ABSTRACT
Tetanus disease, caused by C. tetani, starts with wounds or mucous layer contact. Prevented by vaccination, the lack of booster shots throughout life requires prophylactic treatment in case of accidents. The incidence of tetanus is high in underdeveloped countries, requiring the administration of antitetanus antibodies, usually derived from immunized horses or humans. Heterologous sera represent risks such as serum sickness. Human sera can carry unknown viruses. In the search for human monoclonal antibodies (mAbs) against TeNT (Tetanus Neurotoxin), we previously identified a panel of mAbs derived from B-cell sorting, selecting two nonrelated ones that binded to the C-terminal domain of TeNT (HCR/T), inhibiting its interaction with the cellular receptor ganglioside GT1b. Here, we present the results of cellular assays and molecular docking tools. TeNT internalization in neurons is prevented by more than 50% in neonatal rat spinal cord cells, determined by quantitative analysis of immunofluorescence punctate staining of Alexa Fluor 647 conjugated to TeNT. We also confirmed the mediator role of the Synaptic Vesicle Glycoprotein II (SV2) in TeNT endocytosis. The molecular docking assays to predict potential TeNT epitopes showed the binding of both antibodies to the HCR/T domain. A higher incidence was found between N1153 and W1297 when evaluating candidate residues for conformational epitope.
Subject(s)
Antibodies, Monoclonal , Endocytosis , Molecular Docking Simulation , Neurons , Tetanus Toxin , Animals , Rats , Neurons/metabolism , Humans , Antibodies, Monoclonal/immunology , Tetanus Toxin/immunology , Tetanus Toxin/metabolism , Tetanus/prevention & control , Tetanus/immunology , Epitopes/immunology , Gangliosides/immunology , Gangliosides/metabolism , Cells, Cultured , Computer Simulation , MetalloendopeptidasesABSTRACT
Tetanus disease, caused by C. tetani, starts with wounds or mucous layer contact. Prevented by vaccination, the lack of booster shots throughout life requires prophylactic treatment in case of accidents. The incidence of tetanus is high in underdeveloped countries, requiring the administration of antitetanus antibodies, usually derived from immunized horses or humans. Heterologous sera represent risks such as serum sickness. Human sera can carry unknown viruses. In the search for human monoclonal antibodies (mAbs) against TeNT (Tetanus Neurotoxin), we previously identified a panel of mAbs derived from B-cell sorting, selecting two nonrelated ones that binded to the C-terminal domain of TeNT (HCR/T), inhibiting its interaction with the cellular receptor ganglioside GT1b. Here, we present the results of cellular assays and molecular docking tools. TeNT internalization in neurons is prevented by more than 50% in neonatal rat spinal cord cells, determined by quantitative analysis of immunofluorescence punctate staining of Alexa Fluor 647 conjugated to TeNT. We also confirmed the mediator role of the Synaptic Vesicle Glycoprotein II (SV2) in TeNT endocytosis. The molecular docking assays to predict potential TeNT epitopes showed the binding of both antibodies to the HCR/T domain. A higher incidence was found between N1153 and W1297 when evaluating candidate residues for conformational epitope.
ABSTRACT
Disadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.
The guided selection humanization process enabled the production of 20 human mAbs anti-FGF2;Seven human anti-FGF2 mAbs showed binding to the rFGF2 antigen in the SPR binding assay;Two human anti-FGF2 mAbs inhibited the proliferation and migration of HUVEC and SK-Mel-28 cells and were predicted to contact the FGF2 at a similar patch of residues than the original mAb.
Subject(s)
Antibodies, Monoclonal , Melanoma , Humans , Animals , Mice , Hybridomas , HEK293 Cells , Cell ProliferationABSTRACT
Disadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.
ABSTRACT
Tetanus toxin (TeNT) is produced by C. tetani, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased immune surveillance. Tetanus is most prevalent in low- and medium-income countries, where it remains a health problem. Neutralizing monoclonal antibodies (mAbs) can prevent the severity of illness and death caused by C. tetani infection. We identified a panel of mAbs that bind to TeNT, some of which were investigated in a preclinical assay, showing that a trio of mAbs that bind to different sites of TeNT can neutralize the toxin and prevent symptoms and death in mice. We also identified two mAbs that can impair the binding of TeNT to the GT1b ganglioside receptor in neurons. In this work, to generate a series of cell lines, we constructed vectors containing sequences encoding heavy and light constant regions that can receive the paired variable regions resulting from PCRs of human B cells. In this way, we generated stable cell lines for five mAbs and compared and characterized the antibody produced in large quantities, enabling the characterization experiments. We present the results regarding the cell growth and viability in a fed-batch culture, titer measurement, and specific productivity estimation. The affinity of purified mAbs was analyzed by kinetics and under steady-state conditions, as three mAbs could not dissociate from TeNT within 36,000 s. The binding of mAbs to TeNT was confirmed by ELISA and inhibition of toxin binding to GT1b. The use of the mAbs mixture confirmed the individual mAb contribution to inhibition. We also analyzed the binding of mAbs to FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb.
ABSTRACT
Tetanus toxin (TeNT) is produced by C. tetani, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased immune surveillance. Tetanus is most prevalent in low- and medium-income countries, where it remains a health problem. Neutralizing monoclonal antibodies (mAbs) can prevent the severity of illness and death caused by C.tetani infection. We identified a panel of mAbs that bind to TeNT, some of which were investigated in a preclinical assay, showing that a trio of mAbs that bind to different sites of TeNT can neutralize the toxin and prevent symptoms and death in mice. We also identified two mAbs that can impair the binding of TeNT to the GT1b ganglioside receptor in neurons. In this work, to generate a series of cell lines, we constructed vectors containing sequences encoding heavy and light constant regions that can receive the paired variable regions resulting from PCRs of human B cells. In this way, we generated stable cell lines for five mAbs and compared and characterized the antibody produced in large quantities, enabling the characterization experiments. We present the results regarding the cell growth and viability in a fed-batch culture, titer measurement, and specific productivity estimation. The affinity of purified mAbs was analyzed by kinetics and under steady-state conditions, as three mAbs could not dissociate from TeNT within 36,000 s. The binding of mAbs to TeNT was confirmed by ELISA and inhibition of toxin binding to GT1b. The use of the mAbs mixture confirmed the individual mAb contribution to inhibition. We also analyzed the binding of mAbs to FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb.
ABSTRACT
The use of serum containing polyclonal antibodies from animals immunized with toxins marked the beginning of the application of antibody-based therapy in late nineteenth century. Advances in basic research led to the development of the hybridoma technology in 1975. Eleven years later, the first therapeutic monoclonal antibody (mAb) was approved, and since then, driven by technological advances, the development of mAbs has played a prominent role in the pharmaceutical industry. In this review, we present the developments to circumvent problems of safety and efficacy arising from the murine origin of the first mAbs and generate structures more similar to human antibodies. As of October 2017, there are 61 mAbs and 11 Fc-fusion proteins in clinical use. An overview of all mAbs currently approved is provided, showing the development of sophisticated mAbs formats that were engineered based on the challenges posed by therapeutic indications, including antibody-drug conjugates (ADC) and glycoengineered mAbs. In the field of immunotherapy, the use of immunomodulators, bispecific mAbs and CAR-T cells are highlighted. As an example of promising therapy to treat infectious diseases, we discuss the generation of neutralizing monoclonal-oligoclonal antibodies obtained from human B cells. Scientific and technological advances represent mAbs successful translation to the clinic
Subject(s)
Animals , Mice , Technological Development/classification , Antibodies , Antibodies, Monoclonal/analysis , Mice, Transgenic/classification , Immunotherapy/adverse effectsABSTRACT
The use of serum containing polyclonal antibodies from animals immunized with toxins marked the beginning of the application of antibody-based therapy in late nineteenth century. Advances in basic research led to the development of the hybridoma technology in 1975. Eleven years later, the first therapeutic monoclonal antibody (mAb) was approved, and since then, driven by technological advances, the development of mAbs has played a prominent role in the pharmaceutical industry. In this review, we present the developments to circumvent problems of safety and efficacy arising from the murine origin of the first mAbs and generate structures more similar to human antibodies. As of October 2017, there are 61 mAbs and 11 Fc-fusion proteins in clinical use. An overview of all mAbs currently approved is provided, showing the development of sophisticated mAbs formats that were engineered based on the challenges posed by therapeutic indications, including antibody-drug conjugates (ADC) and glycoengineered mAbs. In the field of immunotherapy, the use of immunomodulators, bispecific mAbs and CAR-T cells are highlighted. As an example of promising therapy to treat infectious diseases, we discuss the generation of neutralizing monoclonal-oligoclonal antibodies obtained from human B cells. Scientific and technological advances represent mAbs successful translation to the clinic.
ABSTRACT
The use of serum containing polyclonal antibodies from animals immunized with toxins marked the beginning of the application of antibody-based therapy in late nineteenth century. Advances in basic research led to the development of the hybridoma technology in 1975. Eleven years later, the first therapeutic monoclonal antibody (mAb) was approved, and since then, driven by technological advances, the development of mAbs has played a prominent role in the pharmaceutical industry. In this review, we present the developments to circumvent problems of safety and efficacy arising from the murine origin of the first mAbs and generate structures more similar to human antibodies. As of October 2017, there are 61 mAbs and 11 Fc-fusion proteins in clinical use. An overview of all mAbs currently approved is provided, showing the development of sophisticated mAbs formats that were engineered based on the challenges posed by therapeutic indications, including antibody-drug conjugates (ADC) and glycoengineered mAbs. In the field of immunotherapy, the use of immunomodulators, bispecific mAbs and CAR-T cells are highlighted. As an example of promising therapy to treat infectious diseases, we discuss the generation of neutralizing monoclonal-oligoclonal antibodies obtained from human B cells. Scientific and technological advances represent mAbs successful translation to the clinic.
ABSTRACT
Glucose, in the presence of reactive oxygen species (ROS), acts as an as an oxidative agent and drives deleterious processes in Diabetes Mellitus. We have studied the mechanism and the toxicological effects of glucose-dependent glycoxidation reactions driven by copper and ROS, using a model peptide based on the exposed sequence of Human Serum Albumin (HSA) and containing a lysine residue susceptible to copper complexation. The main products of these reactions are Advanced Glycation End-products (AGEs). Carboxymethyl lysine and pyrraline condensed on the model peptide, generating a Modified Peptide (MP). These products were isolated, purified, and tested on cultured motor neuron cells. We observed DNA damage, enhancement of membrane roughness, and formation of domes. We evaluated nuclear abnormalities by the cytokinesis-blocked micronucleus assay and we measured cytostatic and cytotoxic effects, chromosomal breakage, nuclear abnormalities, and cell death. AGEs formed by glycoxidation caused large micronucleus aberrations, apoptosis, and large-scale nuclear abnormalities, even at low concentrations.
Subject(s)
Copper/chemistry , Cytotoxins , Glycation End Products, Advanced , Motor Neurons/metabolism , Peptides , Reactive Oxygen Species/chemistry , Serum Albumin , Cell Line , Cell Nucleus/metabolism , Cell Nucleus/pathology , Chromosome Breakage/drug effects , Cytotoxins/chemical synthesis , Cytotoxins/chemistry , Cytotoxins/pharmacology , Glucose/chemistry , Glycation End Products, Advanced/chemical synthesis , Glycation End Products, Advanced/chemistry , Glycation End Products, Advanced/pharmacology , Humans , Motor Neurons/pathology , Peptides/chemical synthesis , Peptides/chemistry , Peptides/pharmacology , Serum Albumin/chemistry , Serum Albumin/pharmacologyABSTRACT
In this work we present a comparative in vitro study of photodynamic activity between hypericin (HYP) and some hypericinates (hypericin ionic pair with lysine or N-methylglucamine) in human mammary adenocarcinoma cells (MCF-7). The toxicity and phototoxicity of hypericin and hypericinates were compared, as well as their cellular uptake and localization and mutagenic, genotoxic and clonogenic capacity. Our results demonstrate that different cationic moieties promote differences in the hypericinate solubility in a biological environment, and can influence the cellular localization and the phototoxicity of the photosensitizer. It was verified that hypericinates have better efficiency to generate singlet oxygen than HYP, and a lower aggregation in biological medium. In vitro assays have shown that HYP and the hypericinates are able to permeate the MCF-7 cell membrane and accumulated in organelles near the nucleus. The difference in location, however, was not determinant to the cell death mechanism, and a higher prevalence of apoptosis for all studied compounds occurred. The photodynamic studies indicated that hypericinates were more effective than HYP and were able to inhibit the formation of cellular colonies, suggesting a possible ability to prevent the recurrence of tumors. It also appears that all compounds have relative safety for mutagenicity and genotoxicity, which opens up a further safe route for application in in vivo studies.
Subject(s)
Perylene/analogs & derivatives , Photosensitizing Agents/chemistry , Anthracenes , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Nucleus/metabolism , Cell Survival/drug effects , Cell Survival/radiation effects , Humans , Light , MCF-7 Cells , Microscopy, Fluorescence , Mitochondria/drug effects , Mitochondria/metabolism , Perylene/chemistry , Perylene/metabolism , Perylene/pharmacology , Photosensitizing Agents/metabolism , Photosensitizing Agents/pharmacology , Singlet Oxygen/chemistry , Singlet Oxygen/metabolismABSTRACT
We report the molecular mechanism for zinc depletion caused by TPEN (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine) in neuroblastoma cells. The activation of p38 MAP kinase and subsequently caspase 3 is not due to or followed by redox imbalance or ROS generation, though these are commonly observed in literature. We found that TPEN is not responsible for ROS generation and the mechanism involves essentially lysosomal disruption caused by intracellular zinc depletion. We also observed a modest activation of Bax and no changes in the Bcl-2 proteins. As a result, we suggest that TPEN causes intracellular zinc depletion which can influence the breakdown of lysosomes and cell death without ROS generation.
Subject(s)
Chelating Agents/pharmacology , Intracellular Space/metabolism , Lysosomes/metabolism , Reactive Oxygen Species/metabolism , Zinc/pharmacology , Apoptosis/drug effects , Blotting, Western , Caspase 3/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Ethylenediamines/pharmacology , Flow Cytometry , Humans , Lysosomes/drug effects , Necrosis , Neuroblastoma/pathology , bcl-2-Associated X Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Highly complex analytical methods with different accuracies of measurement, reproducibilities and ease of analyses are currently being used to quantify metals in cellular media and tissue samples. In this review, the analytical methods commonly used for iron, copper and zinc quantification in mammalian cells are presented and discussed. Herein, we present a literature survey of the most commonly found concentrations of these metals in various mammalian cells in culture and tissues. The aim of this review is to help researchers in metallomic-related areas identify the method that best suits their needs for the accurate quantification of these metals in cells. This accuracy goes beyond simple knowledge of the limit of detection of each technique and needs to be evaluated through comparisons with similar previous studies.