A study on optimization of the CAR-γδ T cell manufacturing process

Jiaxuan ZHAO; Yixuan WANG; Gaohui TIAN; Jiangzhou SHI; Tongcun ZHANG

doi:10.19401/j.cnki.1007-3639.2025.11.004

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A study on optimization of the CAR-γδ T cell manufacturing process

Article | 更新时间：2025-12-31

- A study on optimization of the CAR-γδ T cell manufacturing process
- China Oncology Vol. 35, Issue 11, Pages: 1019-1031(2025)
- 作者机构：
  
  1. 天津科技大学生物工程学院，天津 300457
  2. 武汉科技大学生命科学与健康学院，湖北武汉 430081
- 作者简介：
  
  ZHANG Tongcun E-mail: tony@tust.edu.cn
- 基金信息：
  
  2019 Hubei Province Technology Innovation Special Major Project(2019ACA168)
- DOI：10.19401/j.cnki.1007-3639.2025.11.004
  CLC： R730.51
- Received：31 March 2025，
  
  Revised：2025-08-21，
  
  Published：30 November 2025
- 稿件说明：
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Jiaxuan ZHAO, Yixuan WANG, Gaohui TIAN, et al. A study on optimization of the CAR-γδ T cell manufacturing process[J]. China Oncology, 2025, 35(11): 1019-1031.
DOI：

Jiaxuan ZHAO, Yixuan WANG, Gaohui TIAN, et al. A study on optimization of the CAR-γδ T cell manufacturing process[J]. China Oncology, 2025, 35(11): 1019-1031. DOI： 10.19401/j.cnki.1007-3639.2025.11.004.

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%

90.69%±2.00%）、细胞活力（63.01%±7.05%

89.00%±3.61%）、扩增倍数（876.50±238.35倍

1 627.50±472.15倍）和细胞毒性标志物CD16的表达（4.20%±1.73%

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%

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。

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 antig

en 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

1 627.50±472.15-fold)

cell purity (73.67%±1.53%

90.69%±2.00%)

and cell viability (63.01%±7.05%

89.00%±3.61%). It also increased the expression of the cytotoxicity marker CD16 (4.20%±1.73%

14.66%±0.58%) and reduced the expression of the exhaustion marker programmed death-1 (PD-1) (35.67%±6.26%

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.

关键词

Keywords

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