CAR-γδ T细胞生产工艺的优化研究

doi:10.19401/j.cnki.1007-3639.2025.11.004

摘要/Abstract

摘要：

背景与目的：近年来，嵌合抗原受体T（chimeric antigen receptor T，CAR-T）细胞疗法在肿瘤治疗中取得突破性进展。γδ T细胞具有非主要组织相容性复合体（major histocompatibility complex，MHC）限制性识别抗原、广泛抗肿瘤活性和低移植物抗宿主病（graft-versus-host disease，GVHD）风险等特点，因此CAR-γδ T细胞疗法受到广泛关注，成为肿瘤免疫治疗研究的重要热点之一。然而，CAR-γδ T细胞制备过程中存在体外扩增效率不足、病毒转导效率低等关键问题，严重阻碍了CAR-γδ T细胞的研究和临床应用。本研究通过优化γδ T细胞的体外扩增培养条件和慢病毒转导策略，旨在构建高效制备CAR-γδ T细胞的体系。 方法：首先通过筛选多种细胞因子组合及组合中各细胞因子的浓度优化γδ T细胞的扩增方案，通过评估细胞纯度、细胞活力、扩增倍数、细胞毒性和耗竭标志物表达水平，筛选出γδ T细胞最佳的培养条件。随后，通过确定转导前γδ T细胞的最优活化时间和慢病毒转导感染复数（multiplicity of infection，MOI）进一步优化CAR-γδ T细胞的转导条件。最后，依据优化后的方案制备CAR-γδ T细胞，并利用钙黄绿素释放法和流式细胞术验证其对靶细胞的杀伤活性，同时初步评估CAR-γδ T细胞诱导GVHD的潜在风险。 结果：实验数据显示，相较于单一白细胞介素（interleukin，IL）-2培养组，IL-2+IL-7+IL-15联合培养方案显著提升了γδ T细胞的细胞纯度（73.67%±1.53% vs 90.69%±2.00%）、细胞活力（63.01%±7.05% vs 89.00%±3.61%）、扩增倍数（876.50±238.35倍 vs 1 627.50±472.15倍）和细胞毒性标志物CD16的表达（4.20%±1.73% vs 14.66%±0.58%），显著降低了耗竭标志物程序性死亡蛋白-1（programmed death-1，PD-1）的表达（35.67%±6.26% vs 21.10%±6.49%）。细胞因子浓度梯度正交实验的结果表明，IL-2+IL-7+IL-15联合培养方案中IL-7和IL-15的最优细胞因子浓度为10 ng/mL。此外，在活化96~120 h使用MOI为5~10进行病毒转导，CAR-γδ T细胞具有最优的转导效率（96 h：12.87%±4.35%；120 h：11.37%±2.35%）。经此优化体系制备的CAR-γδ T细胞对靶抗原阳性肿瘤细胞显示出特异性杀伤效应，并且未发现CAR-γδ T细胞诱导GVHD的证据。 结论：IL-2+IL-7（10 ng/mL）+IL-15（10 ng/mL）联合培养方案结合活化96~120 h使用MOI为5~10转导制备的CAR-γδ T细胞在扩增能力、细胞纯度和转导效率等方面均显著优于传统方法。CAR-γδ T细胞疗法通过天然免疫受体与CAR介导的特异性识别实现协同抗肿瘤效应，且未观察到GVHD风险。本研究为CAR-γδ T细胞疗法的临床转化提供了关键的技术支撑，奠定了坚实的理论和实践基础。

关键词: 嵌合抗原受体-γδ T细胞, 细胞因子, 慢病毒, 生产工艺优化, 抗肿瘤活性

Abstract:

Background and purpose: In recent years, chimeric antigen receptor T (CAR-T) cell therapy has achieved breakthrough progress in cancer treatment. γδ T cells, with their non-major histocompatibility complex (MHC)-restricted antigen recognition, broad antitumor activity, and low risk of graft-versus-host disease (GVHD), have garnered significant interest in CAR-γδ T cell therapy. However, critical challenges including suboptimal in vitro expansion and low viral transduction efficiency severely hinder the research and clinical application of CAR-γδ T cells. This study aimed to establish an efficient platform for preparing CAR-γδ T cells by optimizing the in vitro expansion conditions of γδ T cells and refining lentiviral transduction strategies. Methods: We first optimized the expansion protocol for γδ T cells by screening various cytokine combinations and the concentrations of individual cytokines within combination, and evaluating cell purity, viability, fold expansion, and expressions of cytotoxicity and exhaustion markers to identify the optimal culture conditions. Subsequently, the transduction conditions for CAR-γδ T cells were improved by determining the optimal activation duration of γδ T cells prior to gene transfer, as well as the optimal multiplicity of infection (MOI) for lentiviral transduction. Finally, CAR-γδ T cells were successfully generated using the optimized protocol, and their cytotoxic activity against target cells was validated via calcein-release assay and flow cytometry, with a preliminary assessment of the potential risk of GVHD induction. Results: Experimental data demonstrated that, compared with the interleukin (IL)-2-only culture, the IL-2+IL-7+IL-15 combination significantly enhanced the expansion capacity of γδ T cells (876.50±238.35-fold vs 1 627.50±472.15-fold), cell purity (73.67%±1.53% vs 90.69%±2.00%), and cell viability (63.01%±7.05% vs 89.00%±3.61%). It also increased the expression of the cytotoxicity marker CD16 (4.20%±1.73% vs 14.66%±0.58%) and reduced the expression of the exhaustion marker programmed death-1 (PD-1) (35.67%±6.26% vs 21.10%±6.49%). A cytokine concentration gradient orthogonal assay further identified 10 ng/mL IL-7 and 10 ng/mL IL-15 as the optimal concentrations within the IL-2+IL-7+IL-15 combination. Gene transduction performed 96-120 h after activation using a multiplicity of infection (MOI) of 5-10 resulted in the highest transduction efficiency for CAR-γδ T cells (96 h: 12.87%±4.35%; 120 h: 11.37%±2.35%). CAR-γδ T cells generated using the optimized system exhibited specific cytotoxic effects against tumor cells expressing the target antigen, and no evidence of GVHD induction was observed. Conclusion: CAR-γδ T cells produced using the IL-2+IL-7 (10 ng/mL)+IL-15 (10 ng/mL) regimen combined with a 96-120 h activation period prior to transduction using a multiplicity of infection (MOI) of 5-10 significantly outperformed conventional methods in terms of expansion efficiency, cell purity, and transduction efficiency. The synergistic antitumor effects mediated by both natural immune receptors and CAR-specific recognition, along with the initial absence of GVHD risk, provide critical technical support for the clinical translation of CAR-γδ T cell therapy, establishing a solid theoretical and practical foundation.

Key words: Chimeric antigen receptor-γδ T cells, Cytokines, Lentivirus, Manufacturing process optimization, Anti-tumor efficiency

中图分类号:

R730.51

赵家旋, 王伊玄, 田高辉, 史江舟, 张同存. CAR-γδ T细胞生产工艺的优化研究[J]. 中国癌症杂志, 2025, 35(11): 1019-1031.

ZHAO Jiaxuan, WANG Yixuan, TIAN Gaohui, SHI Jiangzhou, ZHANG Tongcun. A study on optimization of the CAR-γδ T cell manufacturing process[J]. China Oncology, 2025, 35(11): 1019-1031.

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Cytokine	Concentration
IL-2^[17-19]	1 000 U/mL
IL-7^[20-21]	10 ng/mL
IL-15^{[9,22 -24]}	10 ng/mL
IL-18^[25]	10 ng/mL
IL-21^[26-27]	10 ng/mL

Group	Expansion fold	Cell purity	Cell viability
IL-2	876.50±238.35	73.67%±1.53%	63.01%±7.05%
IL-2+IL-7	1 863.50±387.16	89.12%±3.61%	89.33%±6.03%
IL-2+IL-15	1 763.50±452.33	91.33%±3.06%	89.67%±6.11%
IL-2+IL-18	1 367.49±367.89	88.50%±2.50%	77.67%±5.51%
IL-2+IL-21	1 233.50±269.59	82.33%±4.51%	68.50%±6.56%
IL-2+IL-7+IL-15	1 627.50±472.15	90.69%±2.00%	89.00%±3.61%
IL-2+IL-7+IL-18	1 221.50±359.75	86.33%±4.04%	81.67%±5.86%
IL-2+IL-7+IL-21	955.50±292.85	83.50%±4.58%	70.67%±1.15%
IL-2+IL-15+IL-18	1 000.50±359.16	85.33%±3.06%	80.33%±3.06%
IL-2+IL-15+IL-21	932.50±254.44	79.67%±4.16%	69.50%±1.50%
IL-2+IL-18+IL-21	988.50±462.76	76.67%±2.52%	56.67%±3.51%
IL-2+IL-7+IL-15+IL-18	743.50±221.05	84.67%±5.86%	60.33%±2.52%
IL-2+IL-7+IL-15+IL-21	700.50±177.34	79.67%±3.51%	64.33%±4.04%
IL-2+IL-7+IL-18+IL-21	601.50±122.26	78.33%±4.73%	60.33%±7.51%
IL-2+IL-15+IL-18+IL-21	554.50±83.35	71.33%±4.04%	52.33%±4.51%
IL-2+IL-7+IL-15+IL-18+IL-21	578.50±132.25	67.50%±3.61%	56.67%±8.33%

Group	Expansion fold	Cell purity	Cell viability
IL-2+IL-7^LOW+IL-15^LOW	905.67±85.81	77.73%±1.35%	75.90%±4.07%
IL-2+IL-7^LOW+IL-15^MEDIUM	1 485.80±129.48	88.73%±3.62%	89.10%±3.70%
IL-2+IL-7^LOW+IL-15^HIGH	1 502.60±159.43	89.70%±1.90%	81.97%±1.33%
IL-2+IL-7^MEDIUM+IL-15^LOW	1 451.40±176.21	88.00%±1.76%	90.47%±5.59%
IL-2+IL-7^MEDIUM+IL-15^MEDIUM	1 465.53±123.03	88.77%±4.19%	88.53%±2.80%
IL-2+IL-7^MEDIUM+IL-15^HIGH	1 535.13±142.60	87.70%±2.69%	81.27%±1.05%
IL-2+IL-7^HIGH+IL-15^LOW	1 579.43±136.85	87.77%±3.32%	87.47%±3.01%
IL-2+IL-7^HIGH+IL-15^MEDIUM	1 454.23±184.48	86.97%±1.75%	88.63%±5.74%
IL-2+IL-7^HIGH+IL-15^HIGH	1 496.57±139.82	83.10%±2.39%	80.30%±4.07%

Group	CD16+	PD-1+
IL-2+IL-7^LOW+IL-15^LOW	6.57%±1.88%	30.37%±7.72%
IL-2+IL-7^LOW+IL-15^MEDIUM	12.63%±2.66%	21.67%±5.37%
IL-2+IL-7^LOW+IL-15^HIGH	15.53%±1.17%	15.50%±4.04%
IL-2+IL-7^MEDIUM+IL-15^LOW	7.35%±1.39%	20.40%±7.14%
IL-2+IL-7^MEDIUM+IL-15^MEDIUM	19.53%±4.52%	15.27%±5.08%
IL-2+IL-7^MEDIUM+IL-15^HIGH	19.77%±3.46%	17.50%±3.85%
IL-2+IL-7^HIGH+IL-15^LOW	10.69%±1.41%	22.93%±5.93%
IL-2+IL-7^HIGH+IL-15^MEDIUM	18.07%±1.36%	17.17%±3.74%
IL-2+IL-7^HIGH+IL-15^HIGH	16.93%±1.99%	14.47%±3.59%