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Thromb Haemost 2005; 94(06): 1131-1137
DOI: 10.1160/TH05-06-0445
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

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

Soheila Gharagozlou
1   Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
2   National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
,
Gholam Ali Kardar
1   Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
3   Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran
,
Hodjattallah Rabbani
4   Monoclonal Antibody Research Center, Avesina Research Center, Tehran, Iran
5   Immune and Gene Therapy Lab, Cancer Center Karolinska, Karolinska Hospital, Stockholm, Sweden
,
Fazel Shokri
1   Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
2   National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
4   Monoclonal Antibody Research Center, Avesina Research Center, Tehran, Iran
› Author Affiliations
Further Information

Publication History

Received 26 June 2005

Accepted after revision 19 September 2005

Publication Date:
07 December 2017 (online)

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Summary

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 (VH) chain gene family usage was identified by RT-PCR usingVH1–6 specific primers. Nucleotide sequences of the VH 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 VH1 (71%) or the VH3 (29%) gene family. Three VH domains were encoded by V1–69 (DP-10),V1–2 (DP-8),andV1–8 (DP-15) genes and two by V1–18 (DP-14) gene, all from the VH1 gene family. Of the VH3-gene family expressing clones, one belonged to V3–66 (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 VH gene segments derived from VH1 gene family. Diversity of the expressed VH genes and their CDR3 length implies that different epitopes are recognized by these mAbs.

Keywords

Hemophilia A - factor VIII - human hybridoma - VH gene
 

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