Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

AbstractObjectiveTo express human vascular endothelial growth factor121 (VEGF121) in insect cells.MethodsA gene construct containing VEGF was cloned in the pFastBac-HTA vector, followed by transformation in DH10BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF.ResultsOur results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells.ConclusionsResults from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes

Cell-specific targeting by engineered M13 bacteriophage expressing VEGFR2 nanobody

Objective(s): Filamentous bacteriophage M13 was genetically engineered to specifically target mammalian cells for gene delivery purpose. Materials and Methods: A vascular endothelial growth factor receptor 2 (VEGFR2)-specific nanobody was genetically fused to the capsid gene III of M13 bacteriophage (pHEN4/3VGR19). A mammalian expression construct containing Cop-green fluorescent protein (Cop-GFP), as a reporter gene, was amplified by PCR and then sub-cloned in the pHEN4/3VGR19 phagemid. The resulting construct was transfected into 293KDR cell. The recombinant phage was extracted and confirmed and then transduced into VEGFR2 expressing cell (293KDR). Results: Seventy-two hr after transfection, green fluorescence was detected in 30% of the cells. About 1% of the cells which transduced by recombinant phages were able to express GFP. Conclusion: It is hoped that the results from this study will help to find potential vectors to improve the efficiency of gene delivery. Taken together, we conclude that this newly-introduced vector can be used in cancer researches

Candidate Biomarkers for Targeting in Type 1 Diabetes; A Bioinformatic Analysis of Pancreatic Cell Surface Antigens

Objective: Type 1 diabetes (T1Ds) is an autoimmune disease in which the immune system invades and destroysinsulin-producing cells. Nevertheless, at the time of diagnosis, about 30-40% of pancreatic beta cells are healthy andcapable of producing insulin. Bi-specific antibodies, chimeric antigen receptor regulatory T cells (CAR-Treg cells), andlabeled antibodies could be a new emerging option for the treatment or diagnosis of type I diabetic patients. The aimof the study is to choose appropriate cell surface antigens in the pancreas tissue for generating an antibody for type Idiabetic patients.Materials and Methods: In this bioinformatics study, we extracted pancreas-specific proteins from two largedatabases; the Human Protein Atlas (HPA) and Genotype-Tissue Expression (GTEx) Portal. Pancreatic-enrichedgenes were chosen and narrowed down by Protter software for the investigation of accessible extracellular domains.The immunohistochemistry (IHC) data of the protein atlas database were used to evaluate the protein expression ofselected antigens. We explored the function of candidate antigens by using the GeneCards database to evaluate thepotential dysfunction or activation/hyperactivation of antigens after antibody binding.Results: The results showed 429 genes are highly expressed in the pancreas tissue. Also, eighteen genes encodedplasma membrane proteins that have high expression in the microarray (GEO) dataset. Our results introduced fourstructural proteins, including NPHS1, KIRREL2, GP2, and CUZD1, among all seventeen candidate proteins.Conclusion: The presented antigens can potentially be used to produce specific pancreatic antibodies that guide CARTreg,bi-specific, or labeling molecules to the pancreas for treatment, detection, or other molecular targeted therapyscopes for type I diabetes

Monomethyl auristatin E Exhibits Potent Cytotoxic Activity against Human Cancer Cell Lines SKBR3 and HEK293

Background: Monomethyl auristatin E (MMAE) is a synthetic analog of dolastatin 10, a compound originally isolated from the marine mollusk. MMAE, as a highly potent microtubule inhibitor, exerts its potent cytotoxic effect by inhibiting microtubule assembly, tubulin-dependent GTP hydrolysis and microtubes polymerization. This molecule, by itself, lacks the tumor specificity required to elicit therapeutic benefit. Nevertheless, the extremely cytotoxic potential of MMAE could be harnessed in the form of MMAE-antibody conjugates. The aim of the present study was to evaluate the cytotoxic activity of MMAE against breast (SKBR3) and kidney (HEK293) cancer cell lines in an in vitro cell-based assay.Materials and Methods: SKBR3 and HEK293 cells were treated with different concentrations ranging from 0.002048, 0.01024, 0.0512, 0.256, 1.28, 6.4, 32, 160, 800 and 4000 nM of MMAE, and cell viability was determined after 72 hours using an MTT colorimetric assay. The effect of MMAE was regularly monitored by direct observation using an invert microscope.Results: Microscopic observation showed that there was a concentration-dependent increase in cell death. Results from the MTT assay revealed a statistically significant loss of viability (P<0.0001) at concentrations ranging from 0.01024 to 4000 nM in SKBR3 cells, and 0.0512 to 4000 nM in HEK293 cells. Our findings showed that MMAE inhibited the growth of SKBR3 and HEK293 cells in a concentration-dependent manner, with IC50 values of 3.27 ± 0.42 and 4.24 ± 0.37 nM, respectively.Conclusion: MMAE was able to significantly inhibit cell growth at nanomolar concentrations, emphasizing its great potential for the development of antibody-drug conjugates

Monomethyl Auristatin E, a Potent Cytotoxic Payload for Development of Antibody-Drug Conjugates against Breast Cancer

Background: Breast cancer is a heterogeneous disease characterized by differential responses to targeted and chemotherapeutic agents. Antibody-drug conjugates are one of the promising strategies for the treatment of breast cancer. Monomethyl auristatin E (MMAE) is a highly potent microtubule inhibitor and a common payload used for development of antibody-drug conjugates. The purpose of this study was to investigate the cytotoxic effects of MMAE on breast cancer cell lines.Materials and Methods: MDA-MB-468 and MDA-MB-453 cells were treated with MMAE at various concentrations (1, 10, 100, and 1000 ng/ml), and cytotoxicity was measured after 48 and 72 hours using an MTT assay.Results: Our findings indicated that MMAE possesses dose- and time-dependent cytotoxic activities against human breast cancer cells. The morphological features of the treated cells were supportive of the cytotoxic activity of MMAE. The results of the MTT assay showed that MMAE has a significant cytotoxicity against MDA-MB-468 and, to a lesser degree, MDA-MB-453 cells.Conclusion: MMAE can be used as a highly cytotoxic payload for development of antibody-drug conjugates against breast cancer

Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function

Objective(s): Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem.   Materials and Methods: Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody), evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody. Results: Our in silico results revealed an identical 3D structure of the humanized nanobody as compare to original nanobody. In vitro studies also demonstrated that the humanization had no significant visible effect on the nanobody affinity or on its biological activity.  Conclusion: The humanized nanobody could be developed and proposed as a promising lead to target pathologic angiogenesis

An overview on application of phage display technique in immunological studies

Phage display is very strong technique in drug discovery and development. Phage display has many applications in improving the immunological studies. Development of monoclonal antibody, peptides, peptidomimetics and epitope mapping are main application of phage display. Selection of monoclonal antibody or peptides that are displayed on the surface of the phages can be occurred through biopanning process. In biopanning process phage library is incubated with antigen and particular phages can be identified and isolated. Increasing the stringency in the biopanning rounds can be help to select phages with high affinity and specificity. Here, we describe an overview of phage display application with focusing on monoclonal antibody production and epitope mapping

A new and simple non-chromatographic method for isolation of drug/linker constructs: vc-MMAE evaluation

Introduction: Auristatin and its derivatives (synthetic analogues of dolastatin 10, an antineoplastic natural product), are highly potent antimitotic agents which have attracted considerable attention because of their cytotoxic activity when targeting tumor cells in the form of antibody-drug conjugates (ADCs). Some sophisticated and expensive equipment such as HPLC are needed for drug/linker isolation. The aim of this study was to provide a simple aqueous work-up procedure for the isolation of such drug/linker constructs. The anti-tumor activity of the extracted drug/linker was also investigated against SKBR3 and HEK293 cancer cell lines, and cell viability was assessed. Methods: In the present study, monomethyl auristatin E (MMAE), a derivative of the cytotoxic tubulin modifier auristatin E, was covalently coupled to maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB), a cathepsin-B-cleavable linker, to obtain MC-vc-PAB-MMAE (vc-MMAE). Afterwards, a non-chromatographic isolation procedure was developed to isolate the drug/linker (vc-MMAE) construct. Silica gel thin-layer chromatography and electrospray ionization mass spectrometry were used to monitor the isolation procedure and to confirm the coupling of the linker to the drug, respectively. Further, the anti-tumor activity of the extracted drug/linker was investigated against SKBR3 and HEK293 cancer cell lines, and cell viability was assessed. Results: After coupling, the isolation process was confirmed as a single spot on the TLC plate. The isolation yield was calculated to be 65%. [M + H]+, [M + 2Na]+ and [M + ACN + 2H]+ species were observed in the mass spectra, showing that the coupling of MMAE to the linker is not adversely affected by the workup method. Our data revealed that the isolated vc-MMAE was highly potent against tumor cell lines, exhibiting that the workup procedure did not affect MMAE-mediated cytotoxicity. Conclusion: The isolation method described in this study can be applied for the development of a wide variety of ADCs

Expression and Purification of Functional Human Vascular Endothelial Growth Factor-A121; the Most Important Angiogenesis Factor

Purpose: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF) and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206). Methods: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside) and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells) cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated. Results: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro. Conclusion: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay

Selection and characterization of specific nanobody against neuropilin-1 for inhibition of angiogenesis

International audienceNeuropilin-1 (NRP-1), non-tyrosine kinase receptor, was initially identified as axonal protein and later recognized as co-receptor for vascular endothelial growth factor (VEGF). Neuropilins (NRPs) are involved in vascular development and tumor angiogenesis. Over the last years, many studies have been accomplished to inhibit angiogenesis. In this study, the nanobody library was panned against immobilized NRP-1 antigen. High affinity and specificity nanobodies were selected through monoclonal ELISA. The selected nanobodies inhibited proliferation and tube formation of HUVEC and MCF-7 cells in vitro and ex vivo. The results highlight potential of anti-NRP1 nanobodies in inhibition of angiogenesis both in vitro and ex vivo and promises development of novel therapeutics against pathologic angiogenesis