Development of Human Serine Protease-based Therapeutics Targeting Fn14 and Identification of Fn14 as a New Target Overexpressed in TNBC

摘要

The cytokine TWEAK and its receptor, Fn14, have emerged as potentially valuable targets for cancer therapy. Granzyme B (GrB)-containing Fn14-targeted constructs were generated containing either the Fn14 ligand TWEAK (GrB-TWEAK) or an anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both constructs showed high affinity and selective cytotoxicity against a panel of Fn14-expressing human tumor cells including triple-negative breast cancer (TNBC) lines. Cellular expression of the GrB inhibitor PI-9 in target cells had no impact on the cytotoxic effect of either construct. Cellular expression of MDR1 showed no cross-resistance to the fusion constructs. GrB-TWEAK and GrB-Fc-IT4 activated intracellular caspase cascades and cytochrome C-related pro-apoptotic pathways consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing established HT-29 xenografts with GrB-TWEAK showed significant tumor growth inhibition compared to vehicle alone (P < 0.05). Both GrB-TWEAK and GrB-Fc-IT4 displayed significant tumor growth inhibition when administered to mice bearing orthotopic MDA-MB-231 (TNBC) tumor xenografts. TCGA analysis revealed that Fn14 mRNA expression was significantly higher in TNBC and in HER2-positive disease (P<0.0001) compared to hormone receptor-positive breast cancer, and in basal-like 2 tumors (P=0.01) compared to other TNBC molecular subtypes. Immunohistochemistry analysis of a 101 patient TNBC tumor microarray showed that 55/101 (54%) of tumors stained positive for Fn14 suggesting that this may be an excellent potential target for precision therapeutic approaches. Targeting Fn14 using fully-human, GrB-containing fusion constructs may form the basis for a new class of novel, potent and highly effective constructs for targeted therapeutic applications.