title: The IgM CH2 domain as covalently linked homodimerization module for the generation of fusion proteins with dual specificity.
abstract: Dimeric assembly of antibody fragments and other therapeutic molecules can result in increased binding and improved bioactivity. Here, we investigated the use of the IgM heavy chain domain 2 (MHD2) as covalently linked homodimerization module. Fusion of single-chain fragment variable (scFv) molecules directed against epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 to the N- and/or C-terminus of the MHD2, respectively, resulted in molecules with single or dual specificity for tumor cells. Bispecific tetravalent molecules were further generated by fusing a bispecific single-chain diabody directed against EGFR and epithelial cell adhesion molecule to the N-terminus of the MHD2. By combining an anti-EGFR scFv with a single-chain derivative of tumor necrosis factor, a tetravalent bifunctional fusion protein was produced. This fusion protein exhibited improved TNF activity, also mimicking the membrane-bound form of TNF, as shown by the activation of TNFR2-mediated cell killing. Furthermore, the scFv moiety allowed for an antigen-dependent delivery of TNF to EGFR-positive cells and an improved stimulatory TNF action on these cells. Thus, we established the MHD2 as a versatile module for the generation of bispecific and bifunctional fusion proteins.
authors.affiliation: Institut für Zellbiologie und Immunologie,  Universität Stuttgart, Allmandring 31, 70569 Stuttgart,  Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Seifert
authors.forename: Aline
authors.initials: A
authors.lastname: Plappert
authors.forename: Nadine
authors.initials: N
authors.lastname: Heidel
authors.forename: Sina
authors.initials: S
authors.lastname: Fellermeier
authors.forename: Sylvia K E
authors.initials: SK
authors.lastname: Messerschmidt
authors.forename: Fabian
authors.initials: F
authors.lastname: Richter
authors.forename: Roland E
authors.initials: RE
authors.lastname: Kontermann
mesh_terms: D000595:Amino Acid Sequence; D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D066246:ErbB Receptors / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007075:Immunoglobulin M / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D066246:ErbB Receptors; D007075:Immunoglobulin M; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C508053:ERBB2 protein, human