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.

Co-stimulation via receptors of the tumor necrosis factor superfamily (TNFSF) emerges as promising strategy to support antitumor immune responses. Targeted strategies with antibody-fusion proteins composed of a tumor-directed antibody part and the extracellular domain of a co-stimulatory ligand of the TNFSF constitute an attractive option to focus the co-stimulatory activity to the tumor site. Since TNFSF members intrinsically form functional units of non-covalently linked homotrimers, the protein engineering of suitable antibody-fusion proteins is challenging. Aiming for molecules of simple and stable configuration, we used TNFSF ligands in a single-chain format (scTNFSF), i.e., three units of the ectodomain connected by polypeptide linkers, folding into an intramolecular trimer. By fusing tumordirected scFv antibody fragments directed against EpCAM or FAP to co-stimulatory scTNFSF molecules (sc4-1BBL, scOX40L, scGITRL or scLIGHT), a set of monomeric scFv-scTNFSF fusion proteins was generated. In comparison to the scFv-TNFSF format, defined by intermolecular homotrimerization via the TNFSF part, scFv-scTNFSF showed equal or enhanced co-stimulatory activity despite reduced avidity in antibody binding. In addition, enhanced serum stability and improved bioavailability in mice were observed. We show that the scFv-scTNFSF format can be applied to various members of the TNFSF, presenting targeting-dependent co-stimulatory activity. Hence, this format exhibits favorable properties that make it a promising choice for further therapeutic fusion protein development.Abbreviations: EpCAM, epithelial cell adhesion molecule; ED-A, fibronectin extradomain A; FAP, fibroblast activation protein; GITRL, glucocorticoid-induced tumor necrosis factor receptor (GITR) ligand; LIGHT, homologous to lymphotoxins, shows inducible expression and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes; mAb, monoclonal antibody; scFv, single-chain fragment variable; TNF, tumor necrosis factor; TNFSF, tumor necrosis factor superfamily; TRAIL, TNF-related apoptosis inducing ligand

Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and E GSH was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis.

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.

Antibody fragments, especially single-chain Fv fragments, have been established for the generation of immunoliposomes for targeted drug delivery in cancer therapy and other applications. Bispecific immunoliposomes should be useful for dual targeting addressing inter- and intratumoral heterogeneity of tumor antigen expression. Here, we established a protocol to generate dual-targeted immunoliposomes using genetically engineered scFv molecules recognizing two different tumor-associated antigens, EGFR and CEA (CEACAM5), applying a step-wise insertion of antibody-coupled micelles into preformed PEGylated liposomes. The dual-targeted immunoliposomes retained binding activity for both antigens and combined the selectivity of both antibodies within one liposome. Thus, these dual-targeted immunoliposomes should be suitable to deliver therapeutic payloads to tumor cells expressing EGFR or CEA, or both antigens