Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology.

Angiogenesis, as a tumor hallmark, plays an important role in the growth and development of the tumor vasculature system. There is a huge amount of evidence suggesting that the vascular endothelial growth factor receptor (VEGFR-2)/VEGF-A axis is one of the main contributors to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2 signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In this study, we employ phage display technology and solution-phase biopanning (SPB) to isolate specific single-chain variable fragments (scFvs) against VEGFR-2 and report on the receptor binding characteristics of the candidate scFvs A semisynthetic phage antibody library to isolate anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations of the VEGFR-2-His tag and VEGFR-2-biotin. After successful expression and purification, the specificity of the selected scFv clones was further analyzed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. The competition assay was undertaken to identify the VEGFR-2 receptor-blocking properties of the scFvs. Furthermore, the molecular binding characteristics of candidate scFvs were extensively studied by peptide-protein docking. Polyclonal ELISA analysis subsequent to four rounds of biopanning showed a significant enrichment of VEGFR-2-specific phage clones by increasing positive signals from the first round toward the fourth round of selection. The individual VEGFR-2-reactive scFv phage clones were identified by monoclonal phage ELISA. The sequence analysis and complementarity-determining region alignment identified the four unique anti-VEGFR-2-scFv clones. The soluble and purified scFvs displayed binding activity against soluble and cell-associated forms of VEGFR-2 protein in the ELISA and flow cytometry assays. Based on the inference from the molecular docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and 3 on the VEGFR-2 protein and displayed competition with VEGF-A for binding to VEGFR-2. The competition assay confirmed that scFvs H1 and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells.

Numerical and analytical analysis of an ultrahigh sensitive surface plasmon resonance sensor based on a black phosphorene/graphene heterostructure.

In this study, a surface plasmon resonance biosensor using angular interrogation based on a black phosphorene (BP) and graphene (G) heterostructure as two-dimensional materials are designed to enhance the sensitivity of conventional biosensors. The proposed structure is composed of eight layers: FK51A coupling prism, silver (Ag) thin film as the plasmonic metal, gold (Au) nanolayer in a protective role, BP nanosheets as an evanescent field enhancer, G monolayer as an immobilization process facilitator, DNA aptamer as biorecognition element, and phosphate buffered saline as a running buffer and sensing medium. To evaluate the performance of the proposed biosensor, analytical parameters such as minimum reflectivity (R m i n ), sensitivity, as well as the full width at half-maximum (FWHM), detection accuracy (DA), and quality factor (QF) are systematically assessed by the use of the transfer matrix method analytically and the finite-difference time-domain method numerically, to validate each other. It is observed that the structure has been optimized with 1.49 (RIU) for the coupling prism and the heterostructure T i O 2/A g/A u/B P/G thicknesses of 65/35/1/3.18/0.34 nm, respectively. It was revealed that the proposed biosensor offered the sensitivity of 356 (°/RIU), QF of 42.4 (R I U -1), R m i n of 0.07 (a.u), FWHM of 8.3 (degree), and DA of 0.22 (unitless) and outperformed those of other results published up to now from the sensitivity point of view.

Theranostic chimeric antigen receptor (CAR)-T cells: Insight into recent trends and challenges in solid tumors.

Cell therapy has reached significant milestones in various life-threatening diseases, including cancer. Cell therapy using fluorescent and radiolabeled chimeric antigen receptor (CAR)-T cell is a successful strategy for diagnosing or treating malignancies. Since cell therapy approaches have different results in cancers, the success of hematological cancers has yet to transfer to solid tumor therapy, leading to more casualties. Therefore, there are many areas for improvement in the cell therapy platform. Understanding the therapeutic barriers associated with solid cancers through cell tracking and molecular imaging may provide a platform for effectively delivering CAR-T cells into solid tumors. This review describes CAR-T cells' role in treating solid and non-solid tumors and recent advances. Furthermore, we discuss the main obstacles, mechanism of action, novel strategies and solutions to overcome the challenges from molecular imaging and cell tracking perspectives.

Recent trends in aptamer-based nanobiosensors for detection of vascular endothelial growth factors (VEGFs) biomarker: A review.

Vascular endothelial growth factor (VEGF) is a remarkable cytokine that plays an important role in regulating vascular formation during the angiogenesis process. Therefore, real-time detection and quantification of VEGF is essential for clinical diagnosis and treatment due to its overexpression in various tumors. Among various sensing strategies, the aptamer-based sensors in combination with biological molecules improve the detection ability VEGFs. Aptamers are suitable biological recognition agents for the preparation of sensitive and reproducible aptasensors (Apt-sensors) due to their low immunogenicity, simple and straightforward chemical modification, and high resistance to denaturation. Here, a summary of the strategies for immobilization of aptamers (e.g., direct or self-assembled monolayer (SAM) attachment, etc.) on different types of electrodes was provided. Moreover, we discussed nanoparticle deposition techniques and surface modification methods used for signal amplification in the detection of VEGF. Furthermore, we are investigating various types of optical and electrochemical Apt-sensors used to improve sensor characterization in the detection of VEGF biomarkers.

Recent Trends in Diagnostic Biomarkers of Tumor Microenvironment.

The tumor microenvironment (TME) play critical roles in tumor survival, progression, and metastasis and can be considered potential targets for molecular imaging of cancer. The targeting agents for imaging of TME components (e.g., fibroblasts, mesenchymal stromal cells, immune cells, extracellular matrix, blood vessels) provide a promising strategy to target these biomarkers for the early diagnosis of cancers. Moreover, various cancer types have similar tumor immune microenvironment (TIME) features that targeting those biomarkers and offer clinically translatable molecular imaging of cancers. In this review, we categorize and summarize the components in TME which have been targeted for molecular imaging. Moreover, this review updated the recent progress in targeted imaging of TIME biological molecules by various modalities for the early detection of cancer.

Implementation of a Design of Experiments to Improve Periplasmic Yield of Functional ScFv Antibodies in a Phage Display Platform.

Purpose: Production of functional recombinant antibody fragments in the periplasm of E. coli is a prerequisite step to achieve sufficient reagent for preclinical studies. Thus, the cost-effective and lab-scale production of antibody fragments demands the optimization of culture conditions. Methods: The culture conditions such as temperature, optical density (OD600) at induction, induction time, and IPTG concentration were investigated to optimize the functional expression of a phage-derived scFv molecule using a design of experiment (DoE). Additionally, the effects of different culture media and osmolyte supplements on the expression yield of scFv were examined. Results: The developed 2FI regression model indicated the significant linear effect of the incubation temperature, the induction time, and the induction OD600 on the expression yield of functional scFv. Besides, the statistical analysis indicated that two significant interactions of the temperature/induction time and the temperature/induction OD600 significantly interplay to increase the yield. Further optimization showed that the expression level of functional scFv was the most optimal when the cultivation was undertaken either in the TB medium or in the presence of media supplements of 0.5 M sorbitol or 100 mM glycine betaine. Conclusion: In the present study, for the first time, we successfully implemented DoE to comprehensively optimize the culture conditions for the expression of scFv molecules in a phage antibody display setting, where scFv molecules can be isolated from a tailor-made phage antibody library known as "Human Single Fold scFv Library I."

An overview on display systems (phage, bacterial, and yeast display) for production of anticancer antibodies; advantages and disadvantages.

Antibodies as ideal therapeutic and diagnostic molecules are among the top-selling drugs providing considerable efficacy in disease treatment, especially in cancer therapy. Limitations of the hybridoma technology as routine antibody generation method in conjunction with numerous developments in molecular biology led to the development of alternative approaches for the streamlined identification of most effective antibodies. In this regard, display selection technologies such as phage display, bacterial display, and yeast display have been widely promoted over the past three decades as ideal alternatives to traditional methods. The display of antibodies on phages is probably the most widespread of these methods, although surface display on bacteria or yeast have been employed successfully, as well. These methods using various sizes of combinatorial antibody libraries and different selection strategies possessing benefits in screening potency, generating, and isolation of high affinity antibodies with low risk of immunogenicity. Knowing the basics of each method assists in the design and retrieval process of antibodies suitable for different diseases, including cancer. In this review, we aim to outline the basics of each library construction and its display method, screening and selection steps. The advantages and disadvantages in comparison to alternative methods, and their applications in antibody engineering will be explained. Finally, we will review approved or non-approved therapeutic antibodies developed by employing these methods, which may serve as therapeutic antibodies in cancer therapy.

Photothermal therapy-mediated autophagy in breast cancer treatment: Progress and trends.

Breast cancer (BC) has different clinical manifestations due to its diverse mechanism of action that has created many challenges to choosing appropriate treatment. Recent findings of the biology of breast cancer including the mechanisms of survival and metastasis, understanding the effective signaling pathways in tumor formation and modeling of cancer cell responses to the therapeutic approaches provided significant advances in BC treatment. In this regard, the use of phototherapy-based approaches such as photothermal therapy (PTT) would be an encouraging alternative for tumor suppression through activating autophagy or suppressing cell signaling that influences the cell cycle to induce cell death. Since autophagy has a dual opposite role consisting of pro-survival and growth inhibition in breast cancer microenvironments, the regulation of autophagy would be playing promising roles in the treatment of BC using PTT. This review updates the molecular mechanisms that PTT could evoke autophagic cell death in breast cancer. Moreover, this article provides insights into the biological effects of autophagy-targeted-PTT as a promising strategy for breast cancer therapy.

A review on targeting tumor microenvironment: The main paradigm shift in the mAb-based immunotherapy of solid tumors.

Monoclonal antibodies (mAbs) as biological macromolecules have been remarked the large and growing pipline of the pharmaceutical market and also the most promising tool in modern medicine for cancer therapy. These therapeutic entities, which consist of whole mAbs, armed mAbs (i.e., antibody-toxin conjugates, antibody-drug conjugates, and antibody-radionuclide conjugates), and antibody fragments, mostly target tumor cells. However, due to intrinsic heterogeneity of cancer diseases, tumor cells targeting mAb have been encountered with difficulties in their unpredictable efficacy as well as variability in remission and durable clinical benefits among cancer patients. To address these pitfalls, the area has undergone two major evolutions with the intent of minimizing anti-drug responses and addressing limitations experienced with tumor cell-targeted therapies. As a novel hallmark of cancer, the tumor microenvironment (TME) is becoming the great importance of attention to develop innovative strategies based on therapeutic mAbs. Here, we underscore innovative strategies targeting TME by mAbs which destroy tumor cells indirectly through targeting vasculature system (e.g., anti-angiogenesis), immune system modulation (i.e., stimulation, suppression, and depletion), the targeting and blocking of stroma-based growth signals (e.g., cancer-associated fibroblasts), and targeting cancer stem cells, as well as, their effector mechanisms, clinical uses, and relevant mechanisms of resistance.

Phage display-derived immunorecognition elements LSPR nanobiosensor for peptide hormone glycine-extended gastrin 17 detection.

The current study intended to evaluate two types of biorecognition element (BRE), namely recombinant antibody fragments and M13 bacteriophage-displayed antibody fragments, where protein L and electrostatic interactions were used to respectively conjugated antibodies and bacteriophages on AuNPs. The functionalization process was examined by DLS to monitor the changes in the size and zeta potential. The formation of the BRE-G17-Gly immunological complexes was manifested by aggregation (confirmed by FE-SEM) and color change from red to dark blue visible to the naked eye. Local refractive index variations of functionalized AuNPs were monitored by a UV - vis spectrophotometer, showing increasing size and decreasing zeta potential in all stages. The calibration plot was developed in the concentration range 1-5 µg/mL and the limit of detection (LOD) was 1 µg/mL. The LSRP nanobiosensor in combination with the phage-based BRE was an affordable and simple approach, as it was able to eliminate the time-consuming and costly step of extracting antibodies. Contrary to the traditional immunoassays, this method does not require additional amplification, e.g., enzymatic, to read the result. The proposed LSPR nanobiosensor model can be adapted to detect a wide range of pathogens, viruses, and biomarkers in the shortest possible time.

Advances in antibody nanoconjugates for diagnosis and therapy: A review of recent studies and trends.

Nowadays, the targeted imaging probe and drug delivery systems are the novel breakthrough area in the nanomedicine and treatment of various diseases. Conjugation of monoclonal antibodies and their fragments on nanoparticles (NPs) have a remarkable impact on personalized medicine, such that it provides specific internalization and accumulation in the tumor microenvironment. Targeted imaging and early detection of cancer is presumably the strong participant to a diminution in mortality and recurrence of cancer disease that will be the next generation of the imaging device in clinical application. These intelligent delivery systems can deliver therapeutic agents that target cancerous tissue with minimal side effects and a wide therapeutic window. Overall, the linkage between the antibody and NPs is a critical subject and requires precise design and development. The attachment of antibody nanoconjugates (Ab-NCs) on the antigen surface shouldn't affect the function of the antibody-antigen binding. Also, the stability of the antibody nanoconjugates in blood circulation is concerned to avoid the release of drug in non-targeted regions and the possible for specific toxicity while disposal to the desired site. Here, we update the recent progress of Ab-NCs to improve early detection and cancer therapy.

Mucin-1 conjugated polyamidoamine-based nanoparticles for image-guided delivery of gefitinib to breast cancer.

PAMAM dendrimers (PAMs) are a group of polymeric macromolecules with distinctive physicochemical features, which can make them multifunctional theranostic nanoparticles (NPs). This study was designed to examine the impact of mucin-1 aptamer-conjugated NPs which were engineered using PAM for image-guided delivery of gefitinib (GEF) in the breast cancer cells/tumor. For this, PAMAM was conjugated with diethylenetriaminepentaacetic acid (DTPA) and modified with PEG2000 to prepare a multi-functionalized NPs. Subsequently, GEF was loaded onto the DTPA-PAM-PEG NPs, which were then armed with MUC-1 aptamer to form the DTPA-PAM-PEG/GEF@MUC-1 nanosystem. Finally, aptamer-conjugated NPs were radiolabeled by gallium-67 as an imaging agent to construct image-guided nanoplatforms. The prepared NPs were characterized by different techniques. The kinetic release models of gefitinib from radiolabeled NPs offer the sustained-release mechanism of the encapsulated drug for over 7 days. In vitro evaluation showed higher cytotoxicity and enhanced uptake of the mucin-grafted NPs in MCF-7 cells. Nuclear medicine imaging and in vivo investigations revealed significant accumulation of 67Ga-DTPA-PAM-PEG/GEF@MUC-1 in the tumor site of the animal models. These data suggest that the engineered NPs are a promising image-guided nanosystem for mucin-expressing breast cells/tumors with the assistance of nuclear medicine.

Bio-assay of the non-amidated progastrin-derived peptide (G17-Gly) using the tailor-made recombinant antibody fragment and phage display method: a biomedical analysis.

In this research, four novel and sensitive immunosensors for electrochemical determination of G17-Gly were designed based on signal amplification and tailor-made recombinant antibody technology. Anti-G17-Gly antibody fragments (i.e. scFv and VL specific to the N- and C-terminal of G17-Gly) were immobilized onto a polymeric nanocomposite comprising poly cetyl trimethyl ammonium bromide (P(CTAB)) as the conductive matrix, chitosan (CS) as a biocompatible agent and gold nanoparticles (AuNPs) as the signal amplification element. The high surface area provided by AuNPs and the small size of scFv/VL establish the basis for immobilizing a high amount of the anti-G17-Gly on the surface of the electrode for detecting G17-Gly in human plasma samples. Under optimal conditions, the designed immunosensors provide an excellent analytical capability for detecting and determining G17-Gly in human plasma samples with a linear range from 0.5 mM to 0.05 pM and a LLOQ of 0.05 pM. The sensitivity order of the immunosensors was Ag/2-mercaptoethanol/phage displaying scFv/P(CTAB-CS)-AuNP/GE, Ag/2-mercaptoethanol/phage displaying VL/P(CTAB-CS)-AuNP/GE, Ag/BSA/scFv/P(CTAB-CS)-AuNP/GE, and Ag/BSA/VL/P(CTAB-CS)-AuNP/GE. The aforementioned characteristics demonstrate that the proposed immune-devices can be used in biological and clinical diagnosis as reliable tools for identifying different oncobiomarkers.

Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies.

Single-chain variable fragments (scFvs) have gained increased attention among researchers in both academic and industrial fields owing to simple production in E. coli. The E. coli periplasm has been the site of choice for the expression of scFv molecules due to its oxidizing milieu facilitating correctly formation of disulfide bonds. Hence, the recovery of high-yield and biologically active species from the periplasmic space is a critical step at beginning of downstream processing. TES (Tris/EDTA/Sucrose) as a simple and efficient extraction method has been frequently used but under varied extraction conditions, over literature. This study, for the first time, aimed to interrogate the effects of four independent variables (i.e., Tris-HCl concentration, buffer's pH, EDTA concentration, and incubation time) and their potential interactions on the functional extraction yield of an scFv antibody from the periplasmic space of E. coli. The results indicated that the Tris-HCl concentration and pH are the most significant variables in the TES method and displayed a positive effect at their lower values on the functional extraction yield. Besides, the statistical analysis revealed 4 significant interactions between different variables. Here is the first report on the successful application of a design of experiment based on a central composite design to establish a generic and optimal TES extraction condition. Accordingly, an optimal condition for TES extraction of scFv molecules from the periplasm of HB2151 at the exponential phase was developed as follows: 50 mM Tris-HCl at pH 7.2, 0.53 mM EDTA, and an incubation time of 60 min.

Immunotargeting and therapy of cancer by advanced multivalence antibody scaffolds.

Monoclonal antibodies (mAbs) are a swiftly growing class of targeted therapeutics for malignancies. After their first advent, the antibody (Ab) engineering trail has shown an evolutionary trajectory - from the rodent-derived Abs to the chimeric, humanised and fully human Abs with higher efficacy and lower/no immunotoxicity. Despite possessing great clinical potentials, several reports have highlighted that monospecific mAbs, even with high-affinity, often fail to induce sufficient immunologic responses. The full activation of the immune system demands cooperative interactions of immunotherapies with target antigen (Ag) towards functional avidity. Although the monospecific mAbs show affinity to a target Ag, they often fail to render sufficient avidity necessary for the activation of intracellular signalling mechanisms and the provocation of the immune system. Thus, various Ab/non-Ab scaffolds with much greater therapeutic impacts have been engineered based on the adjustment of their affinity and avidity balance. Novel multivalent Ab scaffolds (e.g. MDX-447, MT110, CD20Bi, TF2 and FBTA05) and mimetic Abs (e.g. adnectin, DARPins and ecallantide) offer improved pharmacokinetic and pharmacodynamic properties. Here, we discuss the avidity and multivalency and provide comprehensive insights into advanced Ab scaffolds used for immunotargeting and therapy of cancer.

---Successful Application of Whole Cell Panning for Isolation of Phage Antibody Fragments Specific to Differentiated Gastric Cancer Cells.

Purpose: Generation of antibodies which potentially discriminate between malignant and healthy cells is an important prerequisite for early diagnosis and treatment of gastric cancer (GC). Comparative analysis of cell surface protein landscape will provide an experimental basis for biomarker discovery, which is essential for targeted molecular therapies. This study aimed to isolate phage-displayed antibody fragments recognizing cell surface proteins, which were differently expressed between two closely related GC cell lines, namely AGS and MKN-45. Methods: We selected and screened a semisynthetic phage-scFv library on AGS, MKN-45, and NIH-3T3 cell lines by utilizing a tailored selection scheme that was designed to isolate phagescFvs that not only recognize the differentiated AGS cells but also distinguish them from NIH3T3 fibroblasts and the poorly differentiated MKN-45 cells. Results: After four rounds of subtractive whole cell panning, 14 unique clones were identified by ELISA screening and nucleotide sequencing. For further characterization, we focused on four phage-scFvs with strong signals in screening, and their specificity was confirmed by cell-based ELISA. Furthermore, the selected phage-scFvs were able to specifically stain AGS cells with 38.74% (H1), 11.04% (D11), 76.93% (G11), and 69.03% (D1) in flow cytometry analysis which supported the ability of these phage scFvs in distinguishing AGS from MKN-45 and NIH-3T3 cells. Conclusion: Combined with other proteomic techniques, these phage-scFvs can be applied to membrane proteome analysis and, subsequently, identification of novel tumor-related antigens mediating proliferation and differentiation of cells. Furthermore, such antibody fragments can be exploited for diagnostic purposes as well as targeted drug delivery of GC.

Phage display selection of fully human antibody fragments to inhibit growth-promoting effects of glycine-extended gastrin 17 on human colorectal cancer cells.

Glycine-extended gastrin 17 (G17-Gly), a dominant processing intermediate of gastrin gene, has been implicated in the development or maintenance of colorectal cancers (CRCs). Hence, neutralizing G17-Gly activity by antibody entities can provide a potential therapeutic strategy in the patients with CRCs. To this end, we isolated fully human antibody fragments from a phage antibody library through biopanning against different epitopes of G17-Gly in order to obtain the highest possible antibody diversity. ELISA screening and sequence analysis identified 2 scFvs and 4 VL antibody fragments. Kinetic analysis of the antibody fragments by SPR revealed KD values to be in the nanomolar range (87.9-334 nM). The selected anti-G17-Gly antibody fragments were analyzed for growth inhibition and apoptotic assays in a CRC cell line, HCT-116, which is well-characterized for expressing gastrin intermediate species but not amidated gastrin. The antibody fragments exhibited significant inhibition of HCT-116 cells proliferation ranging from 36.5 to 73% of controls. Further, Annexin V/PI staining indicated that apoptosis rates of scFv H8 and VL G8 treated cells were 45.8 and 63%, respectively. Based on these results, we for the first time, demonstrated the isolation of anti-G17-Gly human scFv and VL antibodies with potential therapeutic applications in G17-Gly-responsive tumors.

Phage display-derived antibody fragments against conserved regions of VacA toxin of Helicobacter pylori.

Infection with Helicobacter pylori may result in the emergence of gastric adenocarcinoma. Among various toxins assisting pathogenesis of H. pylori, the vacuolating cytotoxin A (VacA) is one of the most potent toxins known as the major cause of the peptic ulcer and gastric adenocarcinoma. To isolate single-chain variable fragments (scFvs) against two conserved regions of VacA, we capitalized on the phage display technology and a solution-phase biopanning (SPB). Characterization of scFvs was carried out by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and surface plasmon resonance (SPR). Bioinformatics analyses were also performed in order to characterize the structural and functional properties of the isolated scFvs and the interaction(s) between the isolated antibodies (Ab)-antigen (Ag). After four rounds of biopanning, the positive colonies detected by scFv ELISA were harvested to extract the plasmids and perform sequencing. Of several colonies, three colonies showed high affinity to the VacA1 and two colonies for the VacA2. Further complementary examinations (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot, SPR, and flow cytometry) displayed the high affinity and specificity of the isolated scFvs to the VacA. Docking results revealed the interaction of the complementarity-determining regions (CDRs) with the VacA peptide. In conclusion, for the first time, we report on the isolation of several scFvs against conserved residues of VacA toxin with high affinity and specificity, which may be used as novel diagnostic/therapeutic tool in the H. pylori infection.

Efficiency and cytotoxicity analysis of cationic lipids-mediated gene transfection into AGS gastric cancer cells.

In this effort, we provided comparative study on optimization of transfection conditions for AGS human gastric cancer cell line using two commercial non-liposomal cationic lipids. Using reporter vector pEGFP-N1, transfection efficiency of Attractene™ and X-tremeGENE HP™ transfection reagents in terms of cell densities and DNA/reagent ratios was determined in AGS cells by flow cytometry and fluorescence microscopy. In addition, influence of transfection reagents on direct cytotoxicity and cell viability was respectively, measured using lactate dehydrogenase (LDH) leakage and MTT assays. Provided that the transfection rate of 29% and the mean fluorescence intensity of 437.5, the DNA/reagent ratio of 0.4/1.5 was selected as the optimal condition using Attractene™, whereas the optimum condition using X-tremeGENE HP™ was obtained by the ratio of 1/2 with a higher transfection rate of 36.9% and an MFI of 833. Very low direct cytotoxicity (<5% and 6-9% using Attractene™ and X-tremeGENE HP™, respectively) and high cell viability (74.5-95.5% versus 68-75%) showed the biodegradable attribute for both transfection reagents. Altogether, X-tremeGENE HP™ exhibited superiority over Attractene™ as a transfection reagent for AGS cells. In the present research, we have established the optimized protocols for efficient transfection of AGS cells with potential applications in gene function and expression studies as well as gene therapy.

Pleiotropic cytotoxicity of VacA toxin in host cells and its impact on immunotherapy.

Introduction: In the recent decades, a number of studies have highlighted the importance of Helicobacter pylori in the initiation and development of peptic ulcer and gastric cancer. Some potential virulence factors (e.g., urease, CagA, VacA, BabA) are exploited by this microorganism, facilitating its persistence through evading human defense mechanisms. Among these toxins and enzymes, vacuolating toxin A (VacA) is of a great importance in the pathogenesis of H. pylori. VacA toxin shows different pattern of cytotoxicity through binding to different cell surface receptors in various cells. Methods: To highlight attempts in treatment for H. pylori infection, here, we discussed the VacA potential as a candidate for development of vaccine and targeted immunotherapy. Furthermore, we reviewed the related literature to provide key insights on association of the genetic variants of VacA with the toxicity of the toxin in cells. Results: A number of investigations on the receptor(s) binding of VacA toxin confirmed the pleiotropic nature of VacA that uses a unique mechanism for internalization through some membrane components such as lipid rafts and glycophosphatidylinositol (GPI)-anchored proteins (GPI-AP). Considering the high potency of VacA toxin in the clinical presentations in infection and assisting persistence and colonization of H. pylori, it is considered as one of the pivotal components in production vaccines and monoclonal antibodies (mAbs). Conclusion: It is possible to generate mAbs with a considerable potential to convert into secretory immunoglobulins that could penetrate into the niche of H. pylori and inhibit its normal functionalities. Further, conjugation of H. pylori targeting Ab fragments with the toxic agents or drug delivery systems (DDSs) offers new generation of H. pylori treatments.