Protein nanoclusters for delivery of high concentration therapeutic antibodies

用于递送高浓度治疗性抗体的蛋白质纳米簇

• 批准号: 8836948
• 负责人: Keith P. Johnston
• 金额: $ 21.88万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-15 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8836948
• 关键词: Address; Affect; Amino Acid Sequence; Animals; Antibodies; Antibody Response; Autoimmune Diseases; Behavior; Biological; Biological Assay; Biological Availability; Biological Models; Bordetella pertussis; Caliber; Charge; Chronic; Collaborations; Communicable Diseases; Complex; Crowding; Cutaneous; Data; Disaccharides; Disease; Disease model; Dissociation; Dose; Drug Delivery Systems; Drug Formulations; Drug Kinetics; Energy Transfer; Environment; Equilibrium; Event; Exhibits; Faculty; Gel; Health; Human; Image; Immune response; In Vitro; Individual; Inflammatory; Injection of therapeutic agent; Insulin; Isoelectric Point; Kinetics; Label; Leukocytosis; Liquid substance; Malignant Neoplasms; Marketing; Measures; Medical center; Metabolic Clearance Rate; Methods; Modeling; Mus; Nanotechnology; Nature; Outcome; Paper; Patients; Pertussis; Pharmaceutical Preparations; Pharmacologic Substance; Physics; Precipitation; Protein Biochemistry; Proteins; Recovery; Reporting; Research; Research Personnel; Residual state; Rheology; Science; Self Administration; Site; Solutions; Statistical Mechanics; Structure; Students; Subcutaneous Injections; Surface; T cell response; Technology; Testing; Therapeutic; Therapeutic antibodies; Treatment Efficacy; Trehalose; Viscosity; Work; antigen binding; base; comparative efficacy; dosage; high reward; high risk; immunogenicity; in vitro activity; in vivo; insight; interest; interfacial; macromolecule; member; monomer; neutralizing antibody; novel; novel strategies; protein aggregation; protein folding; response; small molecule; subcutaneous; success; technology development; whole animal imaging

DESCRIPTION (provided by applicant): Protein stability at high concentrations (200-400 mg/ml) is of broad interest in science and human health. In cellular environments, a variety of macromolecules crowd proteins into in a compact state, stabilizing folded proteins, as described theoretically and observed experimentally. However, when trying to achieve such high concentrations in vitro, short-ranged attractive and other interactions between closely spaced proteins often produce irreversible aggregation and precipitation. In vitro, these undesirable events dominate, thus novel concepts are needed to mimic nature successfully to stabilize proteins at high concentration for protein drug delivery applications. We have created concentrated antibody protein dispersions in the form of dense, 50-400 nm, reversible clusters of protein molecules, by addition of macromolecular crowding agents. The antibody molecules are conformationally stabilized by crowding, retaining therapeutic activity in vitro and exhibiting expected pharmacokinetics in vivo. Whereas the locally concentrated environment within nanoclusters stabilizes the native state of protein molecules, weak interactions between nanoclusters result in colloidally-stable, translucent dispersions with low viscosities. Not only i the protein native structure maintained within the nanocluster, but upon dilution in vitro, the clusters reversibly dissociate into native monomeric protein molecules with high biological activity. The viscosities of antibody nanocluster dispersions are sufficiently low to allow subcutaneous injection to mice at typical therapeutic antibody dosages. Building on our promising initial work, we aim here to characterize nanocluster fate in vivo, after subcutaneous injection. We will perform three studies crucial for further development of this technology, using large (~300 nm diameter) and small (<100 nm) nanoclusters and solution control antibody. First, we will characterize the mechanism and rate of in vivo cluster dissociation, using fluorescently-labeled antibodies and whole animal imaging. Second, we will compare induction of immune responses in terms of anti-drug antibodies bi-weekly doses of antibody nanoclusters or monomers. Third, we will perform an in vivo efficacy study to compare protection against whooping cough when mice are dosed with nanoclusters or monomers of 1B7, a known protective antibody. The reversible antibody nanoclusters described here may represent a transformative advance to enable patient self-administration of biological pharmaceuticals and provide insight into protein stability in vivo.

描述（由申请人提供）：高浓度（200-400 mg/ml）的蛋白质稳定性引起了科学和人类健康的广泛关注。在细胞环境中，正如理论描述和实验观察到的那样，各种大分子将蛋白质聚集成紧凑状态，稳定折叠蛋白质。然而，当试图在体外达到如此高的浓度时，紧密间隔的蛋白质之间的短程吸引力和其他相互作用通常会产生不可逆的聚集和沉淀。在体外，这些不良事件占主导地位，因此需要新的概念来成功地模拟自然，以稳定高浓度的蛋白质以用于蛋白质药物递送应用。通过添加大分子拥挤剂，我们创建了密集的 50-400 nm 可逆蛋白质分子簇形式的浓缩抗体蛋白质分散体。抗体分子通过拥挤而构象稳定，在体外保留治疗活性并表现出 预期的体内药代动力学。虽然纳米团簇内的局部集中环境稳定了蛋白质分子的天然状态，但纳米团簇之间的弱相互作用导致胶体稳定、半透明的低粘度分散体。纳米簇内不仅保留了蛋白质的天然结构，而且在体外稀释后，纳米簇可逆地解离成具有高生物活性的天然单体蛋白质分子。抗体纳米簇分散体的粘度足够低，可以以典型的治疗抗体剂量对小鼠进行皮下注射。基于我们有前景的初步工作，我们的目标是表征皮下注射后体内纳米簇的命运。我们将使用大（直径约 300 nm）和小（<100 nm）纳米簇和溶液对照抗体进行三项对于进一步开发该技术至关重要的研究。首先，我们将使用荧光标记抗体和整体动物成像来表征体内簇解离的机制和速率。其次，我们将比较抗药物抗体每两周剂量的抗体纳米簇或单体对免疫反应的诱导。第三，我们将进行一项体内功效研究，以比较给小鼠服用 1B7（一种已知的保护性抗体）纳米簇或单体时对百日咳的保护作用。这里描述的可逆抗体纳米簇可能代表了一项革命性的进步，使患者能够自行施用生物药物并提供对体内蛋白质稳定性的深入了解。

项目成果

Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration.

电荷屏蔽可防止高蛋白浓度下增压 GFP 变体的聚集。

• DOI: 10.1021/acs.molpharmaceut.7b00322
• 发表时间: 2017-10-02
• 期刊: Molecular pharmaceutics
• 影响因子: 4.9
• 作者: Laber JR;Dear BJ;Martins ML;Jackson DE;DiVenere A;Gollihar JD;Ellington AD;Truskett TM;Johnston KP;Maynard JA
• 通讯作者: Maynard JA