Zohar Tiran

Peptides modulating conformational changes in secreted chaperones: From in silico design to preclinical proof of concept

Blocking conformational changes in biologically active proteins holds therapeutic promise. Inspired by the susceptibility of viral entry to inhibition by synthetic peptides that block the formation of helix–helix interactions in viral envelope proteins, we developed a computational approach for predicting interacting helices. Using this approach, which combines correlated mutations analysis and Fourier transform, we designed peptides that target gp96 and clusterin, 2 secreted chaperones known to shift between inactive and active conformations. In human blood mononuclear cells, the gp96-derived peptide inhibited the production of TNFα, IL-1β, IL-6, and IL-8 induced by endotoxin by >80%. When injected into mice, the peptide reduced circulating levels of endotoxin-induced TNFα, IL-6, and IFNγ by >50%. The clusterin-derived peptide arrested proliferation of several neoplastic cell lines, and significantly enhanced the cytostatic activity of taxol in vitro and in a xenograft model of lung cancer. Also, the predicted mode of action of the active peptides was experimentally verified. Both peptides bound to their parent proteins, and their biological activity was abolished in the presence of the peptides corresponding to the counterpart helices. These data demonstrate a previously uncharacterized method for rational design of protein antagonists.

A Novel Recombinant Soluble Splice Variant of Met Is a Potent Antagonist of the Hepatocyte Growth Factor/Scatter Factor-Met Pathway

Purpose: The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), are involved in a wide range of biological activities, including cell proliferation, motility, invasion, and angiogenesis. The HGF/SF-Met signaling pathway is frequently activated in a variety of cancers, and uncontrolled Met activation correlates with highly invasive tumors and poor prognosis. In this study, we investigated the inhibitory effect of a novel soluble splice variant of Met on the HGF/SF-Met pathway. Experimental Design: Using our alternative splicing modeling platform LEADS, we have identified a novel splice variant of the Met receptor, which encodes a truncated soluble form of the receptor. This variant was produced as a recombinant Fc-fused protein named Cgen-241A and was tested in various cell-based assays representing different outcomes of the HGF/SF-Met pathway. Results: Cgen-241A significantly inhibited HGF/SF-induced Met phosphorylation as well as cell proliferation and survival. In addition, Cgen-241A showed a profound inhibitory effect on cell scattering, invasion, and urokinase up-regulation. The inhibitory effects of Cgen-241A were shown in multiple human and nonhuman cell types, representing different modes of Met activation. Furthermore, Cgen-241A showed direct binding to HGF/SF. Conclusions: Taken together, our results indicate that Cgen-241A is a potent antagonist of the HGF/SF-Met pathway, underlining its potential as a therapeutic agent for the treatment of a wide variety of human malignancies that are dependent on this pathway.

Abstract B291: Identification of novel immune checkpoints and their implementation as mAb targets for cancer immunotherapy.

Immune checkpoints, such as CTLA4 and PD-1, have emerged as promising drug targets for cancer immunotherapy. We hypothesize that additional novel members of the B7/CD28 family play a role in T cell regulation and thus may serve as targets for therapeutic mAbs. However, the discovery of novel family members is challenging since proteins of the immune system, including proteins of the B7 protein In order to identify novel members of the B7/CD28 protein family, Compugen has developed a discovery approach integrating gene and protein information with extensive expression data, and has identified nine novel membrane proteins that possess characteristics of the B7/CD28 protein family members and are therefore predicted to play a role in T cell co-stimulation. In order to validate our predictive discovery findings, we evaluated the effect of our proteins on immune cells, particularly T cells. For that goal, we expressed the proteins on the cell surface upon ectopic expression, and also produced fusion proteins consisting of the extracellular domain of the predicted proteins, fused to an IgG Fc domain. Here we present results obtained for two of our novel proteins, CGEN-15001T and CGEN-15022. Both display robust inhibition of T cell activation. Interestingly, CGEN-15001, one of the Fc-fused proteins, leads to increased levels of anti-inflammatory cytokines such as IL-4 and IL5, while reducing pro-inflammatory cytokines such as IFN-γ and IL-17. In addition, CGEN-15001 was found to enhance iTregs differentiation. Furthermore, these molecules showed therapeutic efficacy in mouse models of multiple sclerosis and rheumatoid arthritis. To investigate the potential of these membrane proteins as drug targets for treatment of cancer we have performed extensive IHC studies, on a variety of healthy and malignant tissues. Both CGEN-15001T and CGEN-15022 were found to be expressed in numerous types of cancers, each showing a unique pattern of expression. CGEN-15001T, in addition to its expression on tumor cells, was found to be expressed on tumor infiltrating immune cells, especially on Macrophages and Mast cells. Based on the immunomodulatory activities and the expression pattern on malignant and immune cells, CGEN15001T and CGEN15022 may serve as mAb targets for cancer immunotherapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B291. Citation Format: Gady Cojocaru, Ofer Levy, Amir Toporik, Liat Dassa, Iris Hecht, Ilan Vaknin, Sergey Nemzer, Tania Pergan, Amit Novik, Shirley Sameah-Greenwald, Anat Oren, Zohar Tiran, Peter Steinberger, Joseph Podojil, Nora Tarcic, Eyal Neria, Galit Rotman, Zurit Levine. Identification of novel immune checkpoints and their implementation as mAb targets for cancer immunotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B291.

Peptides modulating conformational changes: From in silico design to in vivo proof of concept

From in-Silico Design to in-Vivo Proof of Concept Yossef Kliger, Ofer Levy, Anat Oren, Haim Ashkenazy, Zohar Tiran, Amit Novik, Avi Rosenberg, Anat Amir, Assaf Wool, Amir Toporik, Ehud Schreiber, Dani Eshel, Zurit Levine, Yossi Cohen, Claudia Nold-Petry, Charles A. Dinarello, Itamar Borukhov 1Compugen Ltd. Tel Aviv 69512, Israel. (www.cgen.com) *Correspondence: kliger@cgen.com, itamarb@cgen.com 2The Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel 3University of Colorado Denver, Aurora, CO 80045, USA