Protease dysregulation during response and resistance to immune checkpoint blockade therapy (RNA-seq)

Immune checkpoint blockade (ICB) therapy does not benefit the majority of treated patients, and those who respond to the therapy can become resistant to it. Here we report the design and performance of systemically administered activity sensors conjugated to anti-programmed cell death protein 1 (aPD1) antibodies for the monitoring of antitumour responses to ICB therapy. RNA sequencing analysis revealed that differential expression of tumour and immune proteases underpins ICB response and resistance. We then designed a library of mass-barcoded sensors that, when cleaved by proteases, are released into urine, where they can be detected by mass spectrometry. By using syngeneic mouse models of colorectal cancer, we show that random-forest classification trained on mass-spectrometry signatures can be used to detect early antitumour responses and to discriminate resistance to ICB therapy driven by loss-of-function mutations in either the B2m or Jak1 genes. Our data supports the use of activity-based biomarkers that leverage protease dysregulation for response assessment and classification of refractory tumours. Overall design: 6- to 8-week-old female C57BL/6 mice were subcutaneously inoculated with MC38 tumour cells, including wild type cells and cells harbouring loss-of-function mutations in B2m or Jak1. Mice underwent 3 doses of immune checkpoint therapy with aPD1 antibody or IgG1 isotype control, after which mRNA was isolated from tumors (30 samples, 5 per group) for RNA seq.