HIV-Receptor Interactions and Related Anti-HIV Strategies

HIV 受体相互作用和相关抗 HIV 策略

基本信息

批准号：
10014032
负责人：
Edward Berger
金额：
$ 71.8万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10014032
关键词：
Adopted Animal Model Antibodies B-Lymphocytes BLR1 gene Back Binding Blood CCR5 gene CD28 gene CD8-Positive T-Lymphocytes CXCL13 gene Carbohydrates Cells Child Collaborations Detection Development Genetic Goals HIV HIV Envelope Protein gp120 HIV Infections HIV Receptors HIV-1 Homing Human In Vitro Individual Infection Infection Control Lectin Life Ligands Link Lymphoid Tissue Macaca mulatta Mannose Mannose Binding Lectin Minnesota Molecular Monoclonal Antibodies Mus National Institute of Allergy and Infectious Disease Persons Protocols documentation RNA Research Research Personnel SIV Signal Transduction Site Stains Surface T memory cell T-Lymphocyte Technology Testing Time Tissues United States National Institutes of Health Vaccine Research Variant Virus antiretroviral therapy base c-myc Genes cell type chemokine chimeric antigen receptor chimeric antigen receptor T cells collaboratory comparative design env Glycoproteins experimental study humanized mouse improved in vitro Assay in vivo in vivo Model migration mouse model neutralizing monoclonal antibodies novel novel strategies polypeptide prevent receptor success trafficking

项目摘要

We have continued development of an HIV functional cure strategy based on novel chimeric antigen receptors (CARs). Durable (life-long?) HIV suppression requires the CAR to display not only high potency, but also to approach the ideals of inescapabilty and non-immunogenicity. We have designed bispecific CARs containing CD4 linked to a second moiety that binds to a distinct highly conserved region of Env. The second moiety enhances potency, and also prevents the CD4 from acting as an HIV entry receptor. Particular success was achieved with the carbohydrate binding domain of human mannose binding lectin (MBL), which binds to the universally conserved high-mannose patch on gp120. The CD4-MBL bispecific CAR shows greatly enhanced potency compared to the monospecific CD4 CAR, with wide breadth for genetically diverse HIV-1 isolates; moreover, the lectin moiety inhibits the CD4 from acting as an entry receptor and rendering CAR-expressing CD8+ T cells susceptible to infection (Ghanem et al. 2018). Efforts during the past year have focused on design of improved CAR constructs, and on testing of the CAR-T cells in relevant animal models in collaboration with investigators in the network of Martin Delaney Collboratories for HIV Cure. 1) Comparative potencies of the CD4-MBL CAR to antibody-based alternatives. The HIV field has been greatly stimulated by the development of a wide range of broadly neutralizing monoclonal antibodies (bnAbs) that bind to different conserved regions of Env, and display great breadth and potency against genetical diverse HIV-1 variants. Several research groups are developing corresponding bnAb-based CARs. In collaboration with the group of Dr. Thor Wagner (defeatHIV Collaboratory, Seattle Childrens,), we generated analogous CAR constructs targeted by either CD4-MBL or an scFv of a highly studied bnAb. Our in vitro assay results indicated highly superior activity of the CD4-MBL CAR, likely leading defeatHIV to switch to this CAR. 2) Design of a trispecific CAR. With the goal of further enhancing CAR potency, we examined addition of a third human component of the targeting domain that binds to yet a distinct highly conserved region of HIV-1 Env. Success was obtained using a polypeptide region from the N-terminus of human CCR5, the entry coreceptor used by HIV-1 variants that predominate in infected individuals from the time of initial infection throughout the asymptomatic period. This trispecific CAR, designated CD4-MBL-R5Nt, displayed enhanced potency over CD4-MBL against the two HIV-1 primary isolates tested. Moreover, the presence of the third motif further blocked to undetectable levels the ability of the CD4 moiety from acting as an HIV entry receptor. Additional studies will test the breadth of the CD4-MBL-R5Nt CAR in vitro, and possibly expand to in vivo models. 3) CAR studies in humanized mouse models. Collaborative studies with investigators in the BELIEVE Collaboratory have yielded interesting results. Dr. Harris Goldstein (Yeshiva Med) tested our earlier bispecific construct, the CD4-17b CAR in their intrasplenic humanized mouse. Co-administration of the CAR at the time of HIV-1 challenge resulted in very strong virus suppression. With Dr. Brad Jones (Cornell/Weill), we are following up earlier experiments in his infused memory T cell humanized mouse model. Initial studies showed negligible effects of the CD4-MBL CAR when administered at the time of mouse challenge, whereas potent suppression was observed with an HIV-specific T cell product designated HST-NEETs, developed by Dr. Catherine Bollard (Childrens National). We hypothesized that the difference may be due to the very different mode of CAR-T cell expansion used by the Berger and Bollard labs. Consequently, the CD4-MBL CAR was transduced and the T cells were expanded by the Bollard protocol. In vitro analyses indicating excellent CAR-T cell expansion and CAR surface expression on the expanded cells. These CAR-T cells showed potent HIV suppression in vitro. This sets the stage for direct in vivo comparison of the HST-NEET cells with the similarly expanded CAR-T cells in the Brad Jones humanized mouse. 4. NHP studies. In collaboration with BELIEVE investigator Dr. Pamela Skinner (U. Minnesota) we previously generated an all-rhesus variant of CD4-MBL for co-expression with CXCR5 to facilitate homing of CAR-T cells to B cell follicles, where infected Tfh cells serve as major sites of HIV persistence. Previous findings indicated that CXCR5 promotes selective migration of CAR-T cells toward the chemokine ligand CXCL13 in vitro, as well their accumulation inside B cell follicles in rhesus lymphoid tissue explants ex vivo. Studies in rhesus macaques will require tracking of CAR-T cells in blood and various tissues. Immunostaining of CAR constructs containing c-myc tags in the ectodomains proved unsuitable due to high background staining. The Skinner lab has now adopted RNA-Scope technology, which will enable detection critical cell types in tissues, including CAR-T cells, HIV-infected cells, etc. In a preliminary experiment, the Skinner group observed that 2 of 3 rhesus macaques injected with CD4-MBL CAR-T cells showed significant control of SIV rebound after cessation of antiretroviral therapy, in contrast to 3 controls that did not receive CAR-T cells. This set the stage for examining numerous critical features associated with virus suppression and potential functional cure, including follicular trafficking of CAR/CXCR5-T cells in vivo, and their ability to deplete infected Tfh cells. A second study in rhesus macaques has been initiated with Dr. Mario Roederer (Vaccine Research Center, NIAID, NIH). The goal is to compare a diversity of CAR constructs varying in both targeting domains (CD4-MBL vs. scFvs derived from anti-SIV Env monoclonal antibodies) and also signaling domains (CD28 vs. 4-1BB). The initial focus is to compare the relative persistence of these diverse CAR constructs. Initial results will help focus on optimal designs for durable control of infection.

我们继续开发基于新型嵌合抗原受体（CAR）的HIV功能治疗策略。耐用的（终身？）HIV抑制需要汽车不仅表现出高效力，而且还需要表现出毫无疑问和非免疫原性的理想。我们已经设计了包含与第二个部分的CD4相关的双特异性汽车，该部分与不同的高度保守区域结合。第二个部分提高了效力，还可以防止CD4充当HIV进入受体。通过人类甘露糖结合蛋白（MBL）的碳水化合物结合结构域（MBL）实现了特殊的成功，该结合在GP120上与普遍保守的高甘露糖贴片结合。与单特异性CD4 CAR相比，CD4-MBL双特异性汽车显示出大大提高的效力，对于遗传多样的HIV-1分离株，其宽度很广。此外，凝集素部分抑制CD4充当入口受体，并渲染表达汽车的CD8+ T细胞易受感染的感染（Ghanem等人，2018）。在过去的一年中，与Martin Delaney Collboratories网络中的研究人员合作进行了相关动物模型中的CAR-T细胞的设计，并将其努力用于设计改进的汽车构建体。 1）CD4-MBL汽车与基于抗体的替代方案的比较效力。艾滋病毒场受到了广泛中和的单克隆抗体（BNAB）的发展，这些抗体（BNAB）与不同的保守区域结合，并显示出针对基因多样的HIV-1变体的广度和效能。几个研究小组正在开发相应的基于BNAB的汽车。在与Thor Wagner博士（Defeathiv Collaboratory，Seattle Childrens）的合作中，我们生成了CD4-MBL或经过深入研究的BNAB的SCFV靶向的类似汽车结构。我们的体外测定结果表明CD4-MBL汽车的活动高度出色，可能导致Defeathiv切换到该汽车。 2）设计三角车。为了进一步提高汽车效力，我们检查了靶向域的第三个人类组成部分，该组件与HIV-1 Env的高度保守区域结合。使用来自人类CCR5的N末端的多肽区域获得了成功，这是HIV-1变体所使用的进入的共肽，这些变体从无症状的时期内最初感染开始，这些变体从受感染的个体中占主导地位。这款指定为CD4-MBL-R5NT的Tristexific汽车对CD4-MBL的效力增强了，与测试的两个HIV-1主要分离株相比。此外，第三个基序的存在进一步阻止了无法检测到的水平，CD4部分作为HIV进入受体的能力。其他研究将在体外测试CD4-MBL-R5NT CAR的广度，并可能扩展到体内模型。 3）人性化小鼠模型中的汽车研究。与研究人员合作的合作研究取得了有趣的结果。哈里斯·戈德斯坦（Harris Goldstein）博士（Yeshiva Med）测试了我们早期的双特异性构建体，即在其内部人性化小鼠中的CD4-17B汽车。 HIV-1挑战时汽车的共同给药导致非常强烈的病毒抑制。与布拉德·琼斯（Brad Jones）博士（康奈尔/威尔（Cornell/Weill））一起，我们正在跟踪他注入的记忆T细胞人性化小鼠模型中的早期实验。最初的研究表明，在小鼠挑战时进行给药时，CD4-MBL CAR的作用可忽略不计，而通过Catherine Bollard博士（Childrens National）开发的HIV特异性T细胞产品，观察到有效的抑制作用。我们假设差异可能是由于Berger和Bollard Labs使用的CAR-T细胞膨胀方式非常不同。因此，转导CD4-MBL汽车，并通过Bollard方案扩展T细胞。体外分析表明在扩展的细胞上表明出色的CAR-T细胞膨胀和CAR表面表达。这些CAR-T细胞在体外显示出有效的HIV抑制。这为HST-Neet细胞与Brad Jones人源化小鼠中类似扩展的CAR-T细胞进行直接体内比较奠定了基础。 4。NHP研究。与相信研究者帕梅拉·斯金纳（Pamela Skinner）（U.Minnesota）合作，我们先前生成了与CXCR5共表达CD4-MBL的全逆性变体，以促进CAR-T细胞向B细胞卵泡的归纳，在那里感染的TFH细胞充当HIV持久性的主要部位。先前的发现表明，CXCR5在体外促进了CAR-T细胞向趋化因子配体CXCL13的选择性迁移，并且它们在恒河猴淋巴组织外植体中的B细胞卵泡内的积累。猕猴的研究需要跟踪血液和各种组织中的CAR-T细胞。由于高背景染色，在胞外域中含有C-MYC标签的汽车构建体的免疫染色被证明不适合。 Skinner Lab现在已经采用了RNA-SCOPE技术，该技术将在一个初步的实验中启用组织中的临界细胞类型，包括CAR-T细胞，HIV感染的细胞等。Skinner组观察到，在CD4-MBL CAR-T细胞中注入CD4-MBL CAR-T细胞中的3个颗粒猕猴中的2个表现出对SIV反弹的显着对照，对抗抗逆转录病毒的抗反反率进行了反应，该控制不受3次控制的控制。这为检查与病毒抑制和潜在功能治愈相关的众多关键特征奠定了基础，包括体内CAR/CXCR5-T细胞的卵泡运输，以及它们耗尽感染TFH细胞的能力。马里奥·罗德勒（Mario Roederer）博士（NIAID，NIH）在恒河猕猴中进行了第二项研究。目的是比较两个靶向域（CD4-MBL与源自抗SIV ENV ENV单克隆抗体）和信号域（CD28 vs. 4-1BB）的多种汽车构建体（CD4-MBL与SCFV）。最初的重点是比较这些多样化的汽车结构的相对持久性。初始结果将有助于专注于最佳设计，以持久控制感染。