O-03.03 KLK4 cleaves CXCR3 chemokines and confers immune escape and resistance to cancer immunotherapy

<h3>Background</h3> While immunotherapies such as immune checkpoint blockade against the PD-1/PD-L1 system are already standard of care for many tumor entities, ovarian cancers remains largely resistant. Preliminary work had identified kallikrein-related peptidase (KLK4), which is regularly overexpressed in ovarian cancer, as a possible inactivator of tumor-suppressive immune mechanisms. The aim of the present project was to investigate this role of KLK4 in more detail. <h3>Materials and Methods</h3> Stable overexpression of murine KLK4 in murine ID8–<i>Trp53^(–/–)</i> and ID8–<i>Trp53^(–/–)Brca2^(–/–)</i> ovarian cancer cells; assessment of tumor growth and survival in C57BL6 and athymic nude mice; quantification of tumor-infiltrating immune cells by immunohistochemistry and FACS analysis; identification of murine and human CXCR3 chemokine cleavage and inactivation by in vitro incubation of recombinant proteins, gel blotting, mass spectrometry, migration and glycosaminoglycan binding assays; quantification of murine CXCR3 chemokines in mouse ascites by ELISA; semi-quantitative analysis of KLK4 expression in human high-grade serous ovarian cancer samples (n=237). <h3>Results</h3> Overexpression of KLK4 leads to reduced survival in both the ID8–<i>Trp53^(–/–)</i> and ID8–<i>Trp53^(–/–)Brca2^(–/–)</i> ovarian cancer models. This effect is abrogated in athymic mice, where infiltration of tumor-suppressive T cell subpopulations is significantly reduced by KLK4. In silico and in vitro analyses identify the T cell-recruiting CXCR3 chemokines as high-affinity targets for KLK4-mediated cleavage and inactivation. KLK4-cleaved CXCL9, CXCL10 and CXCL11 lose their chemotactic capabilities and exhibit weaker binding to components of the extracellular matrix. Consequently, CXCR3-dependent anti-tumor therapies, including epigenetic modulators or combined PARP/immune checkpoint inhibition, lose their therapeutic efficacy in vivo. In human ovarian cancer, elevated KLK4 expression correlates with significantly reduced survival. <h3>Conclusions</h3> KLK4-mediated cleavage of CXCR3 chemokines represents a novel immune escape mechanism and identifies KLK4 as a potential new therapeutic target to improve immune intervention in solid tumors. <b>T.F. Dreyer:</b> None. <b>K. Käfinger:</b> None. <b>D. Zolg:</b> None. <b>G. Magno:</b> None. <b>V. Küntzel:</b> None. <b>R. Sekine:</b> None. <b>V. Magdolen:</b> None. <b>H. Bronger:</b> D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; AstraZeneca, Gilead, GSK, AbbVie.