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

Abstract

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.

Structure of the core human NADPH oxidase NOX2

“…To generate monoclonal antibodies, up to 30 mL of blood was drawn from each immunized rabbit and peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation of whole blood over Lympholyte®-M (CL5115, Cedarlane Labs), and IgG+ B cells were enriched by staining with antibody cocktails containing 1:30 of anti-rabbit CD11b and 1:40 of anti-rabbit T-lymphocyte antibody (MCA802GA and MCA800GA respectively, AbD Serotec, BioRad), as well as 1:20 of anti-rabbit IgM (550938, BD Bioscience), and negatively selected through MACS Column (130-042-401, Miltenyi Biotec). The enriched rabbit B cells were stained with mixed APC-labeled mouse anti-rabbit IgG antibody (Purified mouse anti-rabbit IgG, Southern Biotech 4090-01) labeled with APC using Lightning-Link® APC Antibody Labeling Kit (Novus Biotech, 705-0030) and PE-labeled NOX2 enzymatic complex (labeled with Lightning-Link (R) R-PE Antibody Labeling Kit, Novus Biotech 703-0010) in the staining buffer (Phosphate-buffered saline with 0.5% bovine serum albumin and 2 mM EDTA), and single IgG+, NOX2 protein-binding B cells were sorted into 96-well plate with supplemented RPMI 1640 culture medium and EL4-B5 feeder cells for 7 days cultivation at 37 °C, as previously described 78 .…”

“…The B-cell culture supernatants were harvested and screened by ELISA for IgG production and by FACS for binding to cell-surface expressed NOX2. The variable light chain and variable heavy chain sequences from anti-NOX2 B cells were obtained by Sanger sequencing as previously described 78 .…”

Novel Anti-LY6G6D/CD3 T-Cell–Dependent Bispecific Antibody for the Treatment of Colorectal Cancer

“…White New Zealand rabbits were immunized with human LY6G6D and single IgG + huLY6G6D + B cells were isolated and cultured as described previously ( 14 ). The B-cell culture supernatants were assayed by ELISA for binding to human LY6G6D.…”

“…The B-cell culture supernatants were assayed by ELISA for binding to human LY6G6D. Variable regions (VH and VL) of each mAb from rabbit B cells were cloned into expression vectors as previously described ( 14 ). Individual recombinant rabbit antibodies were expressed in Expi293 cells and subsequently purified with protein A. Purified anti-LY6G6D antibodies were then subjected to functional activity assays and kinetic screening.…”

Dual antibody inhibition of KLK5 and KLK7 for Netherton syndrome and atopic dermatitis

“…New Zealand white rabbits or Sprague-Dawley rats were immunized with mouse KLK5, mouse KLK7, hKLK5, or hKLK7. Antigen-specific single B cells were isolated and cultured using a standard protocol ( 64 ). The B cell culture supernatants were assayed by enzyme-linked immunosorbent assay for binding to the relevant KLK5 or KLK7 protein and an irrelevant control protein.…”