PKG

自然杀伤细胞(NK细胞)因其对恶性细胞的高效识别和裂解而被认为是一种特殊的免疫细胞，可以通过基因修饰获得功能强大的效应细胞，用于癌症患者的过继细胞治疗。然而，将基因导入NK细胞相关的生物和技术障碍极大地限制了其进展。最近的技术进步，包括改进的细胞扩增技术、嵌合抗原受体(CAR)、CRISPR/Cas9基因编辑以及增强的病毒转导和电穿孔，已经赋予了全面的转基因NK细胞的产生。最近些年，使用CAR重定向NK细胞为癌症免疫治疗带来了新的治疗手段。使用CAR-NK细胞的各种临床前研究和有限的临床研究显示巨大潜力：既可以有效地消除靶细胞，而且没有CAR-T治疗中出现的细胞因子释放综合征和神经毒性等副作用。NK细胞上表达的功能性CAR分子由三部分组成：胞外域、跨膜区和胞内区(图1)。胞外结构域由信号肽、ScFv和连接该结构与跨膜区的铰链区组成。铰链区域将CAR分子连接到细胞膜上，进而连接到细胞内包含激活信号的胞内域。这些构件与位于开放阅读框架外的调节元件(例如启动子)的组合将决定CAR的功效。成功的CAR设计是通过精心的生物信息学设计和功能测试相结合而实现的。在这里我们我们简要介绍下一个成功的CAR-NK细胞的各个组成部分。图1. CAR-NK细胞构建块的示意图载体骨架和启动子载体骨架包含CAR表达所需的所有元件，如启动子、多聚腺嘌呤信号和转录调控片段。多种启动子被用于驱动CAR的表达。病毒启动子，如CMV、MPSV、MMLV、SFFV等比一些结构活性启动子，如EF1α、CMV和PGK相比，更常用于产生CAR-NK。一项研究表明，EF1α启动子比其他常用的启动子如CMV、UBIC和PGK诱导更高的CAR表达水平；另一项研究表明，MSCV在表达水平和CAR表达稳定性方面优于EF1α和PGK；然而，启动子的有效性可能受到scFv序列本身的影响；一项研究报告指出，当启动子序列位于SIN 5’LTR慢病毒载体内时，与RSV启动子序列相比，CMV启动子产生更高的病毒滴度。CAR-NK信号肽信号肽具有巨大的异质性，这直接转化为不同水平的蛋白质分泌效率。目前，CD8a 信号肽是用于CAR-NK细胞的最常用的多肽序列,其次是免疫球蛋白重链或轻链的信号肽。虽然信号肽序列可能会影响CAR的表达水平，但目前还没有数据支撑关于CAR-NK生产的最佳信号肽的说法。单链抗体 (ScFv)ScFv是抗体重链和轻链可变区的融合蛋白，是CAR的肿瘤抗原结合域。不同的单链抗体具有不同的抗肿瘤能力，但也有不同的副作用，如与正常组织结合。因此，CAR的靶点需要仔细选择，以针对肿瘤相关抗原，同时不识别可能导致严重副作用的自身抗原。由于ScFv不是抗体的自然形式，它是被合成的，因此重链和轻链的顺序是人为确定的。到目前为止，对于CAR-NK设计，大家更喜欢VH-VL方向而不是VL-VH方向。Fujiwara等人的研究证明H链和L链的顺序不影响anti-KDR CAR在T细胞上的表达水平。在CAR中，单链抗体在抗原特异性和结合常数方面的表现有时被发现低于其原始抗体，主要是由于VL和VH结构域的连接发生了变化。这也可能对这两条链的动力学和相对取向产生影响。通过分析VH和VL CDR区的氨基酸序列，并预测ScFv与其靶标的相互作用，计算机辅助设计ScFv可能有助于功能ScFv的开发。NK细胞可以配备多个单链抗体，从而扩大CAR效应细胞的抗原识别能力。有多种选择：CARs可以用带有双顺反子元件的载体转导，诱导两个CAR结构ScFv的表达；两个ScFv可以在一个结构中融合在一起，产生串联的单链抗体。目前大多数临床CAR T细胞试验使用了来自小鼠抗体的单链抗体，从而增加了抗小鼠免疫球蛋白细胞宿主对移植物排斥的风险，从而导致携带CAR细胞的毒性或持续时间缩短。即使人源化的ScFv构建仍然可以诱导宿主抗独特型免疫反应。但到目前为止，在数量有限的CAR-NK临床试验中，没有发现与抗CAR免疫反应有关的主要副作用。连接子重链和轻链之间的连接子有助于ScFv的构象，因此某种程度上决定了CAR识别目标表位的程度。过短的连接子会导致多聚体的形成，因为它们阻止了VH和VL结构域的结合。太长的连接子可能会导致VH和VL结构域之间的蛋白分解或弱结合。对于CAR-NK细胞，五肽GGGGS的多聚体使用最广泛，通常是15-mer (G4S)3。另一个旨在增强蛋白降解稳定性的连接子是Whitlow“218”连接子：GSTGSGSKPGSGEGSTKG。这种连接子增强了ScFv的亲和力，但可能也改变了CAR-NK细胞的功能。铰链区(CD8α，Ig CH2CH3)铰链区，是CAR的细胞外结构区，连接ScFv和跨膜结构域。铰链区通常维持效应细胞中CAR表达和活性所需的稳定性。铰链区也提供了灵活性，以允许容易地接触到目标抗原。大多数CAR-NK结构使用CD8 α或CD28胞外域或IgG为基础的铰链的衍生物(图2)。图2. CAR-NK构建中最常用的铰链区在CD28型和CD8α型铰链域之间的直接比较中，发现CD28型铰链域更有可能促进CAR分子的二聚化。因此，CD28铰链CAR产生的激活刺激更强。虽然这可能是有益的，但这也可能导致细胞因子释放综合征。基于IgG的铰链也被广泛用于CAR结构。其一个主要优点是结构的灵活性，该结构通常由IgG1或IgG4和IgG Fc的CH2/CH3结构域组成。跨膜区(CD3、CD8、CD28、NKG2D、2B4)跨膜(TM)结构域将CAR的胞外区连接到细胞内的激活信号域，并将受体对接到NK细胞膜上。CAR-NK最常用的TM部分是从CD3ζ、CD8和CD28改造而来的，但也探索了其他部分。例如，NKG2D、2B4、DNAM1。目前，在原代CAR-NK细胞中，CD8α和CD28来源的跨膜结构域最受欢迎，而CD28是CAR-NK细胞系的首选跨膜结构域 (图3)。图3. CAR-NK构建中最常用的跨膜区TM结构域的一个重要方面是，最佳的TM区域应该遵循NK细胞上跨膜蛋白的蛋白质的自然取向(N-到C-末端顺序)。然而，天然的NKG2D是一个例外，其具有C-到N-末端的跨膜区，并有一个较短的细胞质尾巴。CAR-NK的激活信号激活结构域负责识别靶抗原后NK细胞的激活。与T淋巴细胞不同，NK细胞使用各种不同的、非重排的受体来激活。这也包括多种细胞因子受体，它们是NK细胞发育、成熟和激活的重要调节因子。第一代CAR-NK细胞和CAR-T细胞一样，只含有CD3ζ信号。第二代和第三代CAR-NK分别带有一个和两个额外的共刺激信号（图4）。共刺激分子通常来源于CD28家族(包括CD28和ICOS)、肿瘤坏死因子受体(TNFR)家族(包括4-1BB、OX40和CD27)或信号转导的淋巴细胞激活分子(SLAM)相关受体家族(包括2B4)。图4. CAR-NK细胞的激活信号CAR-NK细胞中细胞因子信号的修饰可能为CAR-NK细胞的改进提供进一步的可能性。例如，IL-12、IL-15、IL-18等细胞因子可在巨细胞病毒感染后诱导记忆样NK活性，进一步增加了抗肿瘤效应。小编总结CAR-NK细胞作为过继免疫治疗的一种新策略在各种临床试验中显示出了巨大的潜力。成功的CAR设计对于CAR-NK能否展现其优越的抗肿瘤功能至关重要。目前研究人员已经对如何最设计一款成功的CAR-NK付出了大量的研究与努力，但目前的研究仍处于早期阶段，CAR-NK细胞的结构仍需进一步优化，以最大化的发挥其抗肿瘤功效。参考文献1.Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy.2.CAR race to cancer immunotherapy: from CAR T, CAR NK to CAR macrophage therapy.3.CAR-engineered NK cells; a promising therapeutic option for treatment of hematological malignancies.

NEW YORK, June 10, 2022 /PRNewswire/ -- The Lupus Research Alliance (LRA), the world's leading private funder of lupus research, today announced inaugural recipients of Career Development and Postdoctoral Awards to Promote Diversity in Lupus Research. The Diversity in Lupus Research Award funding mechanisms aim to address underrepresentation of minorities in the scientific research profession. Lupus is an autoimmune disease that disproportionally affects underrepresented minorities in both prevalence and disease severity. To encourage the development of talented underrepresented minority early-career scientists and postdoctoral fellows, the LRA launched two new award mechanisms. This year, three outstanding early-career underrepresented minority scientists will receive the Career Development Award to Promote Diversity in Lupus Research, which provides up to $600,000 over four years. The recipients are Ashira Blazer, MD, MSCI, Hospital for Special Surgery (HSS); Andrea Knight, MD, MSCE, The Hospital for Sick Children (SickKids); and Erika Moore, PhD, University of Florida. Two promising postdoctoral research fellows, Ruth Fernandez Ruiz, MD, from Hospital for Special Surgery and Vanessa Wacleche, PhD, from Brigham and Women's Hospital, are the recipients of the Postdoctoral Award to Promote Diversity in Lupus Research. This award provides fellows with $170,000 over two years to support projects and the generation of data needed to become an independent lupus researcher. Career Development Award to Promote Diversity in Lupus Research Recipients: Ashira Blazer, MD, MSCI, Hospital for Special Surgery Dr. Blazer will build upon her preliminary research, which successfully separated lupus nephritis patients from non-lupus nephritis patients through the analysis of the sediment found in the lupus patient urine. The potential clinical use of the non-invasive, urine-based test will be confirmed using a larger multi-national SLE patient group with African ancestry, which will provide a new opportunity to learn more about lupus nephritis in a high-risk and understudied patient group. Andrea Knight, MD, MSCE, The Hospital for Sick Children (SickKids) Dr. Knight is studying ways to detect brain changes in high-risk adolescent patients with childhood-onset lupus by comparing inflammation markers, brain MRIs, and cognitive function tests from healthy adolescents and lupus patients. Since little is known about which adolescents with lupus are most at risk for the structural brain changes that affect cognition, this research aims to find ways to detect these brain changes early and identify potential new treatments. Erika Moore, PhD, University of Florida Dr. Moore will look at monocytes from women of African and European ancestry, with and without lupus, to determine how monocytes promote lupus-related blood vessel inflammation (vasculitis). Understanding how lupus and ancestry affect monocyte functions will help the lupus medical community learn how ancestry influences lupus-related blood vessel inflammation and identify treatments for patients at higher risk of developing lupus-induced cardiovascular disease. Postdoctoral Award to Promote Diversity in Lupus Research Recipients: Ruth Fernandez Ruiz, MD, Hospital for Special Surgery Dr. Fernandez Ruiz will investigate how permanent changes in our genetic makeup, called gene variants, influence lupus development and progression. By studying how genetic variants in the PRKG1 gene affect lupus, the research team may identify new biological pathways that could be targeted to reduce the inflammation in lupus patients caused by high levels of interferon alpha. Vanessa Wacleche, PhD, Brigham and Women's Hospital Dr. Wacleche will study how T peripheral helper (Tph) cells misdirect the immune response seen in systemic lupus erythematosus via an inflammatory molecule CXCL13, which may lead to the production of organ-damaging autoantibodies. The new findings generated by this research could suggest strategies to control CXCL13 production that may eventually lead to the development of novel treatments for lupus patients. Teodora Staeva, PhD, LRA Chief Scientific Officer said, "We congratulate the inaugural recipients of our Diversity in Lupus Research Awards and look forward to seeing how their research findings advance our understanding of lupus and result in the development of new treatment options for lupus patients. The Career Development and Postdoctoral Awards to Promote Diversity in Lupus Research funding mechanisms are our most recent initiatives to alleviate the disparities that are prevalent in both autoimmune diseases and biomedical research." About Lupus Lupus is a chronic, complex autoimmune disease that affects millions of people worldwide. More than 90 percent of people with lupus are women; lupus most often strikes during the childbearing years of 15-45. Ancestrally African, South and Central American, and Native Americans have two to three times higher risk of developing lupus than Americans with European ancestry. In lupus, the immune system, which is designed to protect against infection, creates antibodies that can attack any part of the body including the kidneys, brain, heart, lungs, blood, skin, and joints. About the Lupus Research Alliance The Lupus Research Alliance is the largest non-governmental, non-profit funder of lupus research worldwide. The organization aims to transform treatment by funding the most innovative lupus research, fostering diverse scientific talent, and driving discovery toward better diagnostics, improved treatments and ultimately a cure for lupus. Because the Lupus Research Alliance's Board of Directors funds all administrative and fundraising costs, 100% of all donations goes to support lupus research programs. SOURCE Lupus Research Alliance