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Physical instability of a therapeutic Fc fusion protein: domain contributions to conformational and colloidal stability.

ABSTRACT: Protein therapeutics made up of artificially combined proteins or protein domains, so-called fusion proteins, are a novel and growing class of biopharmaceuticals. We have studied abatacept (Orencia), a fusion protein that is constructed of a modified IgG Fc domain and the soluble part of the T-cell receptor CTLA-4. In accelerated degradation studies conducted at 40 degrees C, a pH shift from 7.5 to 6.0 yields significantly faster aggregation kinetics, as measured by size-exclusion chromatography. To understand how the fusion domains and their interactions contribute to this result, we considered aggregation in light of the modified Lumry-Eyring reaction pathway. Protein conformational stabilities against chaotropes and temperature were measured. The structural consequences of these perturbations were observed by a variety of experimental techniques, including differential scanning calorimetry, circular dichroism, and intrinsic fluorescence. Abatacept's colloidal stability was studied by measuring zeta potentials and osmotic second virial coefficients, as well as by modeling electrostatic potentials on the protein's surface. The domains of abatacept exhibit different conformational stabilities that are highly pH dependent, whereas abatacept was weakly colloidally unstable at pH 6 or 7.5. These results are ascribed to conformational instability of the CTLA-4 and C(H)2 domains, which unfold to form a molten globule-like structure that is aggregation-prone. We suggest the instability against aggregation is determined by the least stable domains.

SUBMITTER: Fast JL

PROVIDER: S-EPMC2835945 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Physical instability of a therapeutic Fc fusion protein: domain contributions to conformational and colloidal stability.

Fast Jonas L JL Cordes Amanda A AA Carpenter John F JF Randolph Theodore W TW

Biochemistry 20091201 49

Protein therapeutics made up of artificially combined proteins or protein domains, so-called fusion proteins, are a novel and growing class of biopharmaceuticals. We have studied abatacept (Orencia), a fusion protein that is constructed of a modified IgG Fc domain and the soluble part of the T-cell receptor CTLA-4. In accelerated degradation studies conducted at 40 degrees C, a pH shift from 7.5 to 6.0 yields significantly faster aggregation kinetics, as measured by size-exclusion chromatography ...[more]

PMID: 19899812

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Project description:BackgroundAlpha-1 antitrypsin (AAT) is a major serine protease inhibitor. AAT deficiency (AATD) is a genetic disorder characterized by early-onset severe emphysema. In well-selected AATD patients, therapy with plasma-derived AAT (pAAT), "augmentation therapy", provides modest clinical improvement but is perceived as cumbersome with weekly intravenous infusions. Using mouse models of emphysema, we compared the effects of a recombinant AAT-IgG1 Fc-fusion protein (AAT-Fc), which is expected to have a longer half-life following infusion, to those of pAAT.MethodsIn an elastase model of emphysema, mice received a single intratracheal instillation of porcine pancreatic elastase (PPE) or human leucocyte elastase (hLE). AAT-Fc, pAAT, or vehicle was administered intraperitoneally 1 day prior to or 3 weeks following elastase instillation. Lung function and histology assessments were performed at 7 and 32 days after elastase instillation. In a cigarette smoke (CS) model of emphysema, mice were exposed to CS daily, 5 days a week, for 6 months and AAT-Fc, pAAT, or vehicle were administered every 10 days during the last 3 months of CS exposure. Assessments were performed 3 days after the last CS exposure. Immune responses to lung elastin peptide (EP) and the effects of AAT-Fc or pAAT treatment on dendritic cell (DC) function were determined ex vivo.ResultsBoth elastase instillation and CS exposure triggered emphysema-like alveolar enlargement, increased lung compliance, and increased markers of inflammation compared to controls. Administration of AAT-Fc either prior to or following elastase instillation or during CS exposure provided greater protection than pAAT against alveolar enlargement, lung dysfunction, and airway inflammation. When challenged ex vivo with EP, spleen mononuclear cells from elastase-exposed mice exhibited dose-dependent production of IFNγ and IL-17, suggesting immune reactivity. In co-culture experiments with splenic CD4+ T cells isolated from elastase-exposed mice, AAT-Fc treatment prior to EP-priming of bone marrow-derived dendritic cells inhibited the production of IFNγ and IL-17.ConclusionsCompared to pAAT, AAT-Fc more effectively prevented or attenuated elastase- and CS-induced models of emphysema. These effects were associated with immunomodulatory effects on DC activity. AAT-Fc may provide a therapeutic option to individuals with AATD- and CS-induced emphysema.

Dataset Information

Physical instability of a therapeutic Fc fusion protein: domain contributions to conformational and colloidal stability.

Publications

Physical instability of a therapeutic Fc fusion protein: domain contributions to conformational and colloidal stability.

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