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

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.

摘要 链霉亲和素（SA）和生物素在自然界中具有最强的已知结合相互作用，其中KD在 飞摩尔范围（4.8 × 10 -14 M）。这种非凡的结合亲和力使其在生物医学中无处不在。 研究和诊断。虽然SA/生物素在许多应用中取得了成功，但大量的内源性生物素 减弱了测定灵敏度。此外，SA是一种高度免疫原性的外源蛋白，这限制了其在免疫治疗中的应用。 治疗应用，如预靶向免疫治疗（PTI）。 双像SA和生物素（D-SA/L-biotin）为这些问题提供了一个优雅的解决方案。例如，D- 蛋白质对L-蛋白酶是惰性的，因此不能被消化以将MHC呈递给免疫系统。 这种性质意味着D-SA的免疫原性大大降低，半衰期增加， L-SA此外，对称性决定了镜像对（D-SA/L-生物素）将具有完全相同的 与天然对（L-SA/D-生物素）一样具有优异的亲和力。重要的是，我们发现D-生物素具有最小的 与D-SA结合。因此，我们提出D-SA/L-生物素可以作为生物素正交链霉亲和素体系 （BOSS）。我们假设BOSS的正交性，沿着D-SA的低免疫原性， 克服了天然SA/生物素的局限性。 我们将采用固相肽合成法，用D-氨基酸和天然的 化学结扎然后，我们将使用BOSS复制以前报告的SA PTI方法。我们会把这个 将D-SA连接到先前研究中使用的抗体片段（scFv）的C末端（scFv-D-SA），并且将 重组表达其L-对应物（scFv-L-SA）。该scFv与GD 2结合，GD 2是一种细胞表面鞘糖脂 在神经母细胞瘤（NB）中上调。我们将首先测量scFv-D-SA在NB细胞中的功效，使用 荧光显微镜我们将把红色荧光团连接到D-生物素上，把品洋红荧光团连接到L-生物素上， 测定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异种移植NB细胞的小鼠中测试我们的BOSS PTI方法 寻找肿瘤周围增强的荧光我们预计BOSS将大幅改善 当前的预定位工作。此外，鉴于生物素/SA为基础的系统在整个生物医学领域的普遍性， 科学，我们还希望BOSS广泛适用于近距离标签，诊断测试， 存在SA免疫原性和生物素干扰的任何方法。