Background The identification of cancer-specific T cell receptor (TCR) sequences is paramount to the advancement of cancer immunotherapies. Recent studies and clinical trials have shown that monoclonal T cell therapy is prone to immune evasion of cancer cells by loss of HLA heterozygosity and low antigen heterogeneity. Cocktail T cell therapy which comprises of TCRs corresponding to multiple HLAs and antigens has been proposed to improve the efficacy of adoptive cell transfer therapy. In addition to CD8+ cytotoxic T cells, neoantigen-specific CD4+ T cells, while identified as important for immunotherapy-induced anti-tumor responses, remain a largely untapped therapeutic resources due to the challenging nature of identification and isolation. Hence, a rapid and high-throughput discovery of both CD8+ and CD4+ TCRs against multiples Class I and II HLAs and cancer antigens is an urgent need. We engineered peptide-bound major histocompatibility complex (pMHC) proteins as capture agents for cancer-specific T cells. The design of these single-chain-trimers (SCTs) enables high-throughput multiplexing for identification and isolation of cancer-targeting CD4+ and CD8+ T cells from multiple patients against large panels of cancer antigens. We applied the technology to identify CD8+ and CD4+ TCRs against oncogenic proteins E6 and E7 from HPV-16, which is the leading cause of cervical cancer.
Methods A panel of 200+ Class I SCTs and 100+ Class II SCTs were designed and expressed in a high-throughput platform. PBMCs from precancerous HPV-16+ patients with cervical lesions were collected and enriched with CD8+ and CD4+ T cells. A large pool of 200+ Class I SCT tetramer pool with barcode as antigen identifier was used to capture cancer-specific CD8+ T cells. A computational analysis pipeline was established to pair TCR α and β. HLA-matching cognate antigen was assigned to each TCR pair after UMI count correction and noise removal. The antigen-specific TCRs are subsequently sequenced, validated for functionality, and analyzed for therapeutic applications.
Results We identified 43 CD8+ TCR pairs against E6 and E7 oncoproteins from HPV-16 and they are in progress for pre-clinical validation.
Conclusions The SCT platform enables rapid identification of cancer-specific CD+ and CD4+ T cells and allows detailed characterization of anti-tumor T cells for which alternative solutions are extremely limited. We applied the technology to PBMCs extracted from HPV-16 related precancerous patients in a clinical trial and discovered cancer-specific TCRs. In summary, the application of the SCT technology is of high value to the fundamental and clinical immune-oncology studies.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.