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Annual Review of Biomedical Engineering 2015The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability,... (Review)
Review
The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody-drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics.
Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Antibody Affinity; Antibody Specificity; Directed Molecular Evolution; Humans; Immunoconjugates; Models, Molecular; Protein Conformation; Protein Engineering; Protein Stability
PubMed: 26274600
DOI: 10.1146/annurev-bioeng-071114-040733 -
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 -
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 -
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 -
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 -
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 -
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 -
Immunological Reviews Nov 2015IgG4, the least represented human IgG subclass in serum, is an intriguing antibody with unique biological properties, such as the ability to undergo Fab-arm exchange and... (Review)
Review
IgG4, the least represented human IgG subclass in serum, is an intriguing antibody with unique biological properties, such as the ability to undergo Fab-arm exchange and limit immune complex formation. The lack of effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, is desirable for therapeutic purposes. IgG4 plays a protective role in allergy by acting as a blocking antibody, and inhibiting mast cell degranulation, but a deleterious role in malignant melanoma, by impeding IgG1-mediated anti-tumor immunity. These findings highlight the importance of understanding the interaction between IgG4 and Fcγ receptors. Despite a wealth of structural information for the IgG1 subclass, including complexes with Fcγ receptors, and structures for intact antibodies, high-resolution crystal structures were not reported for IgG4-Fc until recently. Here, we highlight some of the biological properties of human IgG4, and review the recent crystal structures of IgG4-Fc. We discuss the unexpected conformations adopted by functionally important Cγ2 domain loops, and speculate about potential implications for the interaction between IgG4 and FcγRs.
Topics: Animals; Antibody Affinity; Antibody Specificity; Binding Sites; Complement C1q; Glycosylation; Humans; Hypersensitivity; Immunoglobulin Fab Fragments; Immunoglobulin G; Models, Molecular; Neoplasms; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Protein Multimerization; Receptors, IgG; Structure-Activity Relationship
PubMed: 26497518
DOI: 10.1111/imr.12349 -
International Journal of Molecular... Oct 2020The success of antibody therapeutics is strongly influenced by their multifunctional nature that couples antigen recognition mediated by their variable regions with... (Review)
Review
The success of antibody therapeutics is strongly influenced by their multifunctional nature that couples antigen recognition mediated by their variable regions with effector functions and half-life extension mediated by a subset of their constant regions. Nevertheless, the monospecific IgG format is not optimal for many therapeutic applications, and this has led to the design of a vast number of unique multispecific antibody formats that enable targeting of multiple antigens or multiple epitopes on the same antigen. Despite the diversity of these formats, a common challenge in generating multispecific antibodies is that they display suboptimal physical and chemical properties relative to conventional IgGs and are more difficult to develop into therapeutics. Here we review advances in the design and engineering of multispecific antibodies with drug-like properties, including favorable stability, solubility, viscosity, specificity and pharmacokinetic properties. We also highlight emerging experimental and computational methods for improving the next generation of multispecific antibodies, as well as their constituent antibody fragments, with natural IgG-like properties. Finally, we identify several outstanding challenges that need to be addressed to increase the success of multispecific antibodies in the clinic.
Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Antibody Affinity; Antibody Specificity; Chemical Phenomena; Drug Design; Drug Development; Drug Stability; Humans; Models, Molecular; Protein Engineering; Solubility; Structure-Activity Relationship
PubMed: 33053650
DOI: 10.3390/ijms21207496