Non-viral precision T cell receptor replacement for personalized cell therapy

Nature. 2023 Mar;615(7953):687-696. doi: 10.1038/s41586-022-05531-1. Epub 2022 Nov 10.


T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Publication types

  • Clinical Trial, Phase I
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Neoplasm / genetics
  • Antigens, Neoplasm / immunology
  • Biopsy
  • CRISPR-Cas Systems
  • Cell- and Tissue-Based Therapy* / adverse effects
  • Cell- and Tissue-Based Therapy* / methods
  • Cytokine Release Syndrome / complications
  • Disease Progression
  • Encephalitis / complications
  • Gene Editing*
  • Gene Knock-In Techniques
  • Gene Knockout Techniques
  • Genes, T-Cell Receptor alpha
  • Genes, T-Cell Receptor beta
  • HLA Antigens / immunology
  • Humans
  • Mutation
  • Neoplasms* / complications
  • Neoplasms* / genetics
  • Neoplasms* / immunology
  • Neoplasms* / therapy
  • Patient Safety
  • Precision Medicine* / adverse effects
  • Precision Medicine* / methods
  • Receptors, Antigen, T-Cell* / genetics
  • Receptors, Antigen, T-Cell* / immunology
  • Receptors, Antigen, T-Cell* / metabolism
  • T-Lymphocytes* / immunology
  • T-Lymphocytes* / metabolism
  • Transgenes* / genetics


  • Antigens, Neoplasm
  • Receptors, Antigen, T-Cell
  • HLA Antigens

Associated data