# Structural and epitope characterization of anti-DEFA5 monoclonal antibodies clones 1A8 and 4F5 for inflammatory bowel disease subtype diagnostics

## TL;DR

This study characterizes two antibodies targeting DEFA5, offering insights into their potential for diagnosing inflammatory bowel disease subtypes.

## Contribution

The paper provides novel structural and epitope characterization of anti-DEFA5 monoclonal antibodies for IBD diagnostics.

## Key findings

- 1A8 and 4F5 antibodies bind to DEFA5 with high affinity but differ in epitope recognition and binding domains.
- HDX-MS and SPR reveal conformational stabilization and 1:1 binding stoichiometry for both antibodies.
- Hybridoma sequencing and HADDOCK models show distinct binding interfaces and domain interactions between the antibodies.

## Abstract

Understanding antigen-antibody interactions is key to advancing immunodiagnostics and therapeutic development. Here, we characterize two mouse monoclonal antibodies, 1A8 and 4F5, specific to human DEFA5, an innate immune effector implicated in differentiating colonic inflammatory bowel disease (IBD) subtypes, particularly Ulcerative colitis, Crohn’s colitis and Indeterminate colitis. Although both antibodies target similar regions of DEFA5, they exhibit distinct binding affinities. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) revealed diffuse protection patterns with partial protection upon antibody binding, suggesting conformational stabilization in DEFA5 rather than discrete epitope recognition. Complementary peptide-based epitope mapping confirmed localized linear or semi conformational binding epitopes. Surface Plasmon Resonance (SPR) confirmed 1:1 binding stoichiometry and high-affinity binding of both antibodies for the DEFA5 epitope RATCYCRTGRCAT. Hybridoma sequencing revealed that both antibodies engage DEFA5 by an extended binding interface involving multiple complementarity-determining regions (CDRs) across both heavy and light chains, indicating structurally distributed mode of antigen recognition. Despite identical hybridoma sequencing, HADDOCK models reveal subtle binding pose differences with DEFA5 engages the VH domain in 1A8, while it interacts with the VL domain in 4F5, suggesting phenotypic variations in domain contributions. This integrative approach provides mechanistic insights into their diagnostic potential for distinguishing IBD subtypes, supporting refined tissue-based and serological assays.