Engineering murine GITRL for antibody-mediated delivery to tumor-associated blood vessels

Abstract

Preclinical evidence has suggested that the Glucocorticoid-Induced TNFR-related protein (GITR) may be valuable a target for the development of anticancer therapeutics, but clinical studies with GITR ligand (GITRL) have been disappointing. Here, we report the development of a fusion protein featuring GITR ligand (GITRL) fused to the F8 antibody which targets the alternatively-spliced EDA domain of fibronectin, a tumor-associated antigen often found around the tumor neovasculature. Five different formats for F8-GITRL fusion proteins were cloned and characterized, but quantitative biodistribution studies failed to evidence a preferential accumulation at the tumor site. The in vivo tumor targeting properties of F8-GITRL could be substantially improved by enzymatic deglycosylation or site-directed mutagenesis of the N-glycosylation consensus sequence. However, therapy studies in a murine model of cancer with the glycoengineered F8-GITRL N74S and N157T variant failed to elicit a durable anti-tumor response, both in monotherapy and in combination with PD-1 blockade. HIGHLIGHTSO_LIDifferent formats of fusion proteins featuring Glucocorticoid-induced TNFR-related protein ligand (GITRL) fused to a tumor-targeting antibody were produced. C_LIO_LIThe tumor uptake of the fusion proteins could be increased by enzymatic deglycosylation of the fusion protein or by site-directed mutagenesis of the N-glycosylation consensus sequences. C_LIO_LIThe fusion protein developed in this study failed to show any anti-tumor activity either alone or in combination with PD-1 inhibition. C_LI