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.
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.
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.
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 (Gly
Ser)
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.
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.
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•An insoluble scFv becomes soluble if frameworks are replayed by intrinsically soluble alternatives.•Bulky amino acids in special positions act as a bridge and form a more integrated hydrophobic core.•Composition of surface amino acids in some special positions may influence scFv solubility.•scFv containing frameworks derived from VH3 and VL3 subclasses exhibit a high degree of solubility.
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.
•The ability of diverse metal ions in recovering protein activity following pH-shift inactivation is described in this article.•ZnSO4, KNO3 and MnSO4 help restore protein activity after pH-shift inactivation.•CoCl2 and CuSO4 are more suitable for protein precipitation rather than recovering protein activity.
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.
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.
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.
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.
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.
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.
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