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Characterizing monoclonal antibody structure by carboxyl group footprinting.

ABSTRACT: Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes, and significant correlation of reactivity and solvent accessible surface area.

SUBMITTER: Kaur P

PROVIDER: S-EPMC4622658 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Project description:Amino acid-specific covalent labeling is well suited to probe protein structure and macromolecular interactions, especially for macromolecules and their complexes that are difficult to examine by alternative means, due to size, complexity, or instability. Here we present a detailed account of carbodiimide-based covalent labeling (with GEE tagging) applied to a glycosylated monoclonal antibody therapeutic, which represents an important class of biologic drugs. Characterization of such proteins and their antigen complexes is essential to development of new biologic-based medicines. In this study, the experiments were optimized to preserve the structural integrity of the protein, and experimental conditions were varied and replicated to establish the reproducibility and precision of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include D, E, and the C-terminus, against the experimental surface accessibility data in order to understand the accuracy of the approach in providing an unbiased assessment of structure. Data from the protein were also compared to reactivity measures of several model peptides to explain sequence or structure-based variations in reactivity. The results highlight several advantages of this approach. These include: the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling (indicating that the label does not significantly perturb the structure of the protein), the high reproducibility of replicate experiments (<2 % variation in modification extent), the similar reactivity of the 3 target probe residues (as suggested by analysis of model peptides), and the overall positive and significant correlation of reactivity and solvent accessible surface area (the latter values predicted by the homology modeling). Attenuation of reactivity, in otherwise solvent accessible probes, is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. The results are also compared with data from hydroxyl radical-mediated oxidative footprinting on the same protein, showing that complementary information is gained from the 2 approaches, although the number of target residues in carbodiimide/GEE labeling is fewer. Overall, this approach is an accurate and precise method for assessing protein structure of biologic drugs.

Project description:Her4 is a transmembrane receptor tyrosine kinase belonging to the ErbB-EGFR family. It plays a vital role in the cardiovascular and nervous systems, and mutations in Her4 have been found in melanoma and lung cancer. The kinase domain of Her4 forms a dimer complex, called the asymmetric dimer, which results in kinase activation. Although a crystal structure of the Her4 asymmetric dimer is known, the dimer affinity and the effect of the subsequent phosphorylation steps on kinase domain conformation are unknown. We report here the use of carboxyl-group footprinting MS on a recombinant expressed, Her4 kinase-domain construct to address these questions. Carboxyl-group footprinting uses a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, in the presence of glycine ethyl ester, to modify accessible carboxyl groups on glutamate and aspartate residues. Comparisons of Her4 kinase-domain monomers versus dimers and of unphosphorylated versus phosphorylated dimers were made to map the dimerization interface and to determine phosphorylation induced-conformational changes. We detected 37 glutamate and aspartate residues that were modified, and we quantified their extents of modification by liquid chromatography MS. Five residues showed changes in carboxyl-group modification. Three of these residues are at the predicted dimer interface, as shown by the crystal structure, and the remaining two residues are on loops that likely have altered conformation in the kinase dimer. Incubating the Her4 kinase dimers with ATP resulted in dramatic increase in Tyr-850 phosphorylation, located on the activation loop, and this resulted in a conformational change in this loop, as evidenced by reduction in carboxyl-group modification. The kinase monomer-dimer equilibrium was measured using a titration format in which the extent of carboxyl-group footprinting was mathematically modeled to give the dimer association constant (1.5-6.8 × 10(12) dm(2)/mol). This suggests that the kinase-domain makes a significant contribution to the overall dimerization affinity of the full-length Her4 protein.

Project description:BackgroundIn the era of personalized cancer medicine, identifying techniques for effectively matching patients to efficacious treatments is a critical step in the treatment process. The advent of anti-cancer immunotherapies necessitates novel approaches to biomarker identification beyond traditional genomic profiling. One promising approach is incorporation of nomograms into treatment decisions. Nomograms are prediction tools, based on statistical modeling, designed to predict treatment outcomes. As a first step toward developing a nomogram, we conducted analyses to predict CD137 expression of natural killer cells after monoclonal antibody (mAb) treatment.Patients and methodsPatient, tumor and immune characteristics were collected from 199 patients with breast cancer (N?=?62), head/neck cancers (N?=?46) and non-Hodgkin's lymphoma (NHL) (N?=?91), who were receiving mAb therapy as part of clinical trials. The difference in CD137 expression before and after mAb therapy was assessed by flow cytometry. To evaluate those who respond to mAb therapy via increased CD137 expression, we applied classification and regression trees (CART), multivariable lasso regression tools and Random Forest.ResultsThe CD137 expression was significantly different for each cancer type [mean (SD): Breast: 6.6 (6.5); Head/Neck: 11.0 (7.0); NHL: 7.5 (7.1), P?<?0.0001]. For breast cancer and NHL, FcR polymorphism and baseline CD137 expression were significant predictors of increased CD137 expression; for head/neck cancer, FcR polymorphism and age were significant predictors of increased expression.ConclusionsOur preliminary results suggest that FcR polymorphism, pre-treatment CD137 expression and age are significant predictors of CD137 upregulation in patients. This study demonstrates that the development of a nomogram for therapy response is feasible. Further work validating our models in an independent cohort will provide the next steps in developing a nomogram that may be used to individualize this therapeutic approach for patients (NCT01114256).

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Characterizing monoclonal antibody structure by carboxyl group footprinting.

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