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International audience; Angiogenesis targeting is an attractive approach for cancer treatment. Vascular endothelial growth factor receptor 2 (VEGFR2) is such an important target that is overexpressed in tumor vasculature compared to the endothelium cells of resting blood vessels and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins represent a promising group of targeted therapeutics to combat tumors. They consist of an antibody linked to a toxin and are designed to kill specifically the tumor cells. In this study, we fused a VEGFR2-specific Nanobody, the antigen-binding single-domain fragment derived from functional Heavy-chain antibody of Camelidae, to the truncated form of Pseudomonas exotoxin A and evaluated its ability to bind the VEGFR2 molecule on the cell surface. We demonstrate that this immunotoxin inhibits the proliferation of VEGFR2-expressing cells in vitro. This finding is considered to be a significant achievement in tumor therapy and it forms a basis for further studies in animal models.

Angiogenesis targeting is an attractive approach for cancer treatment. Vascular endothelial growth factor receptor 2 (VEGFR2) is such an important target that is overexpressed in tumor vasculature compared to the endothelium cells of resting blood vessels and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins represent a promising group of targeted therapeutics to combat tumors. They consist of an antibody linked to a toxin and are designed to kill specifically the tumor cells. In this study, we fused a VEGFR2-specific Nanobody, the antigen-binding single-domain fragment derived from functional Heavy-chain antibody of Camelidae, to the truncated form of Pseudomonas exotoxin A and evaluated its ability to bind the VEGFR2 molecule on the cell surface. We demonstrate that this immunotoxin inhibits the proliferation of VEGFR2-expressing cells in vitro. This finding is considered to be a significant achievement in tumor therapy and it forms a basis for further studies in animal models.

International audience; AIMS: A novel chimeric-truncated form of t-PA with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in E.coli (BL21) strain and compare the protein potency in batch and fed-batch processes. METHODS AND RESULTS: The expression cassette for the novel t-PA was prepared in pET-28a(+). The E.coli expression procedure was compared in traditional batch and newly developed fed batch; EnBase® Flo system. The protein was purified in soluble format and potency results were identified using Chromolize t-PA assay kit. The fed-batch fermentation mode, coupled with a Ni-NTA affinity purification procedure under native condition resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46.66 IU mg(-1) ) compared to traditional batch mode (35.8 IU mg(-1) ). CONCLUSIONS: Considering the undeniable advantages of expression in the prokaryotic expression systems such as E.coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed-batch cultivation methods showed the potential to replace miss folded formats of protein with proper folded, soluble form with improved potency. SIGNIFICANCE AND IMPACT OF STUDY: E.coli expression of recombinant proteins, still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system. © 2012The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.