Production and Characterization of Human Immunoglobulin Producing Goats for Diagnostic Reagents and Therapeutics

用于诊断试剂和治疗的人免疫球蛋白生产山羊的生产和表征

• 批准号: 9916705
• 负责人: Irina A. Polejaeva
• 金额: $ 74.31万
• 依托单位: SAB CAPRA, LLC
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-15 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916705
• 关键词: Address; Adjuvant; Advanced Development; Affinity Chromatography; Anaphylaxis; Animals; Antibodies; Antibody Formation; Antigens; Autoimmunity; Bacteria; Biological Assay; Biological Products; Biological Response Modifier Therapy; Birds; CRISPR/Cas technology; Cattle; Cell Line; Cells; Chromosome Transfer; Chromosomes, Artificial, Human; Clinical; Collaborations; Collection; Communicable Diseases; DNA Vaccines; Development; Diagnostic; Diagnostic Reagent; Embryo; Embryo Transfer; Emerging Communicable Diseases; Ensure; Female; Fibroblasts; Filtration; Fractionation; Gene Cluster; Gene Expression; Genes; Genetic; Genetic Engineering; Genus Capra; Goals; Goat; Hemagglutinin; Human; Immunization; Immunoglobulin G; Immunoglobulin Genes; Immunoglobulin M; Immunoglobulins; Immunotherapeutic agent; Immunotherapy; In Vitro; Inbred BALB C Mice; Infection; Inflammation; Influenza A Virus, H7N9 Subtype; Influenza A virus; Knock-out; Light; Live Birth; Mediating; Molecular; Multiple Birth Offspring; Oncology; Peripheral; Phase; Plasma; Plasma Proteins; Pregnancy; Preparation; Procedures; Process; Production; Quality Control; Recombinant Proteins; Recombinants; Research; Risk; Safety; Sampling; Serologic tests; Serum; Serum Sickness; Small Business Technology Transfer Research; System; Technology; Therapeutic; Therapeutic antibodies; Treatment Efficacy; Universities; Utah; Virus; Virus Diseases; Work; Xenobiotics; assay development; base; design; efficacy study; fetal; hyperimmunization; improved; in vivo; in vivo evaluation; influenzavirus; knockout gene; male; mouse model; pandemic influenza; plasmid DNA; polyclonal human antibody; preclinical evaluation; protein expression; reagent standard; response; somatic cell nuclear transfer; vector

Project Summary/Abstract The ultimate goal of this phase II STTR proposal is to expand the capabilities of SAB's diversitAb™ platform by continuing advanced development of Transchromosomic goats (TcGs). This will be accomplished by producing male and female goat endogenous immunoglobulin gene knockout cell lines that contain SAB Capra's human artificial chromosome (HAC) which encodes the entire repertoire of the germline human antibody genes thereby greatly improving the production efficiency of fully human antibodies in TcGs. To further advance the TcG platform towards commercial production of therapeutic and diagnostic antibody products, optimization of the antibody purification process, development of quality control assays, and further development of a purified TcG- derived pandemic influenza antibody product targeting H7N9 by completing in-vitro and in-vivo evaluation is also proposed. To accomplish these goals, endogenous immunoglobulin gene knockouts will be facilitated utilizing the CRISPR/Cas9 system in domestic Nubian/Boar goat fetal fibroblast (GFF) cells, and transfer of the SAB- designed HAC into the knockout cells will be accomplished by microcell mediated chromosome transfer (MMCT). HAC-containing knockout GFF cells will be used to produce embryos by somatic cell nuclear transfer (SCNT). After gestation and birth of multiple TcGs, molecular characterization will be completed, presence of the knockouts and HAC will be confirmed, and human IgG production will be confirmed and evaluated. Two of these TcGs will then be hyperimmunized with a plasmid DNA vaccine targeting H7N9. Plasma will be collected from the hyperimmunized TcGs, and anti-H7N9 fully human IgG will be purified from the TcG plasma. The in-vitro potency and in-vivo efficacy of the purified TcG-derived anti-H7N9 human IgG product will then be evaluated. Optimization of established purification procedures will be undertaken to ensure efficient and effective purification of TcG-derived human IgG from plasma, and development of quality control assays to evaluate product identity, purity, and potency will be completed. SAB's diversitAb™ platform for human antibody production is currently utilized in transchromosomic bovines against a wide range of antigens including viruses, bacteria, oncology targets, recombinant proteins, and DNA vaccines. This proven technology has the advantages of scalability, simplicity, and broad applicability. The addition of TcGs to the platform allows for simpler and cheaper rapid-response production of small volume targeted products as well as diagnostic reagents for serological testing of emerging infectious diseases.

项目摘要/摘要 该II阶段STTR提案的最终目标是扩大SAB的Diversitab™平台的功能 跨染色体山羊（TCGS）的高级发展。这将通过生产来实现 雄性和雌性山羊内源性免疫球蛋白基因基因敲除细胞系，其中包含sab capra的人 人工染色体（HAC），编码人类抗体基因的整个曲目 大大提高了TCG中完全人类抗体的生产效率。进一步推进TCG 用于商业生产治疗和诊断抗体产品的平台，优化 抗体纯化过程，质量控制测定的开发以及纯化的TCG-的进一步发展 通过完成视野内和体内评估，衍生的大流行有影响力抗体产品靶向H7N9也是 建议的。 为了实现这些目标，内源性免疫球蛋白基因敲除将使用 CRISPR/CAS9系统中的nubian/boar山羊胎胎成纤维细胞（GFF）细胞，并转移Sab- 设计的HAC进入基因敲除细胞将通过Microcell介导的染色体转移（MMCT）来完成。 含HAC的敲除GFF细胞将用于通过体细胞核转移（SCNT）产生胚胎。 妊娠和多个TCG的出生后，将完成分子表征，存在 将确认敲除和HAC，并确认并评估人类IgG的产生。其中两个 然后将用靶向H7N9的质粒DNA疫苗过度免疫TCG。血浆将从 高免疫的TCG和抗H7N9完全人类IgG将从TCG等离子体中纯化。视野 然后将评估纯化TCG衍生的抗H7N9人IgG产品的效力和体内效率。 将进行既定纯化程序的优化，以确保有效纯化 来自等离子体的TCG衍生的人IgG，以及开发质量控制测定法以评估产品身份， 纯度和效能将完成。 SAB的Diversitab™人类抗体生产平台目前用于转染色体牛 针对广泛的抗原，包括病毒，细菌，肿瘤靶标，重组蛋白和DNA 疫苗。这项经过验证的技术具有可扩展性，简单性和广泛适用性的优势。这 在平台上添加TCG可以使小体积的快速响应产生更简单，更便宜 有针对性的产品以及用于新兴传染病的血清学测试的诊断试剂。