Zurit Levine

The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity

NK cell cytotoxicity is controlled by numerous NK inhibitory and activating receptors. Most of the inhibitory receptors bind MHC class I proteins and are expressed in a variegated fashion. It was recently shown that TIGIT, a new protein expressed by T and NK cells binds to PVR and PVR-like receptors and inhibits T cell activity indirectly through the manipulation of DC activity. Here, we show that TIGIT is expressed by all human NK cells, that it binds PVR and PVRL2 but not PVRL3 and that it inhibits NK cytotoxicity directly through its ITIM. Finally, we show that TIGIT counter inhibits the NK-mediated killing of tumor cells and protects normal cells from NK-mediated cytoxicity thus providing an “alternative self” mechanism for MHC class I inhibition.

Discovery and Validation of Novel Peptide Agonists for G-protein-coupled Receptors

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.

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.

Activation of Relaxin‐Related Receptors by Short, Linear Peptides Derived from a Collagen‐Containing Precursor

In a screening effort based on algorithmic predictions for novel G‐protein‐coupled receptor (GPCR) peptide activators, we were able to identify and examine two novel peptides (P59 and P74) which are short, linear, and derived from a natural, previously unidentified precursor protein containing a collagen‐like repeat. Both peptides seemed to show an apparent cAMP‐related effect on CHO‐K1 cells transiently transfected with either LGR7 or LGR8, usually after treatment with cAMP‐generating forskolin, compared to the same cells treated with forskolin plus relaxin. This activation was not found for the relaxin‐3 receptor (GPR135). In a set of follow‐up experiments, both peptides were found to stimulate cAMP production, mostly upon initial stimulation of cAMP production by 5 μM forskolin in cells transfected with either LGR7 or LGR8. In a dye‐free cell impedance GPCR activation assay, we were able to show that these peptides were also able to activate a cellular response mediated by these receptors. Although untransfected CHO‐K1 cells showed some cellular activation by both relaxin and at least one of our newly discovered peptides, both LGR7‐ and LGR8‐transfected cells showed a stronger response, indicating stimulation of a cellular pathway through activation of these receptors. In conclusion, we were able to show that these newly discovered peptides, which have no similarity to any member of the relaxin–insulin‐like peptide family, are potential ligands for the relaxin‐related family of receptors and as such might serve as novel candidates for relaxin‐related therapeutic indications. Both peptides are linear and were found to be active after being chemically synthesized.

Systematic antigenic profiling of hematopoietic antigens on ovarian carcinoma cells identifies membrane proteins for targeted therapy development

OBJECTIVE We studied ovarian cancers for the expression of membrane markers of hematopoietic origin. STUDY DESIGN We used flow cytometry to systematically characterize the expression of more than 30 hematologic antigens on ovarian carcinoma cell lines and to assess their stability under estrogen exposure. The expression of the antigens was validated by a bioinformatics survey and immunohistochemical staining of ovarian cancer specimens. RESULTS Several antigens were expressed by the majority of the cells, such as CD15, CD71, and CD138, whereas others were found on small and distinct cellular subpopulations. The expression patterns of the different markers were unaffected by estrogen exposure, indicating their stability. CONCLUSION The antigens described in our work may serve as potential targets for new and existing targeted drugs.

ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain–containing receptor (ILDR)2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer.

S9. Proffered paper: Identification of novel immune checkpoints as targets for cancer immunotherapy

Members of the B7/CD28 family of immune checkpoints, such as CTLA4, PD1 and PDL-1, play critical roles in T cell regulation and have emerged as promising drug targets for cancer immunotherapy. We hypothesize that additional novel members of the B7/CD28 family play a role as negative immune regulators and thus may serve as targets for therapeutic mAbs. Utilising Compugen’ sp redictive discovery platform, we identified nine novel members of this family that may serve as immune checkpoints. The therapeutic relevance of three of these proteins, CGEN-15001T, CGEN-15022, and CGEN-15049, was confirmed following the validation of their immunomodulatory properties and their expression in various cancers. Two of these proteins, CGEN-15001T and CGEN-15022, are the basis of a license and collaboration agreement recently signed with Bayer as targets for cancer immunotherapy. Here we present results obtained for an additional novel immune checkpoint, CGEN-15049. Following its ectopic expression on cancer cell lines, CGEN-15049 inhibits the activity of NK cells and cytotoxic T cells (CTLs). The fusion protein, consisting of the extracellular domain of CGEN-15049 fused to an IgG Fc domain, displays robust inhibition of T cell activation and enhances iTregs differentiation. IHC studies indicate that CGEN15049 is expressed in tumour cells of numerous types of cancers, as well as in tumour infiltrating immune cells. Based on its immunomodulatory activities on several types of immune cells which play key roles in cancer immune evasion, together with its expression pattern,CGEN-15049 may serve as mAb target for cancer immunotherapy.

Abstract 581: Discovery and development of COM701, a therapeutic antibody targeting the novel immune checkpoint PVRIG

Background: While blockade of the CTLA4 and PD1 pathways has emerged as an effective treatment of cancer, the majority of patients do not derive long term benefit. This provides a rationale for identifying and targeting additional checkpoints. Employing our unique computational algorithms, we identified PVRIG, a new member of the B7/CD28 family. We report here the expression pattern, functional characterization, and anti-tumor activity of blocking antibodies targeting PVRIG as well as characterization of PVRIG KO mice. Materials and Methods: PVRIG is expressed by T and NK cells within the tumor microenvironment. We identified PVRL2 as its counterpart and characterized the PVRIG-PVRL2 interaction. Antibody discovery was carried out with phage display and hybridoma platforms and antibodies against the human protein were screened for their ability to enhance T-cell activity in vitro, while surrogate antibodies targeting the mouse protein were assessed in syngeneic models for effects on tumor growth. PVRIG -/- KO mice were generated and characterized including phenotyping and anti-tumor immune response. Results: PVRIG is expressed on different T cell subsets and on NK, NKT and γδ T-cells. Within T cells, memory subsets possess the highest level of PVRIG and its expression is induced upon long term activation with different stimuli. Within tumor microenvironment, PVRIG was found to be expressed on NK and CD8+ T cells in multiple cancers. A high affinity lead Ab was selected, COM701, for further clinical development and demonstrated blockade of the interaction of PVRIG with PVRL2 as well as enhancement of activation of both primary and tumor-derived effector immune cells through a PVRL2-dependent mechanism. Moreover, COM-701 showed notable enhancement of T cell function in-vitro when combined with PD1 or TIGIT Ab blockade. The lead antibody, COM-701, is currently in preclinical development. A surrogate antibody, that blocks PVRIG-PVRL2 interaction, was shown to inhibit growth of colon carcinoma and melanoma in syngeneic models upon combined treatment with anti-PDL1 antibody. Comparative analysis of PVRIG KO versus WT derived T cells revealed enhanced reactivity of PVRIG null T cells upon polyclonal activation in presence of PVRL2-Ig. Accordingly, MC38 tumors grew slower in PVRIG KO than in WT mice and ex vivo analysis pointed to the quantitative and functional differences in anticancer immunity developed in these mice. Conclusion: We describe the identification of PVRIG as a novel T cell immune checkpoint. We further demonstrate that antibody blockade of the PVRIG-PVRL2 interaction has the potential to be efficiently combined with PD1 or TIGIT blockade for enhancing anti-tumor immunity. COM-701 is a high affinity antagonistic antibody that is currently in preclinical development. Taken together, these data demonstrate the utility of targeting PVRIG in addition to other B7 family checkpoints for the treatment of cancer. Citation Format: Ofer Levy, Chris Chan, Gady Cojocaru, Spencer Liang, Eran Ophir, Sudipto Ganguly, Maya Kotturi, Tal Friedman, Benjamin Murter, Liat Dassa, Ling Leung, Shirley Greenwald, Meir Azulay, Sandeep Kumar, Zoya Alteber, Xiaoyu Pan, Andy Drake, Ran Salomon, Arthur Machlenkin, John Hunter, Zurit Levine, Drew Pardoll, Mark White. Discovery and development of COM701, a therapeutic antibody targeting the novel immune checkpoint PVRIG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 581. doi:10.1158/1538-7445.AM2017-581

Abstract 5027: Identification of novel immune checkpoints as potential targets for cancer immunotherapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Members of the B7/CD28 family of immune checkpoints, such as CTLA4, PD1 and PDL-1, play critical roles in immune cell regulation and have emerged as promising drug targets for cancer immunotherapy. We hypothesize that additional immune checkpoints play a role as negative immune regulators and thus may serve as targets for therapeutic mAbs. Utilizing Compugens predictive discovery platform, we identified novel members of this family that may serve as immune checkpoints. The therapeutic relevance of three of these proteins, CGEN-15001T, CGEN-15022, and CGEN-15049, was confirmed following the validation of their immunomodulatory properties and their expression in various cancers. Two of these proteins, CGEN-15001T and CGEN-15022, are the basis of a license and collaboration agreement recently signed with Bayer as targets for cancer immunotherapy. Here we present results obtained for an additional novel immune checkpoint, CGEN-15049. Following its ectopic expression on cancer cell lines, CGEN-15049 inhibits the activity of NK cells and cytotoxic T cells (CTLs). The fusion protein, consisting of the extracellular domain of CGEN-15049 fused to an IgG Fc domain, displays robust inhibition of T cell activation and enhances iTregs differentiation. IHC studies indicate that CGEN-15049 is expressed in tumor cells of numerous types of cancers, as well as in tumor infiltrating immune cells. Based on its immunomodulatory activities on immune cell types with key roles in cancer immune evasion, together with its expression pattern in cancer tissues, CGEN-15049 may serve as mAb target for cancer immunotherapy. Citation Format: Galit Rotman, Ofer Levy, Amir Toporik, Gady Cojocaru, Liat Dassa, Ilan Vaknin, Shirley Sameah-Greenwald, Inbal Barbiro, Jinhong Fan, Susan Watson, John Hunter, Eyal Neria, Zurit Levine. Identification of novel immune checkpoints as potential targets for cancer immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5027. doi:10.1158/1538-7445.AM2014-5027