A novel rabbit model for easy monoclonal antibody production

一种易于单克隆抗体生产的新型兔模型

• 批准号: 10264143
• 负责人: MARILIA Isabel CASCALHO
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10264143
• 关键词: Adoption; Affinity; Animals; Antibodies; Antibody Affinity; Antibody Formation; Antigens; Autoimmune Diseases; B-Lymphocytes; Back; Biology; Biomedical Research; Bioreactors; Breeding; CAMLG gene; Cell Culture Techniques; Clinic; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary DNA; DNA sequencing; Data; Engineered Gene; Engineering; Gene Cluster; Gene Targeting; Generations; Genes; Genetic Engineering; Genome; Genomics; Goals; Hybridomas; Immune system; Immunization; Immunize; Immunoglobulin G; Immunoglobulin Genes; Industry; Infection; Knock-in; Knock-out; Lead; Letters; Life; Malignant Neoplasms; Measures; Mediating; Michigan; Mission; Modeling; Modernization; Molecular Cloning; Monoclonal Antibodies; Mus; Oryctolagus cuniculus; Population Control; Production; Property; Proteins; Reporting; Research; Specificity; Splenocyte; Structure of germinal center of lymph node; Surface; Techniques; Technology; Testing; Therapeutic; Therapeutic Monoclonal Antibodies; Therapeutic antibodies; Time; Translational Research; Transplantation; United States Food and Drug Administration; Universities; Variant; Work; Yang; drug development; genome editing; human disease; hybridoma production; knockout animal; monoclonal antibody production; mutant; novel; plasma cell differentiation; polyclonal antibody; programs; receptor; research and development; response; success; tool; transmission process

PROJECT SUMMARY/ABSTRACT Monoclonal antibodies (mAbs) and mAb-derived antibody therapeutics, are currently widely used to treat human diseases, such as cancer and autoimmune diseases. Although the rabbit as a platform to producing polyclonal antibodies has been utilized for a long time, generation of rabbit monoclonal antibodies has been limited by difficulties in generating stable hybridomas. The adoption of molecular cloning of Ig genes directly from B cells followed by expression in cell culture has introduced new life in the rabbit monoclonal field. However, the efficiency of mAb production is still low and the type of mAbs available from rabbit limited. Recently, we reported that deficiency in the Transmembrane Activator and CAML Interactor (TACI), a receptor that controls plasma cell differentiation, also causes marked expansion of antigen-responsive germinal center (GC) B cells enhancing affinity maturation and facilitating production of high-affinity IgG. TACI-deficiency also increased the yield of monoclonal antibody production by the hybridoma technique. In the present application, we propose to abrogate expression of TACI in the rabbit germline with the goal of enhancing monoclonal antibody production. We have established a highly efficient rabbit genome editing platform using CRISPR/Cas9 technology. We now propose to target the rabbit TACI gene by CRISPR/Cas9 technology to produce a TACI-KO rabbit. We will use the well-established platform to first generate TACI mutant rabbit founders. These founders will be tested for germline transmission, and if so, to establish heterozygous TACI mutant animals, followed by breeding to obtain homozygous TACI mutant animals. We will test the hypothesis that TACI-deficiency in rabbits will enhance mAb production and the affinity and specificity of those antibodies. We propose to establish TACI- deficient rabbits as a superior bio-reactor for mAb production.

项目总结/摘要 单克隆抗体（mAb）和mAb衍生的抗体治疗剂目前广泛用于治疗 人类疾病，如癌症和自身免疫性疾病。虽然兔子作为一个平台， 多克隆抗体已经使用了很长时间，兔单克隆抗体的产生已经 这受到难以产生稳定杂交瘤的限制。采用分子克隆技术直接从大肠杆菌中克隆IG基因， B细胞随后在细胞培养中表达，在兔单克隆领域引入了新的生命。但 单克隆抗体生产的效率仍然很低，并且可从兔获得的单克隆抗体的类型有限。最近，我们报道了 跨膜激活物和CAML相互作用物（TACI）的缺乏， 分化，也引起抗原应答性生发中心（GC）B细胞的显著扩增，增强免疫应答性。 亲和力成熟和促进高亲和力IgG的产生。TACI缺乏还增加了 通过杂交瘤技术生产单克隆抗体。在本申请中，我们建议废除 TACI在兔生殖系中的表达，目的是增强单克隆抗体的产生。 我们利用CRISPR/Cas9技术建立了一个高效的兔基因组编辑平台。 我们现在建议通过CRISPR/Cas9技术靶向兔TACI基因，以产生TACI-KO兔。我们 将使用完善的平台，首先产生TACI突变兔创始人。这些创始人将接受考验 用于生殖系传播，如果是这样，建立杂合TACI突变动物，然后育种， 获得纯合TACI突变动物。我们将检验这一假设，即TACI缺乏的兔子将 增强mAb的产生以及这些抗体的亲和力和特异性。我们建议成立泰慈- 缺陷兔作为mAb生产的上级生物反应器。