Bioconjugate technique for site-specific attachment of IgG onto nanoparticles

描述（由申请人提供）：抗体，最常见的是IgG，由于其广泛的靶点、高特异性和已证实的功效，已在研究和治疗应用中广泛用作靶向配体。这些应用中的许多需要将抗体缀合到表面（例如纳米颗粒和微孔板）上;然而，大多数常规生物缀合技术（例如EDC-NHS）表现出低交联效率，由于非位点特异性标记而阻碍功能性，和/或需要蛋白质工程（例如半胱氨酸柄），这在技术上具有挑战性且耗时。这可能导致成本高、样品不均匀和再现性差。为了克服这些限制，我们将重组表达蛋白Z，其结合IgG的Fc区，在其结合结构域内具有UV活性的非天然氨基酸苯甲酰苯丙氨酸（BPA）。在暴露于长波长UV光时，BPA将被激活并在蛋白Z和IgG的结合Fc区之间形成共价键。这项技术将与表达蛋白连接（EPL）相结合，这将允许引入荧光团和点击相容性。 在重组蛋白纯化步骤期间，将叠氮基转移到蛋白Z的C-末端上。这将使得交联的蛋白质Z-IgG复合物能够通过无铜点击化学有效地和位点特异性地连接到氮杂-二苄基环辛炔修饰的微孔板和纳米颗粒。这种方法具有成本效益，易于扩展，并利用商业上可行的蛋白质生产技术。位点特异性标记已显示不仅改善各种表面结合的抗体缀合物（例如免疫测定和纳米颗粒）的活性，而且改善非表面结合的抗体缀合物（例如抗体-药物缀合物）的活性，因此预期本文提出的方法具有广泛的适用性。该提案的具体目标是（1）优化光反应蛋白Z表达的产量和表达蛋白连接的效率;（2）优化蛋白Z与各种IgG亚型之间的光交联效率。

DESCRIPTION (provided by applicant): Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques (e.g. EDC-NHS) exhibit low crosslinking efficiencies, hinder functionality due to non-site-specific labeling, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging and time consuming. This can result in high costs, heteregeneous samples, and poor reproducibility. To overcome these limitations, we will recombinantly express Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA will be activated and form a covalent link between the Protein Z and the bound Fc region of IgG. This technology will be combined with expressed protein ligation (EPL), which will allow for the introduction of a fluorophore and click compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This will enable crosslinked- Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzycyclooctyne-modified microplates and nanoparticles, via copper-free click chemistry. This approach is cost-effective, easily scalable, and utilizes protein production techniques that are commercially viable. Site-specific labeling has not only been shown to improve the activity of various surface-bound antibody conjugates (e.g. immunoassays and nanoparticles) but also non-surface bound antibody conjugates (e.g. antibody-drug conjugates) and thus the approach proposed here is expected to have broad applicability. The specific aims for the proposal are (1) Optimize the yield of photoreactive Protein Z expression and the efficiency of expressed protein ligation; (2) Optimize the photo-crosslinking efficiency between Protein Z and various IgG subtypes.