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

项目总结/摘要 STTR第二阶段提案的最终目标是通过以下方式扩展SAB的diversitAb™平台的功能： 跨染色体山羊（Transchromosomic Goat，TcGs）的进一步发展。这将通过生产 雄性和雌性山羊内源性免疫球蛋白基因敲除细胞系，其含有SAB Capra's人 人工染色体（HAC），其编码生殖系人抗体基因的整个库，从而 大大提高了TcGs中全人抗体的生产效率。为了进一步推进TcG 平台，以商业化生产治疗和诊断抗体产品，优化 抗体纯化过程，质量控制测定的开发，以及纯化的TcG- 通过完成体外和体内评价， 提出了 为了实现这些目标，将利用免疫球蛋白基因敲除促进内源性免疫球蛋白基因敲除。 CRISPR/Cas9系统在国内努比亚/野猪山羊胎儿成纤维细胞（GFF）中的应用，以及SAB- 将设计的HAC导入敲除细胞将通过微细胞介导的染色体转移（MMCT）来完成。 含有SAC的敲除GFF细胞将用于通过体细胞核移植（SCNT）产生胚胎。 在妊娠和分娩多种TcG后，将完成分子表征， 将确认敲除和HAC，并将确认和评价人IgG产生。两个这样 TcG随后将用靶向H7N9的质粒DNA疫苗进行超免疫。血浆将从 超免疫TcG和抗H7N9全人IgG将从TcG血浆中纯化。体外 然后评价纯化的TcG衍生的抗H7N9人IgG产物的效力和体内功效。 将优化既定的净化程序，以确保高效和有效的净化 从血浆中提取TcG衍生的人IgG，并开发质量控制试验以评价产品特性， 纯度和效力将完成。 SAB用于人抗体生产的diversitAb™平台目前用于转染色体牛 针对广泛的抗原，包括病毒、细菌、肿瘤靶点、重组蛋白和DNA 疫苗。这种成熟的技术具有可扩展性、简单性和广泛适用性的优点。的 将TCG添加到平台允许更简单和更便宜的小批量快速响应生产 目标产品以及用于新发传染病血清学检测的诊断试剂。

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.