免疫学及免疫检测技术论文翻译.doc

Armed and accurate: engineering cytotoxic T cells for eradication of leukemia装备并确认：工程细胞毒性T细胞 根除白血病Marko RadicCorrespondence: Marko Radic Author AffiliationsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractTranslational medicine depends on a rapid and efficient exchange of results between the bench and the bedside. A recent example from the field of cancer immunotherapy highlights the essential nature of this exchange. Methods have been developed to convert a patients cytotoxic T cells into efficient and specific killers of cancer cells in patients with leukemia. By using recombinant DNA techniques, a lentiviral vector was constructed to express chimeric antigen receptors in cytotoxic T cells from patients with advanced chronic lymphocytic leukemia. The purpose of the chimeric receptors was to direct the cytotoxic T cell activity against cells causing the cancer. The effect of infusing the engineered T cells back into the cancer patients was tested in a Phase I trial at the University of Pennsylvania, and the initial results were described in two articles from the research team of Dr. Carl June. The remarkable success of this trial should energize further applications of biotechnology in the development of new cancer immunotherapies.转化医学取决于工作台和病床间的快速，高效的转换的结果。最近的一个例子，从癌症的免疫疗法领域突出这种交换的本质。方法已被开发是将患者的细胞毒性T细胞转换成高效特异的癌细胞杀手。通过使用重组DNA技术，慢病毒载体构建从先进的慢性淋巴细胞白血病的患者的细胞毒性T细胞中表达嵌合抗原受体。嵌合受体的目的是指导细胞毒性T细胞对抗致癌细胞。I期临床试验在美国宾夕法尼亚大学设计的工程T细胞注入到癌症患者的影响进行了测试，卡尔博士的研究小组的两篇文章中描述了初步的结果。这项试验非凡的成功，应该激励在新的癌症免疫疗法的发展中进一步应用生物技术 Main TextRecent advances in cancer immunotherapy have allowed the conversion of T cells from leukemia patients into efficient and specific killers of their own cancer cells. The new technique has been recently tested with remarkable results. The outward signs of the successful therapy were dramatic and clear. The cancer patients shook with fever, chills and pain; their blood pressure precipitously dropped. They drifted in and out of sleep for days following the infusion of their own engineered killer T cells. The internal struggle peaked between two and three weeks after T cell reinfusion. The load of lysed cancer cells stressed and nearly poisoned the patients kidneys. Their immune systems, reinvigorated by the immunotherapy, had destroyed nearly 1 kg of cancer cells. No other available treatment could have achieved a comparable therapeutic success. The new treatment, administered by Dr. Carl June and his colleagues of the Abrahamson Cancer Center at the University of Pennsylvania, achieved an unprecedented complete eradication of the cancer in two of the three chronic lymphocytic leukemia (CLL) patients in whom it was tried. It had been a dream of many: the harnessing of the immune systems ferocious power to eliminate an advanced metastatic cancer.在肿瘤免疫治疗的最新进展已经可以转换白血病患者T细胞变成自己的癌细胞的高效特异的细胞杀手。这项新技术最近已测试成效显着。成功治疗的外在表现是显着的，明确的。因发烧，发冷和疼痛而颤抖的癌症患者，他们的血压会急剧下降。在注入他们自己的工程杀伤性T细胞会使他们在睡醒之间辗转反侧好几天。 T细胞回输后的内部斗争在两至三个星期间达到高峰。裂解肿瘤细胞的负荷加大了病人的肾脏压力并几乎肾中毒。通过免疫治疗恢复活力的免疫系统已经摧毁了近1公斤的肿瘤细胞。没有其他可供选择的治疗已经取得了类似的治疗成功。由卡尔博士和他的在美国宾夕法尼亚大学的亚伯拉罕森癌症研究中心的同事共同执行的新的治疗方法，在试验的3个慢性淋巴细胞白血病（CLL）患者其中两个实现了前所未有的彻底根除癌症。它曾是许多人的梦想：利用免疫系统的惊人的治理力量消除晚期转移性癌症。The stunning results were published in two simultaneous journal articles 1,2, one of the reports presenting data from a single patient 1. The success was beyond expectation. This, after all, was a Phase I clinical trial, one in which the primary objective was to test different doses of the engineered cytotoxic lymphocytes (CTL). Technically, the success could be traced to the fusion of extracellular and intracellular domains into a new, synthetic receptor for the CD8+ T cells 3. In immunotherapy jargon, this type of recombinant protein is called a chimeric antigen receptor (CAR). In this case, the extracellular domain was derived from a mouse monoclonal antibody specific for the CD19 surface protein of human B cells. Because CLL is a B cell cancer, CLL cells express CD19. The anti-CD19 antibody was reduced to its smallest active form, a single-chain variable fragment (scFv), and grafted onto a T cell receptor transmembrane domain. On the inside of the cell, the construct was fused to the cytoplasmic signaling domains of CD137 (41BB) and the chain of CD3 (Figure 1). This particular combination of functional domains is credited for achieving the spectacular success of the therapy令人惊叹的研究结果发表在两个同时进行的期刊文章，其中一个报告数据来自一个单身病人。这个成功的研究是出乎意料的。毕竟，这是第一期临床试验，其主要目的之一是测试不同剂量的工程细胞毒性T细胞（CTL）。从技术上讲，这次成功可以追溯到融合细胞外和细胞内结构域成一个新的，合成受体的CD8+ T细胞。在免疫治疗术语中，这种类型的重组蛋白被称为嵌合抗原受体（CAR）。在这种案例中，胞外结构域是来自特定的人类B细胞CD19表面蛋白的小鼠单克隆抗体。由于CLL是一种B细胞癌，所以白血病细胞表达CD19蛋白。抗-CD19抗体被减少到最小的活性形式，单链可变区片段（scFv），并且被移植到T细胞受体的跨膜结构域。在细胞的里面，该结构被融合到胞质信号域CD137（41BB）和CD3链（图1）。这种功能结构域的特定组合因获得治疗的惊人成功而得名 .Figure 1. The biotechnology of chimeric antigen receptors for cytotoxic T cell-based cancer immunotherapy. A. Map of pELPS 19-BB-3, a lentiviral vector that was approved for clinical trials 1. The open reading frame encoding the chimeric fusion protein CD19-BB contains portions coding for an anti-CD19 single-chain variable region fragment (scFv), the hinge and transmembrane (TM) domain of CD8, and a pair of signaling endodomains (one from CD137, the other from CD3). This fusion protein gene is embedded within a disabled HIV-derived backbone containing truncated gag, pol and env sequences and the 5 and 3 long terminal repeats (LTR). In addition, the construct contains elongation factor 1 (EF-1) coding sequences, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and the bovine growth hormone polyadenylation (GH poly A) tail, as well as the ampicillin resistance gene and a bacterial replication origin. The total size of the construct is 11,556 base pairs. B. Diagram of the chimeric receptor protein in the T cell plasma membrane. The extracellular antibody recognition domain is linked via the CD8 TM domain to the cytoplasmic signaling domains of CD137 and CD3. These endodomains accomplish the activation of transduced T cells and they stimulate T cell survival and proliferation in vivo.图1.细胞毒性T细胞为基础的肿瘤免疫治疗之嵌合抗原受体的生物技术。 A.pELPS19-BB-3图，慢病毒载体被批准用于临床试验的。编码嵌合融合蛋白CD19-BB的开放阅读框，包含抗CD19单链可变区片段（scFv），CD8的铰链和跨膜（TM）域，以及一对内域信号（一个来自CD137，另一个CD3）的部分编码。此融合蛋白的基因被嵌入一个包含删除gag，pol和env序列以及5和3长末端重复序列（LTR）的带有缺陷型获得性HIV脊椎。此外，该结构包含延伸因子1（EF-1）的编码序列，土拨鼠肝炎病毒转录后调控元件（WPRE）和牛生长激素多聚腺苷酸化（生长激素多聚A）尾，以及氨苄青霉素抗性基因和一个细菌的复制起点。该结构的总大小是11556个碱基对。 B.图中的T细胞质膜的嵌合受体蛋白质。通过CD8 TM域实现胞质信号领域的CD137和CD3和细胞外抗体识别域的链接。这些内域完成转导T细胞的激活和刺激T细胞在体内的存活和增殖。 The gene for this CAR was delivered by lentivirus to purified cytotoxic killer T cells from each patient. The HIV-1 virus served as the backbone in the construction of the lentivirus vector. The choice of this virus proved fortunate because the natural host for HIV-1 is a T lymphocyte. Therefore, the vector included sequences that had evolved for replication and expression in T cells. A previous version of this vector was used to treat patients with chronic HIV infections 4. The CLL patients purified T cells were transduced in vitro, then stored for the time required to treat each patient with chemotherapy and reduce the numbers of unmodified lymphocytes. This provided the in vivo space to accommodate the engineered T cells.CAR基因通过慢病毒转入纯化每个病人的细胞毒性杀伤性T细胞。 HIV-1病毒是慢病毒载体的主干。选择这种病毒是幸运的，因为HIV-1的天然宿主是一种T淋巴细胞。因此，这个载体包含在T细胞中复制和表达的序列。这个载体的早期版本被用于治疗艾滋病毒慢性感染。 CLL患者的纯化的T细胞在体外转导，然后记录每个化疗病人治疗和减少未变性淋巴细胞的数目所需要的时间。这提供了适应工程T细胞 在体内的空间Once the patients were ready for the experimental treatment, they received between 15 Million and 1 Billion transduced CTL. The CTL rapidly multiplied to approximately 1000-fold higher numbers in vivo 2. Part of this expansion may be attributed to the presence of target cells. The scientists performing the trial estimated that each engineered CTL destroyed nearly 1000 leukemia cells, until, three weeks after the infusion of the CTL, no detectable cancer cells remained. The peak of CTL proliferation corresponded to the highest level of IFN-, IL-6 and CXCL9 in circulation 2. These cytokines were responsible for the patients high fever. Once the CLL numbers dropped below detection levels, the CTL numbers normalized and the remaining engineered cells acquired the phenotype of effector and memory T cells. At that time, the patients overall wellbeing markedly improved.一旦患者准备进行实验性治疗，他们收到了15亿至10亿之间转导的CTL。的CTL迅速乘以在体内的约1000倍的更高的数字2。这种扩张的部分内容可以归因于靶细胞的存在。进行试验的科学家估计，每个工程的CTL破坏近1000个白血病细胞，直到输注的CTL，三周后，没有检测到癌细胞仍然。的CTL增殖的峰值相对应的最高水平的IFN-，IL-6和CXCL9流通中2。这些细胞因子负责病人的高烧。一旦CLL数字下降到低于检测水平，CTL数归一化和收购余下的工程细胞的表型效应和记忆性T细胞。当时，显着提高患者的整体健康。 One side-effect of the therapy is B cell immunodeficiency because the engineered CTL also kill the patients non-cancerous B cells. This was an anticipated consequence of the therapy because the CTL target antigen, the CD19 receptor, is expressed on all mature B cells and B cell precursors. The transfer of IgG from normal donors (IVIG) can replenish the patients circulating antibodies and partially compensate for the absence of B cells. The IgG injections will be performed at monthly intervals to reduce the risk of infections in treated patients. Two of the patients were, at the time of the reports, free of cancer recurrence for 6 to 11 months 2. The third showed a sustained improvement of the CLL.治疗的一个副作用是B细胞免疫缺陷，因为工程CTL也杀死患者的非癌性的B细胞。这是一个预期的治疗结果，因为CTL的靶抗原，CD19受体被表达于所有成熟B细胞和B细胞的前体。来自正常捐助者（IVIG）的IgG转移可以补充抗体循环并局部弥补缺乏B细胞。将进行间隔每月一次的IgG注射来减少治疗的患者中感染风险。当时的报道，患者中有两位在6个月至11个月无癌症复发。第三个表现出CLL持续改善 Various groups of scientists had attempted to modify CTL to attack cancer cells. In pioneering work, Eshhar and collaborators from the Weizmann Institute of Science in Rehovot (Israel) demonstrated that CTL can express a recombinant surface receptor that links an extracellular recognition domain derived from an antibody to an intracellular signaling domain derived from the T cell receptor 5. Such T cells no longer depend on recognition of HLA-peptide complexes for specific signaling and T cell activation.各种科学家团体曾试图修改CTL来攻击癌细胞。在开创性的工作中，Eshhar和来自以色列魏茨曼科学研究所的合作者表明，CTL表达的重组表面受体连接获得抗体的胞外识别域和获得T细胞受体的细胞内信号域。这种T细胞不再依赖于特定的信号和T细胞活化的HLA-肽复合物识别。 Normally, cytotoxic T cells recognize foreign peptides in the groove of an HLA class I receptor. The peptides may be derived from a virus infecting a host cell, which degrades a portion of the newly forming viral proteins into peptides and loads them into a class I HLA. The recognition of the peptides as foreign to the host triggers the T cell to release perforin and granzyme, proteins that form pores in the target cell and initiate a cascade of enzymatic reactions leading to the programmed death of the infected cells. In this manner, the virus is purged from the body.通常情况下，细胞毒性T细胞在HLA I类的受体的凹槽中识别外源肽。从病毒感染的宿主细胞中可以获得这些降低部分新形成的病毒蛋白形成的多肽并加载它们为I类HAL。从外来得到宿主的多肽的识别触发T细胞释放穿孔素和颗粒酶，蛋白质，形成在目标小区中的孔，并启动酶促反应，导致受感染的细胞程序性死亡的级联。在这种方式中，该病毒是从体内清除An analogous reaction is thought to occur between cytotoxic T cells and tumor cells. The oncogenic transformation of a cancer cell induces the expression of proteins that are not expressed in healthy tissues. The immune system can detect the differences in HLA-associated peptides from cancer cells, and the cytotoxic T cells, in consequence, release their cell-degrading effector molecules. However, in advanced cancers, these cytotoxic T cells are depleted and inefficient. Gradually, the cancer cells gain the upper hand and grow to numbers that become more and more difficult to control.一个类似的反应被认为是发生在细胞毒性T细胞和肿瘤细胞之间。癌症细胞的致癌性转化诱导在健康组织中不表达的蛋白质的表达。免疫系统可以检测癌细胞中与HAL相关的多肽的差异，因此，细胞毒性T细胞释放其降解细胞的效应分子。然而，在晚期癌症，这些细胞毒性T细胞被耗尽，效率不高。渐渐地，癌细胞占据上风和成长的数字变得越来越难以控制 In T cells expressing chimeric antigen receptors, the scFv antibody fragment that binds to antigen substitutes for the TCR binding to HLA plus peptide. The initial success of Gross et al. 5 inspired numerous subsequent studies but obstacles hindered efforts to express recombinant receptors that could bind the desired targets and activate the cytolytic functions of the T cells 6. Various investigators designed and tested second and third generation CAR fusion proteins because single activation domain CAR showed only limited utility for the stimulation of nave T cells 6. The more advanced receptor design incorporated additional endodomains that could simultaneously activate more than one signaling pathway. Experiments by Imai et al. 7 and Finney et al. 8 tested dual activation domains, including the combination of CD3 and CD137 (4-1BB) and found the added activation domains to increase responses of engineered CTL. Subsequently, Milone et al. 3 demonstrated the in vivo efficacy of the new chimeric activation domains in xenogeneic tumor bearing mice. These experiments set the stage for the successful clinical trial at the University of Pennsylvania 1,2.在T细胞中表达嵌合抗原受体，scFv抗体片段绑定的结合HLA加肽的TCR抗原代用品。 Gross等人的初步成功鼓励了众多的后续研究，但可以结合预期的目标和激活的T细胞的杀伤功能的重组受体的表达受到阻碍。各种研究人员设计和测试了第二代和第三代的CAR融合蛋白，因为单激活域CAR仅表现对幼稚T细胞的刺激的有限用途。更先进的受体设计还采用了其它内域能同时激活多个信号转导通路。 Imai等人和芬尼等人的实验测试了双激活域，包括CD3和CD137（4-1BB）相结合的测试，发现增加对工程CTL效应的其他激活域。接着，Milone等表明在异种荷瘤小鼠的新的嵌合激活域在体内的疗效。在宾夕法尼亚大学，这些实验成功的临床试验阶段。 The breakthrough occurred with the discovery that the cytoplasmic domain from CD137 (4-1BB) enabled strong activation of the engineered T cells, provided that it was inserted between the CD8 transmembrane region and the CD3 cytoplasmic domain 3. This tandem configuration of activating domains merged into a single polypeptide chain the task of delivering two signals needed for the activation of nave T cells (Figure 1). In this manner, binding of the extracellular antibody domain to antigen could transmit two signals needed to induce the maturation of nave CD8 T cells into effector T cells. The cells expressing the tandem chimeric antigen receptors received strong signals to proliferate and differentiate into potent cancer-destroying killer T cells 1.这一突破来自CD137（4-1BB）的胞质域中使工程T细胞增强活性的发现，条件是它被插入CD8跨膜区和CD3胞质域之间的。这种串联结构的激活提供幼稚T细胞的活化（图1）所需的两个信号的任务合并成一个单一的多肽链的域。以这种方式，对抗原的抗体的胞外域绑定可以传输需要两个信号诱导幼稚CD8 + T细胞的成熟成效应T细胞。串联嵌合抗原受体的细胞表达获得强烈的信号进行增殖并分化成强力摧毁癌症的杀伤性T细胞 。 Clearly, the new research will stimulate efforts to extend this approach to other refractory cancers. Still, caveats remain. The numbers of patients treated up to now remain very small and the time elapsed since the treatment is short, thus success could still remain elusive. However, hopes that a promising new therapy is within reach increase with every passing day. In all, more than 25 clinical trials currently explore the use of chimeric T cell receptors in various forms of cancer 9. It might be feasible to extend the CLL therapy1,2 to other B lymphocytic neoplasms, as similarly engineered CTL should be capable of attacking any CD19-positive cancer cells. Yet important hurdles remain: It may prove difficult to develop antibodies with exclusive specificity for cancer cells that do not cross-react with healthy tissues. Any cross-reactivity may lead to autoimmunity that could damage vital organs. Furthermore, the violent side-effects associated with the clearance of cancer cells make it desirable to find ways to regulate the rate of CTL activation. For example, there are ways in which a safety switch may be incorporated into engineered T cells that could deactivate them in case that the CTL therapy exposes patients to unacceptable complications 10. The success of the phase I trial with tandem endodomain CARs delivered by lentivirus 1.2 will surely energize the search for additional recombinant immune therapies for advanced cancer.显然，新的研究将刺激拓展这种方法到其他难治性癌症的努力。不过，注意事项依然存在。到现在治好的患者人数仍然非常少并且疗程短，因此成功可能仍然是难以捉摸的。然而，有前途的新疗法的希望是日趋增加。超过25项临床试验，目前正在探索各种形式的癌症。它可能是可行的，延长CLL的治疗到其他B淋巴细胞肿瘤，像类似的工程CTL应该是能够攻击任何CD19阳性癌细胞的。然而，重要的障碍依然存在：它可能难以对不与健康组织发生交叉反应的肿瘤细胞产生专一特异性抗体。任何交叉反应可能会导致自身免疫性疾病，可能会损坏重要器官。此外，与癌细胞的清除的严重副作用使得必须能找到方法来调节CTL的激活率。例如，有一些方法，其中一个能禁止“安全开关”成为工程T细胞一部分以防CTL治疗使患者遭受不可接受的并发症。通过慢病毒传递的 带有串联内域CARs的I期临床试验的成功无疑必将激励寻找对晚期癌症其他的重组免疫疗法。 Competing interestsThe author declares that he has no competing interests.AcknowledgementsThe author acknowledges the expert assistance of Dafne Solera, Ph.D., Executive Editor of BMC Biotechnology, with essential adjustments in style and content. The technical diagram of Mr. Tim Higgins, senior illustrator, is acknowledged.ReferencesPorter D, Levine BL, Kalos M, Bagg A, June CH: Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.N Engl J Med 2011, 365:725-733. PubMed Abstract | Publisher Full Text Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, June CH: T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.Sci Transl Med 2011, 3:95ra73. PubMed Abstract | Publisher Full Text Milone MC, Fish JD, Carpenito C, Carroll RG, Binder GK, Teachey D, Samanta M, Lakhal M, Gloss B, Danet-Desnoyers G, Campana D, Riley JL, Grupp SA, June CH:Chimeric receptors containing CD137 signa