Abstract
Background: T-cell receptor (TCR)-T immunotherapy has emerged as a promising strategy for cancer treatment. However, identifying TCRs that can be used to generate TCR-T cells remains challenging due to tumor heterogeneity, the scarcity of tumor-specific T cells, and the diversity of human leukocyte antigens (HLA). To advance TCR-T immunotherapy, it is crucial to develop an efficient and scalable method to identify tumor-specific TCRs. Methods: To identify tumor-specific TCRs, epitopes, and their corresponding HLA subtypes, we developed a method for rapidly assembling TCRs identified through the single-cell analysis of T cells from various tumors. For each TCR, only two pairs of oligonucleotides corresponding to the CDR3 regions of TCR-α and β chains needed to be synthesized, enabling the construction of a TCR library quickly in a cost-effective manner. Additionally, we engineered HLA-knockout HEK-293T cells as antigen-presenting cells to express patient-specific HLA-I and neoantigens, and a Jurkat NFAT-GFP reporter cell line for screening antigen-reactive TCRs. The efficacy of our TCR-screening system was validated through a small-scale screening of HPV16-specific TCRs from patients with cervical cancer. Results: We successfully developed a TCR assembly method that enables the rapid cloning and construction of TCR libraries within 2 days, significantly accelerating the process and reducing costs. Our antigen-presenting system also allows for flexible expression of patient-specific HLA-I molecules, facilitating personalized screening. The Jurkat reporter cells demonstrated high sensitivity for screening functional TCRs. Using published datasets from patients with HPV16-positive cervical cancer, we successfully used our system to isolate a human papillomavirus (HPV)-specific TCR. Through deletion, alanine scanning, and mass spectrometry analysis, we determined that this TCR specifically recognized an 8-mer peptide (MHGDTPTL) from HPV-E7 presented by HLA-B*15:18. Moreover, T cells expressing this TCR were able to effectively kill HPV-positive cells. Conclusions: We developed an integrated antigen-presenting, TCR assembly, and TCR reporter system for screening tumor-specific TCRs using single-cell sequencing datasets. By using this system, we have successfully identified a functional, HPV-specific TCR, demonstrating the potential of our approach for the efficient screening of tumor-specific TCRs to advance TCR-T immunotherapy.