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

使用新型 CRISPR 工程 B 细胞在体外亲和力成熟平台定向进化 HIV 广泛中和抗体

• 批准号: 10115604
• 负责人: James Even Voss
• 金额: $ 22.19万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10115604
• 关键词: Affinity; Animal Model; Anti-Retroviral Agents; Antibodies; Antibody Binding Sites; Antigen Targeting; B-Lymphocytes; Binding; Binding Sites; Biological Assay; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cell surface; Cells; Characteristics; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Cytidine Deaminase; DNA; Directed Molecular Evolution; Dose; Engineering; Enzyme-Linked Immunosorbent Assay; Epitopes; Flow Cytometry; Generations; Genes; Genetic; Goals; HIV; Half-Life; Harvest; Human; Immune system; Immunoglobulin G; Immunoglobulin M; Immunoglobulin Somatic Hypermutation; Immunoglobulin Variable Region; Immunoglobulins; In Vitro; Individual; Infection; Infection prevention; Intervention; Light; Medical; Methods; Monitor; Monoclonal Antibodies; Mutate; Mutation; Pathway interactions; Pharmaceutical Preparations; Process; Production; Property; Recombinant Proteins; Recombinants; Research Proposals; Resources; Scheme; Solubility; System; Testing; Therapeutic; Therapeutic Monoclonal Antibodies; Therapeutic Uses; Time; Vaccination; Variant; Viremia; Virus; activation-induced cytidine deaminase; antigen binding; biophysical properties; cellular engineering; clinically relevant; cost; experimental study; genetic variant; genome editing; human monoclonal antibodies; improved; in vivo; interest; neutralizing antibody; novel; prophylactic; protein protein interaction; single cell sequencing

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.

抽象的 HIV 广泛中和抗体 (bnAb) 的被动转移有望成为抗逆转录病毒药物的替代方案 药物，因为它们通常具有良好的耐受性，体内半衰期长，并且可以激活宿主免疫 系统。 bnAb 广度和效力的发现后改进应使其作为医疗应用 干预更实际。我们开发了一种新型抗体定向进化平台，我们希望 为此目的而雇用。该平台使用基因组编辑来替换免疫球蛋白可变区 来自人类 B 细胞系中特定单克隆抗体的抗体。内源性激活诱导 胞苷脱氨酶 (AID) 在这些工程基因座处引入突变，产生具有改变的抗体变体 抗原结合特性。新抗体使用内源恒定基因作为细胞表面表达 IgM，允许使用目标抗原选择探针选择具有所需结合特性的变体。我们 已成功使用该平台生成和选择抗体变体，该变体显示出改善的艾滋病毒 中和广度和效力作为概念证明。可以收获工程细胞分泌的 IgM 从细胞上清液中提取并在细胞系定向进化过程中进行表征。单细胞测序 可以对进化的细胞进行研究，以揭示改变结合和产生 改进的单克隆抗体以重组蛋白的形式表达。在此 R21 中，我们建议设计 HIV bnAb VRC01 和 CAP256-VRC26.25 进入我们的 B 细胞系，以进化 CD4 结合位点和 V2-apex 定向 bnAbs 具有改进的中和广度、效力和潜在的生物物理特性，使用多种 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 不仅改善了 HIV bnAb，还改善了任何具有治疗意义的单克隆抗体。