Biology subjects

Mock, J. C.

Publications and source records attributed to Mock, J. C..

3 recordsLinked to original sources

bioRxiv2020

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

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

cancer biology↗

bioRxiv2020

An engineered 4-1BBL fusion protein with ''activity-on-demand''

ABSTRACTEngineered cytokines are gaining importance for cancer therapy but those products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein (termed F8-4-1BBL), which does not exhibit cytokine activity in solution but regains biological activity upon antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively-spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent anti-tumor activity in various mouse models of cancer, without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity-on-demand” properties at the site of disease upon antigen binding and reduce toxicity to normal tissues.Competing Interest StatementDario Neri is a cofounder and shareholder of Philogen SpA (Siena, Italy), the company that owns the F8 and the L19 antibodies. No potential conflicts of interest were disclosed by the other authors.View Full Text

cancer biology↗

bioRxiv2019

A universal reporter cell line for bioactivity evaluation of engineered cytokine products

Engineered cytokine products represent a growing class of therapeutic proteins which need to be tested for biological activity at various stages of pharmaceutical development. In most cases, dedicated biological assays are established for different products, in a process that can be time-consuming and cumbersome. Here we describe the development and implementation of a universal cell-based reporter system for various classes of immunomodulatory proteins. The novel system capitalizes on the fact that the signaling of various types of pro-inflammatory agents (e.g., cytokines, chemokines, Toll-like receptor agonists) may involve transcriptional activation by NF-{kappa}B. Using viral transduction, we generated stably-transformed cell lines of B or T lymphocyte origin and compared the new reporter cell lines with conventional bioassays. The experimental findings with various interleukins and with members of the TNF superfamily revealed that the newly-developed "universal" bioassay method yielded bioactivity data which were comparable to the ones obtained with dedicated conventional methods. The engineered cell lines with reporters for NF-{kappa}B were tested with several antibody-cytokine fusions and may be generally useful for the characterization of novel immunomodulatory products. The newly developed methodology also revealed a mechanism for cytokine potentiation, based on the antibody-mediated clustering of TNF superfamily members on tumor-associated extracellular matrix components.

immunology↗