Tumor-specific drug activation by pericellular proteases

摘要 细胞周围蛋白水解失调是正常上皮细胞恶性转化的主要驱动力 到癌细胞。肿瘤微环境中增加的蛋白水解活性已被用于药物治疗。 发展目的。例如，抗体-药物缀合物（ADC）由高度有效的抗癌化合物组成。 有效载荷缀合至肿瘤靶向抗体。这种联接使得货物不活动，直到其被释放， 在一些实施方案中，抗体在细胞周空间中或在靶细胞内的抗体降解后发生蛋白水解切割。的adc 仍然受到剂量限制性毒性的阻碍，最有可能是由于脱靶细胞结合或药物过早释放 蛋白酶的作用。脱靶结合可以通过工程化蛋白酶激活的前体蛋白来克服。 抗体（pADC），其仅在疾病部位被蛋白酶激活后才能结合，而蛋白酶激活后才能结合。 通过利用在存在下稳定的肽接头序列 血液蛋白酶，但在疾病部位被蛋白酶有效切割。我们的假设是， 需要两个蛋白水解活化步骤，可以提高我们选择性靶向抗癌药物的能力。 肿瘤微环境pADC将通过细胞周蛋白酶转化为ADC，随后 与肿瘤细胞结合。有效载荷将通过细胞外蛋白酶或细胞内蛋白酶释放。 （内体或溶酶体）蛋白酶。本提案旨在设计ADC和pADC， 上级血浆稳定性和改善的癌症相关蛋白酶释放动力学。我们的长期 目的是开发与当前治疗剂相比具有增强的抗癌功效的pADC。 在目标1中，我们将产生由患者来源的类器官分泌的蛋白酶的底物特异性谱， 乳腺癌此外，我们将分离溶酶体和内涵体，并表征蛋白水解活性， 这些细胞器。最后，我们将评估血清和血浆中的蛋白酶活性，以了解循环中的 蛋白水解活性。在目标2中，我们将开发由癌症蛋白酶选择性激活的ADC和pADC 并且在血浆和血清中稳定。

ABSTRACT Dysregulated pericellular proteolysis is a major driver of the malignant transformation of normal epithelial cells to cancer cells. The increased proteolytic activity in the tumor microenvironment has been exploited for drug development purposes. For example, antibody-drug conjugates (ADCs) consist of a highly potent anti-cancer payload conjugated to a tumor-targeting antibody. This coupling renders the cargo inactive until it is released by proteolytic cleavage in the pericellular space or upon degradation of the antibody within the target cell. ADCs are still handicapped by dose-limited toxicities, most likely due to off-target cell binding or premature drug release by proteases in circulation. The off-target binding can be overcome by engineering protease-activated pro- antibodies (pADCs) that can bind only after being activated at the site of disease by a protease while the premature drug release can be improved by utilizing peptide linker sequences that are stable in the presence of blood proteases but are efficiently cleaved by proteases at the site of disease. Our hypothesis is that pADCs that require two proteolytic activation steps, may improve our ability to selectively target anti-cancer drugs to the tumor microenvironment. The pADC will be converted into an ADC by a pericellular protease and subsequently bind to the tumor cell. The payload will be released by either an extracellular protease or an intracellular (endosomal or lysosomal) protease. The objectives of this proposal are to design ADCs and pADCs that have superior plasma stability and improved release kinetics by cancer-associated proteases. Our long-term objectives are to develop pADCs with enhanced anti-cancer efficacy compared to current therapeutics. In Aim 1, we will generate a substrate specificity profile for proteases secreted by patient-derived organoids of breast cancer. In addition, we will isolate lysosomes and endosomes and characterize the proteolytic activity in these organelles. Finally, we will evaluate protease activity in serum and plasma to understand the circulating proteolytic activity. In Aim 2, we will develop ADCs and pADCs that are selectively activated by cancer proteases and stable in plasma and serum.