Biotin Orthogonal Streptavidin System (BOSS) for Drug Pre-Targeting

用于药物预靶向的生物素正交链霉亲和素系统 (BOSS)

• 批准号: 10606180
• 负责人: Steven Draper
• 金额: $ 7.16万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606180
• 关键词: Academia; Acceleration; Affinity; Amino Acids; Animal Disease Models; Antibodies; Attenuated; Binding; Biological; Biological Assay; Biomedical Research; Biotin; Biotinylation; Calorimetry; Cell surface; Cells; Chemicals; Chemistry; Circular Dichroism; Circulation; Communities; Crystallography; Cultured Cells; Diagnostic; Diagnostic tests; Disease; Drug Kinetics; Effectiveness; Fellowship; Fluorescence; Fluorescence Microscopy; Foundations; Future; Generations; Glycosphingolipids; Half-Life; Image; Imaging ligands; Immune system; Immunoassay; Immunoglobulin Fragments; Immunotherapy; Label; Laws; Ligation; Measures; Medical; Methods; Molecular Sieve Chromatography; Mus; Nature; Neuroblastoma; Peptide Hydrolases; Peptide Synthesis; Pharmaceutical Preparations; Phase; Phase I Clinical Trials; Procedures; Process; Property; Proteins; Publishing; Reagent; Recombinants; Reporting; Research; Rosaniline Dyes; Science; Solid; Specificity; Streptavidin; System; Testing; Therapeutic; Time; Titrations; Tumor Antigens; Xenograft procedure; antibody conjugate; cancer therapy; career; chemical conjugate; chemical synthesis; chemotherapy; disease diagnostic; enantiomer; experimental study; fluorophore; immunogenic; immunogenicity; improved; neoplastic cell; neuroblastoma cell; novel; peptide chemical synthesis; side effect; success; tool; tumor; Academia; Acceleration; Affinity; Amino Acids; Animal Disease Models; Antibodies; Attenuated; Binding; Biological; Biological Assay; Biomedical Research; Biotin; Biotinylation; Calorimetry; Cell surface; Cells; Chemicals; Chemistry; Circular Dichroism; Circulation; Communities; Crystallography; Cultured Cells; Diagnostic; Diagnostic tests; Disease; Drug Kinetics; Effectiveness; Fellowship; Fluorescence; Fluorescence Microscopy; Foundations; Future; Generations; Glycosphingolipids; Half-Life; Image; Imaging ligands; Immune system; Immunoassay; Immunoglobulin Fragments; Immunotherapy; Label; Laws; Ligation; Measures; Medical; Methods; Molecular Sieve Chromatography; Mus; Nature; Neuroblastoma; Peptide Hydrolases; Peptide Synthesis; Pharmaceutical Preparations; Phase; Phase I Clinical Trials; Procedures; Process; Property; Proteins; Publishing; Reagent; Recombinants; Reporting; Research; Rosaniline Dyes; Science; Solid; Specificity; Streptavidin; System; Testing; Therapeutic; Time; Titrations; Tumor Antigens; Xenograft procedure; antibody conjugate; cancer therapy; career; chemical conjugate; chemical synthesis; chemotherapy; disease diagnostic; enantiomer; experimental study; fluorophore; immunogenic; immunogenicity; improved; neoplastic cell; neuroblastoma cell; novel; peptide chemical synthesis; side effect; success; tool; tumor

Abstract Streptavidin (SA) and biotin have the strongest known binding interaction in nature, with a KD in the femtomolar range (4.8x10-14 M). This extraordinary binding affinity has led to its ubiquitous use in biomedical research and diagnostics. Though SA/biotin enjoys success in many applications, abundant endogenous biotin attenuates assay sensitivity. Moreover, SA is a highly immunogenic foreign protein, which limits its use in therapeutic applications such as pretargeted immunotherapy (PTI). Mirror-image SA and biotin (D-SA/L-biotin) offer an elegant solution to these problems. For example, D- Proteins are inert to L-proteases and therefore cannot be digested for MHC presentation to the immune system. This property means that D-SA will have greatly decreased immunogenicity and increased half-life compared to L-SA. Additionally, symmetry dictates that the mirror-image pair (D-SA/L-biotin) will have the exact same exceptional affinity as the natural pair (L-SA/D-biotin). Importantly, we have discovered that D-biotin has minimal binding to D-SA. Therefore, we propose that D-SA/L-biotin can be used as a biotin orthogonal streptavidin system (BOSS). We hypothesize that the orthogonality of BOSS, along with D-SA’s low immunogenicity, will overcome the limitations of natural SA/biotin. We will chemically synthesize D-SA using solid-phase peptide synthesis with D-amino acids and native chemical ligation. We will then replicate a previously reported SA PTI method using BOSS. We will attach this D-SA to the C-terminus of the antibody fragment (scFv) used in the previous study (scFv-D-SA) and will recombinantly express its L-counterpart (scFv-L-SA). This scFv binds to GD2, a cell-surface glycosphingolipid that is upregulated in neuroblastoma (NB). We will first measure the efficacy of scFv-D-SA in NB cells using fluorescence microscopy. We will attach a red fluorophore to D-biotin and magenta fluorophore to L-biotin to determine the relative binding of scFv-D-SA and scFv-L-SA to the NB cells and a panel of control cells. We will then test our BOSS PTI method in mice xenografted with NB cells using fluorescence-based full-body imaging to look for enhanced fluorescence localized around the tumor. We expect that BOSS will dramatically improve current pretargeting efforts. Moreover, given the ubiquity of biotin/SA-based systems throughout biomedical science, we also expect BOSS to be widely applicable and relevant to proximity labeling, diagnostic testing, and any method that suffers from SA immunogenicity and biotin interference.

抽象的 链霉亲和素（SA）和生物素在自然界中具有强烈的已知结合相互作用，在 FEMTOLOL范围（4.8x10-14 m）。这种非凡的结合亲和力导致其无处不在的生物医学使用 研究和诊断。尽管SA/生物素在许多应用中都取得了成功，但内源性生物素丰富 减弱测定敏感性。此外，SA是一种高度免疫原性的异物，它限制了其在 治疗应用，例如预先设定的免疫疗法（PTI）。 镜像SA和生物素（D-SA/L-Biotin）为这些问题提供了优雅的解决方案。例如，d- 蛋白质是惰性的，因此不能消化对免疫系统的MHC呈现。 该特性意味着D-SA的免疫原性大大降低，半衰期增加 L-SA。另外，对称性表明镜像对（D-SA/L-Biotin）的具有完全相同的 特殊亲和力为天然对（L-SA/D-Biotin）。重要的是，我们发现D-Biotin的含量很小 与D-SA结合。因此，我们提出D-SA/L-生物素可以用作生物素正交链霉亲和素系统 （老板）。我们假设Boss的正交性以及D-SA的低免疫原性将 克服天然SA/生物素的局限性。 我们将使用与D-氨基酸和天然的固相肽合成化学合成D-SA 化学连接。然后，我们将使用Boss复制先前报道的SA PTI方法。我们将附上这个 D-SA到先前研究中使用的抗体片段（SCFV）的C末端（SCFV-D-SA），并将 重组表达其L-CounterPart（SCFV-L-SA）。该SCFV与GD2结合，GD2是一种细胞表面糖果脂蛋白 在神经母细胞瘤（NB）中进行了更新。我们将首先使用使用NB细胞中SCFV-D-SA的效率 荧光显微镜。我们将将红色荧光团固定在D-Biotin和洋红色荧光团到L-生物素 确定SCFV-D-SA和SCFV-L-SA与NB细胞和一组对照细胞的相对结合。我们将 然后使用基于荧光的全身成像在用NB细胞的小鼠中测试我们的Boss PTI方法 寻找肿瘤周围局部荧光的增强。我们希望老板会大大改善 当前的有预定努力。此外，鉴于生物医学中生物素/基于SA的系统的普遍存在 科学，我们还希望老板将广泛适用，并且与接近标签，诊断测试和 患有SA免疫原性和生物素干扰的任何方法。