NLS-Cholic Acid Conjugation to IL-5Rα-Specific Antibody Improves Cellular Accumulation and In Vivo Tumor-Targeting Properties in a Bladder Cancer Model,Bioconjugate Chemistry

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NLS-Cholic Acid Conjugation to IL-5Rα-Specific Antibody Improves Cellular Accumulation and In Vivo Tumor-Targeting Properties in a Bladder Cancer Model
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2018-02-13 00:00:00 , DOI: 10.1021/acs.bioconjchem.8b00077
Michel Paquette , Simon Beaudoin , Mylene-Annie Tremblay , Steve Jean , Angel F. Lopez ₁ , Roger Lecomte _{2,

3} , Brigitte Guérin _{2,

3} , M’hamed Bentourkia _{2,

3} , Robert Sabbagh , Jeffrey V. Leyton _{2,

3}

Affiliation

Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs. We developed a composite compound (ChAcNLS) that enables ACs to escape endosome entrapment and route to the nucleus resulting in the increased intracellular accumulation as an interleukin-5 receptor α-subunit (IL-5Rα)-targeted agent for muscle invasive bladder cancer (MIBC). We constructed ⁶⁴Cu-A14-ChAcNLS, ⁶⁴Cu-A14-NLS, and ⁶⁴Cu-A14 and evaluated their performance by employing mechanistic studies for endosome escape coupled to nuclear routing and determining whether this delivery system results in improved ⁶⁴Cu cellular accumulation. ACs consisting of ∼20 ChAcNLS or NLS moieties per ⁶⁴Cu-A14 were prepared in good yield, high monomer content, and maintaining high affinity for IL-5Rα. Confocal microscopy analysis demonstrated ChAcNLS mediated efficient endosome escape and nuclear localization. ⁶⁴Cu-A14-ChAcNLS increased ⁶⁴Cu cellular accumulation in HT-1376 and HT-B9 cells relative to ⁶⁴Cu-A14 and ⁶⁴Cu-A14-NLS. In addition, we tested ⁶⁴Cu-A14-ChAcNLS in vivo to evaluate its tissue distribution properties and, ultimately, tumor uptake and targeting. A model of human IL-5Rα MIBC was developed by implanting NOD/SCID mice with subcutaneous HT-1376 or HT-B9MIBC tumors, which grow containing high and low IL-5Rα-positive tumor cell densities, respectively. ACs were intravenously injected, and daily blood sampling, biodistribution at 48 and 96 h, and positron emission tomography (PET) at 24 and 48 h were performed. Region of interest (ROI) analysis was also performed on reconstructed PET images. Pharmacokinetic analysis and biodistribution studies showed that ⁶⁴Cu-A14-ChAcNLS had faster clearance rates from the blood and healthy organs relative to ⁶⁴Cu-A14. However, ⁶⁴Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to ⁶⁴Cu-A14. This resulted in ⁶⁴Cu-A14-ChAcNLS having superior tumor/normal tissue ratios at both 48 and 96 h biodistribution time points. Visualization of AC distribution by PET and ROI analysis confirmed that ⁶⁴Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to ⁶⁴Cu-A14. In addition, ⁶⁴Cu-A14 modified with only NLS had poor tumor targeting. This was a result of poor tumor uptake due to extremely rapid clearance. Thus, the overall findings in this model of human IL-5Rα-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting.

中文翻译：

NLS-胆酸与IL-5Rα特异性抗体的结合可改善膀胱癌模型中的细胞蓄积和体内肿瘤靶向特性。

受体介导的内在化随后通过内体-溶酶体途径转运和降解抗体-偶联物（AC）是在靶肿瘤细胞内递送分子有效载荷的主要机制。尽管在癌症治疗和影像学中使用临床认可的AC来传递有效载荷的主要手段，但肿瘤细胞通常能够降低细胞内有效载荷的浓度，从而降低所需应用的有效性。因此，增加有效载荷细胞内积累已成为设计下一代AC的关注焦点。我们开发了一种复合化合物（ChAcNLS），可使AC逃脱内体包裹，并进入核内，从而作为针对白细胞介素5受体α亚基（IL-5Rα）的肌肉浸润性膀胱癌（MIBC）靶向药物，增加细胞内积累）。我们建造了⁶⁴ Cu-A14-ChAcNLS，⁶⁴ Cu-A14-NLS和⁶⁴ Cu-A14，并通过对结合核途径的内体逃逸进行机理研究并确定该递送系统是否导致改善的⁶⁴ Cu细胞蓄积，评估了它们的性能。制备的AC /每⁶⁴ Cu-A14由20个ChAcNLS或NLS部分组成，收率高，单体含量高，并保持对IL-5Rα的高亲和力。共聚焦显微镜分析表明，ChAcNLS介导的高效核糖体逃逸和核定位。⁶⁴的Cu-A14-ChAcNLS增加⁶⁴在HT-1376和HT-B9细胞的相对的Cu细胞累积至⁶⁴的Cu-A14和⁶⁴铜-A14-NLS。此外，我们在体内测试了^64种Cu-A14-ChAcNLS ，以评估其组织分布特性，并最终评估其对肿瘤的吸收和靶向作用。通过向NOD / SCID小鼠植入皮下HT-1376或HT-B9MIBC肿瘤来开发人IL-5RαMIBC模型，这些皮下肿瘤分别含有高和低的IL-5Rα阳性肿瘤细胞密度。静脉注射AC，并在48和96 h进行每日血液采样，生物分布以及24和48 h进行正电子发射断层扫描（PET）。还对重建的PET图像进行了感兴趣区域（ROI）分析。药代动力学分析和生物分布研究表明，相对于血液和健康器官，⁶⁴ Cu-A14-ChAcNLS具有更快的清除率。⁶⁴ Cu-A14。然而，⁶⁴的Cu-A14-ChAcNLS相对肿瘤积累可比维持⁶⁴的Cu-A14。这导致^64个Cu-A14-ChAcNLS在48和96小时的生物分布时间点具有优异的肿瘤/正常组织比率。由PET和ROI分析AC分布的可视确认⁶⁴的Cu-A14-ChAcNLS改善了对肿瘤相对MIBC靶向⁶⁴的Cu-A14。另外，⁶⁴仅用NLS修饰的Cu-A14的肿瘤靶向性差。这是由于清除速度过快导致肿瘤吸收不良的结果。因此，在该人IL-5Rα阳性MIBC模型中的总体发现描述了一种内体逃逸核定位的胆酸连接肽，该肽实质上增强了AC细胞的积累和肿瘤靶向性。

更新日期：2018-02-13

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