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. 2021 Feb;18(2):427-439.
doi: 10.1038/s41423-020-0515-7. Epub 2020 Sep 16.

Allogeneic Vγ9Vδ2 T-cell immunotherapy exhibits promising clinical safety and prolongs the survival of patients with late-stage lung or liver cancer

Yan Xu #  1   2   3 , Zheng Xiang #  4 , Mohammed Alnaggar #  3   5 , Léonce Kouakanou #  6 , Jiawei Li #  2   3 , Junyi He #  3 , Jiashuang Yang  4 , Yi Hu  3 , Yan Chen  3 , Li Lin  3 , Jianlei Hao  2   3 , Jingxia Li  3 , Jibing Chen  7 , Man Li  3 , Qingling Wu  3 , Christian Peters  6 , Qinghua Zhou  2   3 , Jianshuang Li  2   3 , Yingqing Liang  7 , Xiaohua Wang  7 , Baohui Han  8 , Meili Ma  8 , Dieter Kabelitz  9 , Kecheng Xu  3   7 , Wenwei Tu  4 , Yangzhe Wu  10   11 , Zhinan Yin  12   13
Affiliations

Allogeneic Vγ9Vδ2 T-cell immunotherapy exhibits promising clinical safety and prolongs the survival of patients with late-stage lung or liver cancer

Yan Xu et al. Cell Mol Immunol. 2021 Feb.

Abstract

Vγ9Vδ2 T cells are promising candidates for cellular tumor immunotherapy. Due to their HLA-independent mode of action, allogeneic Vγ9Vδ2 T cells can be considered for clinical application. To apply allogeneic Vγ9Vδ2 T cells in adoptive immunotherapy, the methodology used to obtain adequate cell numbers with optimal effector function in vitro needs to be optimized, and clinical safety and efficacy also need to be proven. Therefore, we developed a novel formula to improve the expansion of peripheral γδ T cells from healthy donors. Then, we used a humanized mouse model to validate the therapeutic efficacy of expanded γδ T cells in vivo; furthermore, the expanded γδ T cells were adoptively transferred into late-stage liver and lung cancer patients. We found that the expanded cells possessed significantly improved immune effector functions, including proliferation, differentiation, and cancer cell killing, both in vitro and in the humanized mouse model. Furthermore, a phase I clinical trial in 132 late-stage cancer patients with a total of 414 cell infusions unequivocally validated the clinical safety of allogeneic Vγ9Vδ2 T cells. Among these 132 patients, 8 liver cancer patients and 10 lung cancer patients who received ≥5 cell infusions showed greatly prolonged survival, which preliminarily verified the efficacy of allogeneic Vγ9Vδ2 T-cell therapy. Our clinical studies underscore the safety and efficacy of allogeneic Vγ9Vδ2 T-cell immunotherapy, which will inspire further clinical investigations and eventually benefit cancer patients.

Keywords: Allogeneic γδ T cells; Cell therapy; Humanized mice; Liver cancer; Lung cancer; New expansion formula.

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Conflict of interest statement

A PCT patent (PCT/CN2019/075491) for the new formula for γδ T-cell expansion was filed. D.K. is a member of the Scientific Advisory Boards of Incysus, Imcheck, and Lava Therapeutics.

Figures

Fig. 1
Fig. 1
New formula (NF) significantly promoted the proliferation and differentiation and reduced the apoptosis of Vγ9Vδ2 T cells. a Absolute cell counts on various indicated days were calculated by using flow cytometry (anti-TCR Vδ2-PE and anti-CD3-FITC antibodies). Here, 5 × 106 PBMCs were used in each group to test two culture medium formulas (six individual donors). The time points analyzed were days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30. b Cell proliferation was evaluated by staining for Ki-67 (seven individual donors). c The new formula significantly promoted cell mitosis, as evidenced by G1-phase reduction and S-phase augmentation (six individual donors). d The new formula (NF) could induce the differentiation of Vγ9Vδ2 T cells, as shown by significant increases in the percentages of effector memory (EM; CD45RA−CD27−) and terminally differentiated (EMRA; CD45RA+CD27−) Vγ9Vδ2 T cells and significant decreases in the percentages of naive (CD45RA+CD27+) and central memory (CD45RA−CD27+) cells, compared with the old formula (OF) (six individual donors). e Cell apoptosis was significantly decreased with the new formula compared to the old formula. Cell apoptosis was assayed on day 14 (normal culture time) and day 21 (long-term culture) (six individual donors). f The new formula enhanced the expression levels of costimulatory molecules on γδ T cells (six individual donors). MFI mean fluorescence intensity. The levels of costimulatory molecules (CD80, CD86, and HLA class-II) on Vγ9Vδ2 T cells were measured by flow cytometry, showing that NF cells expressed significantly higher levels of these costimulatory molecules than OF cells. Error bars represent the standard error of the mean (SEM). *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 2
Fig. 2
NF cells possess stronger cellular energy metabolism, including both mitochondrial respiration and glycolysis, than OF cells. a Metabolic results were obtained with a Seahorse XF analyzer. The ECAR was assessed after the addition of 25 mM glucose (gluc) and in response to the metabolic inhibitors oligomycin (oligo) and 2DG. O2 consumption rates (OCRs) were measured in real time under basal conditions and in response to the indicated mitochondrial inhibitors (three repetitions, mean ± SEM). A1 Quantitative comparisons of mitochondrial functions, including basal respiration, maximum respiration, ATP production and spare respiration capacity, between NF cells and OF cells were performed. A2 Quantitative comparisons of glycolytic metabolism, including glycolysis, the glycolytic capacity and the glycolytic reserve, between NF cells and OF cells were performed. Data are representative of three independent experiments. b Gene set enrichment analysis (GSEA) identified similarities between OF cells and NF cells. The expression of glycolysis-related genes was upregulated in NF cells. c The heat map illustrates the expression of glycolysis-related genes in NF cells versus OF cells. Red: upregulated; blue: downregulated. d The distribution of mitochondria was observed by confocal microscopy, showing that NF cells possessed higher mitochondrial fluorescence than OF cells. Cells were stained by using MitoTracker (red), DAPI (blue) and an anti-Vδ2 antibody (green) for confocal visualization. e The fluorescence intensity of mitochondria was statistically analyzed by flow cytometry (three repetitions, mean ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 3
Fig. 3
New formula upregulates the expression levels of CD107a, NKG2D, IFN-γ, and TNF-α and potentiates the in vitro cytotoxicity of Vγ9Vδ2 T cells. a TNF-α, IFN-γ, CD107a, and NKG2D levels were measured after 14 days following stimulation for 6 h with anti-CD3 (5 μg/mL) and anti-CD28 (1 μg/mL) antibodies. Data are shown as the mean fluorescence intensity (MFI). b The new formula could significantly enhance the in vitro antitumor cytotoxicity of Vγ9Vδ2 T cells, as shown by killing assay results obtained for A549, Jurkat, MCF-7, BJAB, K562, Raji, and Daudi target cells. The Vγ9Vδ2 T cells were cocultured for 6 h with CFSE-labeled tumor cells at a 10:1 ratio. Each linked line between OF and NF in all graphs represents an individual γδ T-cell line. **p < 0.01; ***p < 0.001
Fig. 4
Fig. 4
NF-γδ T cells displayed potent antitumor activity in humanized mice. a The protocol for establishing lung cancer in humanized mice and evaluating the therapeutic effects of NF cells and OF cells. Seven days after inoculation of GFP-549 cells into humanized mice, NF cells and OF cells were adoptively transferred i.v. into the humanized mice. Mice treated with PBS were used as the control. b Macroscopic tumor inspection of tumors from untreated, OF-cell-treated, and NF-cell-treated mice. For the NF-cell-treated group, one mouse had a recurrent tumor on day 125, and two mice were tumor-free when sacrificed on day 238. c Survival curves of the three groups, suggesting that NF cells could significantly prolong survival time of tumor-bearing mice. d Tumor volume comparisons among the untreated, OF-cell-treated, and NF-cell-treated groups, showing that NF cells have optimal therapeutic effects on tumor development. e Live imaging to visualize the colocalization of A549 cancer cells and γδ T cells in humanized mice (views of the lower limb region of mice). NF cells or OF cells were prelabeled with DiR (red color) and then transferred i.v. into mice. The live imaging data indicated that only NF cells (red) visually colocalized with tumor cells (green) when monitored at 48 h post infusion. Data are presented as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 5
Fig. 5
Overall survival of patients with advanced liver cancer or lung cancer infused with allogeneic Vγ9Vδ2 T cells expanded using the new formula. a, b Overall survival curves were plotted by comparing γδ T-cell-treated patients and patients not treated with γδ T cells. The survival time of the γδ T-cell-treated patients was calculated starting from the date of the first infusion until June 2020. For the liver cancer group, the median survival time of the untreated patients was 8.1 months; in contrast, the median survival time of the γδ T-cell-treated patients was 23.1 months. P = 0.0002. For the lung cancer group, the median survival time of the untreated patients was 9.1 months; however, the median survival time was 19.1 months for the γδ T-cell-treated patients. P = 0.0028
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