Rapid identification of anti-idiotypic mAbs with high affinity and diverse epitopes by rabbit single B-cell sorting-culture and cloning technology

• Published in: PloS one Vol. 15; no. 12; p. e0244158
• Main Authors: Lin, WeiYu, Liang, Wei-Ching, Nguy, Trung, Maia, Mauricio, Tyagi, Tulika, Chiu, Cecilia, Hoi, Kam Hon, Chen, Yongmei, Wu, Yan
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 21.12.2020; Public Library of Science (PLoS)
• Subjects: Affinity; Animals; Antibodies; Antibodies, Anti-Idiotypic - genetics; Antibodies, Anti-Idiotypic - immunology; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibody Affinity; Antigenic determinants; Assaying; B cells; B-Lymphocytes - classification; B-Lymphocytes - immunology; Biology and Life Sciences; Cell culture; Cell Separation - methods; Cells, Cultured; Cloning; Cloning, Molecular - methods; Complementarity; Drug development; Engineering; Enzyme-linked immunosorbent assay; Epitopes; Epitopes - genetics; Epitopes - immunology; Euthanasia; Humans; Identification and classification; IgG antibody; Immunization; Immunogenicity; Immunoglobulin G; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Lymphocytes B; Lysates; Medicine and Health Sciences; Monoclonal antibodies; Pharmacokinetics; Physiological aspects; Rabbits; Research and Analysis Methods; Technology; United States; United States > US; South San Francisco California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0244158

The proactive generation of anti-idiotypic antibodies (anti-IDs) against therapeutic antibodies with desirable properties is an important step in pre-clinical and clinical assay development supporting their bioanalytical programs. Here, we describe a robust platform to generate anti-IDs using rabbit single B cell sorting-culture and cloning technology by immunizing rabbits with therapeutic drug Fab fragment and sorting complementarity determining regions (CDRs) specific B cells using designed framework control as a negative gate to exclude non-CDRs-specific B cells. The supernatants of cultured B cells were subsequently screened for binding to drug-molecule by enzyme-linked immunosorbent assay and the positive hits of B cell lysates were selected for cloning of their immunoglobulin G (IgG) variable regions. The recombinant monoclonal anti-IDs generated with this method have high affinity and specificity with broad epitope coverage and different types. The recombinant anti-IDs were available for assay development to support pharmacokinetic (PK) and immunogenicity studies within 12 weeks from the start of rabbit immunization. Using this novel rapid and efficient in-house approach we have generated a large panel of anti-IDs against a series of 11 therapeutic antibody drugs and successfully applied them to the clinical assay development.