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.

摘要 被动转移HIV广泛中和抗体（bnAbs）有望成为抗逆转录病毒的替代方案 因为它们通常耐受性良好，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用途的应用成为可能。 干预更实际。我们已经开发了一种新的抗体定向进化平台，我们希望 为此目的雇用。该平台使用基因组编辑来替换免疫球蛋白可变区， 来自人B细胞系中特异性单克隆抗体的那些。内源性激活诱导 胞苷脱氨酶（AID）在这些工程化基因座处引入突变，产生具有改变的 抗原结合特性。新抗体是利用内源性恒定基因作为细胞表面表达的 IgM，允许使用靶抗原选择探针选择具有所需结合特性的变体。我们 我已经成功地使用这个平台来产生和选择抗体的变体，这些变体显示出改善的HIV 中和广度和效力作为概念的证明。可以收获从工程化细胞分泌的IgM 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改变结合和产生 用于作为重组蛋白表达的改进的mAb。在这个R21中，我们建议工程化HIV bnAbs VRC 01和CAP 256-VRC26.25导入我们的B细胞系中，以进化出CD 4结合位点和V2-顶点定向 使用多种方法制备具有改善的中和宽度、效力和潜在生物物理性质的bnAb， 不同的选择策略。如果成功的话，这个平台将是一个宝贵的资源， 不仅对HIV bnAb，而且对任何有治疗意义的单克隆抗体的改进。