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.

Tetravalent Antibody–scTRAIL Fusion Proteins with Improved Properties

“…Compared with the previously described MHD2 (14), the EHD2 displays superior stability, evident from a strongly increased thermal stability (melting point 80 C vs. 56 C for EHD2 and MHD2, respectively). In addition, this melting point is higher than that of the CH3 domain derived from human IgG1 (GHD3), which is not covalently linked by disulfide bonds, emphasizing the superior properties of the EHD2 for the generation of dimeric fusion proteins.…”

“…In addition, this melting point is higher than that of the CH3 domain derived from human IgG1 (GHD3), which is not covalently linked by disulfide bonds, emphasizing the superior properties of the EHD2 for the generation of dimeric fusion proteins. Similar to MHD2 (14), the use of EHD2 as a dimerization module has the advantage that molecules can be fused to both ends of this domain, allowing great flexibility and modularity in generating bivalent and bifunctional fusion proteins.…”

“…No authentication was done by the authors. EGFR-Fc and HER2-Fc were purified from stably transfected HEK293 cells (14). Bortezomib was purchased from Sigma-Aldrich, and clinical grade bortezomib and cetuximab were kindly provided by Dr. T. M€ urdter (Institute of Clinical Pharmacology, Margarete Fischer-Bosch Foundation, Stuttgart, Germany).…”

“…Purified EHD2 displayed a melting point of 80 C in dynamic light scattering analysis. The biochemical characteristics of EHD2 were compared with those of purified MHD2 (14) and of domain 3 of the human IgG1 heavy chain (GHD3); the latter is routinely used to generate dimeric minibodies ( Fig. 2; ref.…”

“…The dimerization of scTRAIL in the diabody-scTRAIL fusion protein is mediated by the antibody moiety, thus combining targeting and dimerization in the same module. As an alternative, we have recently demonstrated that the heavychain domain 2 of immunoglobulin M (IgM; MHD2) can be applied to generate covalently linked homodimeric fusion proteins, thus using the MHD2 as a functionally independent dimerization module (14). This allows fusion of various targeting modules to the MHD2, as shown for scFv and bispecific scDb, thereby introducing greater flexibility.…”