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.
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Introduction
Cancer is one of the leading lethal diseases worldwide because of its high incidence and mortality. In 2018, 18.1 million new cancer cases and 9.6 million cancer deaths were estimated to occur worldwide.1 Therefore, develo** new antitumor strategies for reducing mortality rates and improving patients’ quality of life are urgently needed. In this respect, the introduction of checkpoint inhibitors to unleash the activity of tumor-reactive T cells has been a milestone in cancer immunotherapy.2 Among various newly emerging treatment strategies, adoptive immune cell transfer therapy (ACT) has attracted great attention. ACT potentially includes αβ T-cell (e.g., CD8+ T cells and CAR-T cells),3,4,5 natural killer (NK) cell-6,7,8 and gamma delta (γδ) T-cell-based9,10,11,12,13,14,15 immunotherapy.
γδ T cells, specifically the Vγ9Vδ2 subset, which is the dominate subset among γδ T cells in human peripheral blood, recognize target cells in a human leukocyte antigen (HLA)-independent manner. Moreover, γδ T cells can directly kill target cells without the involvement of dendritic cells (DCs) and perform dual functional roles in antitumor and anti-infective immunity. Vγ9Vδ2 T cells recognize pyrophosphates secreted by many microbes or overproduced by malignant cells in the context of butyrophilin 3A1 molecules.16,17 Importantly, endogenous production of pyrophosphates can be stimulated by nitrogen-containing aminobisphosphonates, such as zoledronate (ZOL), leading to potent activation of γδ T cells.18 Such advantages enable γδ T cells to rapidly respond against malignant transformation and pathogenic stress.19 For instance, γδ T cells are the earliest producers of IFN-γ in the tumor microenvironment20 and during spinal cord injury.21 γδ T cells utilize a variety of surface receptors and cytokines, such as NKG2D, TRAIL, FASL, TNF-α, IFN-γ, Granzyme B, and perforin, to initiate cytotoxicity against cancer cells.11,12,13,In vivo evaluation of the antitumor activity of Vγ9Vδ2 T cells using humanized mice The establishment of humanized mouse model and related experimental procedures referred to our previously published work in Cancer Cell14,15,23 Prior to 2014, a number of clinical trials were conducted using autologous γδ T cells; however, only limited responses were reported.12,26,27 The main obstacles related to autologous γδ T-cell therapy include the difficulty of expansion, limited cell purity, and impaired cell functions. To obtain adequate cell numbers and optimal effector functions, we developed a NF consisting of ZOL (which selectively activates Vγ9Vδ2 γδ T cells), IL-2, IL-15, and vitamin C (Patent#: PCT/CN2019/075491) to better expand Vγ9Vδ2 T cells from healthy donors in vitro instead of using a gene modification-based methodology. Our results clearly indicated that the NF could promote γδ T-cell proliferation and differentiation, evidenced by significantly increased cell numbers and Ki-67+ proliferating cell numbers, a higher percentage of S-phase cells with a reduced percentage of G1-phase cells, and an elevated percentage of terminally differentiated effector memory (EMRA) cells. Moreover, new formula-expanded γδ T cells (NF cells) had a strikingly lower apoptosis rate than OF-expanded cells. In addition, NF cells expressed significantly higher levels of costimulatory molecules, which implied that the NF-expanded γδ T cells might promote antigen presentation to conventional CD4 and CD8 T cells, and thus correlate with superior antitumor activity. The substantially stronger cellular energy metabolism capability of NF cells was associated with their superior antitumor activity as well. Importantly, NF cells also expressed considerably higher levels of effector molecules (IFN-γ and TNF-α) and exhibited increased degranulation (induction of CD107a expression) associated with enhanced cytotoxicity against various cancer cell lines in vitro. Together, these results demonstrated that NF cells had optimal immune effector functions, as well as stronger in vitro antitumor activity than OF cells. Furthermore, our preclinical in vivo experiments showed that adoptive transfer of NF cells significantly inhibited tumor growth in humanized mice transplanted with human lung tumor cells and prolonged tumor-bearing mouse survival from <3 months to as long as 8 months. It should be mentioned here that the humanized mouse model was established according to our published protocols38,39 and mice that survived GVHD caused by huPBMC pre-engraftment were used for experiments in our work. Notably, we observed that only NF cells visually colocalized with inoculated tumors, implying NF cells had a better ability to migrate to the tumor site than did OF cells. Although OF cells prolonged mouse survival as well, the inhibition of tumor growth was not as efficient as that induced by NF cells, as indicated by the significantly prolonged overall survival of NF-cell-treated mice. It should be remarked here that the variability in outcomes among the five mice per group could be partially related to the differences in immune reconstitution among individual mice or the differences in the antitumor activity of infused γδ T cells derived from different donors. Nevertheless, based on the present in vivo work, which was repeated once previously, we can conclude that NF cells have stronger antitumor activities than OF cells. In addition, although the Vγ9Vδ2 T cells used for adoptive transfer were not 100% pure (≥90%), no allogeneic effects were observed in our mouse experiments. Such results provided scientific support for the initiation of our clinical trials. From our clinical trial investigations, we can draw some clinically important conclusions. First, allogeneic Vγ9Vδ2 T cells (NF cells) are clinically safe. This conclusion is strongly supported by our observation of 414 allogeneic NF-cell infusions in 132 cancer patients, which showed no significant adverse effects (e.g., immune rejection, cytokine storm, or GVHD effects). We emphasized here that the majority of patients received only 1–4 rounds of cell therapy and that the data obtained were thus only used to evaluate the clinical safety of allogeneic Vγ9Vδ2 T cells. It should be mentioned here that we measured cytokine release at 24 h post cell transfer therapy as well and found no evidence of cytokine storm after treatment with allogeneic Vγ9Vδ2 T cells. Nevertheless, a better time point for measuring cytokine release syndrome in patients with solid tumors would be ~1 week post cell therapy. Then, according to clinical observations, cell therapy could improve the quality of life in all patients, including pain relief, appetite, and sleep quality. In our clinical study, among 132 cancer patients, only 8 liver and 10 lung cancer patients received ≥5 cell infusions; therefore, the obtained clinical data were used to preliminarily analyze the efficacy of allogeneic Vγ9Vδ2 T-cell therapy. NF-cell infusions (≥5) prolonged survival in seven of the eight liver cancer patients and eight of the ten lung cancer patients to ≥10 months. Quite excitingly, in our latest follow-up in June 2020, we observed that three liver and two lung cancer patients were still alive (corresponding to survival times between 30 and 35 months) with normal lives. This clearly demonstrates that allogeneic Vγ9Vδ2 T-cell therapy prolonged the survival of most late-stage cancer patients who received ≥5 cell infusions in this small cohort (18 patients) investigation, showing the clinical efficacy of this therapy. In addition, follow-up study of 18 patients preliminarily implied that liver cancer patients might have better clinical responses than lung cancer patients. However, such observations need to be verified by further increasing the patient numbers in extended clinical studies. In conclusion, we developed a NF for the in vitro expansion of Vγ9Vδ2 T cells. The NF could significantly enhance cellular immune functions, including proliferation, differentiation, cellular energy metabolism, effector molecule expression, and cytotoxicity against cancer cell lines, while reducing the apoptotic rate in vitro. Moreover, an in vivo experiment using humanized mice firmly validated the superior antitumor cytotoxicity of NF cells compared with OF cells, which significantly prolonged mouse survival from <3 months to as long as 8 months. Most importantly, our clinical trials for the first time provided scientific evidence that allogeneic Vγ9Vδ2 T cells are clinically safe and preliminarily demonstrated therapeutic efficacy in solid tumor patients. Based on this work, we expect more clinical trials using allogeneic Vγ9Vδ2 T cells to treat malignant tumors to be conducted, which will eventually benefit tumor patients.
Data availability
Detailed clinical data sharing is not applicable to this article as clinical and commercial applications are ongoing.
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Acknowledgements
We thank all patients and investigators involved in the study. This work was supported by the Key Program of the National Natural Science Foundation of China (31830021); Major International Joint Research Program of China (31420103901); “111 project” (B16021); Scientific and Technological Plan of Guangdong Province (201704KW010) (Z.Y.); Fundamental Research Funds for the Central Universities, Natural Science Foundation of Guangdong Province, China (2020A1515010132) (Y.W.); and General Research Fund, Research Grants Council of Hong Kong (17122519, 17121214, 17115015, and 17126317) (W.T.), Hong Kong SAR, China. This work was also partially supported by the National Natural Science Foundation of China (31570898); the Natural Science Foundation of Guangdong Province, China (2016A030313112) (Z.X.); grant Ka 502/19–1 from the German Research Council (Deutsche Forschungsgemeinschaft); and the Cluster of Excellence ExC 306 “Inflammation-at-Interfaces” (Deutsche Forschungsgemeinschaft) (D.K.). Y.H. was supported by the China Postdoctoral Science Foundation (2017M622898). Y.X. was supported by the Postdoctoral Fund of the First Affiliated Hospital of **an University (809008). L.K. was supported by a long-term fellowship from the German Academic Exchange Service (DAAD). C.P. is the recipient of a grant from the Erich und Gertrud Roggenbruck Foundation.
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Protocol design: Z.Y., Y.W., Y.X., Z.X., M.A., W.T., and D.K. In vitro experiments: Y.X., J.H., L.K., Y.H., L.L., C.P., J.W.L., and Y.C. Animal experiments: J.Y., Z.X., Y.X., J.H., and W.T. Clinical therapy for patients: M.A., K.X., and J.C. Immune function testing and statistical analysis: Y.X., J.X.L., J.H., Y.W., Q.W., M.L., J.W.L., J.L.H., and Y.C. Cell culture and quality control for treatment: Y.L., X.W., Y.X., M.A., J.H., M.L., Q.W., and Y.W. Patient recruitment: M.A., K.X., Y.X., Y.W., Z.Y., J.C., and M.L. Patient follow-up: Y.X., M.A., Y.W., Z.Y., M.L., and Q.W. Manuscript writing: Y.W., Z.Y., Y.X., Y.H., and D.K. Manuscript revision: Y.W., Z.Y., Y.H., D.K., W.T., Y.X., and Z.X. All authors contributed to confirmation of the clinical protocol, data analyses, results and discussion and to manuscript proof-reading, and all authors approved the final version.
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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.
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The study protocol received ethical approval from the Regional Ethics Committee of Guangzhou Fuda Cancer Hospital, China. Written informed consent was obtained from the participants in accordance with the Declaration of Helsinki and ClinicalTrials.gov (NCT03183232, NCT03183219, NCT03183206, and NCT03180437). All animal studies were approved and performed in compliance with the guidelines for the use of experimental animals by the Committee on the Use of Live Animals in Teaching and Research, University of Hong Kong.
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Xu, Y., **ang, Z., Alnaggar, M. et al. Allogeneic Vγ9Vδ2 T-cell immunotherapy exhibits promising clinical safety and prolongs the survival of patients with late-stage lung or liver cancer. Cell Mol Immunol 18, 427–439 (2021). https://doi.org/10.1038/s41423-020-0515-7
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DOI: https://doi.org/10.1038/s41423-020-0515-7
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