Effective screening of T cells recognizing neoantigens and construction of T-cell receptor-engineered T cells

Abstract

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.

Induction of Neoantigen-Specific Cytotoxic T Cells and Construction of T-cell Receptor–Engineered T Cells for Ovarian Cancer

“…Probably these two peptides bound equally to HLA-A2 and did not influence the recognition by TCR. We have previously to HLA-A was also similar between mutant and wild-type peptides) (16). On the other hand, amino acid substitutions in RFC5 and BRAP proteins occurred in P4 and P3 positions of the epitope peptides, respectively.…”

“…© 2018 American Association for Cancer clincancerres.aacrjournals.org Downloaded from protocol requires only two weeks from the priming of T cells with neoantigens to the identification of neoantigen-specific TCRs (16). We previously reported that our nextgeneration sequencer-based TCR repertoire analysis is able to accurately identify V-(D)-J combinations including CDR3 sequences in various types of tumor samples (26,29,30,(35)(36)(37)(38)(39)(40)(41).…”

“…Induction of neoantigen-specific T cells were performed following the protocol we developed previously (16 supplemented with CellGro DC/5% ABS with 10 ng/mL IL-7 and 10 ng/mL IL-15. On day 11, neoantigen-specific T cells were assessed using peptide-HLA dextramers (Immudex, Copenhagen, Denmark) for each neoantigen peptide by flow cytometry analysis.…”

“…We established an in-house pipeline to identify neoantigenspecific TCRs using HLA-matched healthy donor-derived peripheral blood mononuclear cells (PBMCs), particularly focusing on the efficiency and timeline for development (16). Our approach takes 2 weeks to induce the neoantigen-specific T cells and obtain TCR information from neoantigen-specific T cells sorted by peptide-loaded HLA-dextramers.…”

Immunopharmacogenomics towards personalized cancer immunotherapy targeting neoantigens

“…As summarized in Table , there are several reports in which neoantigen‐specific T cells were successfully induced. We also developed a rapid pipeline to identify TCRs recognizing neoantigens . The process takes only approximately 2 weeks.…”