Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies

Abstract

Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a wide range of pathologies, including cancer. Protease activity is normally tightly regulated post-translationally and therefore cannot be accurately estimated based on mRNA or protein expression alone. While several types of zymography approaches to estimate protease activity exist, there remains a need for a robust and reliable technique to measure protease activity in biological tissues. We present a novel quantitative ex vivo zymography (QZ) technology based on Probody^® therapeutics (Pb-Tx), a novel class of protease-activated cancer therapeutics that contain a substrate linker cleavable by tumor-associated proteases. This approach enables the measurement and comparison of protease activity in biological tissues via the detection of Pb-Tx activation. By exploiting substrate specificity and selectivity, cataloguing and differentiating protease activities is possible, with further refinement achieved using protease-specific inhibitors. Using the QZ assay and human tumor xenografts, patient tumor tissues, and patient plasma, we characterized protease activity in preclinical and clinical samples. The QZ assay offers the potential to increase our understanding of protease activity in tissues and inform diagnostic and therapeutic development for diseases, such as cancer, that are characterized by dysregulated proteolysis.

Keywords: Probody therapeutics; cancer; diagnostic; in situ zymography; protease activity; therapeutic.

Figure 1

Overview of the in situ quantitative zymography (QZ) assay. Schematic demonstrating QZ methodology for the assessment of in situ Probody therapeutic(s) (Pb-Tx) activation. Labeled Pb-Tx are incubated with a tissue section, and the extent of Pb-Tx activation is measured in conditioned medium using capillary electrophoresis (CE).

Figure 2

Example of capillary electrophoresis (CE)-based Pb-Tx cleavage assessment. Electropherograms of CE-based cleavage assessment of the C225-Sub1 Pb-Tx incubated with (A) recombinant human membrane-type serine protease 1 (MT-SP1) and (B) matrix metalloproteinase-2 (MMP-2) for 4 h. LC, light chain; HC, heavy chain; Pb-Tx, Probody therapeutic.

Figure 3

The effect of experimental variables on the quantitative zymography (QZ) assay. (A) Diagram showing tissue partitioning into halves and quarters prior to QZ activity assessment. (B) QZ assay of a tissue sample assessed at different section sizes using a constant assay volume (100 µL). As the tissue was halved and quartered, decreased activity was observed. (C) QZ assay of a tissue sample assessed in different assay volumes (100 µL and 300 µL) showing correlation of assay volume with activity. (D) QZ assay of tissue sections of different thickness in a fixed volume showing correlation of tissue thickness with activity. (E) QZ assay before and after tissue storage at −80 °C for 4 months showing preservation of activity after storage. (F) QZ assay of tissue samples subjected to different freezing techniques and storage temperatures. Samples frozen using dry ice (CO₂), liquid nitrogen (LN), or −80 °C temperature were stored at −80 °C. Samples frozen using −20 °C temperature were stored at −20 °C. Activity was similar regardless of freezing condition and storage temperature. For data shown in panels (B–E), the Sub1 Probody therapeutic (Pb-Tx) was used, while for panel (F), the Sub2 Pb-Tx was used. The incubation time for data shown in panels (B–D) was 24 h. The incubation time was 48 h for data shown in panels (E,F).

Figure 4

Validation of the quantitative zymography (QZ) assay for different protease specificities in human tumor tissue. Serine protease and matrix metalloproteinase (MMP) activity were measured with 20 µg/mL Probody therapeutics (Pb-Tx) C225-S01 and C225-M01, respectively, after incubation for 48 h at 37 °C with tissue sections. Protease activity was characterized in cryopreserved sections of (A) head and neck squamous cell carcinoma, (B) pancreatic cancer, and (C) prostate cancer in the presence of the following protease inhibitors: serine protease inhibitor aprotinin, MMP protease inhibitor Galardin, and a broad-spectrum protease inhibitor cocktail (BSPI).

Figure 5

Sub1 vs. Sub2 activation in xenograft models and human tumor tissue. (A) Quantitative zymography (QZ) assessment of C225 Probody therapeutics (Pb-Tx) activation with H292 cell line-derived xenograft (CDX) tissue. A total of 20 µg/mL of MC225-Sub1 or MC225-Sub2 was incubated for 48 h at 37 °C with tissue sections from H292 CDX tumors (n = 5). (B) In vivo efficacy at 10 mg/kg (mpk) of the C225 Pb-Tx with Sub1 or Sub2 substrates compared to cetuximab and intravenous immunoglobulin therapy (IVIG) in the H292 xenograft model (n = 8 mice per group). (C) QZ assessment of MC225-Sub1 and MC225-Sub2 Pb-Tx activation with cholangiocarcinoma patient tissues (n = 4). Statistical significance was calculated by Student’s t-test.