Characterization of human hybridoma clones isolated from hemophilia patients with specificity for different domains of coagulating factor VIII

Article type: Research Article

Authors: S. Gharagozlou, ^{a; b} | Ghods, R.^{a; c} | Bahrami, Z. Samadi^b | Roohi, A.^a | Jeddi-Tehrani, M.^{c; d} | Conti-Fine, B.M.^e | Sharifian, R.A.^f | Rabbani, H.^{c; d} | Shokri, F.^{a; b; c; *}

Affiliations: [a] Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran | [b] National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran | [c] Monoclonal Antibody Research Center, Avesina Research Center, Tehran, Iran | [d] Immune and Gene Therapy Lab, Cancer Center Karolinska, Karolinska Hospital, Stockholm, Sweden | [e] Department Of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA | [f] Clinic of Hematology and Oncology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

Correspondence: [*] Address for correspondence: Prof. Fazel Shokri, Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran 14155, Iran. Fax: +98 21 6462267; E-mail: fazshok@yahoo.com.

Abstract: Hemophilia A patients treated with human coagulating factor VIII (FVIII) may develop inhibitory antibodies (inhibitors). Characterization of the inhibitors at the clonal level may help exploring new therapeutic strategies. We have generated lymphoblastoid cell lines (LCLs) producing anti-FVIII antibodies from peripheral blood lymphocytes of hemophilia A patients with high inhibitor titers. We fused the anti-FVIII-positive LCLs with a heteromyeloma, to produce FVIII specific hybridomas. We determined the specificity, isotype, idiotypic and immunoglobulin (Ig) variable region heavy (VH) chain gene family profiles of the secreted antibodies (Ab) by ELISA, immunoblotting and RT-PCR. We established eight hybridomas which produced high titers of anti-FVIII Ab. All hybridomas secreted IgM Ab, associated with either κ(5/8) or λ(3/8) light chain. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized only the VH1 (63%) or the VH3 (37%) gene families. Among the cross-reactive idiotypes (CRIs) we tested, only the VH1 and VK3b-associated CRIs were expressed by 3 hybridomas. Immunoblotting of thrombin-digested FVIII demonstrated distinct patterns of reactivity of the monoclonal Ab (MAb) secreted by the hybridomas, which recognized either the A2 domain of the Fvm heavy chain, or the light chain, or both. Our findings suggest that: a) the isotype of the anti-FVIII Ab secreted by LCLs and hybridoma clones (IgM) differs from that of anti-FVIII Ab in vivo, which are predominantly IgG4: this suggests a negative selection of the isotype-switched FVIII-specific B-cells in the periphery of these patients; b) the anti-FVIII Ab have a biased representation of the VH1 gene family, and c) somatic mutations in the VH genes coding for FVIII specificity occur in the anti-FVIII Ab response, as evidenced by lack of expression of the VH-associated CRI.

Keywords: hemophilia A, factor VIII, monoclonal antibody, inhibitor, human hybridoma, V_H gene, cross-reactive idiotype

DOI: 10.3233/HAB-2003-12303

Journal: Human Antibodies, vol. 12, no. 3, pp. 67-76, 2003