Department of General, Visceral, Thoracic and Vascular Surgery, University Hospital Ruppin-Brandenburg, Neuruppin, Germany; Faculty of Health Sciences Brandenburg, Brandenburg Medical School Theodor Fontane, Neuruppin,Germany.
Zellwerk GmbH - HiPer-Group, Eichstädt, Germany.
Cytotherapy. 2023 May;25(5):537-547. doi: 10.1016/j.jcyt.2023.01.009. Epub 2023 Feb 10.
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 × 10 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).
过继细胞疗法 (ACT) 使用特定的免疫细胞和干细胞，已成为一种很有前途的治疗选择，未来可能会补充传统癌症治疗方法。特别是肿瘤浸润淋巴细胞 (TIL) 在各种临床试验中对实体瘤显示出疗效。尽管结直肠癌 (CRC) 带来了巨大的疾病负担和大量的过早死亡，但对源自 CRC 患者肿瘤组织的 TIL 的研究仍然很少。迄今为止，关于 ACT 的研究通常缺乏受控和可比的扩增过程以及选定的 ACT 相关 T 细胞群体。我们描述了一种生成患者特异性 TIL 的程序，这是在 CRC 中进行 ACT 临床试验的前提。这些 TIL 的制造和特性在重要方面与常用于这种治疗方法的 TIL 不同。从 12 名接受原发性 CRC 手术的患者中获得肿瘤组织样本，主要为低微卫星不稳定性 (pMMR-MSI-L)。切除标本中的肿瘤经病理检查，将批准体积的肿瘤组织转移至一次性灌注生物反应器。使用含有白细胞介素-2 和白细胞介素-12 的起始培养基，在符合 GMP 的封闭灌注生物反应器系统中，对组织样本进行自动控制和高度可重复的培养过程。通过短期补充特定激活鸡尾酒来启动组织样本中 TIL 的生长。在随后的扩增过程中，TIL 在富含白细胞介素-2 的培养基中生长。在 Zellwerk ZRP 生物反应器中在富氧条件下以两阶段低规模过程进行 TIL 扩增，可获得约 2×10 的细胞数。扩增的 TIL 主要由（73%）ACT 相关的 CD3/CD8 效应记忆表型（CD45RO/CCR7）组成。在这些条件下收获的 TIL 表现出高功能潜力，在非特异性刺激（干扰素-γ、肿瘤坏死因子-α细胞因子测定）下得到证实。
