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TCR-T therapy is an ideal cell therapeutic strategy against solid tumors. However, the traditional method of TCR cloning has two major problems. First, we could only develop TCRs against previously discovered tumor antigen epitopes. Second, the affinity maturation of TCR sometimes leads to mortal toxicities due to the unexpected cross-reactivities. Therefore we hereby present a reverse genetic engineering platform to clone tumor specific TCRs from immunotherapies benefit cancer patients. Multi-antigen stimulating cell therapy (MASCT) is a cell-based immunotherapy combining therapeutic DC vaccine and adoptive T cell transfer, the IND application of which was approved in China. We observed dynamic T cell responses against multiple tumor-associated antigens used in MASCT-treated HCC patients, which showed a significant correlation with clinical outcome. Based on this observation, we propose that expanded tumor-specific T cells in MASCT-benefit cancer patients could be good sources to isolate clinical relevant TCRs. A HPV positive metastatic cervical cancer patient has shown partially response after MASCT treatment alone, and survived over 5 years. Moreover, a metastatic lung cancer patient has also shown partially response and survived for longer than 2 years after combination treatment of MASCT and anti-PD1 antibody. Enhanced and persist T cell responses against several TAAs as well as HPV antigens were detected in PBMCs of both patients. We successfully expended and isolated tumor antigen-specific T cells by IFNγ-secreting cell enrichment, and obtained alpha/beta chains paired TCR sequences using single T cell NGS technology. We selected 14 pairs of TCRs isolated from cervical cancer patient for further validation. TCRs from lung cancer patient were currently under synthesis or sequencing. Among 14 TCRs, a TAA-specific TCR and a HPV18E7-specific TCR showed superior antigen specificity and optimum avidity, and would be applied for TCR-T development. Of notes, our reverse genetic engineering of TCR-T (ReGET) platform.can not only identify both HLA class I and II-restricted TCRs, but also identify novel epitopes of tumor antigens, which may provide new targets for TCR-T therapy against solid tumors. Citation Format: Yanyan Han, Xiaoling Liang, Shudan Ou, Xihe Chen, Minjun Ma, Jun Li, Yifan Ma, Xiangjun Zhou. Discovery of tumor-specific T cell receptors (TCRs) by reverse genetic engineering platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5569.

Adoptive transfer of tumor-specific T cell receptor (TCR)-engineered T cells, TCR-T therapy, has shown great efficacy in clinical trials against solid tumors. A cervical cancer patient with bone metastasis was treated with a cell-based immunotherapy, named Multiple-Antigens Stimulating Cellular Therapy (MASCT), which is a combination of multiple tumor antigen peptides loaded dendritic cells (DCs) and autologous T cells stimulated by these DCs. After repeated treatment, the patient showed partially response and remained stable disease for 20 months. Moreover, boosted specific immune responses were detected in patient's periphery blood by IFNγ-Elispot assay against various tumor antigens, such as CEA, RGS5 and HPV18/58. The clinical benefits of this patient indicated that tumor-specific T cells were expanded in vivo and played an important role to control tumor progression. These T cells may be the good sources to isolate tumor-specific TCRs for TCR-T therapies. Given that, patient's PBMCs were stimulated in vitro with selected tumor peptides respectively, such as CEA, RGS5 and HPV18 E7. Tumor-specific T cells were defined as IFNγ-secreting T cells upon peptide stimulation and were further enriched by using magnetic beads. Part of the enriched T cells were analyzed for TCR alpha/beta repertoire via NGS, the other part were sorted into single T cells and analyzed for TCR alpha/beta pairing. The TCR repertoire NGS data revealed that the tumor-specific T cells harbor several hundred unique TCR alpha/beta clonotypes. Some unique TCR alpha/beta clonotypes were considered as tumor-specific TCR candidates, since the copy numbersof these clonotypes were significantly and repeatedly increased after stimulation with CEA, RGS5 or HPV18 E7, and further increased after IFNγ enrichment. After TCR alpha/beta pairing confirmation by single T cell sequencing, alpha and beta chains of these TCRs were sent to synthesis for further investigation. In conclusion we have successfully identified specific TCR clonotypes targeting selected tumor antigens from a MASCT-benefited cervical cancer patient. After functional validations, including epitope specificity, HLA restriction as well as tumor recognition, these tumor-specific TCRs may be good candidates for developing safer and more effective TCR-T therapy targeting multiple tumor antigens. Citation Format: Xiaoling Liang, Shudan Ou, Minjun Ma, Xihe Chen, Xiangjun Zhou, Yanyan Han. To clone tumor-specific TCRs from a cell-based immunotherapy-benefit cervical cancer patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2554.