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

• 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
• 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

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.

总结 广泛中和抗体（bNAb）的长期表达有可能抑制已建立的免疫缺陷。 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。被动输注bNAb的费用高得令人望而却步，并且需要HIV-1阳性个体 每周或每月接受一次输液。通过基因治疗载体递送bNAb几乎总是 产生针对表达的bNAb的抗药物抗体（ADA），所述抗体由于其广泛的免疫原性而具有免疫原性。 超突变最重要的是，没有一组抗体可以充分抑制病毒的范围， 这在很大程度上是因为目前的抗体递送系统无法完成免疫系统所做的事情： 适应不同的不断进化的病原体在这里，我们描述了一系列的技术进步，使我们能够 将bNA B重链和轻链基因引入原代B细胞中它们天然基因座。这些技术使 通过在小鼠和灵长类动物中的亲和力成熟来体内改进bNAb， 体液反应，以迅速增加抗体的效力，广度和生物利用度。它们还允许我们 测试这一提议的核心假设，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。 使这些研究成为可能的主要技术进步是有效的双重编辑的发展 用于同时替换B细胞受体的可变重链和轻链区段的技术。这是 通过使用新表征的Cas 12 a直系同源物和独特的同源定向修复 能够有效替换几乎任何内源性BCR可变区模板设计。净 结果是，与相关的B细胞编辑方法不同，留下了B细胞的完整调节装置 完整的，促进稳健的B细胞发育和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将增加三个良好表征的 bNAb通过体内亲和力成熟。目标2将CRISPR编辑扩展到Fc结构域，引入一个新的CRISPR编辑。 最近描述的IgG 1 Fc结构域中的一组突变，其促进抗体穿过血液的转移- 脑屏障最后，目标3测试表达目标1中改进的bNAb的原代B细胞控制 恒河猴的SHIV感染将执行一系列结构化治疗中断以驱动CAR B增殖并对从储库中出现的病毒产生个体化应答。经过这些 结构性中断，ART将永久停止，以确定CAR B细胞是否可以单独控制 建立感染。