Design and characterization of mouse IgG1 and IgG2a bispecific antibodies for use in syngeneic models.
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ABSTRACT:
SUBMITTER: Wang F
PROVIDER: S-EPMC6927765 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Antibodies (Abs) containing two different antigen-binding sites in one molecule are called bispecific. Bispecific Abs (BsAbs) were first described in the 1960s, the first monoclonal BsAbs were generated in the 1980s by hybridoma technology, and the first article describing the therapeutic use of BsAbs was published in 1992, but the number of papers devoted to BsAbs has increased significantly in the last 10 years. Particular interest in BsAbs is due to their therapeutic use. In the last decade, two BsAbs - catumaxomab in 2009 and blinatumomab in 2014, were approved for therapeutic use. Papers published in recent years have been devoted to various methods of BsAb generation by genetic engineering and chemical conjugation, and describe preclinical and clinical trials of these drugs in a variety of diseases. This review considers diverse BsAb-production methods, describes features of therapeutic BsAbs approved for medical use, and summarizes the prospects of practical application of promising new BsAbs.
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Project description:Fc domain fusion can improve the therapeutic effects of relatively small biological molecules such as peptides, cytokines, and antibody fragments. Fc fusion proteins can also be used to enhance the cytotoxic effects of small bispecific antibodies (bsAbs). However, fragmentation of Fc fusion proteins, which mainly occurs around the hinge regions during production, storage, and circulation in the blood, is a major issue. In this study, we first investigated the mechanisms of fragmentation around the hinge region during storage using Fc-fused bsAbs with specificity for epidermal growth factor receptor and CD3 as a model. The fragmentation peaks generated by gel filtration analysis indicated that both contaminating proteases and dissolved active oxygen should be considered causes of fragmentation. We designed and constructed variants by introducing a point mutation into the upper hinge region, which reduced the cleavage caused by dissolved active oxygen, and shortened the hinge region to restrict access of proteases. These hinge modifications improved fragmentation resistance and did not affect the biological activity of the bsAbs in vitro. We confirmed the versatility of the hinge modifications using another Fc-fused bsAb. Our results show that hinge modifications to the Fc fusion protein, especially the introduction of a point mutation into the upper hinge region, can reduce fragmentation substantially, and these modifications can be used to improve the fragmentation resistance of other recombinant Fc fusion proteins.
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Project description:The cell line-derived xenografts and patient derived xenografts have limited use in cancer immunotherapy evaluation because an immune compromised host is required for xenotransplantation. Syngeneic mouse models are derived by transplanting established mouse cell lines or tumor tissues to strain matched mouse hosts, which are better suited to study the interplay between immune and tumor cells. We investigated the differences as well as similarities of a panel of ten mouse syngeneic models to features of human tumors by proteomics, which will provide valuable information to assist experimental biologists in model selection.
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Project description:In the last decades, antibody-based tumor therapy has fundamentally improved the efficacy of treatment for patients with cancer. Currently, almost all tumor antigen-targeting antibodies approved for clinical application are of IgG1 Fc isotype. Similarly, the mouse homolog mIgG2a is the most commonly used in tumor mouse models. However, in mice, the efficacy of antibody-based tumor therapy is largely restricted to a prophylactic application. Direct isotype comparison studies in mice in a therapeutic setting are scarce. In this study, we assessed the efficacy of mouse tumor-targeting antibodies of different isotypes in a therapeutic setting using a highly systematic approach. To this end, we engineered and expressed antibodies of the same specificity but different isotypes, targeting the artificial tumor antigen CD90.1/Thy1.1 expressed by B16 melanoma cells. Our experiments revealed that in a therapeutic setting mIgG2a was superior to both mIgE and mIgG1 in controlling tumor growth. Furthermore, the observed mIgG2a antitumor effect was entirely Fc mediated as the protection was lost when an Fc-silenced mIgG2a isotype (LALA-PG mutations) was used. These data confirm mIgG2a superiority in a therapeutic tumor model.SignificanceDirect comparisons of different antibody isotypes of the same specificity in cancer settings are still scarce. Here, it is shown that mIgG2a has a greater effect compared with mIgG1 and mIgE in controlling tumor growth in a therapeutic setting.
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Project description:Targeting multiple receptors with bispecific antibodies is a novel approach that may prevent the development of resistance to cancer treatments. Despite the initial promise, full clinical benefit of this technology has yet to be realized. We hypothesized that in order to optimally exploit bispecific antibody technology, thorough fundamental knowledge of their pharmacological properties compared to that of single agent combinations was needed. Therefore, we developed a mathematical model for the binding of bispecific antibodies to their targets that accounts for the spatial distribution of the binding receptors and the kinetics of binding, and is scalable for increasing valency. The model provided an adequate description of internal and literature-reported in vitro data on bispecific binding. Simulations of in vitro binding with the model indicated that bispecific antibodies are not always superior in their binding potency to combination of antibodies, and the affinity of bispecific arms must be optimized for maximum binding potency. Our results suggest that this tool can be used for the design and development of the next generation of anti-cancer bispecific compounds.
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