RESUMO
Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non-small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine-galactose-sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.
Assuntos
Receptores de Morte Celular/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Sequência de Aminoácidos , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Sobrevivência Celular , Predisposição Genética para Doença , Glicosilação , Humanos , Neoplasias Pulmonares , Melanoma , Camundongos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas , RNA Mensageiro/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transplante HeterólogoRESUMO
Recombinant antibodies exhibit low levels of glycation from exposure to reducing sugars during production. As the glycation sites are typically distributed across the entire antibody, the levels at any one site are low and it becomes difficult to detect them in the conventional peptide maps. A model antibody was subjected to forced glycation by incubating with a high concentration of a 1:1 mixture of (12)C(6)/(13)C(6) reducing sugars with the assumption that the same sites in the native antibody will be glycated but to a lower extent. This approach simplified the detection of glycated tryptic peptide elution in the LC/MS analysis by giving a unique signature of two molecular ions with equal intensity and differing by 6.018 Da. An in-house developed script automatically processed large data files to generate a list of such peptide mass pairs. The high mass accuracy of the Orbitrap allowed us to assign the sequences unambiguously by comparison with all possible glycated peptide masses. This sequence list was subsequently used to verify their presence/absence in the digest of the native antibody. This work flow enabled rapid and confident identification of site-specific glycation even when levels are below 0.5%. We found the glycation sites to be distributed across the entire antibody studied.
Assuntos
Anticorpos/química , Carboidratos/química , Glicopeptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sequência de Aminoácidos , Anticorpos/genética , Anticorpos/metabolismo , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão , Glicosilação , Marcação por Isótopo , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
A thermally stressed Fab molecule showed a significant increase of basic variants in imaged capillary isoelectric focusing (iCIEF) analysis. Mass analyses of the reduced protein found an increase in -18Da species from both light chain and heavy chain. A tryptic peptide map identified two isoAsp-containing peptides, both containing Asp-Asp motifs and located in complementarity-determining regions (CDRs) of light chains and heavy chains, respectively. The approaches of hydrolyzing succinimide in H(2)(18)O followed by tryptic digestion were used to label and identify the sites of isomerization. This method enabled identification of the isomerization site by comparing the MS/MS spectra of isomerized peptides with and without (18)O incorporation. The light chain peptide L2 VTITCITSTDID(12)DDMNWYQQKPGK underwent simultaneous isomerization and recemization at residue Asp-12 after thermal stress as evidenced by the coinjection of synthetic peptide L2 with l-Asp-12, l-isoAsp-12, d-Asp-12, and d-isoAsp-12, respectively. A thermal stress study of the synthetic peptide (l-)L2 showed that the isomerization and racemization did not occur, indicating that the Asp degradation in this Asp-Asp motif is more related to the protein conformation than the primary sequence. Another isomerization site was identified as Asp-24 in the heavy chain peptide H5 QAPGQGLEWMGWINTYTGETTYAD(24)DFK. No other isomerizations were detected in CDR peptides containing either Asp-Ser or Asp-Thr motifs.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dipeptídeos/química , Peptídeos/química , Espectrometria de Massas em Tandem/métodos , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Regiões Determinantes de Complementaridade/química , Fragmentos Fab das Imunoglobulinas/química , Focalização Isoelétrica/métodos , Isomerismo , Dados de Sequência Molecular , Mapeamento de Peptídeos/métodos , Proteínas/uso terapêutico , Succinimidas/química , TemperaturaRESUMO
PURPOSE: To investigate the cause of the observed instability of dulanermin in 100 ml polyolefin (PO) infusion bags containing saline. METHODS: Diluted dulanermin in IV bags was collected and frozen prior to analysis by size exclusion chromatography. The UV absorption profiles of the IV bag solutions were characterized by using spectrophotometry. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) measured the metal content. Leachables from IV bags were identified by LC-UV-high resolution MS/MS analysis. RESULTS: An elevated loss of dulanermin monomers was observed only in 100 ml PO bags. These IV bag solutions have a compound that contains zinc and has absorbance at 320 nm. This compound was identified to be 2-Mercaptobenzothiazole, and its zinc salt and was found to come from the stopper used in the 100 ml PO bags. The manufacturer has subsequently corrected this problem by using non-latex components in the 100 ml PO IV bag. CONCLUSIONS: End-users need to be aware that IV bags made from a particular polymer by the same manufacturer may contain components or use a manufacturing process that results in a different product. Analysis of samples after freezing and thawing proved to be useful in identifying potential incompatibility of dulanermin in the IV bags.
Assuntos
Infusões Intravenosas , Proteínas/administração & dosagem , Cloreto de Sódio , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas/métodos , Espectrofotometria UltravioletaRESUMO
Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) via succinimide intermediate is a common route of degradation for proteins that can affect their structural integrity. As Asp/isoAsp is isobaric in mass, it is difficult to identify the site of modification by LC-MS/MS peptide mapping. Here, we describe an approach to label the Asp residue involved in isomerization at the protein level by hydrolyzing the succinimide intermediate in H2¹8O. Tryptic digestion of this labeled protein will result in peptides containing the site of isomerization being 2 Da heavier than the ¹6O-containing counterparts, due to ¹8O incorporation during the hydrolysis process. Comparison of tandem mass spectra of isomerized peptides with and without ¹8O incorporation allows easy identification of the Asp residue involved. This method proved to be especially useful in identifying the sites when isomerization occurs in Asp-Asp motifs.
Assuntos
Ácido Aspártico/química , Ácido Isoaspártico/química , Radioisótopos de Oxigênio/química , Peptídeos/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Hidrólise , Ácido Isoaspártico/isolamento & purificação , Mapeamento de Peptídeos/métodos , Estabilidade Proteica , Proteólise , Coloração e Rotulagem , Succinimidas/química , Espectrometria de Massas em Tandem , Água/químicaRESUMO
We describe here the identification of a stop codon TAA (Stop) â GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC-MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations.
Assuntos
Imunoglobulina G/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Animais , Pareamento Incorreto de Bases/genética , Células CHO , Códon de Terminação/genética , Cricetinae , Cricetulus , Análise Mutacional de DNA , Glutamina/genética , Imunoglobulina G/genética , Cadeias Leves de Imunoglobulina/genética , Espectrometria de Massas , Mutação/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento Pós-Transcricional do RNA/genéticaRESUMO
Recombinant monoclonal antibodies (MAbs) can be heterogeneous due to modifications that can occur during expression, purification or during storage. These large multichain proteins (approximately 150 kDa) are structurally challenging for detailed characterization to identify the sites of modifications. We report the use of LTQ Orbitrap mass spectrometry to accurately measure the average masses of individual glycoforms by direct infusion of an intact antibody. To identify the site-specific modification of methionines in the antibody caused by forced oxidation, we used a 'middle-down' approach. The antibody was subjected to limited digestion using the endoproteinase Lys-C and reduced to generate Fab heavy chain, single chain Fc and light chain fragments (approximately 25 kDa each). These species were subjected to on-line liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis using an LTQ Orbitrap, where these large precursors were dissociated by higher-energy collisions in the C-trap. High resolution and accuracy achieved for resulting fragments allowed us to show in a site-specific manner that only the methionines in the Fc heavy chain were oxidized under the studied conditions.
Assuntos
Anticorpos Monoclonais/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Dados de Sequência Molecular , Peso MolecularRESUMO
A systematic analytical approach combining tryptic and chymotryptic peptide mapping with a Mascot Error Tolerant Search (ETS) has been developed to detect and identify low level protein sequence variants, i.e., amino acid substitutions, in recombinant monoclonal antibodies. The reversed-phase HPLC separation with ultraviolet (UV) detection and mass spectral acquisition parameters of the peptide mapping methods were optimized by using a series of model samples that contained low levels (0.5-5.0%) of recombinant humanized anti-HER2 antibody (rhumAb HER2) along with another unrelated recombinant humanized monoclonal antibody (rhumAb A). This systematic approach's application in protein sequence variant analysis depends upon time and sensitivity constraints. An example of using this approach as a rapid screening assay is described in the first case study. For stable CHO clone selection for an early stage antibody project, comparison of peptide map UV profiles from the top four clone-derived rhumAb B samples quickly detected two sequence variants (M83R at 5% and P274T at 42% protein levels) from two clones among the four. The second case study described in this work demonstrates how this approach can be applied to late stage antibody projects. A sequence variant, L413Q, present at 0.3% relative to the expected sequence of rhumAb C was identified by a Mascot-ETS for one out of four top producers. The incorporation of this systematic sequence variant analysis into clone selection and the peptide mapping procedure described herein have practical applications for the biotechnology industry, including possible detection of polymorphisms in endogenous proteins.
Assuntos
Anticorpos Monoclonais/química , Mapeamento de Peptídeos/métodos , Sequência de Aminoácidos , Animais , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Receptor ErbB-2/imunologia , Proteínas Recombinantes/química , Sensibilidade e Especificidade , Raios UltravioletaRESUMO
Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Glicosilação , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.