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Wei Zhang - One of the best experts on this subject based on the ideXlab platform.
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structural characterization of the maytansinoid monoclonal Antibody immunoconjugate hun901 dm1 by mass spectrometry
Protein Science, 2005Co-Authors: Lintao Wang, Godfrey W Amphlett, Walter A Blattler, John M. Lambert, Wei ZhangAbstract:Immunoconjugates are being explored as novel cancer therapies with the promise of target-specific drug delivery. The immunoconjugate, huN901–DM1, composed of the humanized monoclonal IgG1 Antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901–DM1 contains an average of three to four DM1 drug Molecules per huN901 Antibody Molecule. The drug Molecules are linked to huN901 through random modification of huN901 at e-amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901–DM1 by electrospray time-of-flight mass spectrometry(ESI-TOFMS), which showed that one to six DM1 drug Molecules were attached to an Antibody Molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp-N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp-N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG1, it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.
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analysis of the composition of immunoconjugates using size exclusion chromatography coupled to mass spectrometry
Rapid Communications in Mass Spectrometry, 2005Co-Authors: Alexandru C Lazar, Godfrey W Amphlett, Walter A Blattler, John M. Lambert, Lintao Wang, Wei ZhangAbstract:Recombinant monoclonal Antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and Antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug Molecules per Antibody Molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid Molecules generated charge envelope profiles that differ from those of the non-conjugated Antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug Molecules per Antibody Molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species.
Joseph Sodroski - One of the best experts on this subject based on the ideXlab platform.
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soluble mimetics of human immunodeficiency virus type 1 viral spikes produced by replacement of the native trimerization domain with a heterologous trimerization motif characterization and ligand binding analysis
Journal of Virology, 2005Co-Authors: Marie Pancera, Joseph Sodroski, Jacob Lebowitz, Arne Schon, Ping Zhu, Ernesto Freire, Peter D Kwong, Kenneth H Roux, Richard T WyattAbstract:The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, mediates binding to the viral receptors and, along with the transmembrane glycoprotein gp41, is a major target for neutralizing antibodies. We asked whether replacing the gp41 fusion/trimerization domain with a stable trimerization motif might lead to a more stable gp120 trimer that would be amenable to structural and immunologic analysis. To obtain stable gp120 trimers, a heterologous trimerization motif, GCN4, was appended to the C terminus of YU2gp120. Biochemical analysis indicated that the gp120-GCN4 trimers were superior to gp140 Molecules in their initial homogeneity, and trilobed structures were observable by electron microscopy. Biophysical analysis of gp120-GCN4 trimers by isothermal titration calorimetry (ITC) and ultracentrifugation analyses indicated that most likely two Molecules of soluble CD4 could bind to one gp120-GCN4 trimer. To further examine restricted CD4 stoichiometric binding to the gp120-GCN4 trimers, we generated a low-affinity CD4 binding trimer by introducing a D457V change in the CD4 binding site of each gp120 monomeric subunit. The mutant trimers could definitively bind only one soluble CD4 Molecule, as determined by ITC and sedimentation equilibrium centrifugation. These data indicate that there are weak interactions between the gp120 monomeric subunits of the GCN4-stabilized trimers that can be detected by low-affinity ligand sensing. By similar analysis, we also determined that removal of the variable loops V1, V2, and V3 in the context of the gp120-GCN4 proteins allowed the binding of three CD4 Molecules per trimer. Interestingly, both the gp120-GCN4 variants displayed a restricted stoichiometry for the CD4-induced Antibody 17b of one Antibody Molecule binding per trimer. This restriction was not evident upon removal of the variable loops V1 and V2 loops, consistent with conformational constraints in the wild-type gp120 trimers and similar to those inherent in the functional Env spike. Thus, the gp120-GCN4 trimers demonstrate several properties that are consistent with some of those anticipated for gp120 in the context of the viral spike.
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stoichiometry of Antibody neutralization of human immunodeficiency virus type 1
Journal of Virology, 2005Co-Authors: Xinzhen Yang, Svetla Kurteva, Joseph SodroskiAbstract:The human immunodeficiency virus envelope glycoproteins function as trimers on the viral surface, where they are targeted by neutralizing antibodies. Different monoclonal antibodies neutralize human immunodeficiency virus type 1 (HIV-1) infectivity by binding to structurally and functionally distinct moieties on the envelope glycoprotein trimer. By measuring Antibody neutralization of viruses with mixtures of neutralization-sensitive and neutralization-resistant envelope glycoproteins, we demonstrate that the HIV-1 envelope glycoprotein trimer is inactivated by the binding of a single Antibody Molecule. Virus neutralization requires essentially all of the functional trimers to be occupied by at least one Antibody. This model applies to antibodies differing in neutralizing potency and to virus isolates with various neutralization sensitivities. Understanding these requirements for HIV-1 neutralization by antibodies will assist in establishing goals for an effective AIDS vaccine.
Mariano Sánchez Crespo - One of the best experts on this subject based on the ideXlab platform.
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4 trifluoromethyl derivatives of salicylate triflusal and its main metabolite 2 hydroxy 4 trifluoromethylbenzoic acid are potent inhibitors of nuclear factor κb activation
British Journal of Pharmacology, 1999Co-Authors: Yolanda Bayón, Andres Alonso, Mariano Sánchez CrespoAbstract:1. The effect of two derivatives of salicylate, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) and 2-acetoxy-4-trifluoromethylbenzoic acid (triflusal), on the activation of NF-kappaB elicited by tumour necrosis factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVEC) was tested. 2. The expression of the mRNA of vascular cell adhesion Molecule-1 (VCAM-1) was studied as an example of a gene the expression of which is regulated by NF-kappaB. To extend these findings to other systems, the induction of nitric oxide synthase in rat adherent peritoneal macrophages was studied. 3. Both HTB and triflusal were more potent than aspirin or salicylate as inhibitors of the nuclear translocation of NF-kappaB. The calculation of the IC50 values showed approximately 2 mM for HTB, 4 mM for aspirin and >4 mM for salicylate. 4. Comparison of the potency of these compounds on VCAM-1 mRNA expression showed complete inhibition by both triflusal and HTB at a concentration of 4 mM whereas aspirin and salicylate produced only 36-43% inhibition at the same concentration. 5. Inhibition of NF-kappaB activation was also observed in rat peritoneal macrophages stimulated via their receptors for the Fc portion of the Antibody Molecule with IgG/ovalbumin immune complexes. This was accompanied by a dose-dependent inhibition of nitrite production by the L-arginine pathway via iNOS. IC50 values for this effect were 1.13+/-0.12 mM (triflusal), 1.84+/-0.34 (HTB), 6.08+/-1.53 mM (aspirin) and 9.16+/-1.9 mM (salicylate). 6. These data indicate that the incorporation of a 4-trifluoromethyl group to the salicylate Molecule strongly enhances its inhibitory effect on NF-kappaB activation, VCAM-1 mRNA expression and iNOS induction, irrespective of the presence of the acetyl moiety involved in the inhibition of cyclo-oxygenase.
Walter A Blattler - One of the best experts on this subject based on the ideXlab platform.
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structural characterization of the maytansinoid monoclonal Antibody immunoconjugate hun901 dm1 by mass spectrometry
Protein Science, 2005Co-Authors: Lintao Wang, Godfrey W Amphlett, Walter A Blattler, John M. Lambert, Wei ZhangAbstract:Immunoconjugates are being explored as novel cancer therapies with the promise of target-specific drug delivery. The immunoconjugate, huN901–DM1, composed of the humanized monoclonal IgG1 Antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901–DM1 contains an average of three to four DM1 drug Molecules per huN901 Antibody Molecule. The drug Molecules are linked to huN901 through random modification of huN901 at e-amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901–DM1 by electrospray time-of-flight mass spectrometry(ESI-TOFMS), which showed that one to six DM1 drug Molecules were attached to an Antibody Molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp-N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp-N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG1, it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.
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analysis of the composition of immunoconjugates using size exclusion chromatography coupled to mass spectrometry
Rapid Communications in Mass Spectrometry, 2005Co-Authors: Alexandru C Lazar, Godfrey W Amphlett, Walter A Blattler, John M. Lambert, Lintao Wang, Wei ZhangAbstract:Recombinant monoclonal Antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and Antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug Molecules per Antibody Molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid Molecules generated charge envelope profiles that differ from those of the non-conjugated Antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug Molecules per Antibody Molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species.
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pharmacokinetics and biodistribution of the antitumor immunoconjugate cantuzumab mertansine huc242 dm1 and its two components in mice
Journal of Pharmacology and Experimental Therapeutics, 2004Co-Authors: Charlene Audette, Mary G Hoffee, John M. Lambert, Walter A BlattlerAbstract:The humanized monoclonal Antibody maytansinoid conjugate, cantuzumab mertansine (huC242-DM1) that contains on average three to four linked drug Molecules per Antibody Molecule was evaluated in CD-1 mice for its pharmacokinetic behavior and tissue distribution, and the results were compared with those of the free Antibody huC242. The pharmacokinetics in blood were similar for 125I-labeled conjugate and Antibody with terminal half-lives of 154 and 156 h, respectively. Pharmacokinetic analysis using an enzyme-linked immunosorbent assay (ELISA) method, which measures intact conjugate in plasma samples revealed a faster clearance for the conjugate corresponding to a half-life of 42.2 h. This faster clearance is explained as the result of clearance from circulation and concomitant clearance of drug from circulating conjugate through linker cleavage. An Antibody-specific ELISA allowed the determination of the clearance rate of the Antibody component from circulation. The drug clearance rate from circulating conjugate was then calculated as the difference between the clearance of the conjugate and the clearance of the Antibody component and found to be about three times that of the Antibody component. The above results were confirmed with a conjugate, huC242-[3H]DM1, where the linked DM1 drugs carried a stable tritium label. Tissue distribution studies with 125I-labeled conjugate and Antibody showed Antibody-like behavior for the conjugate; the Antibody of the conjugate did not distribute or bind significantly to any solid tissue.
John M. Lambert - One of the best experts on this subject based on the ideXlab platform.
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structural characterization of the maytansinoid monoclonal Antibody immunoconjugate hun901 dm1 by mass spectrometry
Protein Science, 2005Co-Authors: Lintao Wang, Godfrey W Amphlett, Walter A Blattler, John M. Lambert, Wei ZhangAbstract:Immunoconjugates are being explored as novel cancer therapies with the promise of target-specific drug delivery. The immunoconjugate, huN901–DM1, composed of the humanized monoclonal IgG1 Antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901–DM1 contains an average of three to four DM1 drug Molecules per huN901 Antibody Molecule. The drug Molecules are linked to huN901 through random modification of huN901 at e-amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901–DM1 by electrospray time-of-flight mass spectrometry(ESI-TOFMS), which showed that one to six DM1 drug Molecules were attached to an Antibody Molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp-N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp-N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG1, it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.
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analysis of the composition of immunoconjugates using size exclusion chromatography coupled to mass spectrometry
Rapid Communications in Mass Spectrometry, 2005Co-Authors: Alexandru C Lazar, Godfrey W Amphlett, Walter A Blattler, John M. Lambert, Lintao Wang, Wei ZhangAbstract:Recombinant monoclonal Antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and Antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug Molecules per Antibody Molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid Molecules generated charge envelope profiles that differ from those of the non-conjugated Antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug Molecules per Antibody Molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species.
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pharmacokinetics and biodistribution of the antitumor immunoconjugate cantuzumab mertansine huc242 dm1 and its two components in mice
Journal of Pharmacology and Experimental Therapeutics, 2004Co-Authors: Charlene Audette, Mary G Hoffee, John M. Lambert, Walter A BlattlerAbstract:The humanized monoclonal Antibody maytansinoid conjugate, cantuzumab mertansine (huC242-DM1) that contains on average three to four linked drug Molecules per Antibody Molecule was evaluated in CD-1 mice for its pharmacokinetic behavior and tissue distribution, and the results were compared with those of the free Antibody huC242. The pharmacokinetics in blood were similar for 125I-labeled conjugate and Antibody with terminal half-lives of 154 and 156 h, respectively. Pharmacokinetic analysis using an enzyme-linked immunosorbent assay (ELISA) method, which measures intact conjugate in plasma samples revealed a faster clearance for the conjugate corresponding to a half-life of 42.2 h. This faster clearance is explained as the result of clearance from circulation and concomitant clearance of drug from circulating conjugate through linker cleavage. An Antibody-specific ELISA allowed the determination of the clearance rate of the Antibody component from circulation. The drug clearance rate from circulating conjugate was then calculated as the difference between the clearance of the conjugate and the clearance of the Antibody component and found to be about three times that of the Antibody component. The above results were confirmed with a conjugate, huC242-[3H]DM1, where the linked DM1 drugs carried a stable tritium label. Tissue distribution studies with 125I-labeled conjugate and Antibody showed Antibody-like behavior for the conjugate; the Antibody of the conjugate did not distribute or bind significantly to any solid tissue.