51 Targeted anti-idiotype antibody discovery through functional pre-selection and de novo sequencing

Background Anti-idiotype antibodies bind to the variable regions of other antibodies regulating immune responses and mimicking antigens, making them promising tools for vaccines and therapeutics. The main challenge in anti-idiotypic antibody discovery is finding antibodies that are highly specific to the therapeutic antibody, without cross-reacting with naturally circulating IgGs.Our workflow leverages advanced protein enrichment and depletion strategies to isolate functionally relevant anti-idiotype subtypes before sequencing, significantly boosting discovery efficiency.Using adalimumab as a model, we introduce a mass spectrometry-based proteogenomics approach that maps binding sites and distinguishes blocking from non-blocking variants, crucial for pharmacokinetic applications.Methods Anti-idiotype antibodies were raised against adalimumab F(ab’) 2 via rabbit immunization with one prime and three boosters (figure 1). RNA from splenocytes was used for BCR sequencing on an Illumina MiSeq. Proteomic analysis 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.Results Identified 25 unique heavy chains, 42 kappa light chains and 97 heavy-light chain pairs through proteogenomic analysis.Our adalimumab anti-idiotype enrichment strategy yielded a polyclonal antibody (PD182) with less off-target affinity for human IgG than commercially available anti-idiotypes.Recombinantly expressed antibodies showed high affinity for adalimumab; only one had notable off-target binding to human IgG.Conclusions Our strategic enrichment workflow selectively isolates functional anti-idiotypes before sequencing, streamlining discovery by reducing the candidate pool.Proteomic analysis identifies both blocking (type 1) and non-blocking (type 2) anti-idiotypes through targeted enrichment.Differential heavy-light chain pairing can maintain target specificity and reduce off-target binding.Abstract 51 Figure 1Anti-idiotype antibody discovery workflow with functional pre-selection and de novo polyclonal sequencing. 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. LC-MS/MS was performed on an Orbitrap Exploris 240 using HCD