Kidney Week

Abstract: FR-PO0812

Patient-Derived Anti-HTRA1 Antibody Discovery Using Purified IgG Mass Spectrometry and B-Cell Receptor Sequencing in Membranous Nephropathy

Session Information

• Glomerular Diseases: Practice and New Concepts Shaping Modern Care
  October 23, 2026 | Location: Exhibit Hall A, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1402 Glomerular Diseases: Clinical, Outcomes, and Therapeutics

Authors

• Al-Rabadi, Laith, University of Utah Health, Salt Lake City, Utah, United States

Laith Al-Rabadi, MD

• Almarji, Catreena, University of Utah Health, Salt Lake City, United States

Catreena Almarji, MD

• Sameh, Ahmed, University of Utah Health, Salt Lake City, Utah, United States

Ahmed Sameh, MD, MBChB

• Abuelsamen, Tamer Rami, University of Utah Health, Salt Lake City, Utah, United States

Tamer Rami Abuelsamen, MD

• Mekki, Ossama, University of Utah Health, Salt Lake City, Utah, United States

Ossama Mekki, MD

• Takayama, Suguru, University of Utah Health, Salt Lake City, Utah, United States

Suguru Takayama, MD, PhD

• Hoxha, Elion, Universitatsklinikum Wurzburg, Würzburg, BY, Germany

Elion Hoxha, MD

• Borza, Dorin-Bogdan, Meharry Medical College, Nashville, Tennessee, United States

Dorin-Bogdan Borza, PhD

• Beck, Laurence H., Boston University, Boston, Massachusetts, United States

Laurence H. Beck, MD, PhD

Background

HTRA1 is a recently identified target antigen in a subset of membranous nephropathy (MN), but the antibody sequences responsible for anti-HTRA1 reactivity are unknown. Defining these sequences would enable patient-derived monoclonal antibodies for epitope mapping, assay development, and mechanistic studies. We asked whether purified-IgG mass spectrometry, patient B-cell screening, and iPair/iRepertoire B-cell receptor sequencing could recover functional anti-HTRA1 antibodies from HTRA1-associated MN.

Methods

Anti-HTRA1 IgG was affinity-purified from patient serum using recombinant HTRA1/PDZ-domain antigen and analyzed by mass spectrometry to identify variable-region peptides and infer candidate heavy- and light-chain sequences. In parallel, patient B cells were EBV-immortalized, and culture media were screened for HTRA1 reactivity to identify reactive antibody-secreting cultures and optimize antigen-screening conditions. Antigen-reactive B-cell populations were then analyzed using iPair/iRepertoire sequencing. Candidate antibodies were prioritized based on overlap between antibody proteomics and BCR sequencing, peptide coverage, clonal enrichment, reactive media, and feasible heavy/light-chain pairing. Recombinant IgG candidates were generated and screened for HTRA1 reactivity by western blotting.

Results

Mass spectrometry of purified anti-HTRA1 IgG identified variable-region peptides supporting reconstruction of candidate patient-derived antibody sequences. EBV-immortalized B-cell cultures provided reactive supernatants that helped optimize the screening workflow before iPair/iRepertoire analysis. Integration of serum antibody proteomics, B-cell screening, and BCR sequencing identified a set of candidate anti-HTRA1 clonotypes. We generated 24 recombinant IgG candidates using prioritized heavy- and light-chain pairings; 3 of 24 showed reproducible HTRA1 reactivity by western blot.

Conclusion

Purified-antibody mass spectrometry combined with patient-derived B-cell screening and iPair/iRepertoire sequencing can identify recombinant antibodies that bind HTRA1. Validation of 3 reactive antibodies from 24 candidates supports the feasibility of this approach and provides monoclonal tools to study the anti-HTRA1 immune response in MN. This strategy may help define disease-relevant epitopes and support development of more specific anti-HTRA1 biomarkers.

Funding

• NIDDK Support