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

摘要 广谱中和抗体(BNAbs)的长期表达有可能抑制已建立的 HIV-1感染。然而，目前维持高bNAb浓度的方法对于这种控制是必要的 是不够的。被动输注bNAbs的费用高得令人望而却步，而且需要HIV-1阳性患者 每周或每月接受一次输液。几乎总是通过基因治疗载体运送bNAbs 产生针对表达的bNAbs的抗药抗体(ADA)，由于其广泛的免疫原性 超突变。最重要的是，任何一组抗体都不能充分抑制 人口，很大程度上是因为目前的抗体传递系统不能做免疫系统做得很好的事情： 适应多样化和不断进化的病原体。在这里，我们描述了一系列技术进步，使我们能够 将bNAb重链和轻链基因导入其在原代B细胞中的天然位点。这些技术使 在小鼠和灵长类动物体内通过亲和力成熟来改进bNAbs，利用获得性智慧 迅速提高抗体效力、广度和生物利用度的体液反应。它们还允许我们 检验这一提议的核心假设，即B细胞递送的bNAbs可以永久性地抑制已建立的 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开发高效的双重编辑 同时替换B细胞受体的可变重链和轻链片段的技术。这是 通过使用新鉴定的Cas12a同源同源基因和独特的同源定向修复而成为可能 模板设计能够有效地替换几乎任何内源性bcr可变区。这张网 结果是，与相关的B细胞编辑方法不同，留下了B细胞的全部调节装置 完整，促进B细胞的强健发育和B细胞受体的高效亲和力成熟。 该项目分为三个目标。目标1将增加三个角色的广度和效力 BNAbs在体内通过亲和力成熟。AIM 2将CRISPR编辑扩展到FC域，引入了 最近描述的一组进入IgG1Fc区域的突变促进了抗体在血液中的转移- 大脑屏障。最后，目标3测试原代B细胞表达目标1中改进的bNAbs的能力以对照 恒河猴感染新城疫病毒。将进行一系列结构化治疗中断以驾驶汽车 B增殖，并对从储存库中出现的病毒产生个性化的反应。在这些之后 结构中断，ART将永久撤回，以确定仅CAR B细胞是否可以控制 已确定的感染。

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.