To establish an entirely murine antibody transfer model for Epidermolysis Bullosa Acquisita (EBA) research, we generated a defined array of monoclonal antibodies (mAbs) against the second von Willebrand factor type A-like domain of murine collagen VII (mCOL7vWFA2), the immunodominant epitope in EBA. To identify mCOL7vWFA2 binding antibodies, we generated two single-chain fragment variable (scFv) immune libraries from the spleen and inguinal lymph nodes of a mCOL7vWFA2 immunized mouse with active EBA disease, confirmed by disease affected body area (10%) and anti-mCOL7vWFA IgG serum titer. After two consecutive rounds of panning of with recombinant mCOL7vWFA2, 33 scFv antibodies were Sanger sequenced. We identified 14 individual scFv antibody clones that grouped into four variable heavy chain (VH) clonotypes, of which one was unique to the lymph nodes, two to the spleen, and another one occurred in both tissues. All analyzed VH sequences incorporated the IGHV1-39 V gene and IGHJ3 J gene, but differed in individual point mutations, CDRH3 sequence and length. ScFv antibodies from all VH clonotypes exhibited comparable mCOL7vWFA2 specific binding as measured by ELISA. The six most frequent VH-VL combinations were chosen for recombinant expression as mIgG2b mAbs, in line with the dominant IgG2b subclass in sera from mice immunized with mCOL7vWFA2 in experimental EBA. All mAbs will be further characterized in vitro regarding native antigen binding, complement fixation, neutrophil activation and single and mixed injections into mice will be investigated to passively induce EBA-like disease.

ESDR062 - Phage display-based isolation of collagen VII-specific monoclonal antibodies from mice with actively induced Epidermolysis Bullosa Acquisita

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