article accepted in Protein Engineering Design & Selection (PEDS):
The IgM CH2 domain as covalently linked homodimerization module for the generation of fusion proteins with dual specificity
Oliver Seifert, Aline Plappert, Nadine Heidel, Sina Fellermeier, Sylvia K.E. Messerschmidt,
Fabian Richter & Roland E. Kontermann*
*Correspondence: Professor Dr R Kontermann, Institute of Cell Biology and
Immunology, University of Stuttgart, Allmandring 31, Baden-Württemberg, Stuttgart 70569, Germany.
E-mail: roland.kontermann@izi.uni-stuttgart.de
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 scFv fragments directed against EGFR and HER2 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 (scDb) directed against EGFR and EpCAM to the N-terminus of the MHD2. By combining an anti-EGFR scFv with a single-chain derivative of tumor necrosis factor (scTNF), 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.