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Abstract: TH-PO839

Integrated Proteomics and DNA Analysis of Pathogenic IgG from Patients with Anti-GBM Nephritis

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Foster, Mary H., Duke University Medical Center, Durham, North Carolina, United States
  • Pedchenko, Vadim, Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Clark, Amy G., Duke University Medical Center, Durham, North Carolina, United States
Background

Anti-glomerular basement membrane (anti-GBM) nephritis (Goodpasture’s disease) is marked by immune destruction of kidneys and lungs triggered by IgG autoantibodies that bind the NC1 domain of the α3 chain of collagen IV. Current therapy consists of toxic immunosuppression. Selective therapeutic targeting of pathogenic IgG and B cells is highly desirable, but precluded by the lack of information about their structural basis.

Methods

To address this problem, we used an innovative approach validated in vaccine biology. F(ab’)2 fragments were isolated by pepsin digestion from circulating IgG of two patients with active anti-GBM nephritis and purified on α3(IV)NC1 affinity columns. Antigen-binding (Ag+) and non-binding (NB) flow-through F(ab’)2 were trypsin digested and evaluated by liquid chromatography-tandem mass spectrometry. Peptide spectra were analyzed using a reference library generated from productively rearranged Ig heavy chains (n=11903) in the International ImMunoGeneTics database and formatted for use by the Mascot search engine.

Results

826 unique spectral matches were identified. 41 peptides were identified in both patients’ Ag+ F(ab’)2 and absent in NB flow-through F(ab’)2. All Ag+ matches aligned to Ig heavy chain variable region (VH) genes, primarily in conserved framework regions. Notably, 32% aligned to alleles of a VH gene with an unusual highly hydrophobic heavy chain complementarity determining region 2 (HCDR2) that was previously associated with autoreactivity and reported to encode a human anti-α3(IV)NC1 collagen monoclonal Ig derived from a humanized immune system mouse.

Conclusion

We find evidence of shared and biased gene use among pathogenic IgG that bind α3(IV)NC1 collagen in patients with anti-GBM nephritis. This suggests that patients’ GBM-binding IgG contain unusual targetable motifs in their Ag binding sites. Future interrogation of the spectra using patient-specific Ig DNA reference libraries, generated from patients’ lymphocytes, will permit identification of non-genomic-DNA-templated peptide sequences that are found within HCDR3, a unique region of the Ag binding site created in B cells during VH-DH-JH gene recombination and for which sequences are not present in public databases. Rare motifs will provide additional therapeutic targets for personalized disease management.

Funding

  • NIDDK Support