Characterization of functional heparan sulfate motifs overexpressed on multiple myeloma cells using single chain antibody variable fragments generated by phage display

Abstract

13060 Background: Multiple myeloma (MM) is the most prevalent plasma cell neoplasm and is characterized by the infiltration of the bone marrow by terminally differentiated B lymphocytes. Although novel drugs have recently been introduced, the disease remains incurable. No therapeutic antibody is as yet available for treatment of MM. One of the key molecules involved in multiple myeloma (MM) tumor progression is syndecan-1, a cell surface heparan sulfate (HS) proteoglycan expressed on both MM cells and normal plasma cells. Syndecan-1 regulates cell interactions with soluble molecules and the extracellular matrix through its heparan sulfate chains. These chains allow it to bind multiple growth factors such as Fibroblast Growth Factor type 2 (FGF-2), WNT, Hepatocyte Growth Factor (HGF), midkine, all of which promote survival and metastasis of MM cells. In addition, syndecan-1 on MM cells binds and internalizes osteoprotegerin, the RANKL antagonist, through its HS chains and depletes it from the bone marrow milieu, which triggers osteoclastogenesis. Methods: Using the phage display technique, we have generated a panel of MM-specific human antibody variable fragments (scFvs) against the MM cell line RPMI-8226. These scFvs were tested against a variety of cell lines and primary normal bone marrow or myeloma cells. The biochemical identity of the targeted antigens was determined. Results: Two of the selected scFvs, D4A4 and D6B10, reacted against heparan sulfate motifs that were highly expressed in MM cell lines and patient MM cells but absent (scFv D6B10) or poorly expressed (scFv D4A4) in normal plasma cells. Binding of scFv D6B10 to MM heparan sulfate chains required NS sulfation, 6-O-sulfation and, to a lesser extent, 2-O-sulfation. Additional experiments indicated that FGF-2, midkine and osteoprotegerin competed with scFv D6B10 for binding to HS. Conclusions: This study indicates that the structure of syndecan-1-associated HS is altered in MM cells and may influence its capacity to bind to growth factors and promote tumor progression. Human therapeutic antibodies derived from scFvs D4A4 and D6B10 may be useful for immune targeting of MM cells and interfering with pro-survival signaling pathways. No significant financial relationships to disclose.