免疫學(xué)及免疫檢測技術(shù)論文翻譯

1、Armed and accurate: engineering cytotoxic T cells for eradication of leukemia裝備并確認(rèn)：工程細胞毒性T細胞 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.

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 immunotherap

3、y highlights the essential nature of this exchange. Methods have been developed to convert a patient's 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 ant

4、igen 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

5、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.轉(zhuǎn)化醫(yī)學(xué)取決于工作臺和病床

6、間的快速，高效的轉(zhuǎn)換的結(jié)果。最近的一個例子，從癌癥的免疫療法領(lǐng)域突出這種交換的本質(zhì)。方法已被開發(fā)是將患者的細胞毒性T細胞轉(zhuǎn)換成高效特異的癌細胞殺手。通過使用重組DNA技術(shù)，慢病毒載體構(gòu)建從先進的慢性淋巴細胞白血病的患者的細胞毒性T細胞中表達嵌合抗原受體。嵌合受體的目的是指導(dǎo)細胞毒性T細胞對抗致癌細胞。I期臨床試驗在美國賓夕法尼亞大學(xué)設(shè)計的工程T細胞注入到癌癥患者的影響進行了測試，卡爾博士的研究小組的兩篇文章中描述了初步的結(jié)果。這項試驗非凡的成功，應(yīng)該激勵在新的癌癥免疫療法的發(fā)展中進一步應(yīng)用生物技術(shù) Main TextRecent advances in cancer immunotherapy

7、 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,

8、 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

9、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 Abrah

10、amson 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 system's ferocious power to eliminat

11、e an advanced metastatic cancer.在腫瘤免疫治療的最新進展已經(jīng)可以轉(zhuǎn)換白血病患者T細胞變成自己的癌細胞的高效特異的細胞殺手。這項新技術(shù)最近已測試成效顯著。成功治療的外在表現(xiàn)是顯著的，明確的。因發(fā)燒，發(fā)冷和疼痛而顫抖的癌癥患者，他們的血壓會急劇下降。在注入他們自己的工程殺傷性T細胞會使他們在睡醒之間輾轉(zhuǎn)反側(cè)好幾天。 T細胞回輸后的內(nèi)部斗爭在兩至三個星期間達到高峰。裂解腫瘤細胞的負荷加大了病人的腎臟壓力并幾乎腎中毒。通過免疫治療恢復(fù)活力的免疫系統(tǒng)已經(jīng)摧毀了近1公斤的腫瘤細胞。沒有其他可供選擇的治療已經(jīng)取得了類似的治療成功。由卡爾博士和他的在美國賓夕法尼亞大學(xué)的亞伯拉罕

12、森癌癥研究中心的同事共同執(zhí)行的新的治療方法，在試驗的3個慢性淋巴細胞白血?。–LL）患者其中兩個實現(xiàn)了前所未有的徹底根除癌癥。它曾是許多人的夢想：利用免疫系統(tǒng)的驚人的治理力量消除晚期轉(zhuǎn)移性癌癥。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

13、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 ja

14、rgon, 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 antibo

15、dy 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 combi

16、nation of functional domains is credited for achieving the spectacular success of the therapy令人驚嘆的研究結(jié)果發(fā)表在兩個同時進行的期刊文章，其中一個報告數(shù)據(jù)來自一個單身病人。這個成功的研究是出乎意料的。畢竟，這是第一期臨床試驗，其主要目的之一是測試不同劑量的工程細胞毒性T細胞（CTL）。從技術(shù)上講，這次成功可以追溯到融合細胞外和細胞內(nèi)結(jié)構(gòu)域成一個新的，合成受體的CD8+ T細胞。在免疫治療術(shù)語中，這種類型的重組蛋白被稱為嵌合抗原受體（CAR）。在這種案例中，胞外結(jié)構(gòu)域是來自特定的人類B細胞CD19表面

17、蛋白的小鼠單克隆抗體。由于CLL是一種B細胞癌，所以白血病細胞表達CD19蛋白。抗-CD19抗體被減少到最小的活性形式，單鏈可變區(qū)片段（scFv），并且被移植到T細胞受體的跨膜結(jié)構(gòu)域。在細胞的里面，該結(jié)構(gòu)被融合到胞質(zhì)信號域CD137（41BB）和CD3鏈（圖1）。這種功能結(jié)構(gòu)域的特定組合因獲得治療的驚人成功而得名 .Figure 1. The biotechnology of chimeric antigen receptors for cytotoxic T cell-based cancer immunotherapy. A. Map of pELPS 19-BB-3, a lentivi

18、ral 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 fro

19、m 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 woodch

20、uck 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 p

21、rotein 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.圖1

22、.細胞毒性T細胞為基礎(chǔ)的腫瘤免疫治療之嵌合抗原受體的生物技術(shù)。 A.pELPS19-BB-3圖，慢病毒載體被批準(zhǔn)用于臨床試驗的。編碼嵌合融合蛋白CD19-BB的開放閱讀框，包含抗CD19單鏈可變區(qū)片段（scFv），CD8的鉸鏈和跨膜（TM）域，以及一對內(nèi)域信號（一個來自CD137，另一個CD3）的部分編碼。此融合蛋白的基因被嵌入一個包含刪除gag，pol和env序列以及5'和3'長末端重復(fù)序列（LTR）的帶有缺陷型獲得性HIV脊椎。此外，該結(jié)構(gòu)包含延伸因子1（EF-1）的編碼序列，土撥鼠肝炎病毒轉(zhuǎn)錄后調(diào)控元件（WPRE）和牛生長激素多聚腺苷酸化（生長激素多聚A）尾，以及氨芐青霉

23、素抗性基因和一個細菌的復(fù)制起點。該結(jié)構(gòu)的總大小是11556個堿基對。 B.圖中的T細胞質(zhì)膜的嵌合受體蛋白質(zhì)。通過CD8 TM域?qū)崿F(xiàn)胞質(zhì)信號領(lǐng)域的CD137和CD3和細胞外抗體識別域的鏈接。這些內(nèi)域完成轉(zhuǎn)導(dǎo)T細胞的激活和刺激T細胞在體內(nèi)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 len

24、tivirus 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 infection

25、s 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基因通過慢病毒轉(zhuǎn)入純化每個病人的細胞毒性殺傷性T細胞。 HIV-1

26、病毒是慢病毒載體的主干。選擇這種病毒是幸運的，因為HIV-1的天然宿主是一種T淋巴細胞。因此，這個載體包含在T細胞中復(fù)制和表達的序列。這個載體的早期版本被用于治療艾滋病毒慢性感染。 CLL患者的純化的T細胞在體外轉(zhuǎn)導(dǎo)，然后記錄每個化療病人治療和減少未變性淋巴細胞的數(shù)目所需要的時間。這提供了適應(yīng)工程T細胞 在體內(nèi)的空間Once the patients were ready for the experimental treatment, they received between 15 Million and 1 Billion transduced CTL. The CTL rapidly m

27、ultiplied 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 d

28、etectable 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 remain

29、ing engineered cells acquired the phenotype of effector and memory T cells. At that time, the patients' overall wellbeing markedly improved.一旦患者準(zhǔn)備進行實驗性治療，他們收到了15億至10億之間轉(zhuǎn)導(dǎo)的CTL。的CTL迅速乘以在體內(nèi)的約1000倍的更高的數(shù)字2。這種擴張的部分內(nèi)容可以歸因于靶細胞的存在。進行試驗的科學(xué)家估計，每個工程的CTL破壞近1000個白血病細胞，直到輸注的CTL，三周后，沒有檢測到癌細胞仍然。的CTL增殖的峰值相對應(yīng)的最高水平

30、的IFN-，IL-6和CXCL9流通中2。這些細胞因子負責(zé)病人的高燒。一旦CLL數(shù)字下降到低于檢測水平，CTL數(shù)歸一化和收購余下的工程細胞的表型效應(yīng)和記憶性T細胞。當(dāng)時，顯著提高患者的整體健康。 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

31、 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 inte

32、rvals 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.治療的一個副作用是B細胞免疫缺陷，因為工程CTL也殺死患者的非癌性的B細胞。這是一個預(yù)期的治療結(jié)果，因為CTL的靶抗原，CD19受體被表達于所有成熟B細胞和B細胞的前體。來

33、自正常捐助者（IVIG）的IgG轉(zhuǎn)移可以補充抗體循環(huán)并局部彌補缺乏B細胞。將進行間隔每月一次的IgG注射來減少治療的患者中感染風(fēng)險。當(dāng)時的報道，患者中有兩位在6個月至11個月無癌癥復(fù)發(fā)。第三個表現(xiàn)出CLL持續(xù)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) demonstr

34、ated 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

35、 cell activation.各種科學(xué)家團體曾試圖修改CTL來攻擊癌細胞。在開創(chuàng)性的工作中，Eshhar和來自以色列魏茨曼科學(xué)研究所的合作者表明，CTL表達的重組表面受體連接獲得抗體的胞外識別域和獲得T細胞受體的細胞內(nèi)信號域。這種T細胞不再依賴于特定的信號和T細胞活化的HLA-肽復(fù)合物識別。 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 h

36、ost 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 e

37、nzymatic reactions leading to the programmed death of the infected cells. In this manner, the virus is purged from the body.通常情況下，細胞毒性T細胞在HLA I類的受體的凹槽中識別外源肽。從病毒感染的宿主細胞中可以獲得這些降低部分新形成的病毒蛋白形成的多肽并加載它們?yōu)镮類HAL。從外來得到宿主的多肽的識別觸發(fā)T細胞釋放穿孔素和顆粒酶，蛋白質(zhì)，形成在目標(biāo)小區(qū)中的孔，并啟動酶促反應(yīng)，導(dǎo)致受感染的細胞程序性死亡的級聯(lián)。在這種方式中，該病毒是從體內(nèi)清除An analogous

38、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 cytot

39、oxic 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.一個類似的反應(yīng)被認(rèn)為是發(fā)生在細胞毒性T細胞和

40、腫瘤細胞之間。癌癥細胞的致癌性轉(zhuǎn)化誘導(dǎo)在健康組織中不表達的蛋白質(zhì)的表達。免疫系統(tǒng)可以檢測癌細胞中與HAL相關(guān)的多肽的差異，因此，細胞毒性T細胞釋放其降解細胞的效應(yīng)分子。然而，在晚期癌癥，這些細胞毒性T細胞被耗盡，效率不高。漸漸地，癌細胞占據(jù)上風(fēng)和成長的數(shù)字變得越來越難以控制 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 initia

41、l 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

42、 proteins because single activation domain CAR showed only limited utility for the stimulation of naïve 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

43、 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

44、 mice. These experiments set the stage for the successful clinical trial at the University of Pennsylvania 1,2.在T細胞中表達嵌合抗原受體，scFv抗體片段綁定的結(jié)合HLA加肽的TCR抗原代用品。 Gross等人的初步成功鼓勵了眾多的后續(xù)研究，但可以結(jié)合預(yù)期的目標(biāo)和激活的T細胞的殺傷功能的重組受體的表達受到阻礙。各種研究人員設(shè)計和測試了第二代和第三代的CAR融合蛋白，因為單激活域CAR僅表現(xiàn)對幼稚T細胞的刺激的有限用途。更先進的受體設(shè)計還采用了其它內(nèi)域能同時激活多個信號轉(zhuǎn)導(dǎo)通路。 Im

45、ai等人和芬尼等人的實驗測試了雙激活域，包括CD3和CD137（4-1BB）相結(jié)合的測試，發(fā)現(xiàn)增加對工程CTL效應(yīng)的其他激活域。接著，Milone等表明在異種荷瘤小鼠的新的嵌合激活域在體內(nèi)的療效。在賓夕法尼亞大學(xué)，這些實驗成功的臨床試驗階段。 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 bet

46、ween 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 naïve T cells (Figure 1). In this manner, binding of the extracellular antib

47、ody domain to antigen could transmit two signals needed to induce the maturation of naïve 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.這一突破來自C

48、D137（4-1BB）的胞質(zhì)域中使工程T細胞增強活性的發(fā)現(xiàn)，條件是它被插入CD8跨膜區(qū)和CD3胞質(zhì)域之間的。這種串聯(lián)結(jié)構(gòu)的激活提供幼稚T細胞的活化（圖1）所需的兩個信號的任務(wù)合并成一個單一的多肽鏈的域。以這種方式，對抗原的抗體的胞外域綁定可以傳輸需要兩個信號誘導(dǎo)幼稚CD8 + T細胞的成熟成效應(yīng)T細胞。串聯(lián)嵌合抗原受體的細胞表達獲得強烈的信號進行增殖并分化成強力摧毀癌癥的殺傷性T細胞 。 Clearly, the new research will stimulate efforts to extend this approach to other refractory cancers. St

49、ill, 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 tria

50、ls 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

51、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 de

52、sirable 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 phas

53、e I trial with tandem endodomain CARs delivered by lentivirus 1.2 will surely energize the search for additional recombinant immune therapies for advanced cancer.顯然，新的研究將刺激拓展這種方法到其他難治性癌癥的努力。不過，注意事項依然存在。到現(xiàn)在治好的患者人數(shù)仍然非常少并且療程短，因此成功可能仍然是難以捉摸的。然而，有前途的新療法的希望是日趨增加。超過25項臨床試驗，目前正在探索各種形式的癌癥。它可能是可行的，延長CLL的治療到其他

54、B淋巴細胞腫瘤，像類似的工程CTL應(yīng)該是能夠攻擊任何CD19陽性癌細胞的。然而，重要的障礙依然存在：它可能難以對不與健康組織發(fā)生交叉反應(yīng)的腫瘤細胞產(chǎn)生專一特異性抗體。任何交叉反應(yīng)可能會導(dǎo)致自身免疫性疾病，可能會損壞重要器官。此外，與癌細胞的清除的嚴(yán)重副作用使得必須能找到方法來調(diào)節(jié)CTL的激活率。例如，有一些方法，其中一個能禁止“安全開關(guān)”成為工程T細胞一部分以防CTL治療使患者遭受不可接受的并發(fā)癥。通過慢病毒傳遞的 帶有串聯(lián)內(nèi)域CARs的I期臨床試驗的成功無疑必將激勵尋找對晚期癌癥其他的重組免疫療法。 Competing interestsThe author declares that he

55、 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,

56、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 ant

57、itumor 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 CD13