Engineering CAR-B cells for an HIV-1 functional cure

改造 CAR-B 细胞以实现 HIV-1 功能性治愈

基本信息

批准号：
10514882
负责人：
Michael R. Farzan
金额：
$ 69.5万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10514882
关键词：
Affinity Anatomy Animals Antibodies Antigens Autoantigens B cell repertoire B-Cell Antigen Receptor B-Cell Development B-Lymphocytes Biological Availability Blood - brain barrier anatomy Brain Catabolism Cells Cerebrospinal Fluid Clinical Clustered Regularly Interspaced Short Palindromic Repeats Development Effector Cell Endothelial Cells Engineering Engraftment Epitopes Evolution Excision Face Fc Receptor Fc domain Gene Transduction Agent Genes HIV HIV Infections HIV-1 Half-Life IgG1 Immune Immune Sera Immune system Immunization Immunoglobulin G Immunoglobulin Somatic Hypermutation Immunologics Individual Infection Infection Control Infusion procedures Injections Interruption Intervention Left Light Lymphoid Tissue Macaca Macaca fascicularis Macaca mulatta Methods Modification Monitor Mus Mutation Orthologous Gene Pharmaceutical Preparations Pharmacology Phenotype Population Primates Proliferating Proteins Protocols documentation Proviruses Replacement Therapy Route Self Administration Series Site Structure System T cell response TFRC gene Techniques Technology Testing Time Vaccinated Vaccination Variant Viral Viremia Virus Work antibody transfer antiretroviral therapy arm cerebrovascular chimeric antigen receptor cost delivery vehicle design fitness immunogenic immunogenicity improved in vivo individual response inhibiting antibody neutralizing antibody pathogen repaired response side effect simian human immunodeficiency virus small molecule social stigma transcytosis uptake

项目摘要

SUMMARY Long-term expression of broadly neutralizing antibodies (bNAbs) has the potential to suppress an established HIV-1 infection. However, current methods for maintaining high bNAb concentrations necessary for this control are inadequate. Passive infusion of bNAbs is prohibitively expensive and requires HIV-1 positive individuals to receive infusions on a weekly or monthly basis. Delivery of bNAbs by gene-therapy vectors almost invariably raises anti-drug antibodies (ADA) against expressed bNAbs, which are immunogenic due to their extensive hypermutation. Most importantly, no single set of antibodies can adequately suppress the range of viruses in the population, in large part because current antibody delivery systems fail to do what an immune system does well: adapt to a diverse and evolving pathogen. Here we describe a series of technical advances that allow us to introduce bNAb heavy- and light-chain genes into their native loci in primary B cells. These technologies enable in vivo improvement of bNAbs through affinity maturation in mice and primates, using the acquired wisdom of the humoral response to rapidly increase antibody potency, breadth, and bioavailability. They also allow us to test the core hypothesis of this proposal that B-cell delivered bNAbs can permanently suppress an established infection in the absence of anti-retroviral therapy (ART). The chief technical advance that enables these studies is the development of an efficient double-editing technique for simultaneously replacing the variable heavy and light chain segments of B cell receptors. This is made possible through use of a newly characterized Cas12a ortholog and a unique homology-directed repair template design capable of efficiently replacing nearly any endogenous BCR variable region. The net consequence is that, unlike related B-cell editing approaches, the full regulatory apparatus of the B cell is left intact, facilitating robust B-cell development and efficient affinity maturation of the B-cell receptor. The project is divided into three aims. Aim 1 will increase the breadth and potency of three well characterized bNAbs through affinity maturation in vivo. Aim 2 will extend CRISPR editing to the Fc domain, introducing a recently described set of mutations into the IgG1 Fc domain that facilitate antibody transfer across the blood- brain barrier. Finally, Aim 3 tests the ability of primary B cells expressing the bNAbs improved in Aim 1 to control a SHIV infection in rhesus macaques. A series of structured treatment interrupts will be performed to drive CAR B proliferation and generate an individualized response to virus that emerges from the reservoir. After these structured interruptions, ART will be permanently withdrawn to determine if CAR B cells alone can control an established infection.

概括广泛中和抗体（bNAb）的长期表达有可能抑制已建立的 HIV-1 感染。然而，目前维持这种控制所需的高 bNAb 浓度的方法是不够的。 bNAb 的被动输注非常昂贵，并且需要 HIV-1 阳性个体每周或每月接受输液。几乎总是通过基因治疗载体递送 bNAb 产生针对表达的 bNAb 的抗药物抗体 (ADA)，这些抗体由于其广泛的作用而具有免疫原性超突变。最重要的是，没有一组抗体能够充分抑制体内的病毒范围。人口，很大程度上是因为当前的抗体输送系统无法完成免疫系统擅长的工作：适应多样化且不断进化的病原体。在这里，我们描述了一系列技术进步，使我们能够将 bNAb 重链和轻链基因引入原代 B 细胞中的天然基因座。这些技术使利用小鼠和灵长类动物的亲和力成熟，体内改进 bNAb 快速增加抗体效力、广度和生物利用度的体液反应。它们还让我们能够测试该提案的核心假设，即 B 细胞递送的 bNAb 可以永久抑制已建立的在没有抗逆转录病毒治疗（ART）的情况下感染。实现这些研究的主要技术进步是高效双重编辑的开发同时替换 B 细胞受体的可变重链和轻链片段的技术。这是通过使用新表征的 Cas12a 直向同源物和独特的同源定向修复使之成为可能模板设计能够有效替换几乎任何内源性 BCR 可变区。网结果是，与相关的 B 细胞编辑方法不同，B 细胞的完整调节装置被保留完整，促进 B 细胞的稳健发育和 B 细胞受体的有效亲和力成熟。该项目分为三个目标。目标 1 将增加三个明确特征的广度和效力 bNAb 通过体内亲和力成熟。目标 2 将 CRISPR 编辑扩展到 Fc 结构域，引入最近描述了 IgG1 Fc 结构域的一组突变，这些突变促进抗体跨血液转移脑屏障。最后，目标 3 测试了表达目标 1 中改进的 bNAb 的原代 B 细胞控制的能力恒河猴感染 SHIV。将执行一系列结构化处理中断来驱动 CAR B 增殖并对从储存库中出现的病毒产生个体化反应。在这些之后结构性中断，ART 将永久撤回，以确定 CAR B 细胞是否可以单独控制已确诊感染。