RNF43 p.G659fs 通过 PI3K/AKT/mTOR 信号通路和 HLA-E 上调导致 MSI-high 结直肠癌中 NK 细胞功能障碍
RNF43 p.G659fs leads to natural killer cell dysfunction in MSI-high colorectal cancer through PI3K/AKT/mTOR signaling and HLA-E up-regulation.
CELL INTELLIGENCE · 肿瘤细胞治疗研究
肿瘤细胞治疗研究
基于 CRISPR/Cas9 的细胞因子诱导 SH2 蛋白敲除 TIL（肿瘤浸润淋巴细胞）用于胃肠道肿瘤的临床制备
英文原题：Clinical manufacture of CRISPR/Cas9-based cytokine-induced SH2 protein knock-out tumor-infiltrating lymphocytes for gastrointestinal cancers.
查看英文原题
Clinical manufacture of CRISPR/Cas9-based cytokine-induced SH2 protein knock-out tumor-infiltrating lymphocytes for gastrointestinal cancers.
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研究思路按摘要原文分段
背景
IV期胃肠道（GI）癌的预后较差，结直肠癌的五年生存率为15%。为提高TIL（肿瘤浸润淋巴细胞）的疗效，我们分离了突变反应性自体TIL，并采用CRISPR/Cas9敲除（KO）细胞内检查点蛋白CISH，该蛋白已被证明可增强T细胞扩增、功能亲和力和细胞因子多功能性，从而在动物模型中实现已建立肿瘤的持久消退。材料与方法
方法
TIL培养物从切除的肿瘤碎片中起始，并维持六周后收获和冷冻保存。候选新抗原通过外显子组测序提名，并使用肽段鉴定突变反应性（MR）TIL。选定的MR TIL被解冻，并在含10% AB血清、6000 IU/mL IL-2和5 ng/mL IL-7及IL-15的培养基中恢复24-36小时，随后用板结合抗CD3/可溶性抗CD28刺激4天。CISH KO通过电穿孔Cas9 mRNA和化学修饰的单导RNA进行。将5-7.5百万活细胞加入每个含600 mL扩增培养基（同种异体饲养层MNC:TIL = 100:1）的100 cm² G-Rex容器中，并孵育6-8天。根据细胞浓度标准（和剂量队列）评估并分瓶培养物，并再孵育6-8天。第14天，收获所有细胞，用缓冲液洗涤并冷冻保存（5% DMSO）。批放行检测包括：活力、%CD3+、细胞学审查、革兰染色、无菌性、内毒素、支原体和干扰素γ（IFN-）产生。额外检测包括DNA测序以确定基因组CISH编辑效率和Western blot以确定CISH蛋白缺失。
结果
胃肠道肿瘤患者（结肠[10]、直肠[8]、胰腺[1]和食管[1]）接受了肿瘤采集。20例患者共采集22份肿瘤活检，其中19份进入KO/扩增。最终TIL产品结果（均值[SD]，中位数[范围]）为：活细胞计数（×10^10）-3.25（3.67），1.95（0.018-12.40）；活TIL扩增倍数-327.1（364.8），153.1（8-1454）；%活力-76（13），78（43-92）；%CD3-94.4（5.4），95.8（78.6-99.4）；%CISH KO效率-75（29），87（0-96）；%编辑效率-59.9（24.8），66.9（0.4-86）。5份TIL产品的活力降至70%以下。所有其他批次放行检测均已达到质量标准。13例患者已接受TIL；6例患者因预期输注前疾病进展而未接受治疗。
结论
基于CRISPR/Cas9的CISH KO MR TIL从基础研究实验室到现行药品生产质量管理规范（cGMP）设施的转化已成功完成，从而实现了优化的大规模扩增，以支持一项首次人体临床试验，用于治疗转移性GI癌症患者（ClinicalTrials.gov Identifier: NCT04426669）。
展开英文摘要原文
METHODS
TIL cultures were initiated from resected tumor fragments and maintained for six weeks before harvest and cryopreservation. Candidate neoantigens were nominated by exome sequencing and peptides were used to identify mutation reactive (MR) TIL. Selected MR TIL were thawed and allowed to recover for 24-36 h in media with 10% AB serum, 6000 IU/mL IL-2, and 5 ng/mL IL-7 and IL-15 followed by stimulation with plate-bound anti-CD3/soluble anti-CD28 for 4 days. CISH KO was performed by electroporation of Cas9 mRNA and chemically modified single guide RNA. Between 5 -7.5 million viable cells were added to each 100 cm 2 G-Rex vessel containing 600 mL expansion media (with allogeneic feeder MNC:TIL = 100:1) and incubated for 6-8 days. Cultures were evaluated and split according to cell concentration criteria (and dose cohort) and incubated for an additional 6-8 days. On day 14, all of the cells were harvested, washed with buffer and cryopreserved (5% DMSO). Lot release testing included: viability, %CD3+, cytology review, Gram stain, sterility, endotoxin, mycoplasma, and interferon gamma (IFN- ) production. Additional testing included DNA sequencing to determine genomic CISH editing efficiency and a Western blot for determination of CISH protein loss.
RESULTS
Patients with GI cancers (colon [10], rectal [8], pancreatic [1], and esophageal [1]) underwent tumor collection. Nineteen of 22 tumor biopsies sampled from 20 patients total proceeded to KO/expansion. Final TIL product results (mean [SD], median [range]) were: viable count (x 10 10 ) -3.25 (3.67), 1.95 (0.018-12.40); viable TIL fold expansion -327.1 (364.8), 153.1 (8-1454); % viability - 76 (13), 78 (43-92); % CD3 -94.4 (5.4), 95.8 (78.6-99.4); % CISH KO efficiency - 75 (29), 87 (0-96); % editing efficiency - 59.9 (24.8), 66.9 (0.4-86). Viability fell below 70% for five TIL products. All other lot release testing has met specification. Thirteen patients have received TIL; six patients were not treated due to disease progression prior to anticipated infusion.
CONCLUSION
The translation of CRISPR/Cas9-based CISH KO MR TIL from the basic research lab to current good manufacturing practices The (cGMP) facility was successful, allowing for optimized, large-scale expansion in support of a first-in-human clinical trial to treat patients with metastatic GI cancers (ClinicalTrials.gov Identifier: NCT04426669).
论文信息
- 作者
- Johnson MJ、Sumstad D、Folsom TD、Slipek NJ、DeFeo AP、Growe M、Kadidlo D、Thyagarajan B
- 第一作者单位
- Department of Pediatrics, Division of Hematology and Oncology, University of Minnesota, Minneapolis, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, USA; Center for Genomic Engineering, University of Minnesota, Minneapolis, USA.United States
- 通讯作者单位
- Cell Therapy Laboratory, M Health, Fairview, University of Minnesota Medical Center, Minneapolis, USA; Molecular & Cellular Therapeutics, University of Minnesota, Minneapolis, USA; Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, USA; Stem Cell Institute, University of Minnesota, Minneapolis, USA. Electronic address: mcken020@umn.edu.United States
- 期刊
- Cytotherapy2025 Oct
- 原文标识
- PubMed 40673842 · DOI 10.1016/j.jcyt.2025.06.007