前言
表达嵌合抗原受体（CAR）的基因工程T细胞正在迅速涌现出一种有希望的针对血液和非血液恶性肿瘤的新治疗方法。CAR-T疗法可引起快速而持久的临床反应，但与独特的急性毒性有关。此外，CAR-T细胞易受免疫抑制机制的影响。9月25日上海交通大学医学院附属第九人民医院辅助生殖科与第二军医大学基础医学学院生物物理系团队在Nature Communications查看期刊详情上发表了“CAR exosomes derived from effector CAR-T cells have potent antitumour effects and low toxicity”，文章报道了CAR-T细胞释放细胞外囊泡，主要是在其表面携带CAR的外泌体形式，含CAR的外泌体表达高水平的细胞毒性分子并抑制肿瘤生长。与CAR-T细胞相比，CAR外泌体不表达程序性细胞死亡蛋白1（PD1），并且重组PD-L1处理不能削弱其抗肿瘤作用。在细胞因子释放综合征的临床前体内模型中，与CAR-T疗法相比，CAR外泌体的给药相对安全。这项研究支持将外泌体用作仿生纳米囊泡，这可能在未来的抗肿瘤治疗方法中有用。
研究思路
Fig. 1 Generation and characterization of CAR-T cells. a, Vector maps of tested CAR designs. b, Membrane-bound CAR expression. Forty-eight hours after retroviral transduction, the expression of CAR on human T cells was detected by staining with anti-MYC antibody, followed by flow cytometry analysis.T cells without transduction were used as a negative control. c ,Killing activity of CAR-T cells in response to tumour cells. The cytotoxic activity of CAR-T and control T cells against cancer cell lines was assessed by a 51Cr-release assay at the indicated effector-to-target (E:T) ratios.
Fig. 2 CAR-T cells release extracellular vesicles carrying CAR protein. a, b Schematic (a) of ELISA (b) to measure the CAR concentration on the surface of exosomes isolated from CAR-T cells of different states. c, ELISA of CAR on exosomes from CAR-T, with or without antigen stimulation. d, Immunoblots for CAR expression in whole-cell lysates (W) and purified exosomes from CAR-T cells with CD28/CD3 bead stimulation (B) or cancer cell stimulation (C). All lanes were loaded with the same amount of total protein. e, ELISA of CAR on exosomes from CAR-T with or without different stimulation strategies. f, Antigen binding of exosomes from different cultures with or without blocking antibody cetuximab (CTX) or trastuzumab (TTZ). g, Levels of CAR on the exosomes or microvesicles derived from CAR-T cells as assayed by ELISA. h, Levels of exosomal CAR and microvesicle CAR produced by an equal number of CAR-T cells.
Fig. 3 Characterization of CAR exosomes derived from effector CAR-T cells. a, Schematic representation of the enrichment of CAR-containing exosomes from T cells with repeated antigen stimulation. b, Size distribution of CAR exosomes as measured by NTA, peaking at an 85-nm diameter. c, Transmission electron micrographs of CAR exosomes. d, Immunoblots for CAR expression in exosomes compared with cell markers for endoplasmic reticulum (calregulin), Golgi (Golgi 58 K), mitochondrial (prohibitin), or nuclear (nucleoporin p62) markers. e, Flow cytometry analyses of CAR exosomes linked to latex beads (4-mm diameter) or CAR-T cells and stained with the indicated primary Abs.
Fig. 4 Cytolytic activity of CAR exosomes in vitro. a, Flow cytometry analyses of CAR exosomes linked to latex beads (4-mm diameter) or CAR-T cells stained with the indicated primary Abs. b, Immunoblots for perforin and granzyme B expression in CAR exosomes and CAR-T cells. c, Killing activity of CAR exosomes in response to tumour cells. The cytotoxic activity of CAR exosomes and control T cells against cancer cell lines was assessed by the 51Crrelease assay at the indicated concentration. d, Confocal microscopy analysis of MCF-7 EGFR cells (up) and MCF-7 HER2 cells (down) after incubation with NHS-Rhodamine (Rho)-labelled CAR-EXO-CTX for 2 h.
Fig. 5 CAR exosomes have notable antitumour activity in vivo. a ,Tumour volumes of MDA-MB-231 (left), HCC827 (middle) and SK-BR-3 (right) tumour xenografts after treatment with the indicated treatment. b, c ,Tumour volumes of MDA-MB-231 (b) and SK-BR-3 (c) tumour xenografts after treatment with the indicated CAR exosome treatment with or without blocking recombinant antigen. d, Cancer cell lines or patient-derived tumour tissue fragments established as subcutaneous xenografts and treated with weekly doses of CAR exosomes. Substantial TGI was observed in lung cancer models treated with CAR-EXO-CTX (black bars) and in HER2-positive breast and ovary cancer models treated with CAR-EXO-TTZ (grey bars).
Fig. 6 PD-L1 inhibits CAR-T cells but not CAR exosomes in vitro and in vivo. a, Representative histogram of the expression of granzyme B and Ki-67 after the indicated treatments in CAR-T cells. b, qPCR analyses of IL-2, IFN-γ, and TNF in CAR-T cells after the indicated treatments with or without blocking by recombinant PD-L1 or the anti-PD-L1 antibodies.The relative mRNA expression level was calculated as the ratio to the control cells. c, ELISA of IL-2, IFN-γ, and TNF in CAR-T cells after the indicated treatment with or without blocking by recombinant PD-L1 or the anti-PD-L1 antibodies. d, e, Killing activity of CAR-T cells (d) and CAR exosomes (e) in response to tumour cells. The cytotoxic activity of CAR-T or CAR exosomes against cancer cell lines was assessed by the 51Cr-release assay at the indicated effector-to-target (E:T) ratios or indicated concentrations with or without blocking by recombinant PD-L1 or anti-PD-L1 antibodies. f, Tumour volumes of MDA-MB-231 (up) and SK-BR-3 (down) tumour xenografts after the indicated treatment.
Figure 7, CAR exosomes do not cause cytokine release syndrome in mice. a, Killing activity of CAR-T-T1E cells in response to tumour cells. The cytotoxic activity of CAR-T-T1E and control T cells against cancer cell lines was assessed by the 51Cr-release assay at the indicated effector-to-target (E:T) ratios.b, Tumour volumes of MDA-MB-231 tumour xenografts after the indicated treatment. c, ELISA (left) of CAR on exosomes from CAR-T-T1E and transmission electron micrographs of CAR-EXO-T1E (right). d Cytotoxic activity of CAR exosomes and control T cells against cancer cell lines was assessed by the 51Cr-release assay at the indicated concentration. e, Tumour volumes of MDA-MB-231 tumour xenografts after the indicated treatment. f, Three groups of three tumour-free mice were treated i.p. with CAR-T-T1E cells at the indicated escalating doses. Serial weight measurements were performed and normalized to the starting body weight for each animal. g, Serum levels of human (h) and mouse (m) cytokines, measured at the indicated time points. h, Three groups of three tumour-free mice were treated i.p. with CAR-EXO-T1E cells at the indicated escalating doses. Serial weight measurements were performed and normalized to the starting body weight for each animal.i, Serum levels of human (h) and mouse (m) cytokines, measured at the indicated time points.
结论
基于CAR的过继免疫疗法使用遗传修饰的T淋巴细胞提供肿瘤靶向和免疫应答，可以作为活体药物，对靶细胞持续产生细胞毒攻击。CAR-T细胞的肿瘤杀伤能力取决于细胞自身的寿命以及进一步的体内复制。但是，CAR-T细胞和这些细胞在体内的复制可以促进细胞因子的释放，这是无法控制的。在实体瘤中，CAR-T治疗尚未获得在血液学上的恶性肿瘤中观察到的临床成功。治疗反应差的原因之一是CAR-T细胞无法在不利的肿瘤微环境中积累和复制。此外，CAR-T细胞失活以及可能从肿瘤块中排除，基质细胞与肿瘤之间的直肠相互作用，以及诸如前列腺癌之类的癌症优先向骨骼扩散的倾向可能都是由于缺乏亚实体瘤中大量的CAR介导的T细胞反应。
文章显示了从CAR-T细胞释放的CAR外泌体也具有攻击癌细胞的巨大治疗潜力。使用外泌体作为癌症治疗的直接攻击者可能有几个明显的优势。CAR外泌体可能具有较低的毒性风险。无细胞囊泡的制造过程也比活CAR-T细胞的制造过程更安全，这得到了最近的病例报告的支持，该案例表明，在T细胞制造和生产过程中，CAR基因被无意地引入了单个亮氨酸B细胞中。其产物与白血病细胞表面的CD19表位顺式结合，从而使其无法被CTL019识别并赋予其抵抗力；患者最终死于与进行性白血病相关的并发症。其次，由于外泌体具有纳米级的尺寸，因此它们具有被用于实体瘤治疗的优势。外泌体的适当使用为靶向特定区域中的肿瘤细胞铺平了道路。第三，这两个平台（即CAR-T细胞和CAR外泌体）的组合或交替使用无疑将加强基于CAR的癌症治疗的使用。数据表明，PD-L1治疗最终不会阻止外泌体细胞毒性，这表明CAR外泌体治疗可以克服免疫抑制机制。对于CAR外泌体的临床应用，提出的方案包括从健康供体或癌症患者的外周血中分离T细胞，对T细胞进行基因工程以表达CAR，体外复制和刺激CAR改造的T细胞，分离外泌体，CAR外泌体的纯化，以及向患者体内注入一些外泌体。CAR外泌体可以通过研究中使用的抗原包被的磁珠进一步纯化。
总之，工程化的T细胞免疫疗法用于癌症治疗是有前途的。在癌症的易变性和复杂性的背景下，这种疗法也有一些局限性或缺陷。数据表明，CAR外泌体可能会成为最终的攻击者，从而克服了当前治疗模型的局限性。适当地应用细胞和外泌体平台，基于CAR的治疗将更加有效，并且可能成为靶向癌症治疗的下一个有希望的选择。
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EVs-Exosomes由苏大，浙大, 法国居里研究所数位博士、博后及教授创建。
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