Chimerical antigen receptor for adoptive immunotherapy of cancer.docx

1、Chimerical antigen receptor for adoptive immunotherapy of cancerChimerical antigen receptor for adoptive immunotherapy of cancer: latest research and future prospectsAbstractChimeric antigen receptors (CARs) are recombinant receptors that combine the specificity of an antigen-specific antibody with

2、the T-cells activating functions. Initial clinical trials of genetically engineered CAR T cells have significantly raised the profile of T cell therapy, and great efforts have been made to improve this approach. In this review, we provide a structural overview of the development of CAR technology an

3、d highlight areas that require further refinement. We also discuss critical issues related to CAR therapy, including the optimization of CAR T cells, the route of administration, CAR toxicity and the blocking of inhibitory molecules.Keywords: Adoptive transfer, chimerical antigen receptor, Gene tran

4、sfer, T cells用于癌症过继免疫治疗的Chimerical抗原受体：最新研究和未来前景摘要嵌合抗原受体（CARs）是一种重组受体，它结合了抗原特异性抗体的特异性和T细胞的激活功能。 基因工程CAR T细胞的初步临床试验显着提高了T细胞治疗的特征，并且已经做出很大努力来改进这种方法。 在本次审查中，我们提供了CAR技术发展的结构概述，并突出了需要进一步完善的领域。 我们还讨论了与CAR治疗相关的关键问题，包括CAR T细胞的优化，给药途径，CAR毒性和抑制性分子的阻断。关键词：过继转移，嵌合抗原受体，基因转移，T细胞IntroductionAdoptive cellular thera

5、py (ACT) has received many attentions a realistic technique for cancer treatment 1-3.Tumor-reactive T cells can be isolated from tumorinfiltratinglymphocytes (TILs) and then expanded in vitrobefore re-infusion back into cancer patients 4. The adoptive transfer of TILs yields a durable regression of

6、melanoma tumors 5, 6. However, the process by which tumor-reactive TILs are isolated and expanded is technically difficult, labor-intensive and time-consuming. Moreover, another limitation in the more widespread application of TIL therapy is the difficulty in identifying antigen-specific T cells in

7、other cancer types.介绍过继性细胞疗法（ACT）已经受到许多关注，是一种现实的癌症治疗技术1-3。肿瘤反应性T细胞可以从肿瘤浸润淋巴细胞（TILs）中分离出来，然后在玻璃体内扩大，再输注回癌症患者4。 TIL的过继转移可使黑素瘤肿瘤持续消退5,6。 然而，肿瘤反应性TIL被分离和扩增的过程在技术上是困难的，劳动密集型和耗时的。 此外，更广泛应用TIL疗法的另一个限制是难以鉴定其他癌症类型中的抗原特异性T细胞。To overcome these obstacles and to broaden the applications of ACT, gene-therapeutic

8、approaches for the redirection of T-cells to defined tumor-associated antigens (TAAs) have been developed 7. One sophisticated strategy involves the engineering of antilogous T-cells with a chimerical antigen receptor (CAR) 8, which is composed of a specific antigen-binding moiety that is derived fr

9、om the variable regions of a monoclonal antibody (mAb) and linked through a hinge and a transmembrane (TM) motif to a cytoplasm lymphocyte-signaling moiety 9, 10.The CARs endow Tells antigen-specific recognition, activation and proliferation in an MHC-independent manner. Current clinical trials usin

10、g engineered CAR T cell therapy demonstrate clinical responses in both hematological malignancies and solid tumors 2, 11. Here, we will provide an overview of the recent development of the CAR technology and discuss the challenges and future prospects for this pioneering approach.为了克服这些障碍并拓宽ACT的应用，已

11、经开发出用于将T细胞重定向至确定的肿瘤相关抗原（TAA）的基因治疗方法7。 一种复杂的策略涉及利用嵌合抗原受体（CAR）8设计自体T细胞，该细胞由特异性抗原结合部分组成，该部分来源于单克隆抗体（mAb）的可变区并通过铰链和跨膜（TM）基序到细胞质淋巴细胞 - 信号传导部分9,10。CARs以不依赖于MHC的方式赋予Tcells抗原特异性识别，激活和增殖。使用工程化CAR T细胞疗法的当前临床试验证明了血液恶性肿瘤和实体瘤的临床反应2,11。 在这里，我们将概述CAR技术的最新发展，并讨论这种开创性方法的挑战和未来前景。CAR binding domainThe classic CAR cons

12、ists of an extracellular antigenrecognitiondomain attached to an extracellular spacer/hinge domain, a TM region that anchors the receptor tithe cell surface and a signaling end domain. A scoff derived from the variable heavy chain (VH) and variable light chain (VL) regions of an antigen-specific mob

13、 linked by flexible linker is commonly utilized as the extracellularTAA-binding domain in most CARs (Figure 1A). The scFvretains the same specificity and a similar affinity as the full antibody from which it was derived 12. Moreover, the small molecular size of scoffs facilitates both the genetic ma

14、nipulation and expression of the CAR. Furthermore, it determines the CAR antigen specificity and binds the target protein in an MHC-independent manner. To date, the scoffs of CARs are most often derived from mousemAbs. Human anti-mouse antibody (HAMA) responses can occur within days and can block an

15、tigen recognition by CARs. Therefore, the use of humanized 13 or fully human scoff 14 may be preferable to mouse scoff. In addition, the affinity of scoff must be considered in the design of CARs. The affinity of the scoff selected for designing CAR also should be considered. Hudecek et al. 15 showe

16、d that increasing the affinity of a CAR enhances its-cell effectors function and recognition of tumors. However, the development of higher affinity CARs with greateranti-tumor activity could theoretically increase the risk of on-target toxicity and mandates careful safety studies in relevant model.C

17、AR结合域经典CAR由附着于细胞外间隔区/铰链结构域的细胞外抗原识别结构域，将受体锚定至细胞表面的TM区和信号传导内结构域组成。衍生自通过柔性接头连接的抗原特异性mAb的可变重链（VH）和可变轻链（VL）区的scFv通常用作大多数CAR中的细胞外TAA结合结构域（图1A）。 scFv保留了与其衍生的完整抗体相同的特异性和相似的亲和力12。此外，scFv的小分子大小有利于CAR的遗传操作和表达。此外，它确定CAR抗原特异性并以不依赖MHC的方式结合靶蛋白。迄今为止，CAR的scFv通常源自小鼠mAb。人抗小鼠抗体（HAMA）反应可在数天内发生，并可阻断CAR的抗原识别。因此，使用人源化13或完全

18、人scFv 14可能优于小鼠scFv。此外，在CAR的设计中必须考虑scFv的亲和力。还应考虑选择用于设计CAR的scFv的亲和力。 Hudecek等人。 15表明，增加CAR的亲和力可增强其T细胞效应功能和肿瘤识别。然而，具有更高抗肿瘤活性的更高亲和力的CAR的发展理论上可以增加中心靶毒性的风险并且需要在相关模型中进行仔细的安全性研究。The extracellular antigen-recognition domain of Cars can also be a legend for a receptor that is expressed on tumor cells 11. Non

19、scoff-based legend-binding domains have been utilized in a CAR format (Figure 1B). For example, the CD27 receptor 16, the heregulin molecule (aligned for Her3 and Her4 receptors) 17, interleukin (IL)-13 mute in 18, vascular endothelial growth factor (anti-VEGFR2) 19, and the NKG2D receptor 20-22, ha

20、ve been used successfully for engineered T-cell therapy, resulting in tumor regression in vivo. Recently, a novelchimeric NKp30 CAR targeting the B7-H6 (NKp30 legend) expressing tumor was developed 23.CAR的细胞外抗原识别结构域也可以是在肿瘤细胞上表达的受体的配体11。 基于非scFv的配体结合结构域已经以CAR形式使用（图1B）。 例如，CD27受体16，调蛋白分子（Her3和Her4受体的配

21、体）17，白细胞介素（IL） - 13突变蛋白18，血管内皮生长因子（抗VEGFR2）19， 和NKG2D受体20-22已成功用于工程化T细胞治疗，导致体内肿瘤消退。 最近，开发了一种靶向B7-H6（NKp30配体）表达肿瘤的新型嵌合NKp30 CAR 23。To expand the applications for T cell-based immunotherapy in cancer, Tamara et al. 24 and Urbanska et al.25 constructed similar “universal” CARs (car) that utilize anti-f

22、luoresce in isothiocyanate (FITC) scoff andavidin in either a monomer (mica) or diametric (dace)form as binding domains fused to T-cell signaling domains, respectively (Figure 1C). These car T cells recognize various cancer types when bound to FITClabeledor biotinylated antigen-specific mobs or scFv

23、s, resulting in efficient target lyses, T-cell proliferation, and cytokine production. More recently, Kudos et al. 26 constructed novel uCAR containing the high-affinity CD16(FCGR3A) V158 variant, CD8 hinge and transmembranedomains, along with signaling domains. CD16V-baseduCAR T cells have bound hu

24、manized antibodies withhigher affinity and engagement of the CD16V-uCAR provokedT cell activation, exocytosis of the lytic granules andsustained proliferation. Further, the co-administration ofCD16V uCAR T cells with immunotherapeutic antibodiesexerted considerable antitumor activity in vivo. Import

25、antly,the treatment of immunocompromised mice usingthe novel uCAR T cells plus the labeled mAbs currently inclinical use exhibited potent antitumor activity. The needfor many different immune receptor genes to cover allcancers limits the feasibility of ACT, and the use of uCARsmay address this issue

26、.为扩大癌症中基于T细胞的免疫疗法的应用，Tamada等。 24和Urbanska等人25构建了类似的“通用”CARs（uCAR），其利用异硫氰酸荧光素（FITC）scFv和抗生物素蛋白以单体（mcAv）或二聚体（dcAv）形式作为分别与T细胞信号传导结构域融合的结合域（图1C） 。当与FITC标记的或生物素化的抗原特异性mAb或scFv结合时，这些uCAR T细胞识别各种癌症类型，导致有效的靶裂解，T细胞增殖和细胞因子产生。最近，Kudo等人。 26构建了一种新的uCAR，其含有高亲和力CD16（FCGR3A）V158变体，CD8铰链和跨膜结构域，以及信号结构域。基于CD16V的uCAR T

27、细胞结合了具有更高亲和力的人源化抗体，并且CD16V-uCAR的参与激发了T细胞活化，裂解颗粒的胞吐作用和持续增殖。此外，CD16V uCAR T细胞与免疫治疗性抗体的共同施用在体内发挥了相当大的抗肿瘤活性。重要的是，使用新型uCAR T细胞加上目前临床使用的标记mAb治疗免疫受损小鼠表现出有效的抗肿瘤活性。需要许多不同的免疫受体基因来覆盖所有癌症限制了ACT的可行性，并且使用uCAR可以解决这个问题。CAR targetingMost antigens targeted by CAR-T cells are simply tumorassociatedand not tumor-specif

28、ic. The potential for“on-target, off-organ” toxicity is a serious concern in CART-cell therapy. Thus, the judicious selection of TAAs isthe first step and is critical to the success of CAR-based ACT. CD19 is the widely and successfully utilized target ofCAR-modified T cells 27-29, being universally

29、expressedby acute lymphoblastic leukemia, the most common malignancyof children, whereas its expression on non-tumortissues is restricted to B-cells and their progenitors, butnot hematopoietic stem cells. The toxicity of targeting thisantigen using anti-CD19 CAR-modified T cells is limitedto B cell

30、aplasia and the consequent effects on humoralimmunity, which is considered to be a tolerable side-effectof this therapy 11. In contrast, one colon cancer patienttreated with Her2/neu CAR-T cells died 5 days after theadoptive transfer; this patient died of what appears to havebeen a cytokine storm an

31、d respiratory failure triggered bythe recognition of the low levels of antigens on lung epithelialcells 30. These studies suggest that ideal TAAs arerequired by the tumor cell for survival and should showrestricted expression to the tumor cell surface and otherwisenon-vital tissues.CAR定位CAR-T细胞靶向的大多

32、数抗原只是“肿瘤相关”而不是“肿瘤特异性”。 “靶向，非器官”毒性的可能性是CART细胞疗法中的严重问题。因此，明智地选择TAA是第一步，对CAR-BASEDACT的成功至关重要。 CD19是广泛且成功利用的CAR修饰T细胞27-29的目标，由急性淋巴细胞白血病（儿童最常见的恶性肿瘤）普遍表达，而其在非肿瘤组织中的表达仅限于B细胞及其祖细胞，但不是造血干细胞。使用抗CD19 CAR修饰的T细胞靶向该抗原的毒性仅限于B细胞发育不良及其对体液免疫的影响，这被认为是该疗法的可耐受的副作用11。相反，用Her2 / neu CAR-T细胞治疗的一名结肠癌患者在转移转移后5天死亡;这名患者因肺上皮细胞中低水平抗原识别引起的细胞因子风暴和呼吸衰竭而死亡30。这些研究表明，理想的TAA是肿瘤细胞存活所必需的，并且应该显示出对肿瘤细胞表面和其他重要组织的限制性表达。The effect of antigen density for CAR therapy is notyet well defined. It appears that CAR T cells typicallytarget highly expressed antigens, while low antigenexpr