A platform for parallel T cell receptor cloning and testing enables anti-neoantigen tumor immunotherapy

Alexander M. Rowe; Smriti Chaurasia; Wenzhong Wei; Laura García-Diéguez; Katherine Querry; Johnathon G. Schiebel; Christy Smolak; Alexander G. Muralles; Daniel Wikenheiser; Kevin Quann; Collin Pirner; Kentin Codispot; Mark J. Shlomchik; Warren D. Shlomchik

doi:10.1172/jci.insight.203622

¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

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¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

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¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

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¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

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¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

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¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

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¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

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¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

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¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

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¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

Find articles by Quann, K. in: PubMed | Google Scholar

¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

Find articles by Pirner, C. in: PubMed | Google Scholar

¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

Find articles by Codispot, K. in: PubMed | Google Scholar

¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

Find articles by Shlomchik, M. in: PubMed | Google Scholar |

¹Department of Immunology, University of Pittsburgh, Pittsburgh, United States of America

²Bluesphere Bio, Pittsburgh, United States of America

³Department of Medicine, Division of Oncology and Malignant Hematology, University of Pittsburgh, Pittsburgh, United States of America

Find articles by Shlomchik, W. in: PubMed | Google Scholar

JCI Insight. https://doi.org/10.1172/jci.insight.203622.
Copyright © 2026, Rowe et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Published April 28, 2026 - Version history

Tumor-infiltrating CD8 cells recognize neoantigens created by tumor-specific mutations. Nonetheless, even after checkpoint inhibitor therapy, most patients progress. A deeper understanding of anti-tumor responses could facilitate development of better therapies. To enable such studies, we applied TCXpress, a high throughput platform that clones fully expressible TCRs from single cells into retro- or lenti- viral vectors without sequencing or gene synthesis, to study TCRs from CD8 cells infiltrating mouse MC38 tumors. We expressed cloned TCRs in reporter cells and interrogated TCR specificity by coculturing them with B6WT3 cells transduced with tandem minigenes encoding predicted neoantigens. We isolated TCRs reactive against epitopes from mutant Rpl18, Adpgk, Psmd2, and Zc3h7b along with self-reactive TCRs that recognized normal B6 and MC38 cells. Importantly, we successfully treated MC38-bearing mice with T cells transduced with anti-Rpl18 TCRs. These results establish a system that could be used to study many types of T cell responses and validates a therapeutic approach that could be tested in the clinic.

Version 1 (April 28, 2026): In-Press Preview

A platform for parallel T cell receptor cloning and testing enables anti-neoantigen tumor immunotherapy

Alexander M. Rowe,¹ Smriti Chaurasia,¹ Wenzhong Wei,¹ Laura García-Diéguez,¹ Katherine Querry,¹ Johnathon G. Schiebel,¹ Christy Smolak,¹ Alexander G. Muralles,² Daniel Wikenheiser,¹ Kevin Quann,³ Collin Pirner,³ Kentin Codispot,³ Mark J. Shlomchik,¹ and Warren D. Shlomchik¹

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A platform for parallel T cell receptor cloning and testing enables anti-neoantigen tumor immunotherapy

Alexander M. Rowe,1 Smriti Chaurasia,1 Wenzhong Wei,1 Laura García-Diéguez,1 Katherine Querry,1 Johnathon G. Schiebel,1 Christy Smolak,1 Alexander G. Muralles,2 Daniel Wikenheiser,1 Kevin Quann,3 Collin Pirner,3 Kentin Codispot,3 Mark J. Shlomchik,1 and Warren D. Shlomchik1

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Alexander M. Rowe,¹ Smriti Chaurasia,¹ Wenzhong Wei,¹ Laura García-Diéguez,¹ Katherine Querry,¹ Johnathon G. Schiebel,¹ Christy Smolak,¹ Alexander G. Muralles,² Daniel Wikenheiser,¹ Kevin Quann,³ Collin Pirner,³ Kentin Codispot,³ Mark J. Shlomchik,¹ and Warren D. Shlomchik¹