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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 -
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 -
PLoS Computational Biology May 2021Antibodies are widely used reagents to test for expression of proteins and other antigens. However, they might not always reliably produce results when they do not...
Antibodies are widely used reagents to test for expression of proteins and other antigens. However, they might not always reliably produce results when they do not specifically bind to the target proteins that their providers designed them for, leading to unreliable research results. While many proposals have been developed to deal with the problem of antibody specificity, it is still challenging to cover the millions of antibodies that are available to researchers. In this study, we investigate the feasibility of automatically generating alerts to users of problematic antibodies by extracting statements about antibody specificity reported in the literature. The extracted alerts can be used to construct an "Antibody Watch" knowledge base containing supporting statements of problematic antibodies. We developed a deep neural network system and tested its performance with a corpus of more than two thousand articles that reported uses of antibodies. We divided the problem into two tasks. Given an input article, the first task is to identify snippets about antibody specificity and classify if the snippets report that any antibody exhibits non-specificity, and thus is problematic. The second task is to link each of these snippets to one or more antibodies mentioned in the snippet. The experimental evaluation shows that our system can accurately perform the classification task with 0.925 weighted F1-score, linking with 0.962 accuracy, and 0.914 weighted F1 when combined to complete the joint task. We leveraged Research Resource Identifiers (RRID) to precisely identify antibodies linked to the extracted specificity snippets. The result shows that it is feasible to construct a reliable knowledge base about problematic antibodies by text mining.
Topics: Animals; Antibody Specificity; Data Mining; Humans; Mice; Neural Networks, Computer
PubMed: 34043624
DOI: 10.1371/journal.pcbi.1008967 -
ELife Nov 2023A strategy to identify high-quality commercially available antibodies for research reveals extensive use of non-specific antibodies and offers solutions for future...
A strategy to identify high-quality commercially available antibodies for research reveals extensive use of non-specific antibodies and offers solutions for future large-scale testing.
Topics: Antibodies; Antibody Specificity
PubMed: 37962204
DOI: 10.7554/eLife.93329 -
New Biotechnology Oct 2018Validation of antibodies and other protein binders is a subject of pressing concern for the research community and one which is uppermost in the minds of all who use... (Review)
Review
Validation of antibodies and other protein binders is a subject of pressing concern for the research community and one which is uppermost in the minds of all who use antibodies as research and diagnostic reagents. Assessing an antibody's fitness for purpose includes accurate ascertainment of its target specificity and suitability for the envisaged task. Moreover, standardised procedures are essential to guarantee sample quality in testing procedures. The problem of defining precise standards for antibody validation has engendered much debate in recent publications and meetings, but gradually a consensus is emerging. At the 8th Alpbach Affinity Proteomics workshop (March 2017), a panel of leaders in the antibody field discussed suggestions which could bring this complex but essential issue a step nearer to a resolution. 'Alpbach recommendations' for best practice include tailoring binder validation processes according to the intended applications and promoting greater transparency in publications and in the information available from commercial antibody developers/providers. A single approach will not fit all applications and end users must ensure that the reported validation holds for their specific requirements, highlighting the need for adequate training in the fundamentals of antibody characterisation and validation across the user community.
Topics: Antibodies; Antibody Specificity; Humans; Proteomics; Reproducibility of Results
PubMed: 30086383
DOI: 10.1016/j.nbt.2018.08.002 -
Current Opinion in Biotechnology Dec 2019Despite the recent explosion in the use of monoclonal antibodies (mAbs) as drugs, it remains a significant challenge to generate antibodies with a combination of... (Review)
Review
Despite the recent explosion in the use of monoclonal antibodies (mAbs) as drugs, it remains a significant challenge to generate antibodies with a combination of physicochemical properties that are optimal for therapeutic applications. We argue that one of the most important and underappreciated drug-like antibody properties is high specificity - defined here as low levels of antibody non-specific and self-interactions - which is linked to low off-target binding and slow antibody clearance in vivo and high solubility and low viscosity in vitro. Here, we review the latest advances in characterizing antibody specificity and elucidating its molecular determinants as well as using these findings to improve the selection and engineering of antibodies with extremely high, drug-like specificity.
Topics: Antibodies, Monoclonal; Antibody Specificity; Solubility; Viscosity
PubMed: 30822699
DOI: 10.1016/j.copbio.2019.01.008 -
Chembiochem : a European Journal of... Sep 2022Antibodies recognize their cognate antigens with high affinity and specificity, but the prediction of binding sites on the antigen (epitope) corresponding to a specific...
Antibodies recognize their cognate antigens with high affinity and specificity, but the prediction of binding sites on the antigen (epitope) corresponding to a specific antibody remains a challenging problem. To address this problem, we developed AbAdapt, a pipeline that integrates antibody and antigen structural modeling with rigid docking in order to derive antibody-antigen specific features for epitope prediction. In this study, we systematically assessed the impact of integrating the state-of-the-art protein modeling method AlphaFold with the AbAdapt pipeline. By incorporating more accurate antibody models, we observed improvement in docking, paratope prediction, and prediction of antibody-specific epitopes. We further applied AbAdapt-AF in an anti-receptor binding domain (RBD) antibody complex benchmark and found AbAdapt-AF outperformed three alternative docking methods. Also, AbAdapt-AF demonstrated higher epitope prediction accuracy than other tested epitope prediction tools in the anti-RBD antibody complex benchmark. We anticipate that AbAdapt-AF will facilitate prediction of antigen-antibody interactions in a wide range of applications.
Topics: Antibodies; Antibody Specificity; Antigens; Binding Sites, Antibody; Epitopes
PubMed: 35893479
DOI: 10.1002/cbic.202200303 -
RNA Biology Sep 2017Nucleotide modifications constitute marks in RNA and DNA that contribute to gene regulation, development and other cellular processes. The understanding of their... (Review)
Review
Nucleotide modifications constitute marks in RNA and DNA that contribute to gene regulation, development and other cellular processes. The understanding of their intricate molecular roles has been hampered by the high number of different modifications, the lack of effective methods and tools for their detection and quantification as well as by their complex structure-function relationship. The recent development of RNA and DNA immunoprecipitation followed by high-throughput sequencing (RIP- and DIP-seq) initiated detailed transcriptome- and genome-wide studies. Both techniques depend on highly specific and sensitive antibodies to specifically enrich the targeted modified nucleotides without background or potential biases. Here, we review the challenges and developments when generating and validating antibodies targeting modified nucleotides. We discuss antibody-antigen interactions, different strategies of antigen generation and compare different binder formats suitable for state-of-the-art high resolution mapping and imaging technologies.
Topics: Animals; Antibodies; Antibody Specificity; Antigens; Carrier Proteins; Epigenesis, Genetic; Humans; Nucleic Acids; Protein Binding; Reproducibility of Results
PubMed: 28277931
DOI: 10.1080/15476286.2017.1295905 -
MAbs 2020In the wake of the reproducibility crisis and numerous discussions on how commercially available antibodies as research tool contribute to it, The Antibody Society...
In the wake of the reproducibility crisis and numerous discussions on how commercially available antibodies as research tool contribute to it, The Antibody Society developed a series of 10 webinars to address the issues involved. The webinars were delivered by speakers with both academic and commercial backgrounds. This report highlights the problems, and offers solutions to help the scientific community appropriately identify the right antibodies and to validate them for their research and development projects. Despite the various solutions proposed here, they must be applied on a case-by-case basis. Each antibody must be verified based on the content of the product sheet, and subsequently through experimentation to confirm integrity, specificity and selectivity. Verification needs to focus on the precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly. The various approaches discussed here all have caveats, so a combination of solutions must be considered.
Topics: Antibodies, Monoclonal; Antibody Specificity; Humans; Protein Engineering; Validation Studies as Topic
PubMed: 32748696
DOI: 10.1080/19420862.2020.1794421