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.

Designing and Developing Enzyme-Linked Immunosorbent Assay Sandwich Kit for Measuring Placental Growth Factor Concentration.

Placental growth factor (PlGF) is an angiogenic factor belonging to vascular endothelial growth factor family. This factor is mainly expressed in the placenta and have important role in blood supply to embryonic tissues and fetal. According to accumulated data after 10th week of gestational age the expression of PlGF is increased. The peak of this factor is seen in the 30th week of pregnancy. The abnormal expression of PlGF have been seen in some diseases such as preeclampsia, eclampsia, cancer, and atherosclerotic lesions. Preeclampsia is a pregnancy complication characterized by high blood pressure and signs of damage to another organ system, most often the liver and kidneys. As noted the level of PlGF decreased in preeclampsia is, therefore, timely and accurate measurement of this factor could help in diagnosing preeclampsia. In this study, we worked on development of sandwich enzyme-linked immunosorbent assay (ELISA) kit for measurement of PlGF, to this end, bivalent single-domain monoclonal antibody with high affinity binding was used as detection antibody and rabbit polyclonal antibody with strong signal to PlGF was used as capture antibody. Both types of antibodies were produced in the laboratory. Therefore, this study showed that the designed kit can measure PlGF up to 7.5 pg/mL. Intra-assay accuracy was <10% and interassay accuracy was <15%. The ELISA sandwich kit had the appropriate sensitivity and accuracy in measuring human PlGF.

Generating the Engineered Form of a Nanobody Against Placenta Growth Factor with High Antiangiogenesis Potential.

Antibody engineering is a dynamic field in antibody industry. Over 30% of novel monoclonal antibodies (mAbs) in R&D and clinical trials are engineered forms. Affinity enhancement contributes to the development of new binders that are not only effective in low dose and cost but also improve some drawbacks of antibody production. After previous successful work on in silico affinity maturation of nanobody against placenta growth factor and finding the best engineered nanobodies (Mut2:S31D and Mut4:R45E), according to bioinformatic parameters and molecular dynamics (MD) simulation results, in this study we focused on experimental confirmation of affinity enhancement of a mutant form of nanobody. So, we cloned and expressed two of four mutant forms in pHEN6c vector. Affinity binding was assayed by enzyme-linked immunosorbent assay on purified mutants, with results showing that 10-time enhancement in affinity compared with the native form associated with MD simulation results. We checked the effectiveness of these mutant nanobodies in angiogenesis inhibition by human umbilical vein endothelial cell proliferation and 3D capillary tube formation. EC50 of mut2, mut4, and native in proliferation assay was 110, 140, and 190 ng/mL, respectively, and that in 3D capillary tube formation was 80, 83, and 100 ng/mL. The results of functional studies revealed strong effectiveness of Mut2 followed by Mut4 compared with the native form. Our study confirmed that in silico approach could facilitate development of novel versions of mAb with better characteristics, which could save cost and time.

Web-based tools for miRNA studies analysis.

In recent years, thousands of microRNAs (miRNA) have been found in numerous species. The huge data resulting from miRNA researches are ordered in several levels including miRNA discovery, the sequence of mature and precursor miRNAs, role in diseases, pathway interaction, target validation, and prediction, etc. To prepare a simple approach to this information, numerous databases for miRNA records have been established. The selection of the correct database can be laborious and prolonged, especially for scientists who do not have experience in this field. To facilitate access to these resources, we have classified the available databases into seven categories and have described each one. The different categories of miRNA databanks contain miRNA sequence, target validation, target prediction, expression, function, role in pathways and networks, and their roles in diseases. Moreover, examples are included to describe the prototype databases for the most common applications. Hence, this review introduces available miRNA databases and presents a convenient overview of these databases that will enable researchers with different backgrounds to find suitable miRNA-related bioinformatics web tools and relevant microRNA information rapidly.

Development of a Recombinant Monospecific Anti-PLGF Bivalent Nanobody and Evaluation of it in Angiogenesis Modulation.

During the past two decades, tumor therapy based on monoclonal antibody has been found as a confident therapeutic approach in solid tumors and hematologic malignancies. Nanobodies are the smallest fragment of an antigen-binding domain in heavy chain-only antibody originated from the Camelidae family. Accordingly, they are being recently developed rapidly as diagnostic and therapeutic agents. In this regard, targeting of angiogenic factors like Placenta growth factor (PLGF) via nanobodies show a high effectiveness. In the current study, we developed a recombinant anti-PLGF bivalent nanobody based on the affinity enhancement mutant form of anti-PLGF nanobody to suppress the angiogenesis progression. Thereafter, the bivalent nanobody (bi-Nb) was cloned and then expressed into a bacterial system. Afterward, the purity was authorized using western blot assay and the affinity was assessed using ELISA. In this regard, proliferation, 3D capillary tube formation, and migration assays were employed as functional assays. The obtained data were analyzed using t-test and P < 0.05 was considered as statistically significant. The results indicate that the bivalent nanobody could inhibit proliferation, mobility, and formation of endothelial cell capillary-like structure. Moreover, the EC50 was estimated for endothelial cell's proliferation and capillary tube's formation to be about 100 ng/ml and 65 ng/ml, respectively. Migration of MCF-7 was inhibited as about 69%, rather than the control. Accumulation of data have shown that targeting of angiogenic factors like VEGF via monoclonal antibodies or nanobodies can be useful in the suppression of tumor progression. Also, the inhibition of PLGF with monoclonal antibody indicated that it is significant in angiogenesis suppression. However, due to intrinsic properties of nanobodies, they are suggested to be used. Since the small size is rapidly removed through liver or kidney system, so it is important to use bivalent or polymeric forms for extending the half-life. Our findings indicated that the inhibition of PLGF can prevent growth and proliferation of endothelial cells and tumor cells through the bivalent nanobody. So, it is suggested as a novel therapeutic agent for angiogenesis suppression.

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.

Rational MD simulations for improvement the affinity of nanobody against PlGF (placenta growth factor): mutagenesis based on electrostatic interactions.

AbbreviationsCOMcenter of mass distanceMDmolecular dynamicsMM-PBSAMolecular Mechanics Poisson-Boltzmann Surface AreaNbnanobodyPlGFplacenta growth factorRgradius of gyrationRMSDroot mean-square deviationSASAsolvent-accessible surface areaVEGFvascular endothelial growth factor.

Reducing effect of insulin resistance on alpha-synuclein gene expression in skeletal muscle.

BACKGROUND: Alpha-synuclein (SNCA) as the presynaptic protein is expressed in different tissues and prevents insulin-resistance (IR) through increasing glucose-uptake by adipocytes and muscles. However, the effect of insulin metabolism on SNCA expression has scarcely elucidated. In present study we assessed the probable effect of insulin resistance on SNCA expression in muscle C2C12 cells and also skeletal muscle tissues of type 2 diabetic mice. MATERIALS AND METHODS: Sixteen male C57BL/6 mice were divided into two experimental groups, including control and type 2 diabetic mice with IR (induced by high-fat diet + low-dose streptozotocin). The animals of the study involved the measurements of fasting blood glucose, oral-glucose-tolerance-test, as well as fasting plasma insulin. Moreover, insulin-resistant and insulin-sensitive muscle C2C12 cells were prepared. The insulin-resistance was confirmed by the glucose-uptake assay. Comparative quantitative real time PCR was used to assess the SNCA expression. RESULTS: The obtained results have showed a significant ~ 27% decrease in SNCA expression level in muscle tissue of diabetic mice (P = 0.022). Moreover, there was a significant change of SNCA expression in insulin-resistant C2C12 cells (P < 0.001). CONCLUSION: Type 2 diabetes due to insulin-resistance can decrease SNCA gene expression in muscles. In addition to the role of SNCA in cell susceptibility to insulin and glucose uptake, the SNCA expression can also be affected by insulin metabolism.

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.

Rational affinity enhancement of fragmented antibody by ligand-based affinity improvement approach.

Antibody engineering is now a noteworthy area in biopharmaceuticals as the next generation of marketed antibodies is engineered antibodies such as affinity- or stability-improved antibodies, fragmented or fused antibodies, antibody drug conjugates (ADCs), and PEGylated antibody fragments. In the current study, affinity enhancement of Nb against PlGF was performed by an in silico affinity maturation and molecular dynamics (MD) simulation. First, 300 single-point mutants were designed by identifying the residues involved in interaction with PlGF and different energy distributions. An energy based screening was performed to select best single-point mutants. Additionally, one variant containing two mutations was designed based on the selected single-point mutants. Finally, mutants-PlGF complexes were analyzed in details by all atom MD simulation. Trajectory analysis revealed that in both single (L112H, S31D, A97K, and R45E) and double (S31D & R45E) mutants, the free binding energies and the stability of complexes were significantly improved. The highest increment in affinity was observed for S31D mutant due to substantial increase in polar and electrostatic interactions. The secondary structure of Nb was intact in all variants and a shrinkage of PlGF over Nb was observed in all mutant-PlGF complexes during simulation. In addition, contact area and hydrogen-bond analysis as well as distance measurement in mutants-PlGF complexes also confirmed the affinity enhancement of variants relative to the native form. Our study showed that ligand-based affinity improvement could be considered as a promising approach for designing high affinity fragmented antibodies.

Synthesis, characterization and in vivo evaluation of cadmium telluride quantum dots toxicity in mice by toxicometabolomics approach.

Quantum dots (QDs) have widespread application in many fields such as medicine and electronics. The need for understanding the potentially harmful side effects of these materials becomes clear. In this study, the toxicity of cadmium telluride quantum dots (CdTe-QDs) and bulk Cd2+ has been investigated and compared by applying metabolomics methods. The datasets were 1H-NMR data from mice plasma which had been taken from four groups of mice in different time intervals. Then, the data were analyzed by applying chemometrics methods and the metabolites were found from Human Metabolome Database (HMDB). The results showed the significant change in the level of some metabolites especially estrogenic steroids in different groups with different amounts of received Cd. The findings also indicated that steroid hormone biosynthesis, lysine biosynthesis and taurine and hypotaurine metabolism are the most affected pathways by CdTe-QDs especially in estrogenic steroids. The over-representation analysis indicated that endoplasmic reticulum, gonads, and hepatocytes are most affected. Since the pattern of metabolite alteration of CdTe-QDs with equivalent Cd2+ was similar to those of CdCl2, it was postulated that beside Cd2+ effects, the toxicity of CdTe-QDs is associated with other factors.

In Vitro Evaluation of Vegf-Pseudomonas Exotoxin: A Conjugated on Tumor Cells.

BACKGROUND: Angiogenesis which occurs mandatory in solid tumors, is a critical step in malignancy progression. Vascular endothelial growth factor (VEGF) is mainly responsible for angiogenesis process and facilitates the formation of new vessels. Distribution of monoclonal antibodies against VEGF or VEGF receptor (VEGFR) into the solid tumors is limited because of their huge dimensions. Moreover, many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors. MATERIALS AND METHODS: We designed, expressed and evaluated the cytotoxicity of a novel nano-immunotoxin composed of VEGF splice variant containing 121 amino acids (VEGF121) and truncated the exotoxin A of Pseudomonas aeruginosa (PE38-KDEL). The fusion protein VEGF121-PE38 was successfully cloned and expressed in Escherichia coli, purified by Ni+ 2 affinity chromatography. The fusion protein was subsequently subjected to refolding using the reduced and oxidized glutathione. RESULTS: The expression level of the fusion protein reached to 1 mg/ml. The VEGF121-PE38 immunotoxin showed a 59 KDa MW which had cytotoxic effect on HUVEC and 293/KDR cells as low and high expressing VEGFR2 cells, respectively. But the cytotoxicity on 293/KDR was 100 folds more than that of VEGFR2 low expressing cell HUVEC. CONCLUSION: The designed immunotoxin showed more selectivity for higher VEGFR2 expressing cells in vitro.

Nanobodies As Novel Agents for Targeting Angiogenesis in Solid Cancers.

Solid cancers are dependent on angiogenesis for sustenance. The FDA approval of Bevacizumab in 2004 inspired many scientists to develop more inhibitors of angiogenesis. Although several monoclonal antibodies (mAbs) are being administered to successfully combat various pathologies, the complexity and large size of mAbs seem to narrow the therapeutic applications. To improve the performance of cancer therapeutics, including those blocking tumor angiogenesis, attractive strategies such as miniaturization of the antibodies have been introduced. Nanobodies (Nbs), small single-domain antigen-binding antibody fragments, are becoming promising therapeutic and diagnostic proteins in oncology due to their favorable unique structural and functional properties. This review focuses on the potential and state of the art of Nbs to inhibit the angiogenic process for therapy and the use of labeled Nbs for non-invasive in vivo imaging of the tumors.

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.

Identification and characterization of a novel nanobody against human placental growth factor to modulate angiogenesis.

Placental growth factor (PlGF), a member of vascular endothelial growth factors (VEGF) family, is considered as an important antigen associated with pathological conditions such as cancer cell growth, and metastasis. PlGF-targeting via nanobody (Nb) therefore could be beneficial to modulate these pathologies. In this work, phage-display and computational approach was employed to develop a high affinity PlGF-specific Nb. An Nb library was constructed against human recombinant PlGF (rPlGF). After panning on immobilized rPlGF the periplasmic-extract (PE) of individual colonies were screened by ELISA (PE-ELISA). The 3D structures of selected Nbs were then homology modeled and energy minimized using the AMBER force field. Binding score calculations were also assessed to reveal possible Nb-PlGF interactions. Via ELISA-based affinity/specificity determinations, the best-qualified Nb was further evaluated by proliferation, migration, 3D capillary formation, invasion assays and on Chick chorioallantoic membrane (CAM) model. An immune library of 1.5×107 individual Nb clones was constructed. By PE-ELISA 12 clones with strong signals were selected. Three out of 12 sequenced Nbs (Nb-C13, Nb-C18 and Nb-C62) showed high binding scores ranging between -378.7 and -461kcal/mol. Compared to a control Nb, Nb-C18 significantly inhibited proliferation, migration and the 3D-capillary formation of HUVEC cells (p<0.05) with an EC50 of 35nM, 42nM and 24nM and invasion of MDA-MB231was significantly suppressed (p<0.05) with an EC50 of57nM. The result of the CAM assay shows that Nb-C18 could inhibit the vascular formation in the chicken chorioallantoic membrane. This Nb can be used as anti-angiogenesis agent in future.

Design and expression of fusion protein consists of HBsAg and Polyepitope of HCV as an HCV potential vaccine.

BACKGROUND: Hepatitis C virus (HCV) infection is a serious public health threat worldwide. Cellular immune responses, especially cytotoxic T-lymphocytes (CTLs), play a critical role in immune response toward the HCV clearance. Since polytope vaccines have the ability to stimulate the cellular immunity, a recombinant fusion protein was developed in this study. MATERIALS AND METHODS: The designed fusion protein is composed of hepatitis B surface antigen (HBsAg), as an immunocarrier, fused to an HCV polytope sequence. The polytope containing five immunogenic epitopes of HCV was designed to induce specific CTL responses. The construct was cloned into the pET-28a, and its expression was investigated in BL21 (DE3), BL21 pLysS, BL21 pLysE, and BL21 AI Escherichia coli strains using 12% gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the identity of expressed fusion protein was confirmed by Western blotting using anti-His monoclonal antibody and affinity chromatography was applied to purify the expressed protein. RESULTS: The accuracy of the construct was confirmed by restriction map analysis and sequencing. The transformation of the construct into the BL21 (DE3), pLysS, and pLysE E. coli strains did not lead to any expression. The fusion protein was found to be toxic for E. coli DE3. By applying two steps inhibition, the fusion protein was successfully expressed in BL21 (AI) E. coli strain. CONCLUSION: The HBsAg-polytope fusion protein expressed in this study can be further evaluated for its immunogenicity in animal models.

Camel Heavy Chain Polyclonal Antibody Raised Against Recombinant Murine Placental Growth Factor Expressed in Escherichia coli.

Angiogenesis, a neovascularization process, is the most important occurrence in developmental and pathological conditions. Key factors involve belonging to the vascular endothelial growth factor family. Recently, placental growth factor (PlGF) has been considered in medicine because of its pathological importance in solid tumor invasion and metastasis. Therefore, PlGF targeting plays a promising role in the hindrance of tumor progress. In this study, murine PlGF was cloned, expressed in an Escherichia coli system, and used for development of polyclonal camel antibody. Codon-optimized mouse PlGF (mPlGF) cDNA was synthesized and cloned in to pET-28a. The expression was performed in BL21 (DE3) E. coli strain and purified by immobilized metal affinity column chromatography. A camel was subcutaneously immunized six times over a one-week interval using purified protein. IgGs were purified by applying the serum on two sequential column affinity chromatography using proteins A and G. Then, anti-PlGF IgG was identified by Western bolt analysis and ELISA using commercial and expressed PlGF. Synthesized mPlGF was cloned successfully into the pET-28a expression vector, and the accuracy of construct was confirmed by map restriction analysis and sequencing. The expression was induced by 1 mM IPTG and confirmed by SDS-PAGE and Western blot using anti-His monoclonal antibody. The camel was immunized using recombinant mPlGF, and IgG was purified by two-step affinity chromatography. Identification of specific IgG against mPlGF was confirmed by ELISA assay.

Inhibition of angiogenesis in human endothelial cell using VEGF specific nanobody.

Angiogenesis is an important step in tumor development and metastasis. Vascular endothelial growth factor (VEGF) plays an important role in progression of angiogenesis. VEGF121 and VEGF165 are the most relative forms of VEGF family which contain the full biological activity. Nanobodies derived from camelidae are the smallest biding site of antigen. Unique characteristic of nanobodies make them as a useful candidate for research. In this report, we describe the isolation of VEGF specific nanobodies from dromedaries immunized with purified VEGF antigen using phage display. Four clones that showed the highest signal value in ELISA experiment were selected and expressed as a His-tagged fusion protein. Four selected nanobodies were reacted strongly to VEGF in cross-reactivity assay. The binding affinity of selected nanobodies named Nb22, Nb23, Nb35 and Nb42 were differed from 0.1 to 60nM. The nanobodies inhibited endothelial cell proliferation or tube formation in response of VEGF in a dose-dependent manner. These results indicate the potential of nanobodies in inhibition of VEGF and represent a promising candidate for cancer research and therapeutics.

Generation and characterization of nanobodies targeting PSMA for molecular imaging of prostate cancer.

Nanobodies show attractive characteristics for tumor targeting in cancer diagnosis and therapy. A radiolabeled nanobody binding the prostate-specific membrane antigen (PSMA) could offer a noninvasive strategy to select prostate cancer patients eligible for PSMA-targeted therapies. We here describe the generation, production and in vivo evaluation of anti-PSMA nanobodies. Nanobodies were derived from heavy-chain-only antibodies, raised in immunized dromedaries. Binding characteristics were evaluated through ELISA and flow cytometry. Selected nanobodies were radiolabeled with (99m) Tc at their hexahistidine tail, after which cell binding capacity and internalization were evaluated on PSMA(pos) LNCaP and PSMA(neg) PC3 cell lines. In vivo tumor targeting was analyzed in both LNCaP and PC3 xenografted mice through SPECT/microCT and tissue sampling. A panel of 72 generated clones scored positive on ELISA, all contributing to three nanobody groups, of which group 3 dominated with 70 clones. ELISA and FACS analysis led to the selection of two dominant nanobodies. (99m) Tc-labeled PSMA6 and PSMA30 both showed specific binding on LNCAP cells, but not on PC3 cells. (99m) Tc-PSMA30 internalized significantly more in LNCaP cells compared to (99m) Tc-PSMA6. Higher absolute tumor uptake and tumor-to-normal organ ratios were observed for (99m) Tc-PSMA30 compared with (99m) Tc-PSMA6 and a (99m) Tc-control nanobody in LNCaP but not in PC3 tumor-bearing mice. PSMA30 nanobody has improved targeting characteristics both in vitro as well as in vivo compared with PSMA6 and the control nanobody, and was therefore selected as our in-house-developed lead compound for PSMA targeting.

Recombinant expression and purification of human placental growth factor 1 and specific camel heavy chain polyclonal antibody preparation.

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family. Unlike VEGF, PlGF is dispensable for normal cell development as well as playing various roles in pathological angiogenesis which occurs in tissue ischemia, inflammation, and malignancy. The PlGF-1 has been considered as a potential candidate for the diagnosis and targeting of pathological angiogenesis. Camelidae serum contains an important fraction of functional antibodies, called heavy-chain antibodies (HcAbs) that are naturally devoid of light chains. Camelid HcAbs recognize their cognate antigens by a single variable-domain, referred to as VHH or Nanobody. Here, we describe the expression and purification of recombinant human PlGF-1 (rhPlGF-1). This protein was subsequently used for the preparation of camel heavy chain polyclonal antibody against rhPlGF-1. The recombinant expression plasmid pET-26b-hPlGF-1 was introduced into Escherichia coli BL21 cells to express the rhPlGF-1 protein. Purified rhPlGF-1 was used to immunize camel, the specific reactivity of HcAb was determined with ELISA and western blot. Western blot analysis indicated that the antiserum specifically reacted to the recombinant protein. The rhPlGF-1 protein and its antibody may be used for the development of detection assays needed for clinical research.