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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要单克隆抗体 (mAb) 和 mAb 衍生抗体疗法目前广泛用于治疗人类疾病，例如癌症和自身免疫性疾病。虽然以兔子为平台来生产多克隆抗体已被使用很长时间，兔单克隆抗体的产生已由于难以产生稳定的杂交瘤而受到限制。采用分子克隆直接从 Ig 基因 B 细胞在细胞培养物中表达为兔单克隆领域带来了新的活力。然而，单克隆抗体的生产效率仍然较低，并且可从兔获得的单克隆抗体类型有限。近日，我们报道了跨膜激活因子和 CAML 相互作用因子 (TACI) 的缺陷，TACI 是一种控制浆细胞的受体分化，还会引起抗原反应性生发中心 (GC) B 细胞的显着扩增，从而增强亲和力成熟并促进高亲和力 IgG 的产生。 TACI 缺乏也增加了产量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 在兔种系中的表达，目的是增强单克隆抗体的产生。我们利用CRISPR/Cas9技术建立了高效的兔基因组编辑平台。我们现在提出通过CRISPR/Cas9技术靶向兔子TACI基因来生产TACI-KO兔子。我们将利用完善的平台首先产生TACI突变兔创始人。这些创始人将受到考验用于种系传播，如果是这样，则建立杂合的 TACI 突变动物，然后进行育种获得纯合TACI突变动物。我们将检验以下假设：兔子的 TACI 缺陷会导致增强 mAb 的产生以及这些抗体的亲和力和特异性。我们建议建立 TACI- 缺陷兔作为单克隆抗体生产的优质生物反应器。