Production of a Ribosome-Displayed Mouse scFv Antibody Against CD133, Analysis of Its Molecular Docking, and Molecular Dynamic Simulations of Their Interactions.

The pentaspan transmembrane glycoprotein CD133, prominin-1, is expressed in cancer stem cells in many tumors and is promising as a novel target for the delivery of cytotoxic drugs to cancer-initiating cells. In this study, we prepared a mouse library of single-chain variable fragment (scFv) antibodies using mRNAs isolated from mice immunized with the third extracellular domain of a recombinant CD133 (D-EC3). First, the scFvs were directly exposed to D-EC3 to select a new specific scFv with high affinity against CD133 using the ribosome display method. Then, the selected scFv was characterized by the indirect enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (ICC), and in silico analyses included molecular docking and molecular dynamics simulations. Based on ELISA results, scFv 2 had a higher affinity for recombinant CD133, and it was considered for further analysis. Next, the immunocytochemistry and flow cytometry experiments confirmed that the obtained scFv could bind to the CD133 expressing HT-29 cells. Furthermore, the results of in silico analysis verified the ability of the scFv 2 antibody to bind and detect the D-EC3 antigen through key residues employed in antigen-antibody interactions. Our results suggest that ribosome display could be applied as a rapid and valid method for isolation of scFv with high affinity and specificity. Also, studying the mechanism of interaction between CD133's scFv and D-EC3 with two approaches of experimental and in silico analysis has potential importance for the design and development of antibody with improved properties.

Physicochemical Stimulus-Responsive Systems Targeted with Antibody Derivatives.

The application of monoclonal antibodies and antibody fragments with the advent of recombinant antibody technology has made notable progress in clinical trials to provide a regulated drug release and extra targeting to the special conditions in the function site. Modification of antibodies has facilitated using mAbs and antibody fragments in numerous models of therapeutic and detection utilizations, such as stimuli-responsive systems. Antibodies and antibody derivatives conjugated with diverse stimuli-responsive materials have been constructed for drug delivery in response to a wide range of endogenous (electric, magnetic, light, radiation, ultrasound) and exogenous (temperature, pH, redox potential, enzymes) stimuli. In this report, we highlighted the recent progress on antibody-conjugated stimuli-responsive and dual/multi-responsive systems that affect modern medicine by improving a multitude of diagnostic and treatment strategies.

Green Synthesis of Silver Nanoparticles Using the Plant Extracts of Vitex Agnus Castus L: An Ecofriendly Approach to Overcome Antibiotic Resistance.

Background: These days, silver nanoparticles (Ag NPs) have been given considerable attention and applied in medical technology due to their great antimicrobial and antioxidant features. In the present study, we aimed to synthesize Ag NPs through the reduction of silver nitrate in the presence of Vitex agnus castus L fruit extract. Methods: After collecting fruits, their extract was prepared and added to Ag NO3 to produce Ag NPs. The effect of different parameters like AgNO3 concentration (0.5, 1, 3, and 5 mM), sunlight exposure, and sunlight irradiation time (10, 20, 30, and 40 min) was investigated in the synthesis of Ag NPs. The features of Ag NPs were characterized using UV-visible spectroscopy, scanning electron microscope (SEM), X-ray diffraction (XRD) analysis, and dynamic light scattering analysis. Moreover, antimicrobial function of Ag NPs was evaluated using Escherichia coli and Bacillus cereus bacteria species and minimal inhibitory concentration (MIC) of Ag NPs against these two pathogens was measured. Results: The results showed that the synthesized nanoparticles had a spherical shape and the range size of 30-60 nm. For the first time, the antimicrobial activity of synthesized Ag NPs of Vitex agnus castus L fruit extract was shown. Conclusions: It can be stated that the biosynthesis of Ag NPs using fruit extract of this plant is an environmentally friendly, economic and harmless method without any use of poisonous substances and no side effects. These Ag NPs can be considered as suitable antibacterial agents and replacements for antibiotics.

Antibody Fragment and Targeted Colorectal Cancer Therapy: A Global Systematic Review.

BACKGROUND AND AIMS: Antibody-based therapeutics have been shown to be promising for the treatment of colorectal cancer patients. However, the size and long-circulating half-lives of antibodies can limit their reproducible manufacture in clinical studies. Consequently, in novel therapeutic approaches, conventional antibodies are minimized and engineered to produce fragments like Fab, scFv, nanobody, bifunctional antibody, bispecific antibody, minibody, and diabody to preserve their high affinity and specificity to target pharmaceutical nanoparticle conjugates. This systematic review for the first time aimed to elucidate the role of various antibody fragments in colorectal cancer treatment. METHODS: A systematic literature search in the web of sciences, PubMed, Scopus, Google Scholar, and ProQuest was conducted. Reference lists of the articles were reviewed to identify the relevant papers. The full-text search included articles published in English during 19902021. RESULTS: Most of the 53 included studies were conducted in vitro and in most conducted studies singlechain antibodies were among the most used antibody fragments. Most antibodies targeted CEA in the treatment of colorectal cancer. Moreover, a large number of studies observed apoptosis induction and tumor growth inhibition. In addition, few studies implicated the role of the innate immune system as an indirect mechanism of tumor growth by enhancing NK-cell killing. CONCLUSION: Antibody-based therapy was demonstrated to be of great promise in the treatment of colorectal cancer rather than common treatments such as radiotherapy, chemotherapy, and surgical operations. This type of specified cancer treatment can also induce the activation of the innate and specific immune systems to eradicate tumor cells.

An In-silico Approach and Experimental Analysis Combination: Two Strategies for Selecting the third Extracellular Domain (D-EC3) of Human CD133 Marker as a Target for Detection of Cancer Stem Cells.

The selection of the appropriate fragment of the cell surface receptors as an antigen is significant for the production of antibodies. CD133, as a suitable biomarker candidate in the cancer stem cells (CSCs), is a glycosylated protein. The antibodies used for analyzing it recognize glycosylated epitopes of CD133. Since the glycosylated motifs have a dynamic nature over the lifetime of a protein, they limit the detection of CD133. In this study, to access a specific antibody against the antigenic, accessible, and non-glycosylated fragment of the native CD133, we performed an in-silico analysis. Then, we expressed the third domain (D-EC3) (serine641-leucine710) in E. coli BL21 (DE3), then the purified recombinant antigen immunized BALB/c mice. Finally, the dignity of an epitope of pure recombinant antigen has been approved by the interactions of antibody and antigen with the use of mice immunized sera via ELISA and flow cytometry experimentation. The results showed that the selected non-glycosylated fragment can compete well with the commercial antibody against the glycosylated epitopes to identify the native cell surface markers. The results can be considered for diagnosis and target therapy development of CD133+ cancer cells.

Recent developments in antibody derivatives against colorectal cancer; A review.

Colorectal cancer (CRC) is the fourth most common cause of cancer and mortality worldwide and is the third most common cancer in men and women. Surgery, radiotherapy, and chemotherapy are conventionally used for the treatment of colorectal cancer. However, these methods are associated with various side effects on normal cells. Thus, new studies are moving towards more effective and non-invasive methods for treatment of colorectal cancer. Targeted therapy of CRC is a promising new approach to enhance the efficiency and decrease the toxicity of the treatment. In targeted therapy of CRC, antibody fragments can directly inhibit tumor cell growth and proliferation. They also can act as an ideal carrier for targeted delivery of anticancer drugs. In the present study, the structure and function of different formats of antibody fragments, immune-targeted therapy of CRC using antibody fragments will be discussed.

Current status of COVID-19 pandemic; characteristics, diagnosis, prevention, and treatment.

Humans have always been encountered to big infectious diseases outbreak throughout the history. In December 2019, novel coronavirus (COVID-19) was first noticed as an agent causing insidious pneumonia in Wuhan, China. COVID-19 was spread rapidly from Wuhan to the rest of the world. Until late June 2020, it infected more than 10,000,000 people and caused more than 500,000 deaths in almost all of countries in the world, creating a global crisis worse than all previous epidemics and pandemics. In the current review, we gathered and summarized the results of various studies on characteristics, diagnosis, treatment, and prevention of this pandemic crisis.

Bioinformatics prediction and experimental validation of VH antibody fragment interacting with Neisseria meningitidis factor H binding protein.

OBJECTIVES: We previously conducted an in silico research on the interactions between the ribosome display-selected single chain variable fragment (scFv) and factor H binding protein (fHbp) of Neisseria meningitidis. We found that heavy chain variable (VH) fragment of this scFv had considerable affinity to fHbp. These results led us to evaluate the ability of this small antibody fragment in binding and detection of fHbp antigen. MATERIALS AND METHODS: In this study, at first, the three-dimensional structure of VH fragment was simulated by Kotai Antibody Builder web server. By using ClusPro 2.0 web server, the 3D structure of the soluble form of fHbp (PDB: 2KC0) was docked to the modeled VH fragment to extract the structure of the complex's binding. Molecular dynamics (MD) simulation was carried out using GROMACS 4.5.3 package for 65 ns. Secondly, coding sequence of VH fragment was cloned separately and expressed in Escherichia coli. After purification of the VH fragment, its binding activity to fHbp protein was analyzed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) method. RESULTS: Important amino acids involved in antigen- antibody interaction were identified by analyzing the fHbp-VH complex. The ability of the VH antibody fragment to bind and detect fHbp antigen has been confirmed by the results of in silico analysis, ELISA and SPR methods. CONCLUSION: These results showed that this small fragment of antibody could be used for designing diagnostic kits.

Antimicrobial peptides: a promising strategy for lung cancer drug discovery?

INTRODUCTION: Antimicrobial peptides (AMPs), also called host defense peptides (HDPs), are identified in almost any form of life, which play an important role in innate immune systems. They have a broad spectrum of antifungal, antiviral, antibacterial, and anticancer activities. Lung cancer remains the leading cause of global cancer-related death. Unfortunately, lung cancer chemotherapy is accompanied by serious side effects, nonspecific toxicity, and multidrug resistance. Hence, to overcome these drawbacks, anticancer peptides (ACPs) derived from AMPs may represent a potential promising synergistic treatment strategy for lung cancer. AREAS COVERED: In this review, the authors provide the recent advancements in the use of AMPs for the treatment of lung cancer. Furthermore, the anti-lung cancer modes of action of these peptides have been fully reviewed. Importantly, various strategies for increasing the efficiency and safety of AMPs have been discussed. EXPERT OPINION: The combination of AMPs and other cancer treatment approaches such as chemotherapy, nanoparticle-based delivery systems, and photodynamic therapy can be used as a promising revolutionary strategy for the treatment of lung cancer. The most significant limitations of this strategy that need to be focused on are low efficiency and off-target events.

An update on antiviral antibody-based biopharmaceuticals.

Due to the vastness of the science virology, it is no longer an offshoot solely of the microbiology. Viruses have become as the causative agents of major epidemics throughout history. Many therapeutic strategies have been used for these microorganisms, and in this way the recognizing of potential targets of viruses is of particular importance for success. For decades, antibodies and antibody fragments have occupied a significant body of the treatment approaches against infectious diseases. Because of their high affinity, they can be designed and engineered against a variety of purposes, mainly since antibody fragments such as scFv, nanobody, diabody, and bispecific antibody have emerged owing to their small size and interesting properties. In this review, we have discussed the antibody discovery and molecular and biological design of antibody fragments as inspiring therapeutic and diagnostic agents against viral targets.

Dendritic Cell-Based Therapy Using LY6E Peptide with a Putative Role Against Colorectal Cancer.

INTRODUCTION: Albeit early stage gastrointestinal (GI) carcinomas have a good prognosis if treated with surgery, diagnosis is often confirmed at a late stage and efficacious drugs are lacking. Recent progress in immune-based therapies has focused on dendritic cells (DCs), aiming to elicit tumor-specific responses by inducing immunological memory. Our previous microarray study indicated that a biomarker, termed lymphocyte antigen-6E (LY6E), is commonly overexpressed in two potentially lethal GI cancers: those of colon and stomach. In this study, we examined the antigenic potency of LY6E in stimulating DCs. METHODS: Following isolation, differentiation, and maturation of mononuclear cells, DCs were pulsed with LY6E peptide, a protein related to major histocompatibility complex (MHC) class I/II. Subsequently, DCs were co-cultured with mouse splenocytes to assess antigen-specific T-cell proliferation. Elucidated cytotoxic T-lymphocyte responses were assessed using subcutaneous colorectal murine tumor models. RESULTS: Our in vitro results suggest that DCs loaded with LY6E peptide antigen are capable of stimulating and inducing proliferation of murine T-cells. Furthermore, our in vivo results demonstrate that LY6E peptide has a substantial impact on provoking immune responses against induced colon cancer in mice. DISCUSSION: In conclusion, based on the overexpression of LY6E in colorectal, gastric, and pancreatic cancers, the role of this peptide should be further investigated with a goal of developing new therapies for these challenging diseases.

Antibody-drug therapeutic conjugates: Potential of antibody-siRNAs in cancer therapy.

Codelivery is a promising strategy of targeted delivery of cytotoxic drugs for eradicating tumor cells. This rapidly growing method of drug delivery uses a conjugate containing drug linked to a smart carrier. Both two parts usually have therapeutic properties on the tumor cells. Monoclonal antibodies and their derivatives, such as Fab, scFv, and bsAb due to targeting high potent have now been attractive candidates as drug targeting carrier systems. The success of some therapeutic agents like small interfering RNA (siRNA), a small noncoding RNAs, with having problems such as enzymatic degradation and rapid renal filtration need to an appropriate carrier. Therefore, the aim of this study is to review the recent enhancements in development of antibody drug conjugates (ADCs), especially antibody-siRNA conjugates (SRCs), its characterizations and mechanisms in innovative cancer therapy approaches.

Identification and characterization of a novel single-chain variable fragment (scFv) antibody against Neisseria meningitidis factor H-binding protein (fHbp).

Purpose. Neisseria meningitidis is the leading global cause of meningitis and sepsis. Detection, followed by identification, of bacterial pathogens is important in medicine and public health. In the present study, we used the ribosome display technique to select single-chain variable fragments (scFv) that are specific to the surface-exposed fHbp antigen of N. meningitidis. Methodology. The recombinant fHbp protein was used as the antigen for the immunization of BALB/c mice. Anti-fHbp VH/k chain ribosome display libraries were assembled by joining VH and k into the VH/k chain with a specially constructed linker by PCR overlap extension. The scFv library was panned against the recombinant fHbp protein by using a single round of the ribosome display method via a rabbit reticulocyte lysate system.Results/Key findings. The selected anti-fHbp antibody exhibited high affinity and specificity in the enzyme-linked immunosorbent assay (ELISA) and the whole bacterial cell enzyme-linked immunosorbent assay (Bact-ELISA).Conclusion. The affinity of the selected scFv was ~8.65×109 M-1. The isolated scFv can provide the basis for developing a diagnostic kit.

Targeted cancer therapy through antibody fragments-decorated nanomedicines.

Active targeting in cancer nanomedicine, for improved delivery of agents and diagnose, has been reviewed as a successful way for facilitating active uptake of theranostic agents by the tumor cells. The application of a targeting moiety in the targeted carrier complexes can play an important role in differentiating between tumor and healthy tissues. The pharmaceutical carriers, as main part of complexes, can be polymeric nanoparticles, micelles, liposomes, nanogels and carbon nanotubes. The antibodies are among the natural ligands with highest affinity and specificity to target pharmaceutical nanoparticle conjugates. However, the limitations, such as size and long circulating half-lives, hinder reproducible manufacture in clinical studies. Therefore, novel approaches have moved towards minimizing and engineering conventional antibodies as fragments like scFv, Fab, nanobody, bispecific antibody, bifunctional antibody, diabody and minibody preserving their functional potential. Different formats of antibody fragments have been reviewed in this literature update, in terms of structure and function, as smart ligands in cancer diagnosis and therapy of tumor cells.

Cloning, expression and purification of the factor H binding protein and its interaction with factor H.

BACKGROUND AND OBJECTIVE: Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. The factor H binding protein (fHBP) is a key virulence factor of Neisseria meningitidis that is able to selectively bind to human factor H, the key regulator of the alternative complement pathway, which it has important implications for meningococcal pathogenesis and vaccine design. The aims of present research were cloning, expression, purification of fHbp and confirmation of the interaction between serum factor H (fH) and produced factor H binding protein. MATERIALS AND METHODS: A 820 base pairs fhbp gene fragment was amplified by PCR and cloned into expression vector pET28a (+) in Bam HI and SalI restriction enzymes sites. Recombinant DNA was expressed in BL21 (DE3) cell. fHBP protein was purified by Ni-NTA agarose resin. Coupling of recombinant protein into CNBr activated Sepharose 4B resin was carried out for application in serum fH protein purification. (fH-fHBP) interaction was confirmed by SDS-PAGE and far-western blotting. RESULTS AND CONCLUSIONS: SDS-PAGE results showed a 35 kDa protein band. 150 kDa fH protein was purified by designed Sepharose 4B resin. Far-western blotting confirmed (fH-fHBP) interaction and proper folding of factor H binding protein.

Comparative study of the reactivity of natural and mutated streptokinase with total antistreptokinase antibodies in human sera.

Streptokinase is widely used as an anticoagulant drug for the treatment of heart attacks. Because of antibody production against injected drug in individuals consuming streptokinase and causing allergic reactions, streptokinase treatment effects become neutral. Recombinant mutant type of streptokinase was prepared by removing of 42 amino acids from the C terminal region. ELISA plates were coated by natural and mutant streptokinase as antigen. Ninety-six normal serum samples as well as 27 streptokinase consumer serum samples (patients with acute myocardial infraction) were analyzed. The results showed that serum antibodies against natural streptokinase were three times more than those against the mutated streptokinase. In case of preserving thrombolytic activity, mutated streptokinase can be used as an alternative of the natural form.

Construction of Plasmid Insulin Gene Vector Containing Metallothionein IIA (pcDNAMTChIns) and Carbohydrate Response Element (ChoRE), and Its Expression in NIH3T3 Cell Line.

BACKGROUND: Type 1 diabetes mellitus is one of the metabolic diseases that cause insulin-producing pancreatic ß cells be destroyed by immune system self-reactive T cells. Recent-ly, new treatment methods have been developed including use of the stem cells, ß islet cells transplantation and gene therapy by viral and non-viral gene constructs. OBJECTIVES: The aim of this project was preparing the non-viral vector containing the glucose inducible insulin gene and using it in the NIH3T3 cell line. MATERIALS AND METHODS: Cloning was carried out by standard methods. Total RNA was extracted from pancreatic tissue, RNA was converted to cDNA using RT-PCR reaction and preproinsulin gene was amplified using specific primers. PNMTCH plasmid was extract-ed and digested by NotI, HindIII, and MTIIA and ChoRE genes were purified and cloned into pcDNA3.1 (-) plasmid and named pcDNAMTCh. Finally, the preproinsulin genes were cloned into pcDNA3.1 (-) plasmid and pcDNAMTChIns was built. RESULTS: The cloned gene constructs were evaluated by restriction enzyme digestion and RT-PCR. The NIH3T3 cells were transfected by plasmid naked DNA containing preproinsu-lin gene and expression was confirmed by Reverse Transcriptase PCR and Western Blot-ting Techniques. CONCLUSIONS: Gel electrophoresis of PCR products confirmed that cloning was per-formed correctly. The expression of preproinsulin gene in recombinant plasmid in NI-H3T3 cell line was observed for the first time. The findings in this study can be the basis of further research on diabetes mellitus type 1 gene therapy on animals.

Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania.

BACKGROUND: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. METHODS: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. RESULTS: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6-biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. CONCLUSION: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully.