T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor-reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen-specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single-cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor-reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted.
Bibliographical noteFunding Information:
We thank Anna Panek and Zoe Schmiechen for experimental assistance. We thank Dr. Philip D. Greenberg and his team, in particular Dr. Thomas Schmitt, for generously providing the Gateway‐compatible pLRRSIN vector and CD8 6 + Jurkat cell line, and Dr. Rachel Perret for her expertise on human T cell culture. We are grateful for the NIH Tetramer Core for generating human tetramers. We thank Jerry Daniel and the single‐cell sequencing team at the University of Minnesota Genomics core for performing single‐cell sequencing. We thank Dr. Stephen Shen at the University of Minnesota Institute for Health Bioinformatics for his continued assistance with bioinformatics analyses. Figure was created with BioRender.com under a subscription to M.R.R. I.M.S. is supported by an AACR Pancreatic Cancer Action Network Career Development Award (17‐20‐25‐STRO), an AACR Pancreatic Cancer Action Network Catalyst Award (19‐35‐STRO), an American Cancer Society Institutional Research Grant, and pilot awards from the Masonic Cancer Center and University of Minnesota Medical School Translational Working Group for GI malignancies.
- adoptive cell therapy
- engineered T cells
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't