The Experts below are selected from a list of 1656 Experts worldwide ranked by ideXlab platform
Max Woisetschlager - One of the best experts on this subject based on the ideXlab platform.
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in vivo and in vitro activity of an Immunoglobulin Fc Fragment Fcab with engineered her 2 neu binding sites
Biotechnology Journal, 2014Co-Authors: Max Woisetschlager, Manuela Kainer, Bernhard Antes, Susanne Wiederkum, Gordana Wozniakknopp, Florian Ruker, Radha Borrowdale, Herta Steinkellner, Kevin Moulder, Geert C MuddeAbstract:Antigen-binding Fc Fragments (Fcabs) are a new unique class of immunotherapeutics. They are small (50 kD) fully functional antibody alternatives that bind antigen and elicit effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity. Since Fcabs carry the natural FcRn binding site of antibodies, they have very favorable pharmacokinetics. We showed recently that Fcab H10-03-6 is a high-affinity binder of Her-2/neu (ErbB2/neu) mediating killing of Her-2/neu-overexpressing tumor cells in the presence of immune effector cells, strongly suggesting that the mechanism of killing is due to ADCC. The present study further confirms ADCC as the mechanism by which H10-03-6 mediates tumor cell killing, since H10-03-6 was shown to interact simultaneously with Her-2/neu and the Fc receptor CD16a. The epitope recognized by H10-03-6 overlaps with that of the clinically used monoclonal antibody trastuzumab. However, unlike trastuzumab, Fcab H10-03-6 did not inhibit proliferation of human tumor cells in vitro even under conditions favoring Her-2/neu crosslinking. Treatment of mice harboring human BT-474 cell xenograft tumors with Fcab H10-03-6 led to statistically significant retardation of tumor growth. For the first time, in vivo properties of an Fcab are presented, supporting the view that Fcabs could become highly efficacious immunotherapeutics for human use.
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correlation between cd16a binding and immuno effector functionality of an antigen specific Immunoglobulin Fc Fragment Fcab
Archives of Biochemistry and Biophysics, 2012Co-Authors: Manuela Kainer, Bernhard Antes, Susanne Wiederkum, Gordana Wozniakknopp, Anton Bauer, Florian Ruker, Max WoisetschlagerAbstract:Antigen binding Immunoglobulin Fc Fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions.
Margherita Ruoppolo - One of the best experts on this subject based on the ideXlab platform.
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hierarchical formation of disulfide bonds in the Immunoglobulin Fc Fragment is assisted by protein disulfide isomerase
Journal of Biological Chemistry, 2004Co-Authors: Floriana Vinci, Silvia Catharino, Stephen Frey, Johannes Buchner, Gennaro Marino, Piero Pucci, Margherita RuoppoloAbstract:Abstract Antibodies provide an excellent system to study the folding and assembly of all β-sheet proteins and to elucidate the hierarchy of intra/inter chain disulfide bonds formation during the folding process of multimeric and multidomain proteins. Here, the folding process of the Fc Fragment of the heavy chain of the antibody MAK33 was investigated. The Fc Fragment consists of the CH3 and CH2 domains of the Immunoglobulin heavy chain, both containing a single S-S bond. The folding process was investigated both in the absence and presence of the folding catalyst protein-disulfide isomerase (PDI), monitoring the evolution of intermediates by electrospray mass spectrometry. Moreover, the disulfide bonds present at different times in the folding mixture were identified by mass mapping to determine the hierarchy of disulfide bond formation. The analysis of the uncatalyzed folding showed that the species containing one intramolecular disulfide predominated throughout the entire process, whereas the fully oxidized Fc Fragment never accumulated in significant amounts. This result suggests the presence of a kinetic trap during the Fc folding, preventing the one-disulfide-containing species (1S2H) to reach the fully oxidized protein (2S). The assignment of disulfide bonds revealed that 1S2H is a homogeneous species characterized by the presence of a single disulfide bond (Cys-130–Cys-188) belonging to the CH3 domain. When the folding experiments were carried out in the presence of PDI, the completely oxidized species accumulated and predominated at later stages of the process. This species contained the two native S-S bonds of the Fc protein. Our results indicate that the two domains of the Fc Fragment fold independently, with a precise hierarchy of disulfide formation in which the disulfide bond, especially, of the CH2 domain requires catalysis by PDI.
Jinhai Huang - One of the best experts on this subject based on the ideXlab platform.
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porcine Immunoglobulin Fc fused p30 p54 protein of african swine fever virus displaying on surface of s cerevisiae elicit strong antibody production in swine
Virologica Sinica, 2020Co-Authors: Chen Chen, Deping Hua, Jingxuan Shi, Zheng Tan, Min Zhu, Kun Tan, Lilin Zhang, Jinhai HuangAbstract:African swine fever virus (ASFV) infects domestic pigs and European wild boars with strong, hemorrhagic and high mortality. The primary cellular targets of ASFV is the porcine macrophages. Up to now, no commercial vaccine or effective treatment available to control the disease. In this study, three recombinant Saccharomyces cerevisiae (S. cerevisiae) strains expressing fused ASFV proteins-porcine Ig heavy chains were constructed and the immunogenicity of the S. cerevisiae-vectored cocktail ASFV feeding vaccine was further evaluated. To be specific, the P30-Fcγ and P54-Fcα fusion proteins displaying on surface of S. cerevisiae cells were produced by fusing the Fc Fragment of porcine Immunoglobulin IgG1 or IgA1 with p30 or p54 gene of ASFV respectively. The recombinant P30-Fcγ and P54-Fcα fusion proteins expressed by S. cerevisiae were verified by Western blotting, flow cytometry and immunofluorescence assay. Porcine Immunoglobulin Fc Fragment fused P30/P54 proteins elicited P30/P54-specific antibody production and induced higher mucosal immunity in swine. The absorption and phagocytosis of recombinant S. cerevisiae strains in IPEC-J2 cells or porcine alveolar macrophage (PAM) cells were significantly enhanced, too. Here, we introduce a kind of cheap and safe oral S. cerevisiae-vectored vaccine, which could activate the specific mucosal immunity for controlling ASFV infection.
Manuela Kainer - One of the best experts on this subject based on the ideXlab platform.
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in vivo and in vitro activity of an Immunoglobulin Fc Fragment Fcab with engineered her 2 neu binding sites
Biotechnology Journal, 2014Co-Authors: Max Woisetschlager, Manuela Kainer, Bernhard Antes, Susanne Wiederkum, Gordana Wozniakknopp, Florian Ruker, Radha Borrowdale, Herta Steinkellner, Kevin Moulder, Geert C MuddeAbstract:Antigen-binding Fc Fragments (Fcabs) are a new unique class of immunotherapeutics. They are small (50 kD) fully functional antibody alternatives that bind antigen and elicit effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity. Since Fcabs carry the natural FcRn binding site of antibodies, they have very favorable pharmacokinetics. We showed recently that Fcab H10-03-6 is a high-affinity binder of Her-2/neu (ErbB2/neu) mediating killing of Her-2/neu-overexpressing tumor cells in the presence of immune effector cells, strongly suggesting that the mechanism of killing is due to ADCC. The present study further confirms ADCC as the mechanism by which H10-03-6 mediates tumor cell killing, since H10-03-6 was shown to interact simultaneously with Her-2/neu and the Fc receptor CD16a. The epitope recognized by H10-03-6 overlaps with that of the clinically used monoclonal antibody trastuzumab. However, unlike trastuzumab, Fcab H10-03-6 did not inhibit proliferation of human tumor cells in vitro even under conditions favoring Her-2/neu crosslinking. Treatment of mice harboring human BT-474 cell xenograft tumors with Fcab H10-03-6 led to statistically significant retardation of tumor growth. For the first time, in vivo properties of an Fcab are presented, supporting the view that Fcabs could become highly efficacious immunotherapeutics for human use.
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correlation between cd16a binding and immuno effector functionality of an antigen specific Immunoglobulin Fc Fragment Fcab
Archives of Biochemistry and Biophysics, 2012Co-Authors: Manuela Kainer, Bernhard Antes, Susanne Wiederkum, Gordana Wozniakknopp, Anton Bauer, Florian Ruker, Max WoisetschlagerAbstract:Antigen binding Immunoglobulin Fc Fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions.
Floriana Vinci - One of the best experts on this subject based on the ideXlab platform.
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hierarchical formation of disulfide bonds in the Immunoglobulin Fc Fragment is assisted by protein disulfide isomerase
Journal of Biological Chemistry, 2004Co-Authors: Floriana Vinci, Silvia Catharino, Stephen Frey, Johannes Buchner, Gennaro Marino, Piero Pucci, Margherita RuoppoloAbstract:Abstract Antibodies provide an excellent system to study the folding and assembly of all β-sheet proteins and to elucidate the hierarchy of intra/inter chain disulfide bonds formation during the folding process of multimeric and multidomain proteins. Here, the folding process of the Fc Fragment of the heavy chain of the antibody MAK33 was investigated. The Fc Fragment consists of the CH3 and CH2 domains of the Immunoglobulin heavy chain, both containing a single S-S bond. The folding process was investigated both in the absence and presence of the folding catalyst protein-disulfide isomerase (PDI), monitoring the evolution of intermediates by electrospray mass spectrometry. Moreover, the disulfide bonds present at different times in the folding mixture were identified by mass mapping to determine the hierarchy of disulfide bond formation. The analysis of the uncatalyzed folding showed that the species containing one intramolecular disulfide predominated throughout the entire process, whereas the fully oxidized Fc Fragment never accumulated in significant amounts. This result suggests the presence of a kinetic trap during the Fc folding, preventing the one-disulfide-containing species (1S2H) to reach the fully oxidized protein (2S). The assignment of disulfide bonds revealed that 1S2H is a homogeneous species characterized by the presence of a single disulfide bond (Cys-130–Cys-188) belonging to the CH3 domain. When the folding experiments were carried out in the presence of PDI, the completely oxidized species accumulated and predominated at later stages of the process. This species contained the two native S-S bonds of the Fc protein. Our results indicate that the two domains of the Fc Fragment fold independently, with a precise hierarchy of disulfide formation in which the disulfide bond, especially, of the CH2 domain requires catalysis by PDI.
