Kato et al. established a two-week protocol using partially HLA-matched healthy donors to rapidly identify TCR sequences recognizing patient-specific neoantigen epitopes, and demonstrated that these TCRs were target specific following transfer to third-party PBMCs. A single round of in vitro stimulation, dextramer sorting, and NGS determined the paired α and β TCR chains. Interestingly, a TCR identified against a neoepitope for which the anchor residue was mutated reacted with both mutant and wild-type peptide:MHC, warranting increased scrutiny of potential neoepitopes.

Neoantigens are the main targets of tumor-specific T cells reactivated by immune checkpoint-blocking antibodies or when using tumor-infiltrating T cells for adoptive therapy. While cancers often accumulate hundreds of mutations and harbor several immunogenic neoantigens, the repertoire of mutation-specific T cells in patients might be restricted. To bypass suboptimal conditions, which impede the reactivation of existing T cells or the priming of neoantigen-specific T cells in a patient, we employ T cells of healthy donors with an overlapping HLA repertoire to target cancer neoantigens. In this study, we focus on streamlining the process of in vitro-induction of neoantigen-specific T cells and isolating their T cell receptors (TCRs) to establish a time-efficient protocol that will allow the patient to benefit from subsequent therapy. We first optimized the priming of T cells to omit multiple restimulations and extended culturing. Neoantigen-specific T cells were enriched using specific dextramers and next-generation sequencing was applied to determine the TCR repertoire. This allowed us to circumvent the laborious process of expanding T cell clones. Using this protocol, we successfully identified HLA-A-restricted TCRs specific for neoantigens found in an esophageal cancer cell line (TE-8) and a primary ovarian cancer. To verify TCR specificity, we generated TCR-engineered T cells and confirmed recognition of the tumor-derived neoantigens. Our results also emphasize the importance of neoepitope selection in order to avoid cross-reactivity to corresponding wild-type peptide sequences. In conclusion, we established a 2-week protocol for generating and identifying neoantigen-specific TCRs from third-party donors making this strategy applicable for clinical use.

Author Info: (1) Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. (2) Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. (3)

Author Info: (1) Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. (2) Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. (3) Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. (4) Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. (5) Institute of Immunology - Campus Buch, Charite - Universitatsmedizin Berlin, Berlin 13125, Germany. Berlin Institute of Health, Berlin 10117, Germany. (6) OncoTherapy Science Inc., Kawasaki, Kanagawa 213-0012, Japan. (7) OncoTherapy Science Inc., Kawasaki, Kanagawa 213-0012, Japan. (8) OncoTherapy Science Inc., Kawasaki, Kanagawa 213-0012, Japan. (9) Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. (10) Department of Gynecologic Oncology, Saitama Medical University International Medical Center, Hidaka, Saitama 350-1298, Japan. (11) Department of Gynecologic Oncology, Saitama Medical University International Medical Center, Hidaka, Saitama 350-1298, Japan. (12) Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. (13) Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. Department of Surgery, The University of Chicago, Chicago, IL 60637, USA.

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