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Journal of Neurochemistry Feb 2008High morbidity, enormous socioeconomic costs, and lack of specific treatments emphasize the importance of research on protective therapies against Alzheimer's disease.... (Review)
Review
High morbidity, enormous socioeconomic costs, and lack of specific treatments emphasize the importance of research on protective therapies against Alzheimer's disease. The efficacy of anti-amyloid immunization strategies has been demonstrated preclinically, prompting the design of clinical studies. However, the detailed mechanisms of action of therapeutic antibodies, especially their influence on the complex amyloid beta peptide (Abeta) metabolism and various Abeta-equilibria present both within and outside the CNS, are far from being clear. Furthermore, physiological Abeta metabolism is poorly understood and the analytical tools to characterize and quantify treatment effects on Abeta metabolism are suboptimal. Thus, the design of immunization strategies with optimized benefit-to-risk ratios for patients is subjected to significant obstacles. Indeed, an active immunization trial with Abeta was discontinued because of severe adverse effects. Anti-Abeta immunization protocols designed to attain high blood levels of antibodies bear the potential to induce brain inflammation and/or hemorrhage, thus directing the biomedical research towards development of more predictable therapies for minimizing the risk of adverse effects. The focus of this review is to summarize current knowledge of Abeta metabolism under physiological and antibody-based therapeutic conditions and to introduce a promising approach, namely the passive immunization using antibody fragments, which are characterized by entirely different pharmacokinetic and pharmacodynamic properties compared with conventional monoclonal antibodies.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antibodies; Antibody Specificity; Humans; Immunization, Passive
PubMed: 17986215
DOI: 10.1111/j.1471-4159.2007.05064.x -
Frontiers in Immunology 2019B-cells can neutralize pathogenic molecules by targeting them with extreme specificity using receptors secreted or expressed on their surface (antibodies). This is...
B-cells can neutralize pathogenic molecules by targeting them with extreme specificity using receptors secreted or expressed on their surface (antibodies). This is achieved via molecular interactions between the paratope (i.e., the antibody residues involved in the binding) and the interacting region (epitope) of its target molecule (antigen). Discerning the rules that define this specificity would have profound implications for our understanding of humoral immunogenicity and its applications. The aim of this work is to produce improved, antibody-specific epitope predictions by exploiting features derived from the antigens and their cognate antibodies structures, and combining them using statistical and machine learning algorithms. We have identified several geometric and physicochemical features that are correlated in interacting paratopes and epitopes, used them to develop a Monte Carlo algorithm to generate putative epitopes-paratope pairs, and train a machine-learning model to score them. We show that, by including the structural and physicochemical properties of the paratope, we improve the prediction of the target of a given B-cell receptor. Moreover, we demonstrate a gain in predictive power both in terms of identifying the cognate antigen target for a given antibody and the antibody target for a given antigen, exceeding the results of other available tools.
Topics: Algorithms; Amino Acid Sequence; Animals; Antibodies; Antibody Specificity; Antigen-Antibody Complex; Binding Sites, Antibody; Epitope Mapping; Epitopes, B-Lymphocyte; Humans; Models, Molecular; Monte Carlo Method; Neural Networks, Computer; Protein Domains; Reproducibility of Results
PubMed: 30863406
DOI: 10.3389/fimmu.2019.00298 -
Journal of Neurochemistry Jan 2020Antibodies raised against defined phosphorylation sites of the microtubule-associated protein tau are widely used in scientific research and being applied in clinical...
Antibodies raised against defined phosphorylation sites of the microtubule-associated protein tau are widely used in scientific research and being applied in clinical assays. However, recent studies have revealed an alarming degree of non-specific binding found in these antibodies. In order to quantify and compare the specificity phospho-tau antibodies and other post-translational modification site-specific antibodies in general, a measure of specificity is urgently needed. Here, we report a robust flow cytometry assay using human embryonic kidney cells that enables the determination of a specificity parameter termed Φ, which measures the fraction of non-specific signal in antibody binding. We validate our assay using anti-tau antibodies with known specificity profiles, and apply it to measure the specificity of seven widely used phospho-tau antibodies (AT270, AT8, AT100, AT180, PHF-6, TG-3, and PHF-1) among others. We successfully determined the Φ values for all antibodies except AT100, which did not show detectable binding in our assay. Our results show that antibodies AT8, AT180, PHF-6, TG-3, and PHF-1 have Φ values near 1, which indicates no detectable non-specific binding. AT270 showed Φ value around 0.8, meaning that approximately 20% of the binding signal originates from non-specific binding. Further analyses using immunocytochemistry and western blotting confirmed the presence of non-specific binding of AT270 to non-tau proteins found in human embryonic kidney cells and the mouse hippocampus. We anticipate that the quantitative approach and parameter introduced here will be widely adopted as a standard for reporting the specificity for phospho-tau antibodies, and potentially for post-translational modification targeting antibodies in general. Cover Image for this issue: doi: 10.1111/jnc.14727.
Topics: Animals; Antibodies, Phospho-Specific; Antibody Specificity; Flow Cytometry; Fluorescent Dyes; Glycogen Synthase Kinase 3 beta; Green Fluorescent Proteins; HEK293 Cells; Hippocampus; Humans; Immunohistochemistry; Kidney; Mice; Phosphorylation; Transfection; tau Proteins
PubMed: 31325178
DOI: 10.1111/jnc.14830 -
Protein Science : a Publication of the... Sep 2009Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of...
Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three-fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications.
Topics: Amino Acid Sequence; Antibodies; Antibody Specificity; Binding Sites, Antibody; Epitopes; Molecular Sequence Data; Peptide Library
PubMed: 19610073
DOI: 10.1002/pro.203 -
Science Advances Aug 2019Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance...
Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Cell Line, Tumor; Clustered Regularly Interspaced Short Palindromic Repeats; Genomics; Hybridomas; Immunoglobulin Fab Fragments; Mice; Mice, Inbred C57BL; Recombinant Fusion Proteins
PubMed: 31489367
DOI: 10.1126/sciadv.aaw1822 -
Molecules (Basel, Switzerland) Feb 2017Owing to the widespread application value, monoclonal antibodies (MAbs) have become a tool of increasing importance in modern bioscience research since their emergence.... (Review)
Review
Owing to the widespread application value, monoclonal antibodies (MAbs) have become a tool of increasing importance in modern bioscience research since their emergence. Recently, some researchers have focused on the production of MAbs against medical plant-derived natural products (MPNP), the secondary metabolites of medical plants. At the same time, various immunoassay methods were established on the basis of these MPNP MAbs, and then rapidly developed into a novel technique for medical plant and phytomedicine research in the area of quality control, pharmacological analysis, drug discovery, and so on. Dependent on the research works carried out in recent years, this paper aims to provide a comprehensive review of MAbs against MPNP and the application of various immunoassay methods established on the basis of these MAbs, and conclude with a short section on future prospects and research trends in this area.
Topics: Antibodies, Monoclonal; Antibody Specificity; Biological Products; Drug Industry; Humans; Plants, Medicinal
PubMed: 28245640
DOI: 10.3390/molecules22030355 -
Proceedings of the National Academy of... Feb 2013Monoclonal antibodies are used in numerous therapeutic and diagnostic applications; however, their efficacy is contingent on specificity and avidity. Here, we show that...
Monoclonal antibodies are used in numerous therapeutic and diagnostic applications; however, their efficacy is contingent on specificity and avidity. Here, we show that presentation of antibodies on the surface of nonspherical particles enhances antibody specificity as well as avidity toward their targets. Using spherical, rod-, and disk-shaped polystyrene nano- and microparticles and trastuzumab as the targeting antibody, we studied specific and nonspecific uptake in three breast cancer cell lines: BT-474, SK-BR-3, and MDA-MB-231. Rods exhibited higher specific uptake and lower nonspecific uptake in all cells compared with spheres. This surprising interplay between particle shape and antibodies originates from the unique role of shape in determining binding and unbinding of particles to cell surface. In addition to exhibiting higher binding and internalization, trastuzumab-coated rods also exhibited greater inhibition of BT-474 breast cancer cell growth in vitro to a level that could not be attained by soluble forms of the antibody. The effect of trastuzumab-coated rods on cells was enhanced further by replacing polystyrene particles with pure chemotherapeutic drug nanoparticles of comparable dimensions made from camptothecin. Trastuzumab-coated camptothecin nanoparticles inhibited cell growth at a dose 1,000-fold lower than that required for comparable inhibition of growth using soluble trastuzumab and 10-fold lower than that using BSA-coated camptothecin. These results open unique opportunities for particulate forms of antibodies in therapeutics and diagnostics.
Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibody Specificity; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Endocytosis; Female; Humans; Nanoparticles; Temperature; Trastuzumab
PubMed: 23401509
DOI: 10.1073/pnas.1216893110 -
BMC Biotechnology Jul 2013Monoclonal antibody therapeutics are rapidly gaining in popularity for the treatment of a myriad of diseases, ranging from cancer to autoimmune diseases and neurological...
BACKGROUND
Monoclonal antibody therapeutics are rapidly gaining in popularity for the treatment of a myriad of diseases, ranging from cancer to autoimmune diseases and neurological diseases. Multiple forms of antibody therapeutics are in use today that differ in the amount of human sequence present in both the constant and variable regions, where antibodies that are more human-like usually have reduced immunogenicity in clinical trials.
RESULTS
Here we present a method to quantify the humanness of the variable region of monoclonal antibodies and show that this method is able to clearly distinguish human and non-human antibodies with excellent specificity. After creating and analyzing a database of human antibody sequences, we conducted an in-depth analysis of the humanness of therapeutic antibodies, and found that increased humanness score is correlated with decreased immunogenicity of antibodies. We further discovered a surprisingly similarity in the immunogenicity of fully human antibodies and humanized antibodies that are more human-like based on their humanness score.
CONCLUSIONS
Our results reveal that in most cases humanizing an antibody and confirming the humanness of the final form may be sufficient to eliminate immunogenicity issues to the same extent as using fully human antibodies. We created a public website to calculate the humanness score of any input antibody sequence based on our human antibody database. This tool will be of great value during the preclinical drug development process for new monoclonal antibody therapeutics.
Topics: Animals; Antibodies, Monoclonal, Humanized; Antibody Specificity; Databases, Factual; Humans; Immunogenetic Phenomena; Immunoglobulin Variable Region; Mice; Rats
PubMed: 23826749
DOI: 10.1186/1472-6750-13-55 -
Journal of Immunology Research 2016Central to the humoral theory of transplantation is production of antibodies by the recipient against mismatched HLA antigens in the donor organ. Not all mismatches... (Review)
Review
Central to the humoral theory of transplantation is production of antibodies by the recipient against mismatched HLA antigens in the donor organ. Not all mismatches result in antibody production, however, and not all antibodies are pathogenic. Serologic HLA matching has been the standard for solid organ allocation algorithms in current use. Antibodies do not recognize whole HLA molecules but rather polymorphic residues on the surface, called epitopes, which may be shared by multiple serologic HLA antigens. Data are accumulating that epitope analysis may be a better way to determine organ compatibility as well as the potential immunogenicity of given HLA mismatches. Determination of the pathogenicity of alloantibodies is evolving. Potential features include antibody strength (as assessed by antibody titer or, more commonly and inappropriately, mean fluorescence intensity) and ability to fix complement ( by C1q or C3d assay or by IgG subclass analysis). Technical issues with the use of solid phase assays are also of prime importance, such as denaturation of HLA antigens and manufacturing and laboratory variability. Questions and controversies remain, and here we review new relevant data.
Topics: Antibody Specificity; Epitopes; HLA Antigens; Histocompatibility Testing; Humans; Isoantibodies; Kidney Transplantation
PubMed: 28070526
DOI: 10.1155/2016/5197396 -
Theranostics 2019Therapeutic antibodies are one most significant advances in immunotherapy, the development of antibodies against disease-associated MHC-peptide complexes led to the... (Review)
Review
Therapeutic antibodies are one most significant advances in immunotherapy, the development of antibodies against disease-associated MHC-peptide complexes led to the introduction of TCR-like antibodies. TCR-like antibodies combine the recognition of intracellular proteins with the therapeutic potency and versatility of monoclonal antibodies (mAb), offering an unparalleled opportunity to expand the repertoire of therapeutic antibodies available to treat diseases like cancer. This review details the current state of TCR-like antibodies and describes their production, mechanisms as well as their applications. In addition, it presents an insight on the challenges that they must overcome in order to become commercially and clinically validated.
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Humans; Immunotherapy; Neoplasms; Receptors, Antigen, T-Cell
PubMed: 31695801
DOI: 10.7150/thno.35486