The erythropoietin-producing hepatoma A2 receptor (EphA2) is a tyrosine kinase overexpressed by tumor stroma and cancer cells. A high expression level of EphA2 predicts poor prognosis, correlating with disease progression and metastasis. Therefore, EphA2 is a relevant therapeutic target for human cancer. Antibodies, selectively bound to EphA2, can induce rapid receptor phosphorylation that results in antibody internalization and degradation. This internalization mechanism has been exploited with the development of antibody-drug conjugates (ADCs) for cancer chemotherapy. In this study, we used PET imaging to study the pharmacokinetics and tumor delivery of a panel of anti-EphA2 monoclonal antibodies (mAbs) with and without drug conjugates. Methods: A library of human anti-EphA2 mAbs were screened and evaluated for EphA2 internalization rate, binding affinity, epitope binding, and hydrophobicity. We chose 3 of these antibodies, denoted as 1C1, 3B10, and 2H7, which recognize different epitopes, for further evaluation. ADCs were generated by S239C mutation to give a ratio of 2 drug molecules per antibody. Native mAbs and ADCs were characterized, after conjugation to a DFO chelator and 89Zr radiolabeling, in assays including cell uptake, internalization, hydrophobicity, and in vivo imaging using PET. Results: All 3 mAbs had high affinities for EphA2 but exhibited different internalization rates following the order of 1C1 > 3B10 > 2H7. Internalization rate is only 1 factor that affects in vitro cell uptake and in vivo tumor accumulation. Interestingly, the hydrophobicity of the mAbs, which followed the order of 2H7 > 1C1 > 3B10, had a strong correlation with in vivo tumor uptake measured by PET, with the least hydrophobic antibody, 3B10, showing the highest tumor uptake. ADC significantly reduced the in vivo uptake of all 3 mAbs. Conclusion: Tumor uptake of mAb is a complex process that is affected by multiple parameters, including internalization, hydrophobicity, and chemical modification. Our results suggest that the addition of drug molecules to mAb increases the clearance of the mAb presumably due to the increased hydrophobicity. Understanding the complexity of antibody-based tumor delivery may help improve ADC engineering for better tumor targeting and reduced side effects.

中文翻译：

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PET引导的评估和优化针对产生促红细胞生成素的肝癌A2受体的内在抗体药物结合物。

产生促红细胞生成素的肝癌A2受体（EphA2）是一种被肿瘤基质和癌细胞过度表达的酪氨酸激酶。EphA2的高表达水平预后不良，与疾病进展和转移相关。因此，EphA2是人类癌症的相关治疗靶标。与EphA2选择性结合的抗体可以诱导受体快速磷酸化，从而导致抗体内在化和降解。随着抗体-药物偶联物（ADC）的开发，已开发出这种内在化机制用于癌症化学疗法。在这项研究中，我们使用PET成像技术来研究一组有或没有药物结合物的抗EphA2单克隆抗体（mAb）的药代动力学和肿瘤传递。方法：筛选人抗EphA2单克隆抗体库并评估EphA2内在化速率，结合亲和力，表位结合和疏水性。我们选择了其中三种识别为不同表位的抗体，分别表示为1C1、3B10和2H7，以进行进一步评估。通过S239C突变产生ADC，以使每个抗体具有2个药物分子的比率。与DFO螯合剂和89Zr放射性标记缀合后，天然mAb和ADC在包括细胞摄取，内在化，疏水性和使用PET的体内成像在内的测定中得以表征。结果：所有3个mAb对EphA2都具有高亲和力，但按照1C1> 3B10> 2H7的顺序表现出不同的内在化速率。内部化率只是影响体外细胞摄取和体内肿瘤积累的1个因素。有趣的是，mAb的疏水性遵循2H7> 1C1> 3B10的顺序，与PET测得的体内肿瘤吸收密切相关，疏水抗体最少的3B10表现出最高的肿瘤吸收。ADC显着降低了所有3种mAb的体内吸收。结论：mAb的肿瘤吸收是一个复杂的过程，受多种参数影响，包括内在化，疏水性和化学修饰。我们的结果表明，向mAb添加药物分子可能是由于疏水性增加而增加了mAb的清除率。了解基于抗体的肿瘤递送的复杂性可能有助于改善ADC工程性，从而更好地靶向肿瘤并减少副作用。mAb的肿瘤吸收是一个复杂的过程，受多种参数影响，包括内在化，疏水性和化学修饰。我们的结果表明，向mAb添加药物分子可能是由于疏水性增加而增加了mAb的清除率。了解基于抗体的肿瘤递送的复杂性可能有助于改善ADC工程性，从而更好地靶向肿瘤并减少副作用。mAb的肿瘤吸收是一个复杂的过程，受多种参数影响，包括内在化，疏水性和化学修饰。我们的结果表明，向mAb添加药物分子可能是由于疏水性增加而增加了mAb的清除率。了解基于抗体的肿瘤递送的复杂性可能有助于改善ADC工程性，从而更好地靶向肿瘤并减少副作用。