Molecular analysis of the heavy chain variable region genes of human hybridoma clones specific for coagulation factor VIII

Hemophilia A is a X-linked hematologic disorder characterized by undetectable or low amounts of functional coagulation factor VIII (FVIII). Replacement therapy induces FVIII neutralizing antibody (Ab) (inhibitor) in a proportion of patients which makes further treatment of these patients ineffective and costly. To envisage mechanisms underlying inhibitor development, seven hybridoma clones specific for FVIII were generated from two hemophiliaA patients with high titer of inhibitor.Specificity and isotype of the monoclonal antibodies (mAbs) were determined by ELISA. Immunoglobulin (Ig) variable region heavy (V H ) chain gene family usage was identified by RT-PCR usingV H 1–6 specific primers. Nucleotide sequences of the V H gene of FVIII specific clones were determined and aligned to the most homologous germ line genes in the GenBank. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized either the V H 1 (71%) or the V H 3 (29%) gene family.Three VH domains were encoded byV1–69 (DP-10),V1–2 (DP-8),andV1–8 (DP-15) genes and two by V1–18 (DP-14) gene, all from the V H 1 gene family. Of the V H 3-gene family expressing clones, one belonged toV3–66 (DP-86) and the other one toV3–21 (DP-77) germline genes. The CDR3 length was found to be highly different amongst these clones ranging from 11 to 22 amino acid residues. These data suggest that FVIII-specific Abs preferentially use V H gene segments derived fromV H 1 gene family.Diversity of the expressed VH genes and their CDR3 length implies that different epitopes are recognized by these mAbs.