038\/384134a0"},{"key":"2026010510301585000_2026.01.02.697397v1.52","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2017.06.010"},{"key":"2026010510301585000_2026.01.02.697397v1.53","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-021-03819-2"},{"key":"2026010510301585000_2026.01.02.697397v1.54","doi-asserted-by":"publisher","DOI":"10.1126\/science.add2187"},{"key":"2026010510301585000_2026.01.02.697397v1.55","doi-asserted-by":"publisher","DOI":"10.1101\/gr.849004"},{"key":"2026010510301585000_2026.01.02.697397v1.56","doi-asserted-by":"publisher","DOI":"10.1007\/s00251-001-0408-6"},{"key":"2026010510301585000_2026.01.02.697397v1.57","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btv552"},{"key":"2026010510301585000_2026.01.02.697397v1.58","doi-asserted-by":"publisher","DOI":"10.1093\/protein\/gzg087"},{"key":"2026010510301585000_2026.01.02.697397v1.59","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444906029799"},{"key":"2026010510301585000_2026.01.02.697397v1.60","unstructured":"SADABS v. Version 2016\/2 (Bruker AXS Inc., Madison, Wisconsin, USA, 2016)."},{"key":"2026010510301585000_2026.01.02.697397v1.61","unstructured":"SAINT v. Version 8.40B (Bruker AXS Inc., Madison, Wisconsin, USA, 2019)."},{"key":"2026010510301585000_2026.01.02.697397v1.62","doi-asserted-by":"publisher","DOI":"10.1107\/S0021889807021206"},{"key":"2026010510301585000_2026.01.02.697397v1.63","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444910007493"},{"key":"2026010510301585000_2026.01.02.697397v1.64","doi-asserted-by":"publisher","DOI":"10.1107\/S2059798319011471"},{"key":"2026010510301585000_2026.01.02.697397v1.65","unstructured":"Kinjo, T. & Kuhlman, B. Bioorthogonal t cell receptor molecules and methods of making and using the same. International (WIPO) patent WO2024036166A1 (2024)."}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.01.02.697397","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,5]],"date-time":"2026-01-05T18:30:26Z","timestamp":1767637826000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.01.02.697397"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,2]]},"references-count":65,"URL":"https:\/\/doi.org\/10.64898\/2026.01.02.697397","relation":{},"subject":[],"published":{"date-parts":[[2026,1,2]]},"subtype":"preprint"}}