Abstract Number: OC 55.2
Meeting: ISTH 2021 Congress
Background: Though inhibitors against FVIII are the major cause of morbidity and mortality in hemophilia A (HA), there is only a limited understanding of the monoclonal composition of anti-FVIII antibodies from HA patients.
Aims: To develop a FVIII-specific immunoproteomic method for the characterization of the circulating anti-FVIII antibody repertoire in HA patients.
Methods: Paired full-length VH-VL amplicons were generated from activated B cells and sequenced. Anti-FVIII IgG was purified from patient plasma by binding to an optimized biotinylated full-length FVIII (FVIII-SS-biotin) immobilized on magnetic streptavidin beads. FVIII-specific IgG and non-specific IgG were analyzed by LC-MS/MS, and spectra were searched against patient-specific VH-VL sequences. Anti-FVIII IgG were defined as high-quality CDRH3 peptides identified in replicates of the FVIII-specific IgG pool and not present in replicates of the non-specific IgG pool.
Results: FVIII-SS-biotin was able to efficiently capture a panel of 12 monoclonal antibodies, targeting all five immunogenic domains of FVIII, spiked into normal human plasma, demonstrating that FVIII-SS-biotin captures anti-FVIII antibodies without an epitope bias. Monoclonal antibodies were identified by LC-MS/MS at concentrations of 10-100 fmol in the input, comparable to the sensitivity of LC-MS/MS alone.
As an initial evaluation of our approach, we performed two replicate anti-FVIII IgG purifications from two time points from a pediatric HA patient with a high-titer inhibitor and a F8 early-stop mutation. We purified anti-FVIII IgG comprising only 0.5% of the total IgG pool while maintaining ≥97% capture of all anti-FVIII IgG and complete capture of all neutralizing antibodies in all replicates. Searching LC-MS/MS spectra against VH sequences identified 10 putative anti-FVIII antibody clones from different germline VH genes.
Conclusions: We developed an unbiased, efficient, and highly sensitive strategy to isolate and identify anti-FVIII antibodies from HA inhibitor patients. This approach has the potential to elucidate the immune repertories responsbile for the human anti-FVIII response.
To cite this abstract in AMA style:Chen R, Doshi B, Lopez S, Pan X, Fahad AS, Gutierrez Gonzalez MF, Dekosky B, Arruda V, Bhoj V, Samelson-Jones B. Purifying the Needle in the Haystack: A Novel Immunoproteomic Pipeline for the Characterization Anti-FVIII Antibodies in Hemophilia A Inhibitor Patients [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/purifying-the-needle-in-the-haystack-a-novel-immunoproteomic-pipeline-for-the-characterization-anti-fviii-antibodies-in-hemophilia-a-inhibitor-patients/. Accessed March 4, 2024.
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