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; 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的被动转移广泛中和抗体（BNAB）有望作为抗逆转录病毒的替代品 药物通常耐受性良好，体内半衰期长，并且可以激活宿主免疫 系统。 BNAB宽度和效力的分配后改善应成为医疗的应用 干预更实用。我们已经开发了一个新颖的抗体定向的进化平台 为此目的雇用。该平台使用基因组编辑来代替免疫球蛋白可变区域 来自人类B细胞系中特定单克隆抗体的那些。内源激活诱导的诱导 胞苷脱氨酶（AID）在这些工程基因座引入突变，产生了变化的抗体变体 抗原结合特性。新抗体使用内源常数基因作为细胞表面表达 IgM，允许使用目标抗原选择探针具有所需结合特性的选择变体。我们 成功地使用了该平台生成和选择抗体的变体，这些变体显示了改善的HIV 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 来自细胞上清液，并在细胞系的定向演变中进行表征。单细胞测序的 可以进行进化的细胞以发现改变结合的遗传基础和生成 改进的mAb作为重组蛋白的表达。在此R21中，我们建议工程艾滋病毒bnabs VRC01和CAP256-VRC26.25进入我们的B细胞系，以进化CD4结合位点，并定向V2-apex 使用多种 不同的选择策略。如果成功的话，该平台将是后发现的宝贵资源 不仅要改善艾滋病毒bnabs，而且还要改善任何治疗兴趣的单克隆。