Current trends in targeted therapy for drug-resistant infections.

Escalating antibiotic resistance is now a serious menace to global public health. It may be led to the emergence of "postantibiotic age" in which most of infections are untreatable. At present, there is an essential need to explore novel therapeutic strategies as a strong and sustainable pipeline to combat antibiotic-resistant infections. This review focuses on recent advances in this area including therapeutic antibodies, antimicrobial peptides, vaccines, gene therapy, genome editing, and phage therapy for tackling drug-resistant infections.

Development of a Novel Human scFv Against EGFR L2 Domain by Phage Display Technology.

Epidermal growth factor receptor (EGFR) as a transmembrane tyrosine kinase receptor frequently overexpresses in tumors with epithelial origin. The L2 domain from extracellular part of EGFR is involved in ligand binding and the blockage of this domain prevents activation of related signaling pathways. This study was aimed to develop a novel human scFv against EGFR L2 domain as a promising target for cancer therapy. The L2 recombinant protein was purified and used for panning a human scFv phage library (Tomlinson I). In this study, a novel screening strategy was applied to select clones with high binding and enrichment of rare specific phage clones of the L2 protein. After five biopanning rounds several specific clones were isolated which among them one phage clone with high binding was purified for further analysis. The specific interaction of selected clone against target antigen was confirmed by ELISA and western blotting. Immunofluorescence staining showed that purified scFv binds to A431 cells surface, displaying EGFR surface receptor. In the present study, we isolated for the first time a novel human scFv against EGFR L2 domain. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFR overexpressing cancers using this novel human anti-L2 ScFv.

Evolution of phage display technology: from discovery to application.

Phage display technology as a selection-based system is an attractive method for evolution of new biological drugs. Unique ability of phage libraries for displaying proteins on bacteriophage surfaces enable them to make a major contribution in diverse fields of researches related to the diagnosis and therapy of diseases. One of the great challenges facing researchers is the modification of phage display technology and the development of new applications. This article reviews the molecular basis of phage display library, and summarizes the novel and specific applications of this technique in the field of biological drugs development including therapeutic antibodies, peptides, vaccines, and catalytic antibodies.

Invert biopanning: A novel method for efficient and rapid isolation of scFvs by phage display technology.

Phage display is a prominent screening technique for development of novel high affinity antibodies against almost any antigen. However, removing false positive clones in screening process remains a challenge. The aim of this study was to develop an efficient and rapid method for isolation of high affinity scFvs by removing NSBs without losing rare specific clones. Therefore, a novel two rounds strategy called invert biopanning was developed for isolating high affinity scFvs against EGFRvIII antigen from human scFv library. The efficiency of invert biopanning method (procedure III) was analyzed by comparing with results of conventional biopanning methods (procedures I and II). According to the results of polyclonal ELISA, the second round of procedure III displayed highest binding affinity against EGFRvIII peptide accompanied by lowest NSB comparing to other two procedures. Several positive clones were identified among output phages of procedure III by monoclonal phage ELISA which displayed high affinity to EGFRvIII antigen. In conclusion, results of our study indicate that invert biopanning is an efficient method for avoiding NSBs and conservation of rare specific clones during screening of a scFv phage library. Novel anti EGFRvIII scFv isolated could be a promising candidate for potential use in treatment of EGFRvIII expressing cancers.

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.

Development and evaluation of a single domain antibody against human epidermal growth factor receptor (EGFR).

Epidermal growth factor receptor (EGFR) plays an important role in cell growth, multiplication and differentiation. Over expression of EGFR is associated with carcinogenesis and seen in variety of cancers. Anti-EGFR monoclonal antibodies can block EGFR downstream signaling pathway resulting in inhibition of uncontrolled cell proliferation. Antibody fragments have a variety of advantages. In comparison to full length antibodies they have smaller size and therefor exhibit better tumor penetration ability. The aim of this study was to prepare a single domain antibody to target extracellular domain of EGFR. mRNA was extracted from C225 hybridoma cells producing anti-EGFR antibody and subjected to reverse transcription reaction (RT-PCR) to obtain cDNA molecules encoding VH domain of mAb C225. The cDNA encoded VH domain was in frame introduced into the pET-22b(+) vector and expressed in BL21 (DE3) bacterial cells. The resultant antibody was purified via Ni- NTA column and its reactivity was assessed by ELISA and western blot techniques using A431 cell lysate. Analysis by ELISA revealed that this single domain antibody was able to bind EGFR on A431cells. This result was further confirmed by western blotting. In conclusion, the results of this study indicated that single domain antibody can identify and bind to EGFR of A431 carcinoma cells. This recombinant fragment antibody would potentially be used for targeting of cancer cells with high EGFR expression.

Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli.

PURPOSE: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. METHODS: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. RESULTS: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. CONCLUSION: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies.

Development trends for generation of single-chain antibody fragments.

Recombinant antibodies are increasingly being employed as therapeutic agents especially in combination with anti-cancer drugs. The single-chain antibody fragments are small antigen-binding proteins which provide the most commonly used antibody formats for diagnostic and therapeutic purposes. These antibody fragments have more rapid tumor penetration and clearance from the serum relative to full-length monoclonal antibodies. There are in vitro antibody-display technologies such as phage display, cell surface display, ribosome display and mRNA display that can be used to isolate high specificity and affinity single-chain antibodies against a wide variety of targets. We review these strategies for generation of stable and active antibody fragments in the present article.

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.