Construction and characterization of recombinant human C9 or C7 linked to single Chain Fv directed to CD25

• Published in: Molecular immunology Vol. 55; no. 3-4; pp. 400 - 408
• Main Authors: Raitses Gurevich, Maria, Fishelson, Zvi
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-10-2013
• Subjects: CD25; Cell Line, Tumor; Complement; Complement C7 - chemistry; Complement C7 - genetics; Complement C7 - metabolism; Complement C9 - chemistry; Complement C9 - genetics; Complement C9 - metabolism; Complement Membrane Attack Complex - chemistry; Complement Membrane Attack Complex - genetics; Complement Membrane Attack Complex - metabolism; Cytotoxicity, Immunologic; HEK293 Cells; Humans; Immunoconjugates - chemistry; Immunoconjugates - genetics; Immunotherapy; Interleukin-2 Receptor alpha Subunit - metabolism; Neoplasms - immunology; Neoplasms - pathology; Neoplasms - therapy; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; scFv; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - genetics; Single-Chain Antibodies - metabolism; HIS; C7; C5b-9; scFv; C9; NHS; Complement; CD25; MAC
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2013.03.013

•Cancer cells resist cytotoxicity by the Complement membrane attack complex, MAC.•Complement resistance reduces potency of therapeutic antibodies.•Anti-CD25 humanized single chain Fv was fused to Complement protein C9, ScFv-C9.•Recombinant scFv-C9 increases MAC deposition on and death of CD25-positive cells.•Antibody-C9 fusion protein may be useful for cancer immunotherapy.
Complement-dependent cytotoxicity (CDC) is a potent promoter of tumor clearance during monoclonal antibody therapy. Complement activation on antibody-bearing tumor cells results in formation of the membrane attack complex (MAC), which activates cell death. The complement activation cascade that bridges between antibody binding and MAC formation is regulated by complement inhibitors that are over-expressed on tumor cells. In order to bypass those complement regulators, we have designed an immunoconjugate composed of a humanized single chain Fv of an anti-Tac (CD25) monoclonal antibody fused at its C terminus either to complement protein C9 (scFv-C9) or to complement C7 (scFv-C7) and tagged with six histidines at the C terminal end. Recombinant scFv-C9 and scFv-C7 were expressed in 293T cells and purified. Both are shown to efficiently bind to CD25-positive tumor cells. In addition, scFv-C9, but not scFv-C7, increases MAC deposition on the cells and enhances complement-mediated cell death of target CD25-positive cells. Thus, scFv-C9 fusion protein is potentially a novel reagent for application in cancer immunotherapy.