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PDF file - 126K, Supplementary Figure 2. In vitro cytotoxicity of EHD2-scTRAIL and scFv-EHD2-scTRAIL in comparison with scTRAIL

PDF file - 110K, Supplementary Figure 3. In vitro cytotoxicity of scFv-EHD2-scTRAIL in comparison with Db-scTRAIL.

PDF file - 41K

PDF file - 132K, Supplementary Figure 1. Determination of melting points of the different fusion proteins by dynamic light scattering.

PDF file - 85K, Supplementary Table 1: In vitro cytotoxicity; Supplementary Table 2: Pharmacokinetic properties of EHD2 fusion proteins

We applied the immunoglobulin E (IgE) heavy-chain domain 2 (EHD2) as the covalently linked homodimerization module to generate antibody–scTRAIL fusion proteins. By fusing a humanized single-chain fragment variable (scFv) directed against EGFR to the N-terminus of the EHD2 and a single-chain derivative of TRAIL (scTRAIL) to the C-terminus of the EHD2, we produced a dimeric, tetravalent fusion protein. The fusion protein retained its binding activity for EGFR and TRAIL receptors. In vitro, the targeted antibody–scTRAIL fusion protein exhibited an approximately 8- to 18-fold increased cytotoxic activity compared with the untargeted EHD2-scTRAIL fusion protein. This resulted in increased antitumor activity in a subcutaneous Colo205 xenograft tumor murine model. In summary, the scFv-EHD2-scTRAIL fusion protein combines target cell selectivity with an increased TRAIL activity leading to improved antitumor activities. Mol Cancer Ther; 13(1); 101–11. ©2013 AACR.

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.