Tumour induction in BALB/c mice for imaging studies: An improved protocol.

Dealing with nude mice, which lack thymus and therefore are sensitive to unsterile conditions, needs special care and laboratory conditions. For preclinical studies, especially tumour imaging purposes, in which therapeutic properties of drugs or therapeutic compounds are not studied, mice with normal immune system can be a favourable alternative if they carry tumours of interest. In the current study, we introduce an optimized protocol for induction of human tumours in BALB/c mice for preclinical studies. Immune system of BALB/c mice was suppressed by administration of cyclosporine A (CsA), ketoconazole and cyclophosphamide. The tumours of MDA-MB-231, A-431 and U-87-MG human cancer cells were induced by subcutaneous injection of the cells to the immunosuppressed mice. Tumour size was calculated weekly. Histopathological and metastatic analyses were performed using haematoxylin and eosin staining. The combination of the three drugs was found to suppress immune system and decrease the numbers of white blood cells, including lymphocytes. At the eighth week, tumours with a dimension of approximately 1400 mm3 developed. Large atypical nuclei with scant cytoplasm were found to exist using histopathological analysis. No metastasis was observed in the tumour-bearing mice. A combination of CsA, ketoconazole and cyclophosphamide can be used to suppress the immune system in BALB/c mice and induce tumours with significant size.

Near-Infrared Fluorescence Imaging of EGFR-Overexpressing Tumors in the Mouse Xenograft Model Using scFv-IRDye800CW and Cetuximab-IRDye800CW.

EGFR (epidermal growth factor receptor) is overexpressed in a variety of human cancers (including squamous cell carcinoma of head and neck, colon cancer, and some breast cancers) and therefore is regarded as an ideal target for cancer therapy or imaging purposes. In the current study, we produced a scFv-based near-infrared probe (called cet.Hum.scFv-IRDye-800CW) and evaluated its ability in recognizing and imaging of EGFR-overexpressing tumors in a mouse model. Like the molecular probe consisting of its parental antibody (cetuximab, an FDA-approved monoclonal antibody) and IRD800CW, cet.Hum.scFv-IRDye-800CW was able to recognize EGFR-overexpressing tumors in mice. cet.Hum.scFv-IRDye-800CW was found to be superior to the cetuximab-based probe in imaging of mouse tumors. The tumor-to-background ratio and blood clearance rate were higher when cet.Hum.scFv-IRDye-800CW was used as an imaging probe.

Obtaining active recombinant proteins from bacterial inclusion bodies using salt solutions under neutral pH conditions.

Eukaryotic recombinant proteins expressed in bacterial cells usually aggregate within the cells as inclusion bodies. Despite the widely-accepted theory considering inclusion bodies as inactive materials, inclusion bodies may contain large amounts of correctly-folded active recombinant proteins. Proteins trapped in inclusion bodies can be released using a high pH solution (pH ≥ 11); however, they may undergo structural changes in such pH conditions that may lead to their inactivation. Shifting in pH alongside the use of metal ions can help recover protein activity. The model protein we used in this study, 9R-Nimo.scFv, is highly active when extracted from bacterial inclusion bodies at high pH condition (pH 12) but loses its activity when pH is reduced to pH 7. We evaluated the capacity of nine salt solutions in terms of recovering protein activity in neutral pH conditions and found that ZnSO4 solution was the best one for this purpose. KNO3 and MnSO4 were also found to have a good capacity for recovering protein activity, as well.

In-Situ Gel-Free Plasmid Reassembling for Rapid Gene Subcloning and Truncation

Abstract Gene subcloning, a process in which the nucleotide sequence of interest is excised from on plasmid and inserted into another, seems to be an easy task to done. However, not all subcloning attempts are successful, even when the insert sequence and the double digested target plasmid are successfully purified form agarose gel and thought to be ready for subsequent ligation. In the current study we introduce a reliable, easy, and time consuming method for gene subcloning and also truncation. The stages are all carried out in a single microtube without any running on a gel, making it possible to accomplish a successful gene subcloning or truncation even with low concentrations of DNA molecules. Summarily, subcloning is achieved by mixing the plasmids of interest in a microtube and subjecting to restriction enzymes whose restriction sites flank the segment that is going to be subcloned. Digestion mixture is precipitated in the same microtube using isopropanol and the resultant DNA molecules are allowed to take part in a ligation reaction. The recombinant plasmids of interest are screened by colony PCR. Truncation is achieved by double- digestion of the plasmid of interest using a restriction enzyme whose restriction site flanks the segment that is going to be cut out.

Test bacterial inclusion body for activity prior to start denaturing and refolding processes to obtain active eukaryotic proteins.

One of a major drawbacks correlated with expressing antibody fragments in bacterial cells is insolubility, which is often regarded as an obstacle in obtaining active molecules. Recombinant proteins aggregated as inclusion bodies within bacterial cells are thought to be unfolded or misfolded, and therefore inactive. So, denaturing and refolding strategies, which are laborious and sometime inefficient, are used to obtain correctly-folded active proteins. In the current study, we show that large quantities of correctly folded and completely active scFv molecules are there in bacterial inclusion bodies; they only need to be isolated from inclusion bodies.

Engineering of single chain antibodies for solubility.

In the current study, we introduce a method to design intrinsically soluble single chain antibodies (scFvs) that can be easily produced in bacterial expression systems in a soluble form. Summarily, CDR loops are grafted on framework regions derived from intrinsically soluble subclass 3 (VH3 and VL3) human germline sequences. Framework-donor sequences should carry CDR loops of interest (in terms of canonical classes) and contain special residues in their hydrophobic cores. Recombinant variable fragments resultant from CDR grafting are subjected to 3D modeling, mutated (if necessary), and superposed to parental variable domains. Recombinant type 3 variable domains with the least RMSD (Root-Mean-Square Deviation) values are chosen to constitute scFv moieties. The scFv designed using this method was shown to be soluble when expressed in bacterial cells.

Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells.

The aim of this study was to produce a humanized single chain antibody (scFv) as a potential improved product design to target EGFR (Epidermal Growth Factor Receptor) overexpressing cancer cells. To this end, CDR loops of cetuximab (an FDA-approved anti-EGFR antibody) were grafted on framework regions derived from type 3 (VH3 and VL3 kappa) human germline sequences to obtain recombinant VH and VL domainslinked together with a flexible linker [(Gly4Ser)3] to form a scFv. Codon optimized synthetic gene encoding the scFv (with NH2-VH-linker-VL-COOH orientation) was expressed in E. coli Origami™ 2(DE3) cells and the resultant scFv purified by using Ni-NTA affinity chromatography. The scFv, called cet.Hum scFv, was evaluated in ELISA and immunoblot to determine whether it can recognize EGFR. The scFv was able to recognize EGFR over-expressing cancer cells (A-431) but failed to detect cancer cells with low levels of EGFR (MCF-7 cells). Although the affinity of the scFv forA-431 cells was 9 fold lower than that of cetuximab, it was strong enough to recognize these cells. Considering its ability to bind EGFR molecules, the scFv may exhibit a potential application for the detection of EGFR-overexpressing cancer cells.

Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae.

Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet-visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use.

Use of single chain antibody derivatives for targeted drug delivery.

Single chain antibodies (scFvs), which contain only the variable domains of full-length antibodies, are relatively small molecules that can be used for selective drug delivery. In this review, we display how scFv antibodies help improve the specificity and efficiency of drugs. Small interfering RNA (siRNA) delivery using scFv-drug fusion peptides, siRNA delivery using scFv-conjugated nanoparticles, targeted delivery using scFv-viral peptide- fusion proteins, use of scFv in fusion with cell penetrating peptides for effective targeted drug delivery, scFv-mediated targeted delivery of inorganic nanoparticles, scFv-mediated increase of tumor killing activity of granulocytes, use of scFv for tumor imaging, site-directed conjugation of scFv molecules to drug carrier systems, use of scFv to relieve pain, use of scFv for increasing drug loading efficiency are among the topics that are discussed here.

CD20-targeting in B-cell malignancies: novel prospects for antibodies and combination therapies.

Expression of CD20 antigen by the most of transformed B cells is believed to be the driving force for targeting this molecule by using anti-CD20 monoclonal antibodies. While it is true that most lymphoma/leukemia patients can be cured, these regimens are limited by the emergence of treatment resistance. Based on these observations, development of anti-CD20 monoclonal antibodies and combination therapies have been recently proposed, in particular with the aim to optimize the cytotoxic activity. Here we outline a range of new experimental agents concerning the CD20 positive B-cell tumors which provide high benefit from conventional therapy.

Antioxidant Activity of Different Fractions of Methanolic Extract of the Golden Chanterelle Mushroom Cantharellus cibarius (Higher Basidiomycetes) from Iran.

Natural compounds with antioxidant activity can be useful for treatment of reactive oxygen species-related diseases, comprising atherosclerosis, inflammatory injury, cardiovascular disease, cancer, diabetes, Alzheimer's disease, cataracts, autism, and aging. The current study was perform to assay the antioxidant activity different fractions of methanolic extract of golden chanterelle mushroom Cantharellus cibarius, a mushroom found in the north of Iran. Different fractions of methanolic extract of this mushroom, including n-hexane, chloroform, ethyl acetate, n-butanol, and water, were evaluated for antioxidant activity using six in vitro assay systems. Mushroom fruit was obtained from the local market, Sari (northern Iran). The n-hexane fraction had higher amounts of flavonoids contents (40.01 ± 1.30 mg quercetin equivalent g-1 of extract) and the highest exhibition of nitric oxide scavenging activity (77.21 ± 1.48%). The highest content of phenol was observed in the n-butanol fraction, which was 40.97 ± 0.99 mg gallic acid equivalent g-1 of extract. Among all the fractions, the ethyl acetate fraction was found to show higher DPPH scavenging activity (33.43 ± 1.30%) and the aqueous fraction to display the most reducing power. The highest Fe2+ chelating activity was observed in the chloroform fraction and then in the n-hexane fraction (86.13 ± 1.61 and 80.68 ± 2.07, respectively). The results all together signify C. cibarius as a valuable source of natural bioactive compounds with antioxidant activity.

Natural inhibitors of PI3K/AKT signaling in breast cancer: emphasis on newly-discovered molecular mechanisms of action.

Epidermal growth factor receptor (EGFR) plays a critical role in the initiation and progression of a variety of human cancers, including breast cancer. An important signaling pathway downstream of EGFR is the PI3K/AKt pathway, which regulates cellular processes as diverse as cell growth, survival, proliferation and migration. Deregulated activity of this pathway may lead to uncontrolled cell growth, survival, migration and invasion, contributing to tumor formation. In this review, we evaluate natural compounds that, in vitro (breast cancer cell lines) and/or in vivo (animal model, clinical) studies, suppress breast cancer cells or tumors mainly by suppressing the PI3K/AKT signaling pathway. The effect of these compounds on cell cycle arrest, inhibition of cell migration and invasion, tumor angiogenesis and metastasis in breast cancer are discussed.

Recent advances in head and neck squamous cell carcinoma--a review.

The current review presents the results of the most recent studies performed on different aspects of human head and neck squamous cell carcinoma, including radiosensitivity induction, efficiency improvement of monoclonal antibodies using low-intensity ultrasound, chemical compounds such as toll-like receptor (TLC) agonists, dasatinib, resveratrol and niclosamide, nuclear inhibition of cancer using STAT3 decoy oligonucleotide, efficiency of anti-EGFR monoclonal antibodies in detection of head and neck cancers and other related issues.

Optimized condition for enhanced soluble-expression of recombinant mutant anabaena variabilis phenylalanine ammonia lyase.

PURPOSE: Recently discovered Anabaena variabilis phenylalanine ammonia lyase (AvPAL) proved to be a good candidate for enzyme replacement therapy of phenylketonuria. Outstanding stability properties of a mutant version of this enzyme, produced already in our laboratory, have led us to the idea of culture conditions optimization for soluble expression of this therapeutically valuable enzyme in E. coli. METHODS: In the present study, the gene encoding mutant version of AvPAL was cloned into the pET28a expression vector. Different concentrations of IPTG, induction period, growth temperature, shaking speed, as well as different types of culture media were examined with respect to the amount of recombinant protein produced and specific activity of the enzyme. RESULTS: Based upon our findings, maximum amount of active mutant enzyme was attained by addition of 0.5 mM IPTG at 150 rpm to the TB culture media. The yield of active enzyme at cluture tempreature of 25 °C and induction period of 18 hour was the highest. CONCLUSION: The results of this study indicated that the yield of mutant AvPAL production in E. coli can be affected mainly by culture temperature and inducer concentration.

Affinity measurement of single chain antibodies: a mathematical method facilitated by statistical software SigmaPlot.

Because they are monovalent for antigen, single chain antibodies display a different antibody-antigen interaction pattern from that of full-length antibodies. Using the law of mass action and considering the antibody-antigen binding pattern at OD-100% and OD-50% points, we introduced a formula for estimating single chain antibody affinity. Sigmoid curves of optical density values versus antibody concentrations were drawn and used to determine antibody concentrations at OD-50% points using statistical software SigmaPlot. The OD-50% points were then used to calculate the affinity via the mathematical formula. A software-adapted format of the equation is also presented for further facilitation of the calculation process. The accuracy of this method for affinity calculation was proved by surface plasma resonance. This method offers a precise evaluation of antibody affinity without requiring special material or apparatus, making it possible to be performed in any biological laboratory with minimum facilities.

Antihaemolytic activity of thirty herbal extracts in mouse red blood cells.

Reactive oxygen species (ROS) can lead to haemolysis and eventually to diseases such as thalassemia and sickle cell anaemia. Their action can be counteracted by the antihaemolytic activity of therapeutic agents. The aim of our study was to identify plants that most efficiently counteract ROS-caused haemolysis. From ten plants known for their antioxidant activity (Orobanche orientalis G. Beck, Cucumis melo L., Albizzia julibrissin Durazz, Galium verum L., Scutellaria tournefortii Benth, Crocus caspius Fischer & Meyer, Sambucus ebulus L., Danae racemosa L., Rubus fruticsos L., and Artemisia absinthium L.) we prepared 30 extracts using three extraction methods (percolation, Soxhlet, and ultrasound-assisted extraction) to see whether the extraction method affects antihaemolytic efficiency, and one extraction method (polyphenol extraction) to see how much of this action is phenol-related. Extract antihaemolytic activity was determined in mice red blood cells and compared to that of vitamin C as a known antioxidant. Nine of our extracts were more potent than vitamin C, of which G. verum (aerial parts/percolation) and S. tournefortii (aerial parts/polyphenol) extracts were the most potent, with an IC50 of 1.32 and 2.08 µg mL⁻¹, respectively. Haemolysis inhibition depended on extract concentration and the method of extraction. These plants could provide accessible sources of natural antioxidants to the pharmaceutical industry.

humMR1, a highly specific humanized single chain antibody for targeting EGFRvIII.

Production of an efficient humanized single chain antibody is reported here to specifically target EGFRvIII, a truncated receptor expressed in a wide variety of human cancers. CDR loops of MR1, a phage display-derived murine single chain antibody developed against this mutant receptor, were grafted on human frameworks that had been selected based on similarity to MR1 in terms of two distinct parameters, variable domain protein sequence and CDR canonical structures. Moreover, two point mutations were introduced in CDR-H2 and CDR-H3 loops of the humanized antibody to destroy its cross-reactivity to wild-type EGFR. The resultant antibody, referred to as humMR1, was found by MTT assay, ELISA and western blot techniques to be highly specific for EGFRvIII. The affinity of this antibody for EGFRvIII-specific 14-amino acid synthetic peptide and HC2 cells were measured to be 1.87 × 10(10) and 2.17 × 10(10)/M respectively. This humanized antibody leads to 78.5% inhibition in proliferation of EGFRvIII-overexpressing cells.

Protein L: a robust enzyme-conjugated molecule for detection of humanized single chain antibodies.

The current study was aimed at introducing an efficient enzyme-conjugated molecule for use in the detection of humanized single chain antibodies. Because they are composite in their variable domain sequences and also devoid of constant domains, humanized single chain antibodies require a suitable secondary enzyme-conjugated antibody (more precisely enzyme-conjugated protein molecule) to be efficiently recognized and detected in ELISA, dot blot, and other detection tests, guaranteeing a more precise evaluation of their quantity, affinity, and other features. In the current study we examined the ability of three HRP-conjugated protein molecules including protein L, anti-human IgG antibody, and anti-mouse IgG antibody in the detection of three humanized single chain antibodies (anti-CD20, anti-EGFRvIII, and anti-EGFR) in ELISA and dot blot tests. The results signify the HRP-conjugated protein L as the most efficient molecule for detecting humanized single chain antibody.

Antibody humanization methods - a review and update.

This article reviews recent advances achieved during recent years on various aspects of antibody humanization theories and techniques. Common methods for producing humanized antibodies including framework-homology-based humanization, germline humanization, complementary determining regions (CDR)-homology-based humanization and specificity determining residues (SDR) grafting, as well as advantages and disadvantages of each of these methods and their applications are discussed.