The Experts below are selected from a list of 28947 Experts worldwide ranked by ideXlab platform
Barry L Karger - One of the best experts on this subject based on the ideXlab platform.
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affinity probe capillary electrophoresis analysis of recombinant human growth hormone with a fluorescent labeled Antibody Fragment
Analytical Chemistry, 1994Co-Authors: Kiyohito Shimura, Barry L KargerAbstract:A new highly sensitive microscale analytical procedure called affinity probe capillary electrophoresis (APCE) is presented. One of the two species, which can form a biospecific complex, is labeled with a fluorescent dye. The affinity probe is used to detect the analyte as a complex after the separation of the excess free probe by capillary electrophoresis. As an example of this approach, an FAB' Fragment of a mouse monoclonal Antibody (anti-human growth hormone) was labeled with tetramethylrhodamine-iodoacetamide at a hinge region thiol group. Samples were mixed with the purified labeled Antibody Fragment, and the associated complex was separated by capillary isoelectric focusing with detection by laser-induced fluorescence
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affinity probe capillary electrophoresis analysis of recombinant human growth hormone with a fluorescent labeled Antibody Fragment
Analytical Chemistry, 1994Co-Authors: Kiyohito Shimura, Barry L KargerAbstract:A new highly sensitive microscale analytical procedure called affinity probe capillary electrophoresis (APCE) is presented. One of the two species, which can form a biospecific complex, is labeled with a fluorescent dye. The affinity probe is used to detect the analyte as a complex after the separation of the excess free probe by capillary electrophoresis. As an example of this approach, an FAB' Fragment of a mouse monoclonal Antibody (anti-human growth hormone) was labeled with tetramethylrhodamine-iodoacetamide at a hinge region thiol group. Samples were mixed with the purified labeled Antibody Fragment, and the associated complex was separated by capillary isoelectric focusing with detection by laser-induced fluorescence. In these preliminary results, methionyl recombinant human growth hormone (met-rhGH) could be successfully determined down to detection levels of approximately 5 x 10(-12) M. Mono- and dideamidated variants of met-rhGH were detected simultaneously with the nondeamidated form of the antigen as separate peaks.
Jianwu Dai - One of the best experts on this subject based on the ideXlab platform.
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a collagen binding egfr Antibody Fragment targeting tumors with a collagen rich extracellular matrix
Scientific Reports, 2016Co-Authors: Hui Liang, Bin Wang, Bing Chen, Yannan Zhao, Jie Sun, Yan Zhuang, Jiajia Shi, He Shen, Zhijun Zhang, Jianwu DaiAbstract:Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR Antibody Fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The Antibody Fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn’t showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as Antibody–drug conjugates (ADC) or immunotoxins.
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a collagen binding egfr single chain fv Antibody Fragment for the targeted cancer therapy
Journal of Controlled Release, 2015Co-Authors: Hui Liang, Bin Wang, Bing Chen, Yannan Zhao, Yan Zhuang, He Shen, Zhijun Zhang, Jianwu DaiAbstract:Collagen, a primary component of the extracellular matrix (ECM), is highly expressed in a variety of cancers and influences the tumor microenvironment by increasing the recruitment of macrophages and endothelial cells. Therefore, collagen is a highly promising target for cancer therapy. The collagen-binding domain (CBD) can dynamically bind to collagen and achieve the sustained release of CBD-fused protein in the collagen network. Here, we developed a collagen-binding epidermal growth factor receptor (EGFR) Antibody Fragment for targeting the collagen-rich ECM in tumors. The single chain Fragment variable (scFv) of cetuximab was fused to CBD (CBD-scFv) and expressed in Pichia pastoris. CBD-scFv preserved the antigen binding domain and anti-tumor activity of cetuximab in vitro. Moreover, CBD-scFv displayed a collagen binding ability due to the function of CBD. In vivo experiments revealed that CBD-scFv bound to collagen and achieved sustained release in tumors. Furthermore, CBD-scFv significantly suppressed the growth of tumors in A431 xenografts. Therefore, CBD-scFv had a potential therapeutic value for the collagen-rich carcinomas. The specific target and sustained release of CBD-scFv in tumors could be a new approach for targeted drug delivery in cancer therapy.
Kimberly M Bonger - One of the best experts on this subject based on the ideXlab platform.
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dual site specific chemoenzymatic Antibody Fragment conjugation using crispr based hybridoma engineering
Bioconjugate Chemistry, 2021Co-Authors: Camille Le M Gall, Johan M S Van Der Schoot, Ivan Ramostomillero, Melek Parlak Khalily, Floris J Van Dalen, Zacharias Wijfjes, Liyan Smeding, Duco Van Dalen, Anna Cammarata, Kimberly M BongerAbstract:Functionalized antibodies and Antibody Fragments have found applications in the fields of biomedical imaging, theranostics, and Antibody-drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab' Fragments bearing two distinct site-specific labeling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab' molecule (DTFab'), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab' with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theranostics, and next-generation ADCs.
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dual site specific chemoenzymatic Antibody Fragment conjugation using crispr based hybridoma engineering
bioRxiv, 2020Co-Authors: Camille Le M Gall, Johan M S Van Der Schoot, Ivan Ramostomillero, Melek Parlak Khalily, Floris J Van Dalen, Zacharias Wijfjes, Liyan Smeding, Duco Van Dalen, Anna Cammarata, Kimberly M BongerAbstract:Functionalized antibodies and Antibody Fragments have found applications in the fields of biomedical imaging, theragnostics, and Antibody-drug conjugates (ADC). Antibody functionalization is classically achieved by coupling payloads onto lysine or cysteine residues. However, such stochastic strategies typically lead to heterogenous products, bearing a varying number of payloads. This affects bioconjugate efficacy and stability, as well as its in vivo biodistribution, and therapeutic index, while potentially obstructing the binding sites and leading to off-target toxicity. In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labelling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogenous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab Fragments bearing two distinct site-specific labelling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab molecule (DTFab), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theragnostics, and next-generation ADCs.
Dario Neri - One of the best experts on this subject based on the ideXlab platform.
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Synergistic Therapeutic Effects of a Tumor Targeting Antibody Fragment, Fused to Interleukin 12 and to Tumor Necrosis Factor α
Cancer Research, 2003Co-Authors: Cornelia Halin, Verena Gafner, Maria Elena Villani, Alexander Berndt, Hartwig Kosmehl, Luciano Zardi, Laura Borsi, Dario NeriAbstract:The potent antitumor activity of certain cytokines is often achieved at the expense of unacceptable toxicity. One avenue to improve the therapeutic index of cytokines in cancer therapy consists of fusing them to monoclonal antibodies capable of a selective localization at the tumor site. We have constructed fusion proteins of interleukin-12 (IL-12) and tumor necrosis factor (TNF-α) with L19, an Antibody Fragment specific to the extradomain B of fibronectin which has been shown to target tumors in animal models and in patients with cancer. These fusions display a potent antitumor activity in several immunocompetent murine models of cancer but do not lead to complete remissions of established aggressive tumors. In this article, we have evaluated the tumor-targeting properties and the anticancer activities of combinations of the two Antibody-cytokine fusion proteins, as well as of a triple fusion protein between IL-12, L19, and TNF-α. Although all fusion proteins were active in vitro , the triple fusion protein failed to localize to tumors in vivo and to show significant therapeutic effects. By contrast, the combination of IL-12-L19 and L19-TNF-α displayed potent synergistic anticancer activity and led to the eradication of F9 teratocarcinomas grafted in immunocompetent mice. When cured mice were rechallenged with tumor cells, a delayed onset of tumor growth was observed, indicating the induction of a partial antitumor vaccination effect. Potent anticancer effects were achieved at doses of IL-12-L19 and L19-TNF-α (2 μg + 2 μg/mouse), which were at least 5-fold lower than the maximal-tolerated dose. The combined administration of the two fusion proteins showed only a modest increase in toxicity, compared with treatments performed with the individual fusion proteins. These results show that the targeted delivery of cytokines to the tumor environment strongly potentiates their antitumor activity and that the combination treatment with IL-12-L19 and L19-TNF-α appears to be synergistic in vivo .
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Selective targeting and photocoagulation of ocular angiogenesis mediated by a phage-derived human Antibody Fragment
Nature Biotechnology, 1999Co-Authors: Manfred Birchler, Bernhard Spiess, Francesca Viti, Luciano Zardi, Dario NeriAbstract:Molecules that selectively target and occlude new blood vessels would be useful for diagnosis and treatment of pathologies associated with angiogenesis. We show that a phage-derived human Antibody Fragment (L19) with high affinity for the ED-B domain of fibronectin, a marker of angiogenesis, selectively localizes to newly formed blood vessels in a rabbit model of ocular angiogenesis. The L19 Antibody, chemically coupled to a photosensitizer and irradiated with red light, mediates complete and selective occlusion of ocular neovasculature and promotes apoptosis of the corresponding endothelial cells. These results demonstrate that new ocular blood vessels can be distinguished immunochemically from preexisting ones and suggest that the targeted delivery of photosensitizers may be effective in treating angiogenesis-related pathologies.
Kiyohito Shimura - One of the best experts on this subject based on the ideXlab platform.
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affinity probe capillary electrophoresis analysis of recombinant human growth hormone with a fluorescent labeled Antibody Fragment
Analytical Chemistry, 1994Co-Authors: Kiyohito Shimura, Barry L KargerAbstract:A new highly sensitive microscale analytical procedure called affinity probe capillary electrophoresis (APCE) is presented. One of the two species, which can form a biospecific complex, is labeled with a fluorescent dye. The affinity probe is used to detect the analyte as a complex after the separation of the excess free probe by capillary electrophoresis. As an example of this approach, an FAB' Fragment of a mouse monoclonal Antibody (anti-human growth hormone) was labeled with tetramethylrhodamine-iodoacetamide at a hinge region thiol group. Samples were mixed with the purified labeled Antibody Fragment, and the associated complex was separated by capillary isoelectric focusing with detection by laser-induced fluorescence
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affinity probe capillary electrophoresis analysis of recombinant human growth hormone with a fluorescent labeled Antibody Fragment
Analytical Chemistry, 1994Co-Authors: Kiyohito Shimura, Barry L KargerAbstract:A new highly sensitive microscale analytical procedure called affinity probe capillary electrophoresis (APCE) is presented. One of the two species, which can form a biospecific complex, is labeled with a fluorescent dye. The affinity probe is used to detect the analyte as a complex after the separation of the excess free probe by capillary electrophoresis. As an example of this approach, an FAB' Fragment of a mouse monoclonal Antibody (anti-human growth hormone) was labeled with tetramethylrhodamine-iodoacetamide at a hinge region thiol group. Samples were mixed with the purified labeled Antibody Fragment, and the associated complex was separated by capillary isoelectric focusing with detection by laser-induced fluorescence. In these preliminary results, methionyl recombinant human growth hormone (met-rhGH) could be successfully determined down to detection levels of approximately 5 x 10(-12) M. Mono- and dideamidated variants of met-rhGH were detected simultaneously with the nondeamidated form of the antigen as separate peaks.