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.
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