Directed Evolution of HIV Broadly Neutralizing Antibodies Using a Novel CRISPR-Engineered B cell in Vitro Affinity Maturation Platform

摘要 被动转移HIV广谱中和抗体有望成为抗逆转录病毒的替代方案 药物一般耐受性好，体内半衰期长，并能激活宿主免疫。 系统。发现后bNab广度和效力的改进应使其作为医学应用 干预更切合实际。我们已经开发了一个新的抗体定向进化平台，我们希望 为此目的而雇用。该平台使用基因组编辑来取代免疫球蛋白可变区 这些来自人类B细胞系中的特定单抗。内源性激活诱导 胞苷脱氨酶(AID)在这些产生抗体变异体的工程基因座上引入突变 抗原结合特性。新抗体是用内源性恒定基因作为细胞表面来表达的。 免疫球蛋白，允许使用靶抗原选择探针选择具有所需结合特性的变异体。我们 已经成功地利用这个平台产生和选择了一种抗体的变种，这种抗体显示出改进的艾滋病毒 中和广度和效力，作为概念的证明。从工程细胞中分泌的免疫球蛋白可以被收获 来自细胞上清液，并在细胞系的定向进化过程中表征。单细胞测序 进化的细胞可以用来揭示结合改变的遗传基础和产生 以重组蛋白形式表达的改良单抗。在这个R21中，我们建议对HIV bNAbs进行工程 VRC01和CAP256-VRC26.25进入我们的B细胞系，以进化出一个CD4结合位点和V2-顶端定向 具有改进的中和广度、效力和潜在生物物理特性的bNAbs，使用各种 不同的选择策略。如果成功，该平台将成为发现后的宝贵资源 不仅是HIV bNAbs的改进，而且是对任何具有治疗意义的单抗的改进。

Abstract Passive transfer of HIV broadly neutralizing antibodies (bnAbs) is promising as an alternative to antiretroviral drugs because they are generally well tolerated, have long in vivo half-lives, and can activate the host immune system. Post-discovery improvement of bnAb breadth and potency should make their application as a medical intervention more practical. We have developed a novel antibody directed evolution platform we would like to employ for this purpose. This platform uses genome editing to replace immunoglobulin variable regions with those from specific monoclonal antibodies in a human B cell line. Endogenous activation-induced induced cytidine deaminase (AID) introduces mutations at these engineered loci generating antibody variants with altered antigen binding properties. The new antibody is expressed using endogenous constant genes as cell surface IgM, allowing for selection variants with desired binding properties using target antigen selection probes. We have successfully used this platform to generate and select variants of an antibody which show improved HIV neutralizing breadth and potency as a proof of concept. IgM secreted from engineered cells can be harvested from cell supernatants and characterized during the directed evolution of cell lines. Single cell sequencing of evolved cells can be performed to uncover the genetic basis for altered binding and for the generation of improved mAbs for expression as recombinant proteins. In this R21, we propose engineering the HIV bnAbs VRC01 and CAP256-VRC26.25 into our B cell line in order to evolve a CD4 binding site and V2-apex directed bnAbs with improved neutralizing breadth, potency and potentially biophysical properties, using a variety of different selection strategies. If successful, this platform would be a valuable resource for post-discovery improvement of not only HIV bnAbs, but for any monoclonal of therapeutic interest.