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EBioMedicine Feb 2020DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was...
BACKGROUND
DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A.
METHODS
T-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys.
FINDINGS
DuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable.
INTERPRETATION
Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies.
FUNDING
Genmab.
Topics: Animals; Antibodies, Bispecific; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Antigens, CD20; Antineoplastic Agents, Immunological; CD3 Complex; Cell Line, Tumor; Cytotoxicity, Immunologic; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Humans; Leukemia, B-Cell; Lymphocyte Activation; Lymphoma, B-Cell; Macaca fascicularis; Mice; Mutation; Recombinant Proteins; T-Lymphocytes; Xenograft Model Antitumor Assays
PubMed: 31981978
DOI: 10.1016/j.ebiom.2019.102625 -
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 -
Molecular Cancer Therapeutics Sep 2019Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more...
Although treatment advances over recent decades have significantly improved survival of patients with multiple myeloma, there is still an unmet medical need for more effective treatments. In this study, we identified G-protein-coupled receptor family C group 5 member D (GPRC5D) expression on the surface of malignant cells involved in multiple myeloma, but except for plasma cells and B cells, not at appreciable levels on normal hematopoietic cells and bone marrow progenitors, including hematopoietic stem cells. In addition, we constructed IgG-based anti-GPRC5D/CD3 bispecific T-cell-redirecting antibodies (GPRC5D TRAB), which suppressed the tumor growth of GPRC5D-positive myeloma cells through the activation of T cells and in xenograft models. Collectively, these findings suggest that GPRC5D is an antigen specific to multiple myeloma and a potential target of TRAB therapy.
Topics: Animals; Antibodies, Bispecific; Antibody Specificity; CD3 Complex; CHO Cells; Cell Line, Tumor; Cricetulus; Female; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Multiple Myeloma; Receptors, G-Protein-Coupled; Tumor Burden; Xenograft Model Antitumor Assays
PubMed: 31270154
DOI: 10.1158/1535-7163.MCT-18-1216 -
Cold Spring Harbor Protocols Jun 2020Immunoblotting allows detection of a protein antigen immobilized on the protein-retaining membrane support such as nitrocellulose or polyvinylidene fluoride (PVDF). The...
Immunoblotting allows detection of a protein antigen immobilized on the protein-retaining membrane support such as nitrocellulose or polyvinylidene fluoride (PVDF). The detection of the protein of interest relies on the binding of an antibody that specifically recognizes the protein of interest exposed on the membrane. The protein of interest can be purified or mixed with other proteins as in cell or tissue extracts. Usually immunoblotting combines the resolution of proteins by gel electrophoresis with immunochemical detection and is referred to as "western blotting." Immunoblotting can be used to determine the presence and the steady-state level of the protein of interest in the sample, its relative molecular weight, and the distribution of the protein between cellular fractions. Immunoblotting can be performed using the antibodies raised against synthetic peptide antigens modified to mimic posttranslational modifications of proteins, such as phosphorylation and acetylation, to study these modifications in the protein of interest in vivo. When antibodies against the protein of interest are not available, immunoblotting can be performed using antibodies that specifically recognize the recombinant epitope tags (hemagglutinin [HA]-, Flag-, cMyc-, or glutathione--transferase [GST]) fused to the protein of interest using recombinant DNA techniques. Immunoblotting has a variety of research, clinical, and forensic medicine applications. It is also one of the standard techniques for characterization of antibodies from different samples of polyclonal sera or hybridoma supernatants.
Topics: Animals; Antibodies; Antibody Specificity; Antigens; Electrophoresis; Epitopes; Humans; Immunoblotting; Membranes, Artificial; Molecular Weight; Peptides; Proteins
PubMed: 32482904
DOI: 10.1101/pdb.top098392 -
Current Opinion in Organ Transplantation Aug 2020Despite significant improvement in pancreas allograft survival, rejection continues to be a major clinical problem. This review will focus on emerging literature related... (Review)
Review
PURPOSE OF REVIEW
Despite significant improvement in pancreas allograft survival, rejection continues to be a major clinical problem. This review will focus on emerging literature related to the impact of pretransplant and de-novo DSA (dnDSA) in pancreas transplant recipients, and the diagnosis and treatment of T-cell-medicated rejection (TCMR) and antibody-mediated rejection (ABMR) in this complex group of patients.
RECENT FINDINGS
Recent data suggest that pretransplant DSA and the emergence of dnDSA in pancreas transplant recipients are both associated with increased risk of ABMR. The pancreas allograft biopsy is essential for the specific diagnosis of TCMR and/or ABMR, distinguish rejection from other causes of graft dysfunction, and to guide-targeted therapy. This distinction is important especially in the setting of solitary pancreas transplants but also in simultaneous pancreas-kidney transplants where solid evidence has now emerged demonstrating discordant biopsy findings. Treatment of rejection in a functioning pancreas can prolong allograft survival.
SUMMARY
The accurate and timely diagnosis of active alloimmune destruction in pancreas transplant recipients is paramount to preserving graft function in the long term. This review will discuss new, rapidly evolving information that is valuable for the physician caring for these patients to achieve optimal immunological outcomes.
Topics: Allografts; Antibodies; Antibody Specificity; Graft Rejection; HLA Antigens; Histocompatibility; Humans; Pancreas Transplantation; Risk Factors; T-Lymphocytes; Transplantation Immunology; Transplantation, Homologous
PubMed: 32692039
DOI: 10.1097/MOT.0000000000000776 -
NPJ Systems Biology and Applications Aug 2020Mosunetuzumab, a T-cell dependent bispecific antibody that binds CD3 and CD20 to drive T-cell mediated B-cell killing, is currently being tested in non-Hodgkin lymphoma....
Mosunetuzumab, a T-cell dependent bispecific antibody that binds CD3 and CD20 to drive T-cell mediated B-cell killing, is currently being tested in non-Hodgkin lymphoma. However, potent immune stimulation with T-cell directed therapies poses the risk of cytokine release syndrome, potentially limiting dose and utility. To understand mechanisms behind safety and efficacy and explore safety mitigation strategies, we developed a novel mechanistic model of immune and antitumor responses to the T-cell bispecifics (mosunetuzumab and blinatumomab), including the dynamics of B- and T-lymphocytes in circulation, lymphoid tissues, and tumor. The model was developed and validated using mosunetuzumab nonclinical and blinatumomab clinical data. Simulations delineated mechanisms contributing to observed cell and cytokine (IL6) dynamics and predicted that initial step-fractionated dosing limits systemic T-cell activation and cytokine release without compromising tumor response. These results supported a change to a step-fractionated treatment schedule of mosunetuzumab in the ongoing Phase I clinical trial, enabling safer administration of higher doses.
Topics: Antibody Specificity; Antigens, CD20; CD3 Complex; Clinical Trials, Phase I as Topic; Cytokine Release Syndrome; Humans; Lymphoma, Non-Hodgkin; Models, Biological; Risk; Translational Research, Biomedical
PubMed: 32859946
DOI: 10.1038/s41540-020-00145-7 -
Analytical Chemistry Feb 2022Antibody-antigen (Ab-Ag) interactions are canonically described by a model that exclusively accommodates noninteraction (0) or reproducible interaction (RI) states, yet...
Antibody-antigen (Ab-Ag) interactions are canonically described by a model that exclusively accommodates noninteraction (0) or reproducible interaction (RI) states, yet this model is inadequate to explain often-encountered nonreproducible signals. Here, by monitoring diverse experimental systems using a peptide-protein hybrid microarray, we observed that Ab-probe interactions comprise a substantial proportion of nonreproducible antibody-based results. This enabled our discovery and capacity to reliably identify nonreproducible Ab-probe interactions (NRIs), as well as our development of a powerful explanatory model ("0-NRI-RI-Hook four-state model") that is mAb concentration-dependent, regardless of specificity, which ultimately shows that both nonspecific interactions and NRIs are not predictable yet certain to happen. Our discoveries challenge the centrality of Ab-Ag interaction specificity data in serology and immunology.
Topics: Antibodies; Antibody Specificity; Antigens; Peptides
PubMed: 35044162
DOI: 10.1021/acs.analchem.1c03264 -
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 -
Lab on a Chip Sep 2021Antibodies (Abs) are among the most important class of biologicals, showcasing a high therapeutic and diagnostic value. In the global therapeutic Ab market, fully-human... (Review)
Review
Antibodies (Abs) are among the most important class of biologicals, showcasing a high therapeutic and diagnostic value. In the global therapeutic Ab market, fully-human monoclonal Abs (FH-mAbs) are flourishing thanks to their low immunogenicity and high specificity. The rapidly emerging field of single-cell technologies has paved the way to efficiently discover mAbs by facilitating a fast screening of the antigen (Ag)-specificity and functionality of Abs expressed by B cells. This review summarizes the principles and challenges of the four key concepts to discover mAbs using these technologies, being confinement of single cells using either droplet microfluidics or microstructure arrays, identification of the cells of interest, retrieval of those cells and single-cell sequence determination required for mAb production. This review reveals the enormous potential for mix-and-matching of the above-mentioned strategies, which is illustrated by the plethora of established, highly integrated devices. Lastly, an outlook is given on the many opportunities and challenges that still lie ahead to fully exploit miniaturized single-cell technologies for mAb discovery.
Topics: Antibodies, Monoclonal; Antibody Specificity; Antineoplastic Agents, Immunological; Humans
PubMed: 34505611
DOI: 10.1039/d1lc00243k -
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