Generation and characterization of three monoclonal IgM antiphospholipid antibodies recognizing different phospholipid antigens.

Antiphospholipid antibodies (APLs) might be involved in the pathogenesis of the antiphospholipid syndrome (APS). This study analyzes the structural characteristics of monoclonal APLs derived from patients with this disease. Patient-derived B cells were immortalized using Epstein-Barr virus transformation and subsequent fusion to the myeloma cell line CB-F7. APL-producing hybridomas were cloned to obtain cell lines producing monoclonal APL. DNA encoding the variable region of heavy and light chains of the antibodies was sequenced and analyzed regarding their usage within the V-gene family and the existence of somatic hypermutation. Binding patterns of APL to various phospholipids and beta-2-glycoprotein-I (beta2-GPI) were determined using ELISA, with special regard to beta2-GPI dependency. As a result, three APL-producing hybridoma cell lines from patients with APS were established: JGG9, HVA2, and HLC9. APLs were of the IgM isotype and showed different binding patterns toward phospholipids and beta2-GPI. One of them, JGG9, showed extensive somatic hypermutations in both the CDR3 region and a framework region of the heavy chain. JGG9 bound to cardiolipin in the presence of the protein cofactor beta2-GPI. In contrast, the antibodies HVA2 and HLC9 (which also showed somatic hypermutations in the CDR3 region) presented polyreactivity to several phospholipids-cardiolipin, phosphatidyl-serine, -ethanolamine, -inositol, -choline, and sphingomyelin-but not to beta2-GPI. In conclusion, JGG9 presents a high degree of mutation in the CDR3 and framework region resulting from the deletions of nucleotides, and affects amino acid composition. Polyreactivity and the absence of cofactor dependency present HLC9- and HVA2-like natural antibodies that have no contact with any antigen. Nonetheless, these natural antibodies show somatic hypermutation of the heavy chain, indicating antigen-driven maturation. Regarding the possible role of APL in infection, HVA2 in particular may represent a pathogen-maturated antibody showing cross-reactivity between phospholipids and infectious agents. Further experiments are needed to reveal the functional activity of these antibodies.