B-149 From Polyclonal to Recombinant: A Proteomics Workflow for Anti-Idiotype Antibody Development

Abstract

Anti-idiotype antibodies are essential tools for therapeutic drug monitoring of biologic therapies in cancer, inflammatory bowel disease, and rheumatoid arthritis. While several platforms exist for their development, including B-cell immortalization and phage display, we employ a proteomics-based mass spectrometry approach. The primary challenge lies in identifying antibodies highly specific to the therapeutic biologic while maintaining no affinity toward endogenous human IgG. Using adalimumab as proof-of-concept, we generated recombinant anti-idiotype candidates and validated their specificity through ELISA and surface plasmon resonance (SPR). Selected candidates will undergo additional characterization by HDX-MS to map epitope binding sites and confirm target specificity. Discovery: Anti-idiotype antibodies were raised against adalimumab F(ab')2 via rabbit immunization with one prime and three boosters. RNA from splenocytes was used for BCR sequencing on an Illumina MiSeq. Proteomic analysis of immune serum involved native PAGE, IEF, and 2D electrophoresis, followed by in-gel and multi-enzyme in-solution digestion. LC-MS/MS was performed on an Orbitrap Exploris 240 using HCD fragmentation. Validation: ELISA-based affinity screening and competition assays identified several candidate anti-idiotype antibodies. Competition with TNF-a revealed both Type ß candidates (blocking adalimumab-TNF-a interaction) and Typecandidates (non-blocking). Following ELISA validation, full kinetic profiling of recombinant anti-idiotype antibodies was performed by Surface Plasmon Resonance (SPR). Single-cycle kinetic analysis and epitope binning were conducted on a Biacore 8K using CM5 carboxyl sensors with amine-coupling chemistry for adalimumab immobilization. Identified 25 unique heavy chains, 42 kappa light chains, and 97 heavy-light chain pairs through proteogenomic analysis. A total of 30 recombinant antibodies were expressed. Our adalimumab-specific enrichment strategy yielded polyclonal antibodies with significantly reduced off-target affinity for human IgG compared to commercially available anti-idiotypes. Recombinantly expressed antibodies demonstrated high-affinity binding to adalimumab with minimal cross-reactivity to endogenous IgG. Strategic heavy-light chain pairing maintains target specificity while minimizing off-target binding. Proteomic characterization of the immune response identified both blocking (Type ß) and non-blocking (Type ?) anti-idiotype populations. Kinetic analysis and epitope binning revealed significant diversity in binding profiles within each functional cluster, demonstrating a level of complexity not detected by ELISA alone. Combined ELISA and SPR screening narrowed the candidate pool to four antibodies—two Type ß and two Type ?—for further characterization by HDX-MS. This study demonstrates successful conversion of polyclonal anti-idiotypes into functionally equivalent recombinants with thorough characterization by ELISA and SPR. Selected candidates will undergo HDX-MS analysis to map epitope binding sites, providing a comprehensive platform for anti-idiotype development.