采用特定的免疫细胞和干细胞的采用细胞疗法（ACT）已经成为一个有前途的治疗选择，未来可能会成为传统肿瘤治疗的补充选择。具体而言，治疗肿瘤浸润淋巴细胞（TILs）在各种临床试验中已被证明对实体瘤有很好的疗效。尽管大肠癌（CRC）所造成的疾病负担和早逝人数非常高，但对来自CRC患者肿瘤组织中分离TILs的研究仍然很少。迄今为止，ACT的研究往往缺乏可控制和可比较的扩增过程以及精选的ACT相关T细胞群体。我们描述了一种生成患者特异性TILs的程序，这是进行CRC ACT临床试验的前提条件。这些TILs的制造和特征在重要模块中与通常用于此治疗方法的TILs不同。从12名接受原发CRC手术治疗的患者中获取肿瘤组织样本，主要为低微卫星不稳定性（pMMR-MSI-L）的肿瘤。在送审的切除标本中检查了肿瘤，把一定量的肿瘤组织移至一次性灌流生物反应器中。采用含有白细胞介素2和白细胞介素12的起始培养基，在符合GMP标准的闭环灌流生物反应器系统中对组织样本进行自动控制和高度重复的培养过程。短期加入特定的激活混合物启动TIL的培育生长。在随后的扩增中，TILs在富含白细胞介素2的培养基中生长。在Zellwerk ZRP生物反应器中的低规模双相过程中扩张TILs，在高氧环境下结果产生约20亿个细胞。扩展的TILs主要由ACT相关CD3+/CD8+效应记忆表型（CD45RO+/CCR7−）组成（73％）。在非特异性刺激（干扰素γ，肿瘤坏死因子α细胞因子分析）下证实其高功能潜力。Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.

Adoptive cell therapy (ACT) using specific immune cells and stem cells has emerged as a promising treatment option that could complement traditional cancer therapies in the future. In particular, tumor-infiltrating lymphocytes (TILs) have been shown to be effective against solid tumors in various clinical trials. Despite the enormous disease burden and large number of premature deaths caused by colorectal cancer (CRC), studies on TILs isolated from tumor tissue of patients with CRC are still rare. To date, studies on ACT often lack controlled and comparable expansion processes as well as selected ACT-relevant T-cell populations. We describe a procedure for generating patient-specific TILs, which are prerequisites for clinical trials of ACT in CRC. The manufacturing and characteristics of these TILs differ in important modalities from TILs commonly used for this therapeutic approach. Tumor tissue samples were obtained from 12 patients undergoing surgery for primary CRC, predominantly with low microsatellite instability (pMMR-MSI-L). Tumors in the resected specimens were examined pathologically, and an approved volume of tumor tissue was transferred to a disposable perfusion bioreactor. Tissue samples were subjected to an automatically controlled and highly reproducible cultivation process in a GMP-conform, closed perfusion bioreactor system using starting medium containing interleukin-2 and interleukin-12. Outgrowth of TIL from tissue samples was initiated by short-term supplementation with a specific activation cocktail. During subsequent expansion, TILs were grown in interleukin-2-enriched medium. Expansion of TILs in a low-scaled, two-phase process in the Zellwerk ZRP bioreactor under hyperoxic conditions resulted in a number of approximately 2 × 109 cells. The expanded TILs consisted mainly (73%) of the ACT-relevant CD3+/CD8+ effector memory phenotype (CD45RO+/CCR7-). TILs harvested under these conditions exhibited high functional potential, which was confirmed upon nonspecific stimulation (interferon-γ, tumor necrosis factor-α cytokine assay).Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.