Cytotherapy Laboratory, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; the First Affiliated Hospital, Southern University of Science and Technology), Shenzhen Guangdong, China.
Institute of Clinical Oncology, The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong, China.
Oncoimmunology. 2021 May 25;10(1):1929726. doi: 10.1080/2162402X.2021.1929726.
Adoptive cell therapy using TCR-engineered T cells (TCR-T cells) represents a promising strategy for treating relapsed and metastatic cancers. We previously established methods to identify neoantigen-specific TCRs based on patients' PBMCs. However, in clinical practice isolation of PBMCs from advanced-stage cancer patients proves to be difficult. In this study, we substituted blood-derived T cells for tumor-infiltrating lymphocytes (TILs) and used an HLA-matched cell line of antigen-presenting cells (APCs) to replace autologous dendritic cells. Somatic mutations were determined in head and neck squamous cell carcinoma resected from two patients. HLA-A*02:01-restricted neoantigen libraries were constructed and transferred into HLA-matched APCs for stimulation of patient TILs. TCRs were isolated from reactive TIL cultures and functionality was tested using TCR- T cells and . To exemplify the screening approach, we identified the targeted neoantigen leading to recognition of the minigene construct that stimulated the strongest TIL response. Neoantigen peptides were used to load MHC-tetramers for T cell isolation and a TCR was identified targeting the KIAA1429 mutation. TCR-T cells were activated, exhibited cytotoxicity, and secreted cytokines in a dose-dependent manner, and only when stimulated with the mutant peptide. Furthermore, comparable to a neoantigen-specific TCR that was isolated from the patient's PBMCs, KIAA1429-specific TCR T cells destroyed human tumors in mice. The established protocol provides the required flexibility to methods striving to identify neoantigen-specific TCRs. By using an MHC-matched APC cell line and neoantigen-encoding minigene libraries, autologous TILs can be stimulated and screened when patient PBMCs and/or tumor material are not available anymore. Abbreviations: Head and neck squamous cell carcinoma (HNSCC); adoptive T cell therapy (ACT); T cell receptor (TCR); tumor-infiltrating lymphocytes (TIL); cytotoxic T lymphocyte (CTL); peripheral blood mononuclear cell (PBMC); dendritic cell (DC); antigen-presenting cells (APC).
采用 TCR 工程化 T 细胞（TCR-T 细胞）的过继细胞疗法代表了治疗复发和转移性癌症的一种很有前途的策略。我们之前建立了基于患者外周血单核细胞（PBMC）鉴定新抗原特异性 TCR 的方法。然而，在临床实践中，从晚期癌症患者中分离 PBMC 被证明是困难的。在这项研究中，我们用血液来源的 T 细胞代替肿瘤浸润淋巴细胞（TIL），并用 HLA 匹配的抗原呈递细胞（APC）细胞系代替自体树突状细胞。从两名患者切除的头颈部鳞状细胞癌中确定了体细胞突变。构建了 HLA-A*02:01 限制的新抗原文库，并将其转移到 HLA 匹配的 APC 中，以刺激患者 TIL。从反应性 TIL 培养物中分离 TCR，并使用 TCR-T 细胞和进行功能测试。为了举例说明筛选方法，我们鉴定了导致识别刺激最强 TIL 反应的 minigene 构建体的靶向新抗原。新抗原肽用于加载 MHC-四聚体以分离 T 细胞，并鉴定靶向 KIAA1429 突变的 TCR。TCR-T 细胞以剂量依赖性方式激活、表现出细胞毒性并分泌细胞因子，并且仅在刺激突变肽时才如此。此外，与从患者 PBMC 中分离的新抗原特异性 TCR 相比，KIAA1429 特异性 TCR-T 细胞在小鼠中破坏了人类肿瘤。该建立的方案为旨在鉴定新抗原特异性 TCR 的方法提供了所需的灵活性。通过使用 MHC 匹配的 APC 细胞系和新抗原编码 minigene 文库，当患者 PBMC 和/或肿瘤材料不再可用时，可以刺激和筛选自体 TIL。缩写：头颈部鳞状细胞癌（HNSCC）；过继性 T 细胞疗法（ACT）；T 细胞受体（TCR）；肿瘤浸润淋巴细胞（TIL）；细胞毒性 T 淋巴细胞（CTL）；外周血单核细胞（PBMC）；树突状细胞（DC）；抗原呈递细胞（APC）。
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