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Methods in Molecular Biology (Clifton,... 2020The hemagglutination inhibition (HI) assay for influenza A virus has been used since the 1940s. The assay may be utilized to detect or quantify antibodies to influenza A...
The hemagglutination inhibition (HI) assay for influenza A virus has been used since the 1940s. The assay may be utilized to detect or quantify antibodies to influenza A viruses and can be used to characterize differences in antigenic reactivity between influenza isolates. In addition, data from HI assays are routinely used for antigenic cartography, influenza virus surveillance, epidemiology, and vaccine-seed strain selection. For antibody quantification, the HI assay is a fast and inexpensive method; other than a source of red blood cells, no expensive or unusual lab equipment is needed, and results can be obtained within a few hours. Historically, the HI assay has also served as a primary method of subtype identification and is still used widely. However, as gene sequencing technology has evolved to be cheaper and faster, it is replacing the HI assay for this purpose.
Topics: Animals; Antibodies, Viral; Antibody Specificity; Antigens, Viral; Chickens; Erythrocytes; Hemagglutination Inhibition Tests; Immune Sera
PubMed: 32170677
DOI: 10.1007/978-1-0716-0346-8_2 -
Science (New York, N.Y.) May 2021
Topics: Animals; Antibodies, Bispecific; Antibody Affinity; Antibody Specificity; Binding Sites, Antibody; CD3 Complex; Cytokines; Humans; Immunotherapy; Lymphocyte Activation; Neoplasms; Protein Engineering; T-Lymphocytes
PubMed: 34045345
DOI: 10.1126/science.abg1209 -
Frontiers in Immunology 2020Monoclonal antibodies (mAbs) have become one of the most important classes of biopharmaceutical products, and they continue to dominate the universe of biopharmaceutical... (Review)
Review
Monoclonal antibodies (mAbs) have become one of the most important classes of biopharmaceutical products, and they continue to dominate the universe of biopharmaceutical markets in terms of approval and sales. They are the most profitable single product class, where they represent six of the top ten selling drugs. At the beginning of the 1990s, an antibody selection technology known as antibody phage display was developed by John McCafferty and Sir. Gregory Winter that enabled the discovery of human antibodies for diverse applications, particularly antibody-based drugs. They created combinatorial antibody libraries on filamentous phage to be utilized for generating antigen specific antibodies in a matter of weeks. Since then, more than 70 phage-derived antibodies entered clinical studies and 14 of them have been approved. These antibodies are indicated for cancer, and non-cancer medical conditions, such as inflammatory, optical, infectious, or immunological diseases. This review will illustrate the utility of phage display as a powerful platform for therapeutic antibodies discovery and describe in detail all the approved mAbs derived from phage display.
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antineoplastic Agents, Immunological; Cell Surface Display Techniques; Clinical Trials as Topic; Drug Development; Drug Evaluation, Preclinical; Genetic Engineering; High-Throughput Screening Assays; Humans; Molecular Targeted Therapy; Translational Research, Biomedical; Treatment Outcome
PubMed: 32983137
DOI: 10.3389/fimmu.2020.01986 -
Cell Sep 2021Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain...
Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAP) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAP and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.
Topics: Animals; Antibody Specificity; Base Sequence; Brain; CRISPR-Cas Systems; Cell Membrane; Cells, Cultured; Glycoproteins; Glycosaminoglycans; Glycosylation; Host-Pathogen Interactions; Humans; LDL-Receptor Related Protein-Associated Protein; Ligands; Low Density Lipoprotein Receptor-Related Protein-1; Membrane Glycoproteins; Mice; Protein Binding; Protein Denaturation; Rift Valley Fever; Rift Valley fever virus; Virus Internalization
PubMed: 34559985
DOI: 10.1016/j.cell.2021.09.001 -
Nature Oct 2022Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins...
Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras (PROTACs), have highlighted clinically important advantages of target degradation over inhibition. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.
Topics: Animals; Antibodies; Antibody Specificity; Colorectal Neoplasms; Ligands; Membrane Proteins; Proteolysis; Receptors, Cell Surface; Substrate Specificity; Ubiquitin-Protein Ligases
PubMed: 36131013
DOI: 10.1038/s41586-022-05235-6 -
Frontiers in Immunology 2021Bispecific antibodies (BsAbs) are antibodies with two binding sites directed at two different antigens or two different epitopes on the same antigen. The clinical...
Bispecific antibodies (BsAbs) are antibodies with two binding sites directed at two different antigens or two different epitopes on the same antigen. The clinical therapeutic effects of BsAbs are superior to those of monoclonal antibodies (MoAbs), with broad applications for tumor immunotherapy as well as for the treatment of other diseases. Recently, with progress in antibody or protein engineering and recombinant DNA technology, various platforms for generating different types of BsAbs based on novel strategies, for various uses, have been established. More than 30 mature commercial technology platforms have been used to create and develop BsAbs based on the heterologous recombination of heavy chains and matching of light chains. The detailed mechanisms of clinical/therapeutic action have been demonstrated with these different types of BsAbs. Three kinds of BsAbs have received market approval, and more than 110 types of BsAbs are at various stages of clinical trials. In this paper, we elaborate on the classic platforms, mechanisms, and applications of BsAbs. We hope that this review can stimulate new ideas for the development of BsAbs and improve current clinical strategies.
Topics: Animals; Antibodies, Bispecific; Antibody Specificity; Binding Sites, Antibody; Biotechnology; Drug Design; Epitopes; Humans; Immunotherapy; Protein Engineering; Recombinant Proteins; Translational Research, Biomedical
PubMed: 34025638
DOI: 10.3389/fimmu.2021.626616 -
Cell Nov 2021Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental...
Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.
Topics: Animals; Antibody Specificity; Bacteria; Bacterial Proteins; Biofilms; Cell Line; Chinchilla; DNA, Bacterial; DNA, Cruciform; Deoxyribonucleases; Extracellular Matrix; Extracellular Space; Extracellular Traps; Humans; Tetradecanoylphorbol Acetate
PubMed: 34735796
DOI: 10.1016/j.cell.2021.10.010 -
Theranostics 2022T-cell-redirecting bispecific antibodies (bsAbs) and trispecific antibodies (tsAbs) designed to recognize different epitopes or antigens have emerged as promising...
T-cell-redirecting bispecific antibodies (bsAbs) and trispecific antibodies (tsAbs) designed to recognize different epitopes or antigens have emerged as promising cancer therapies. Current approaches are all designed to include another antibody specific to the site of the primary antibody, and the molecular structures are generally established. However, the dimensions of target molecule and epitope location play a key role in the efficiency of the immunological synapse (IS) formation and subsequent T-cell-redirecting activities, therefore the connection flexibility of these antibodies determines the geometries of different formats of these molecules and will have a major impact on the efficacy. We describe a novel recombination strategy using various linker designs to site-specifically fuse anti-Her2 (2Rs15) or anti-VEGFR2 (3VGR19) nanobodies to different positions of the anti-CD3 antibody fragment (Fab, SP34). Based on the comparison among the various antigen-specific bsAbs, we could determine the desired fusion site of each nanobody to SP34, and further ensure the optimal structure of tsAbs with synergistic dual-antigen enhanced T-cell-redirecting activities. This approach allows precise control of the formation of IS between Her2- and/or VEGFR2-expressing cancer cells and T cells, to obtain the optimal structure of the Her2/VEGFR2/CD3 tsAb without the need to map antibody-binding epitopes. Optimization of Her2/VEGFR2/CD3 tsAb results in enhanced T-cell-redirecting and antitumor efficacy compared with the corresponding bsAbs alone or in combination, and the potency to overcome tumor relapse due to antigen escape or resistance to Herceptin and Cyramza therapy. The novel design strategy for developing tsAbs using a site-specific recombination approach represents a promising platform for immuno-oncology and in applications other than cancer therapy.
Topics: T-Lymphocytes; Antibodies, Bispecific; Lymphocyte Activation; Epitopes; Antibody Specificity
PubMed: 36451856
DOI: 10.7150/thno.75037 -
International Journal of Biological... 2021() encodes a tumor suppressor that is frequently mutated in familial breast and ovarian cancer patients. BRCA1 functions in multiple important cellular processes...
() encodes a tumor suppressor that is frequently mutated in familial breast and ovarian cancer patients. BRCA1 functions in multiple important cellular processes including DNA damage repair, cell cycle checkpoint activation, protein ubiquitination, chromatin remodeling, transcriptional regulation, as well as R-loop formation and apoptosis. A large number of BRCA1 antibodies have been generated and become commercially available over the past three decades, however, many commercial antibodies are poorly characterized and, when widely used, led to unreliable data. In search of reliable and specific BRCA1 antibodies (Abs), particularly antibodies recognizing mouse BRCA1, we performed a rigorous validation of a number of commercially available anti-BRCA1 antibodies, using proper controls in a panel of validation applications, including Western blot (WB), immunoprecipitation (IP), immunoprecipitation-mass spectrometry (IP-MS), chromatin immunoprecipitation (ChIP) and immunofluorescence (IF). Furthermore, we assessed the specificity of these antibodies to detect mouse BRCA1 protein through the use of testis tissue and mouse embryonic fibroblasts (MEFs) from Brca1 and Brca1 mice. We find that Ab1, D-9, 07-434 (for recognizing human BRCA1) and 287.17, 440621, BR-64 (for recognizing mouse BRCA1) are specific with high quality performance in the indicated assays. We share these results here with the goal of helping the community combat the common challenges associated with anti-BRCA1 antibody specificity and reproducibility and, hopefully, better understanding BRCA1 functions at cellular and tissue levels.
Topics: Animals; Antibodies; Antibody Specificity; BRCA1 Protein; Blotting, Western; Cell Line, Tumor; Chromatin Immunoprecipitation; Humans; Male; Mass Spectrometry; Mice; Real-Time Polymerase Chain Reaction
PubMed: 34421362
DOI: 10.7150/ijbs.63115 -
Life Sciences Jun 2024Antibody-drug conjugates (ADCs) are immunoconjugates that combine the specificity of monoclonal antibodies with a cytotoxic agent. The most appealing aspects of ADCs... (Review)
Review
Antibody-drug conjugates (ADCs) are immunoconjugates that combine the specificity of monoclonal antibodies with a cytotoxic agent. The most appealing aspects of ADCs include their potential additive or synergistic effects of the innate backbone antibody and cytotoxic effects of the payload on tumors without the severe toxic side effects often associated with traditional chemotherapy. Recent advances in identifying new targets with tumor-specific expression, along with improved bioactive payloads and novel linkers, have significantly expanded the scope and optimism for ADCs in cancer therapeutics. In this paper, we will first provide a brief overview of antibody specificity and the structure of ADCs. Next, we will discuss the mechanisms of action and the development of resistance to ADCs. Finally, we will explore opportunities for enhancing ADC efficacy, overcoming drug resistance, and offer future perspectives on leveraging ADCs to improve the outcome of ADC therapy for cancer treatment.
Topics: Humans; Immunoconjugates; Neoplasms; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Drug Resistance, Neoplasm; Antibody Specificity
PubMed: 38688384
DOI: 10.1016/j.lfs.2024.122676