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Arvind Rajpal - One of the best experts on this subject based on the ideXlab platform.
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production of soluble and active microbial transglutaminase in escherichia coli for site specific antibody Drug Conjugation
Protein Science, 2016Co-Authors: Mathias Rickert, Santiago E. Farias, Pavel Strop, David L Shelton, Jaume Pons, Jody A Meltonwitt, Davide Foletti, Arvind RajpalAbstract:Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody Drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody Drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.
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Production of soluble and active microbial transglutaminase in Escherichia coli for site‐specific antibody Drug Conjugation
Protein science : a publication of the Protein Society, 2015Co-Authors: Mathias Rickert, Santiago E. Farias, Pavel Strop, David L Shelton, Jaume Pons, Davide Foletti, Victor Lui, Jody A. Melton-witt, Arvind RajpalAbstract:Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody Drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody Drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.
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Mass Spectrometric Characterization of Transglutaminase Based Site-Specific Antibody–Drug Conjugates
Bioconjugate Chemistry, 2014Co-Authors: Santiago E. Farias, Kathy Delaria, Meritxell Galindo Casas, Pavel Strop, David L Shelton, Magdalena Dorywalska, Jaume Pons, Arvind RajpalAbstract:Antibody Drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (Drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical Conjugation methods that yield heterogeneous mixtures of varying number of Drugs attached at different positions. The potential benefits of site-specific Drug Conjugation in terms of stability, manufacturing, and improved therapeutic index has recently led to the development of several new site-specific Conjugation technologies. However, detailed characterization of the degree of site specificity is currently lacking. In this study we utilize mass spectrometry to characterize the extent of site-specificity of an enzyme-based site-specific antibody–Drug Conjugation technology that we recently developed. We found that, in addition to Conjugation of the engineered site, a small amount of aglycosylated antibody present in starti...
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Mass spectrometric characterization of transglutaminase based site-specific antibody-Drug conjugates.
Bioconjugate chemistry, 2014Co-Authors: Santiago E. Farias, Kathy Delaria, Meritxell Galindo Casas, Pavel Strop, David L Shelton, Magdalena Dorywalska, Jaume Pons, Arvind RajpalAbstract:Antibody Drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (Drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical Conjugation methods that yield heterogeneous mixtures of varying number of Drugs attached at different positions. The potential benefits of site-specific Drug Conjugation in terms of stability, manufacturing, and improved therapeutic index has recently led to the development of several new site-specific Conjugation technologies. However, detailed characterization of the degree of site specificity is currently lacking. In this study we utilize mass spectrometry to characterize the extent of site-specificity of an enzyme-based site-specific antibody–Drug Conjugation technology that we recently developed. We found that, in addition to Conjugation of the engineered site, a small amount of aglycosylated antibody present in starti...
Santiago E. Farias - One of the best experts on this subject based on the ideXlab platform.
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production of soluble and active microbial transglutaminase in escherichia coli for site specific antibody Drug Conjugation
Protein Science, 2016Co-Authors: Mathias Rickert, Santiago E. Farias, Pavel Strop, David L Shelton, Jaume Pons, Jody A Meltonwitt, Davide Foletti, Arvind RajpalAbstract:Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody Drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody Drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.
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Production of soluble and active microbial transglutaminase in Escherichia coli for site‐specific antibody Drug Conjugation
Protein science : a publication of the Protein Society, 2015Co-Authors: Mathias Rickert, Santiago E. Farias, Pavel Strop, David L Shelton, Jaume Pons, Davide Foletti, Victor Lui, Jody A. Melton-witt, Arvind RajpalAbstract:Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody Drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody Drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.
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Mass Spectrometric Characterization of Transglutaminase Based Site-Specific Antibody–Drug Conjugates
Bioconjugate Chemistry, 2014Co-Authors: Santiago E. Farias, Kathy Delaria, Meritxell Galindo Casas, Pavel Strop, David L Shelton, Magdalena Dorywalska, Jaume Pons, Arvind RajpalAbstract:Antibody Drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (Drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical Conjugation methods that yield heterogeneous mixtures of varying number of Drugs attached at different positions. The potential benefits of site-specific Drug Conjugation in terms of stability, manufacturing, and improved therapeutic index has recently led to the development of several new site-specific Conjugation technologies. However, detailed characterization of the degree of site specificity is currently lacking. In this study we utilize mass spectrometry to characterize the extent of site-specificity of an enzyme-based site-specific antibody–Drug Conjugation technology that we recently developed. We found that, in addition to Conjugation of the engineered site, a small amount of aglycosylated antibody present in starti...
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Mass spectrometric characterization of transglutaminase based site-specific antibody-Drug conjugates.
Bioconjugate chemistry, 2014Co-Authors: Santiago E. Farias, Kathy Delaria, Meritxell Galindo Casas, Pavel Strop, David L Shelton, Magdalena Dorywalska, Jaume Pons, Arvind RajpalAbstract:Antibody Drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (Drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical Conjugation methods that yield heterogeneous mixtures of varying number of Drugs attached at different positions. The potential benefits of site-specific Drug Conjugation in terms of stability, manufacturing, and improved therapeutic index has recently led to the development of several new site-specific Conjugation technologies. However, detailed characterization of the degree of site specificity is currently lacking. In this study we utilize mass spectrometry to characterize the extent of site-specificity of an enzyme-based site-specific antibody–Drug Conjugation technology that we recently developed. We found that, in addition to Conjugation of the engineered site, a small amount of aglycosylated antibody present in starti...
Harald Kolmar - One of the best experts on this subject based on the ideXlab platform.
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efficient site specific antibody Drug Conjugation by engineering a nature derived recognition tag for microbial transglutaminase
ChemBioChem, 2019Co-Authors: Aileen Ebenig, Norbert E. Juettner, Lukas Deweid, Olga Avrutina, Hans-lothar Fuchsbauer, Harald KolmarAbstract:Microbial transglutaminase (mTG) has recently emerged as a powerful tool for antibody engineering. In nature, it catalyzes the formation of amide bonds between glutamine side chains and primary amines. Being applied to numerous research fields from material sciences to medicine, mTG enables efficient site-specific Conjugation of molecular architectures that possess suitable recognition motifs. In monoclonal antibodies, the lack of native transamidation sites is bypassed by incorporating specific peptide recognition sequences. Herein, we report a rapid and efficient mTG-catalyzed bioConjugation that relies on a novel recognition motif derived from its native substrate Streptomyces papain inhibitor (SPIP ). Improved reaction kinetics compared to commonly applied sequences were demonstrated for model peptides and for biotinylation of Her2-targeting antibody trastuzumab variants. Moreover, an antibody-Drug conjugate assembled from trastuzumab that was C-terminally tagged with the novel recognition sequence revealed a higher payload-antibody ratio than the reference antibody.
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Efficient Site‐Specific Antibody–Drug Conjugation by Engineering a Nature‐Derived Recognition Tag for Microbial Transglutaminase
ChemBioChem, 2019Co-Authors: Aileen Ebenig, Norbert E. Juettner, Lukas Deweid, Olga Avrutina, Hans-lothar Fuchsbauer, Harald KolmarAbstract:Microbial transglutaminase (mTG) has recently emerged as a powerful tool for antibody engineering. In nature, it catalyzes the formation of amide bonds between glutamine side chains and primary amines. Being applied to numerous research fields from material sciences to medicine, mTG enables efficient site-specific Conjugation of molecular architectures that possess suitable recognition motifs. In monoclonal antibodies, the lack of native transamidation sites is bypassed by incorporating specific peptide recognition sequences. Herein, we report a rapid and efficient mTG-catalyzed bioConjugation that relies on a novel recognition motif derived from its native substrate Streptomyces papain inhibitor (SPIP ). Improved reaction kinetics compared to commonly applied sequences were demonstrated for model peptides and for biotinylation of Her2-targeting antibody trastuzumab variants. Moreover, an antibody-Drug conjugate assembled from trastuzumab that was C-terminally tagged with the novel recognition sequence revealed a higher payload-antibody ratio than the reference antibody.
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Site-Specific Antibody-Drug Conjugation Using Microbial Transglutaminase.
Methods of Molecular Biology, 2019Co-Authors: Stephan Dickgiesser, Lukas Deweid, Harald Kolmar, Roland Kellner, Nicolas RascheAbstract:Antibody-Drug conjugates (ADCs) are a relatively young class of cancer therapeutics that combine the superior selectivity of monoclonal antibodies (mAbs) with the high potency of cytotoxic agents. In the first generation of ADCs, the toxic payload is attached to the mAb via chemical Conjugation to endogenous lysine or cysteine residues providing only limited control over site specificity and Drug-to-antibody ratio (DAR). The resulting product is a heterogeneous population of different ADC species, each with individual characteristics concerning pharmacokinetics, toxicology, and efficacy. Such diverse ADC mixtures are not only difficult to develop but are potentially also accompanied by a suboptimal therapeutic window. To overcome these limitations, alternative Conjugation technologies have been developed that allow the production of tailor-made homogeneous ADCs. Due to its high specificity and robust applicability, microbial transglutaminase (mTG), a protein-glutamine γ-glutamyltransferase isolated from Streptomyces mobaraensis, emerged as a versatile tool for ADC manufacturing. Herein, we report a protocol for the site-specific, mTG-mediated modification of antibodies that allows the production of homogeneous and defined ADCs. Moreover, analytical methods for ADC characterization are provided.
Fredric S. Jacobson - One of the best experts on this subject based on the ideXlab platform.
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Trisulfide modification impacts the reduction step in antibody-Drug Conjugation process.
Bioconjugate chemistry, 2013Co-Authors: Katherine Cumnock, Timothy Tully, Christopher Cornell, Matthew Hutchinson, Jeffrey Gorrell, Ken Skidmore, Yan Chen, Fredric S. JacobsonAbstract:Antibody–Drug conjugates (ADCs) utilizing cysteine-directed linker chemistry have cytotoxic Drugs covalently bound to native heavy–heavy and heavy–light interchain disulfide bonds. The manufacture of these ADCs involves a reduction step followed by a Conjugation step. When tris(2-carboxyethyl)phosphine (TCEP) is used as the reductant, the reaction stoichiometry predicts that for each molecule of TCEP added, one interchain disulfide should be reduced, generating two free thiols for Drug linkage. In practice, the amount of TCEP required to achieve the desired Drug-to-antibody ratio often exceeds the predicted, and is variable for different lots of monoclonal antibody starting material. We have identified the cause of this variability to be inconsistent levels of interchain trisulfide bonds in the monoclonal antibody. We propose that TCEP reacts with each trisulfide bond to form a thiophosphine and a disulfide bond, yielding no net antibody free thiols for Conjugation. Antibodies with higher levels of trisul...
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Trisulfide modification impacts the reduction step in antibody-Drug Conjugation process.
Bioconjugate chemistry, 2013Co-Authors: Katherine Cumnock, Timothy Tully, Christopher Cornell, Matthew Hutchinson, Jeffrey Gorrell, Ken Skidmore, Yan Chen, Fredric S. JacobsonAbstract:Antibody-Drug conjugates (ADCs) utilizing cysteine-directed linker chemistry have cytotoxic Drugs covalently bound to native heavy-heavy and heavy-light interchain disulfide bonds. The manufacture of these ADCs involves a reduction step followed by a Conjugation step. When tris(2-carboxyethyl)phosphine (TCEP) is used as the reductant, the reaction stoichiometry predicts that for each molecule of TCEP added, one interchain disulfide should be reduced, generating two free thiols for Drug linkage. In practice, the amount of TCEP required to achieve the desired Drug-to-antibody ratio often exceeds the predicted, and is variable for different lots of monoclonal antibody starting material. We have identified the cause of this variability to be inconsistent levels of interchain trisulfide bonds in the monoclonal antibody. We propose that TCEP reacts with each trisulfide bond to form a thiophosphine and a disulfide bond, yielding no net antibody free thiols for Conjugation. Antibodies with higher levels of trisulfide bonds require a greater TCEP:antibody molar ratio to achieve the targeted Drug-to-antibody ratio.
Jaume Pons - One of the best experts on this subject based on the ideXlab platform.
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production of soluble and active microbial transglutaminase in escherichia coli for site specific antibody Drug Conjugation
Protein Science, 2016Co-Authors: Mathias Rickert, Santiago E. Farias, Pavel Strop, David L Shelton, Jaume Pons, Jody A Meltonwitt, Davide Foletti, Arvind RajpalAbstract:Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody Drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody Drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.
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Production of soluble and active microbial transglutaminase in Escherichia coli for site‐specific antibody Drug Conjugation
Protein science : a publication of the Protein Society, 2015Co-Authors: Mathias Rickert, Santiago E. Farias, Pavel Strop, David L Shelton, Jaume Pons, Davide Foletti, Victor Lui, Jody A. Melton-witt, Arvind RajpalAbstract:Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody Drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody Drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.
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Mass Spectrometric Characterization of Transglutaminase Based Site-Specific Antibody–Drug Conjugates
Bioconjugate Chemistry, 2014Co-Authors: Santiago E. Farias, Kathy Delaria, Meritxell Galindo Casas, Pavel Strop, David L Shelton, Magdalena Dorywalska, Jaume Pons, Arvind RajpalAbstract:Antibody Drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (Drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical Conjugation methods that yield heterogeneous mixtures of varying number of Drugs attached at different positions. The potential benefits of site-specific Drug Conjugation in terms of stability, manufacturing, and improved therapeutic index has recently led to the development of several new site-specific Conjugation technologies. However, detailed characterization of the degree of site specificity is currently lacking. In this study we utilize mass spectrometry to characterize the extent of site-specificity of an enzyme-based site-specific antibody–Drug Conjugation technology that we recently developed. We found that, in addition to Conjugation of the engineered site, a small amount of aglycosylated antibody present in starti...
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Mass spectrometric characterization of transglutaminase based site-specific antibody-Drug conjugates.
Bioconjugate chemistry, 2014Co-Authors: Santiago E. Farias, Kathy Delaria, Meritxell Galindo Casas, Pavel Strop, David L Shelton, Magdalena Dorywalska, Jaume Pons, Arvind RajpalAbstract:Antibody Drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (Drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical Conjugation methods that yield heterogeneous mixtures of varying number of Drugs attached at different positions. The potential benefits of site-specific Drug Conjugation in terms of stability, manufacturing, and improved therapeutic index has recently led to the development of several new site-specific Conjugation technologies. However, detailed characterization of the degree of site specificity is currently lacking. In this study we utilize mass spectrometry to characterize the extent of site-specificity of an enzyme-based site-specific antibody–Drug Conjugation technology that we recently developed. We found that, in addition to Conjugation of the engineered site, a small amount of aglycosylated antibody present in starti...
