CytomX Therapeutics, Inc.; 151 Oyster Point Blvd. Suite 400, South San Francisco, California, 94080, USA.
Department of Pharmaceutical Chemistry, University of California, 600 16th Street, San Francisco, CA, 94143, USA.
Sci Rep. 2020 Apr 3;10(1):5894. doi: 10.1038/s41598-020-62339-7.
Proteases have been implicated in the development of many pathological conditions, including cancer. Detection of protease activity in diseased tissues could therefore be useful for diagnosis, prognosis, and the development of novel therapeutic approaches. Due to tight post-translational regulation, determination of the expression level of proteases alone may not be indicative of protease activities, and new methods for measuring protease activity in biological samples such as tumor biopsies are needed. Here we report a novel zymography-based technique, called the IHZ assay, for the detection of specific protease activities in situ. The IHZ assay involves imaging the binding of a protease-activated monoclonal antibody prodrug, called a Probody therapeutic, to tissue. Probody therapeutics are fully recombinant, masked antibodies that can only bind target antigen after removal of the mask by a selected protease. A fluorescently labeled Probody molecule is incubated with a biological tissue, thereby enabling its activation by tissue endogenous proteases. Protease activity is measured by imaging the activated Probody molecule binding to antigen present in the sample. The method was evaluated in xenograft tumor samples using protease specific substrates and inhibitors, and the measurements correlated with efficacy of the respective Probody therapeutics. Using this technique, a diverse profile of MMP and serine protease activities was characterized in breast cancer patient tumor samples. The IHZ assay represents a new type of in situ zymography technique that can be used for the screening of disease-associated proteases in patient samples from multiple pathological conditions.
蛋白酶在许多病理状况的发展中都有牵连，包括癌症。因此，在患病组织中检测蛋白酶活性对于诊断、预后和新的治疗方法的开发可能是有用的。由于翻译后调控紧密，单独确定蛋白酶的表达水平可能不能指示蛋白酶活性，因此需要新的方法来测量生物样品（如肿瘤活检）中的蛋白酶活性。在这里，我们报告了一种新的基于酶谱的技术，称为 IHZ 测定法，用于原位检测特定蛋白酶活性。IHZ 测定法涉及到成像蛋白酶激活的单克隆抗体前药（称为 Probody 治疗剂）与组织的结合。Probody 治疗剂是完全重组的、被掩蔽的抗体，只有在选定的蛋白酶去除掩蔽后才能与靶抗原结合。用荧光标记的 Probody 分子孵育生物组织，从而使其能够被组织内源性蛋白酶激活。通过成像激活的 Probody 分子与样品中存在的抗原结合来测量蛋白酶活性。该方法在异种移植肿瘤样本中使用了特定的蛋白酶底物和抑制剂进行了评估，并与各自的 Probody 治疗剂的功效相关联。使用该技术，在乳腺癌患者肿瘤样本中表征了多种 MMP 和丝氨酸蛋白酶活性。IHZ 测定法代表了一种新的原位酶谱技术，可用于筛选来自多种病理状况的患者样本中的疾病相关蛋白酶。
