Monoclonal Antibodies In Terms Research Articles

Cell Surface Vimentin Detection in Cancer Cells by Peptide-Based Monoclonal Antibody

Vimentin is a prominent Intermediate Filaments (IFs) protein expressed in different mesenchymal origin cell types. Besides a wide range of cellular function roles associated with vimentin expression, its dysregulation and cell surface expression in the induction of malignancy properties have been reported extensively, making it a promising cancer-specific target. Therefore, this study aimed to generate and characterize anti-vimentin monoclonal antibodies. A 14-mer synthetic peptide from vimentin was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of Blab/C mice and monoclonal production by conventional hybridoma technology. The monoclonal antibody was purified using affinity chromatography of supernatants from the selected hybridoma cells. ELISA, Immunoprecipitation-Western blotting (IP-WB), Immunocytochemistry (ICC), and flow cytometry were employed to characterize the produced monoclonal antibody in terms of interaction with vimentin immunizing peptide as well as vimentin protein. Amid the several obtained producing anti-vimentin antibody hybridomas, the 7C11-D9 clone (IgG1 isotype with kappa light chain) showed higher reactivity with the immunizing peptide, and led to its selection for purification and characterization. The purified antibody could detect vimentin protein in IP-WB, ICC and flow cytometry of the normal and cancerous cells with different origin. No vimentin expression was found in normal healthy Peripheral Blood Mononuclear Cell (PBMC). Taken together, 7C11-D9 anti-vimentin monoclonal antibody might be used as immune diagnostic or immune therapeutic tool where detection or targeting of vimentin in a wide range of organisms is required.

The art of suicidal molecular seduction for targeting drug resistance

The development of drug resistance is one of the most significant challenges of the current century in the pharmaceutical industry. Superinfections, cancer chemoresistance, and resistance observed in many non-infectious diseases are nullifying the efforts and monetary supplies, put in the advent of new drug molecules. Millions of people die because of this drug resistance developed gradually through extensive use of the drugs. Inherently, some drugs are less prone to become ineffective by drug resistance than others. Covalent inhibitors bind to their targets via a biologically permanent bound with their cognate receptor and therefore display more potent inhibiting characteristics. Suicide inhibitors or mechanism-based inhibitors are one of the covalent inhibitors, which require a pre-activation step by their targeting enzyme. This step accrues their selectivity and specificity with respect to other covalent inhibitors. After that pre-activation step, they produce an analogue of the transition state of the catalytic enzyme, which is practically incapable of dissociating from the enzyme. Suicide inhibitors, due to their high intrinsic affinity toward the related enzyme, are resistant to many mechanisms involved in the development of drug resistance and can be regarded as one of the enemies of this scientific hurdle. These inhibitors compete even with monoclonal antibodies in terms of their cost-effectiveness and efficacy.

Linear and dendrimeric antiviral peptides: design, chemical synthesis and activity against human respiratory syncytial virus.

Respiratory syncytial virus (RSV) is one of the most common viral pathogens. It is especially dangerous for newborns and young children. In some cases it could lead to severe bronchiolitis, pneumonia with hospitalization or even a lethal outcome. Despite decades of investigation of RSV biology, effective and safe therapeutics are still under development. Certain natural peptides have been found to exhibit antiviral activity against respiratory viruses, but their implementation is limited by low stability in biological media. One of the current approaches to enhance the peptide therapeutic opportunities is chemical synthesis of peptide dendrimers with hyperbranched structures. Taking into account the recent data of bioactive cationic and helical regions of natural peptides and the structure features of nucleolin identified as an RSV cellular receptor, the main goal of this study was to design relatively short linear and dendrimeric cationic peptides and to test their antiviral activity against RSV. As a result 3 linear cationic peptides and 4 peptide dendrimers were synthesized and compared with known LL-37 (cathelicidin family) and anti-F0 monoclonal antibodies in terms of cytotoxicity and antiviral activity. Their affinity to the supposed molecular target - nucleolin (C23) - was estimated in silico by molecular docking analysis. Four synthesized peptides demonstrated a cytotoxic effect, two of them were even more cytotoxic than LL-37, which could be explained by a combination of a high amount of positive charge and amphipathicity. Contrariwise, non-hydrophobic dendrimer peptides did not exhibit cytotoxicity in mammalian cells in the studied concentration range. Two of the seven synthesized peptides, LTP (dendrimer) and SA-35 (linear), used in this study had a stronger antiviral effect than natural peptide LL-37, and three others showed slightly lower activity than anti-F0 monoclonal antibodies. The data obtained in this study suggest that evenly distributed positive charge, and low or medium amphipathicity play a key role in the antiviral activity of the studied peptides. Moreover, the calculated free energy values of the peptide/nucleolin complex for the most active peptides supported the idea that the peptide ability of nucleolin interaction promotes the anti-RSV properties of the molecules.

The Role of Anti-calcitonin Gene-related Peptide in Migraine and its Implication in Developing Countries: A Reasonable Option to Consider Despite Higher Cost.

Migraine is among the commonest causes of headache in all ages. It is the second most common cause of neurological disability. Currently, antihypertensive, antidepressants and antiepileptic drugs are reasonable preventable medications for chronic migraine. Despite the higher levels of stigma associated with this disease, fewer attempts were made in past regarding the treatment options for chronic migraine. Recently, a novelty treatment was introduced known as calcitonin gene-related peptide (CGRP) monoclonal antibodies as a possible mechanism for the prevention of migraine attacks. CGRP played an important role in the etiology of migraine headaches and was considered as the major peptide behind the cause of the headache disorders. We have reviewed the benefits of these monoclonal antibodies in terms of their efficacy and adverse effects with the available treatment choices. These drugs showed superior results when compared to the placebo and were considered generally safe in the majority of clinical trials. Earlier versions of CGRP antagonists, known as gepants, were less tolerable due to their tendency to cause liver and cardiovascular complications. Thus, in comparison to the earlier gepants, these CGRP monoclonal antibodies were safer and demonstrated excellent tolerability. Short-term side effects were only limited to mild-moderate injection site rash or pruritus, however, their long-term side effects are still unknown. Despite the higher cost of these drugs, we have analyzed the applicability of this drug in the developing countries. Although the quality-adjusted life year (QALY) gained per cost of the drug is still expensive and majority of people may not afford, its excellent tolerability and less adverse effects should also be considered a reason to implement this drug, particularly for resource-limited countries. Moreover, these medications could also become a prototype for future inventions and creations (cost-effective versions for resource-limited countries). In conclusion, this review suggests that CGRP monoclonal antibodies are safer and excellent alternate option for patients with chronic migraine as it has better efficacy, tolerability, and provides a hope to reduce the stigma associated with migraine. All these benefits should be the deciding factors when opting for this treatment and the decision should not be made solely on the socioeconomic status.

Systemic treatments for metastatic cutaneous melanoma.

Systemic treatments for metastatic cutaneous melanoma.

Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.

Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.

Induction Therapies in Kidney Transplantation: The Experience of Hospital Pablo Tobon Uribe, Medellín, Colombia 2005–2010

Background Induction therapies in kidney transplantation have led to prescriptions of lower doses of maintenance immunosuppression and fewer acute rejection episodes. We sought to assess the use of an affordable monoclonal antibody in terms of the incidences of rejection episodes as well as graft and patient survivals and cytomegalovirus (CMV) and opportunistic infections among our kidney transplant recipients between August 2005 and December 2010. Data were obtained for patients who had more than 20 months' follow-up. Materials and methods We retrospectively analyzed data from kidney recipients between August 2005 and December 2010, using descriptive statistics and Kaplan-Meier survival analysis. We performed a multivariate analysis with logistic regression for the dependent variables of rejection episodes and death. Results Among 425 transplant patients graft survival was 89.2% and patient survival was 94.1% after 76.2% of patients received alemtuzumab, 10.7% daclizumab, 3.6% basiliximab, 2.4% thymoglobulin, and 7%, no induction therapy. Rejection incidence in general in the first year was 10.8% and CMV incidence 10%. There was an increased risk of rejection among subjects without any us with alemtuzumab induction therapy. Conclusion Induction therapies show an important reduction in kidney transplant rejection incidence during the first year, allowing low doses of maintenance immunosuppressants, thereby diminishing long-term adverse effects. Alemtuzumab seemed to be a safe alternative with similar results to those obtained with standard immunosuppression.

Monoclonal anti-epidermal growth factor receptor (ior EGF/r3) antibody pharmacokinetic studies on nude mice I: a radio-receptor analysis applied to drug serum quantification.

Due to its antagonistic properties upon ligand-epidermal growth factor receptor (EGFr) interaction, the monoclonal antibody anti-EGFr ior EGF/r3 is considered a potential therapeutic agent against several epithelium-derived tumours. This paper affords further analysis of the relevant corporal interaction of this monoclonal antibody in terms of its pharmacodynamic properties, using nude mice, following a single bolus intravenous dose administration. The radio-receptor assay allows quantification of the serum ior EGF/r3 level. The dose selection procedure, according to the Kolmogorov-Smirnov test, suggested using doses of 12.5-16 mg kg(-1) for pharmacokinetic assessments. The experimental data were best fitted to a bi-exponential function (r2 = 0.985), through the classical two-compartment open modelling approach. The model selection was corroborated by the AKAIKE information criteria, and also the SCHWARTZ and ESTRIP test were used. The estimated pharmacokinetic parameters (e.g. t 1/2 beta = 34.65 h, Vc = 2.84 mL, Vss = 4.21 mL and CL = 0.09 mL h(-1)) bear out the strategies for the evaluation of the therapeutic application of this drug. Finally, the radio-receptor analysis has provided a rationale for the proposed serum monoclonal antibody ior EGF/r3 quantification to characterize its concentration-time course.

SIGNS OF APOPTOSIS IN THE PITUITARY, THYROID AND OVARIES OF FEMALE RATS AFTER A SINGLE DOSE OF POTASSIUM IODIDE

The aim of this work was to study the effect of a single dose of iodide on the expression of apoptosis mediating enzymes (caspase 32 and caspase 8) as well as of antiapoptopic protein Bcl-2 in the anterior pituitary, thyroid and ovaries in rats. A total of 24 inbred rat females of local laboratory strain weighing 250-300 g at the stage of diestrus and/or metestrus were used. Pituitary, thyroid and ovaries were dissected from the first group (8 control rats) at the same time as these from the second group (8 rats) and third group (8 rats) which were given potassium iodide (KI; 4 microg/100g body weight by gavage) 48 hours and 120 hours prior to the sacrifice, respectively. Streptavidin-biotin method was used to define the expression of caspase 8 and 32 as well as that of Bcl-2 with the aid of specific monoclonal antibodies in terms of the percentage of positive immunostained area (% PA). Histological examinations of endocrine glands did not show any signs of necrosis. In the thyroid a significant decrease of both caspases 32 and 8 was found at 120 hrs after KI administration. In contrast, however, in the pituitary a significant increase of caspase 32 appeared at 120 hrs after KI administration, although a considerable, but not significant increase of both caspases 32 and 8 already appeared after 48 hrs. In the ovaries a considerable and about the same increase of both caspases 32 and 8 appeared at 120 hrs after KI, though the difference was significant only for caspase 8. In addition, however, a considerable, but not significant, increase of caspase 32 at 48 hrs also appeared in the ovaries. The apoptotic activity was induced in the pituitary and ovaries of female rats by a single dose of KI, as shown by considerable increase of both the effectory caspase 32 and initiatory caspase 8 at 48 and 120 hrs after iodide administration. In contrast, however, in the thyroid any considerable changes in caspase 32 and caspase 8 were not observed after 48 hrs, while a significant decrease of both appeared after 120 hrs. Although no changes in Bcl-2 were found in the pituitary and thyroid, considerable and significant decrease appeared in the ovaries after 120 hrs.

Development of a High-Affinity Anti-Domoic Acid Sheep scFv and its Use in Detection of the Toxin in Shellfish

The potential of immunoassays as high-throughput screening tools for the detection of harmful substances in foods will only be realized when convenient methods are available for production of the high affinity antibodies needed for sensitive assay development. Recombinant antibodies offer advantages over traditional monoclonal antibodies in terms of ease of production, much greater antibody repertoire for selection, and versatility. We describe here the development of recombinant antibodies against the common shellfish toxin, domoic acid (DA), utilizing the sheep immunoglobulin system as an effective method for generating high affinity anti-hapten recombinant antibody fragments. A single-chain antibody fragment (scFv) library was generated from a sheep immunized with DA-bovine serum albumin conjugate, and anti-DA scFvs were isolated by phage-display. Three selected scFvs gave I50s of 2.6 to 58 ng/mL (8.3-186 nM) in competitive enzyme-linked immunosorbent assay (ELISA). Assay optimization with one of these scFvs gave a very reproducible standard curve with a range of 0.3 to 5.6 ng/mL (1.0 to 17.9 nM), a mean limit of quantification (LOQ, defined as the I20) of 0.5 ng/mL (1.6 nM), and a mean I50 of 1.2 ng/mL (3.9 nM). When the assay was used for the analysis of crude methanolic extracts of scallop tissues, results obtained correlated well with standard HPLC assay results (R2, 0.90, n = 40; R2, 0.81, n = 34), although ELISA results were lower than HPLC results. Adjusting the cutoff point for DA concentration accordingly from the regulatory 20 mg/kg, the potential of the sheep scFv-based ELISA for use as a screening assay for DA in shellfish extracts was demonstrated.

Light-chain shuffling results in successful phage display selection of functional prokaryotic-expressed antibody fragments to N-glycolyl GM3 ganglioside

Phage display technology makes it possible to introduce and rapidly screen diversity in antibody binding sites. Chain shuffling has been successfully used to humanize murine antibody fragments and also to obtain affinity matured variants. Here we report a different application of this method: the use of chain shuffling to overcome improper prokaryotic expression behavior of a hybridoma-derived single-chain antibody fragment. Construction and expression of such recombinant antibody fragments remain as empirical entities, hampered by the inability to express some antibody genes coming from eukaryotic cells in bacterial expression systems. Such problems are different for each combination of variable regions and can be serious enough to preclude the use of some hybridomas as sources of V regions to obtain recombinant antibody fragments. The particular binding properties and potential usefulness of some monoclonal antibodies make it highly desirable to bypass these technical limitations in order to develop smaller size therapeutic agents in the form of antibody fragments. The 14F7 mouse monoclonal antibody is one such attractive candidate due to its high specificity for the N-glycolyl GM3 ganglioside overexpressed in tumor cells and its ability to distinguish this antigen from closely related gangliosides like N-acetyl GM3. Our goal was to construct a phage-displayed single-chain Fv antibody fragment derived from 14F7. After cloning the original variable regions from the 14F7 hybridoma in a phagemid vector, we were unable to detect either binding activity or even expression of antibody fragments in bacteria, despite repetitive efforts. We constructed light-chain shuffling libraries, from which functional antibody fragments were readily selected. These combined the original 14F7 heavy chain variable region with a wide variety of unrelated murine and human light-chain variable regions. New antibody fragments retained the valuable properties of the monoclonal antibody in terms of fine specificity, affinity and tumor recognition. They were readily produced by bacteria, either in phage-displayed form or as soluble molecules, and provided a panel of potentially useful variants for cancer diagnosis and immunotherapy. Chain shuffling and phage display were found to be useful strategies for selecting antibody fragments on the basis of both prokaryotic expression and antigen binding criteria.

The Functional Effects of Biotinylation of Anti-angiotensin-Converting Enzyme Monoclonal Antibody in Terms of Targeting in Vivo

The effect of modification with biotin N-hydroxysuccinimide ester of mouse monoclonal antibody to angiotensin-converting enzyme, anti-ACE Mab 9B9, on its targeting to endothelial cells has been studied in vitro and in vivo. By in vitro assay, Mab 9B9 biotinylated at a biotin/IgG molar ratio in reaction mixture (B/IgG ratio) of 0.7-2.2 bound streptavidin monovalently and retained antigen-binding capacity. Mab 9B9 biotinylated at a B/IgG ratio of 20 and higher bound streptavidin polyvalently. Extensive biotinylation (B/IgG ratio of 60 and higher) led to dramatic reduction of Mab 9B9 Ag-binding capacity and to reduction of Mab 9B9 recognition by goat polyclonal antibody to mouse IgG. Radiolabeled Mab 9B9 biotinylated at a B/IgG ratio of 6 (b6-Mab 9B9) bound effectively to cultured vascular endothelium, with affinity characteristics similar to nonbiotinylated Mab 9B9. Endothelial cells internalized both Mab 9B9 and b6-Mab 9B9 to the same extent (60% internalization at 3 h incubation at 37°C). Degradation of cell surface-associated Mab 9B9 or b6-Mab 9B9 was very low (<1% as measured by TCA solubility of radiolabel). In contrast, degradation of internalized b6-Mab 9B9 was more profound than that of Mab 9B9 (20 ± 3% vs 6 ± 1%, P <0.01). After injection in rats, radiolabeled b6-Mab 9B9 had a biodistribution pattern similar to that of radiolabeled Mab 9B9. Both preparations effectively accumulated in the lung (15-20% of injected dose/g of tissue vs 2% of injected dose/g of blood). Extensive biotinylation led to both a reduction of specific pulmonary uptake of Mab 9B9 and an enhanced blood clearance of Mab 9B9. Streptavidin binding to b6-Mab 9B9 did not alter biodistribution or pulmonary targeting of biotinylated antibody. We conclude that extensive biotinylation induces complex alterations of the functional properties of Mab 9B9. In contrast, b6-Mab 9B9 may serve as an affinity carrier for targeting of biotinylated compounds to the pulmonary endothelium.

The measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal anti-DNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody

The affinities of 9 IgG1 monoclonal anti-dinitrophenol (DNP) antibodies for 3H-ϵ-DNP- L-lysine, 125I-HOP-DNP- L-lysine and 125I-DNP-human serum albumin (HSA) were determined. 3H-DNP-lysine was used in equilibrium dialysis and ammonium sulphate globulin precipitation assays; 125I-HOP-DNP-lysine was used in equilibrium dialysis and polyethylene glycol precipitation; and 125I-DNP 5-HSA in the polyethylene glycol precipitation assay for affinity. The ranking order of the monoclonal antibodies in terms of affinity by the assays was significantly correlated. Of particular importance was the observation that the simple and widely applicable globulin precipitation assay utilizing a protein antigen produced affinity values which showed concordance with the least equivocal but cumbersome assay, equilibrium dialysis. Mixing of antibodies of high and low affinity demonstrated that even a low proportion of high affinity antibody had marked effect on measurements of the amount and affinity of a predominantly low affinity antibody preparation.