Negin Ashki

Rationale and Preclinical Efficacy of a Novel Anti-EMP2 Antibody for the Treatment of Invasive Breast Cancer

Despite significant advances in biology and medicine, the incidence and mortality due to breast cancer worldwide is still unacceptably high. Thus, there is an urgent need to discover new molecular targets. In this article, we show evidence for a novel target in human breast cancer, the tetraspan protein epithelial membrane protein-2 (EMP2). Using tissue tumor arrays, protein expression of EMP2 was measured and found to be minimal in normal mammary tissue, but it was upregulated in 63% of invasive breast cancer tumors and in 73% of triple-negative tumors tested. To test the hypothesis that EMP2 may be a suitable target for therapy, we constructed a fully human immunoglobulin G1 (IgG1) antibody specific for a conserved domain of human and murine EMP2. Treatment of breast cancer cells with the anti-EMP2 IgG1 significantly inhibited EMP2-mediated signaling, blocked FAK/Src signaling, inhibited invasion, and promoted apoptosis in vitro. In both human xenograft and syngeneic metastatic tumor monotherapy models, anti-EMP2 IgG1 retarded tumor growth without detectable systemic toxicity. This antitumor effect was, in part, attributable to a potent antibody-dependent cell-mediated cytotoxicity response as well as direct cytotoxicity induced by the monoclonal antibody. Together, these results identify EMP2 as a novel therapeutic target for invasive breast cancer. Mol Cancer Ther; 13(4); 902–15. ©2014 AACR.

Peroxynitrite Upregulates Angiogenic Factors VEGF-A, BFGF, and HIF-1α in Human Corneal Limbal Epithelial Cells

PURPOSE Corneal neovascularization (NV) is a sight-threatening condition often associated with infection, inflammation, prolonged contact lens use, corneal burns, and acute corneal graft rejection. Macrophages recruited to the cornea release nitric oxide (NO) and superoxide anion (O2(-)), which react together to form the highly toxic molecule peroxynitrite (ONOO(-)). The role of ONOO(-) in upregulating multiple angiogenic factors in cultured human corneal limbal epithelial (HCLE) cells was investigated. METHODS Human corneal limbal epithelial cells were incubated with 500 μM of ONOO(-) donor for various times. VEGF-A, BFGF, and hypoxic-inducible factor-alpha (HIF-1α) were investigated via Western blot and RT-PCR was performed for VEGF. Functional assays using human umbilical vein endothelial cells (HUVEC) used conditioned media from ONOO(-)-exposed HCLE cells. Secreted VEGF from conditioned media was detected and analyzed using ELISA. RESULTS Increased angiogenic factors were observed as early as 4 hours after HCLE exposure to ONOO(-). HIF-1 expression was seen at 4, 6, and 8 hours post-ONOO(-) exposure (P < 0.05). BFGF expression was elevated at 4 hours and peaked at 8 hours after treatment with ONOO(-) (P < 0.005). Increased VEGF-A gene expression was observed at 6 and 8 hours post-ONOO(-) treatment. Functional assays using conditioned media showed increased HUVEC migration and tube formation. CONCLUSIONS Exposure to elevated extracellular concentrations of ONOO(-) results in upregulation of angiogenic factors in HCLE cells. It is possible that, in the setting of inflammation or infection, that exposure to ONOO(-) could be one contributor to the complex initiators of corneal NV. Validation in vivo would identify an additional potential control point for corneal NV.

Abstract 3965: Epithelial membrane protein-2 is a novel regulator of immune editing in breast cancer

Cancer immuno-editing is a process that describes the interaction between immune cells and tumor cells. This interaction can result in elimination of a developing tumor, tumor dormancy, or tumor cells that are capable of surviving in an immune-competent host. We have recently uncovered a novel mechanism of immunoediting by the tetraspan protein Epithelial membrane protein or EMP2. We propose that EMP2 serves as a bridge between innate and adaptive immunity via tumor mediated type I interferon expression. Most tumor cells evade the immune system through suppression or ignorance. EMP2 levels promote type I interferon expression which was found to correlate with PDL1 expression in a number of cell lines. Concordantly, in vivo, in multiple breast cancer cell lines, EMP2 promoted tumor growth. In contrast, reduction in EMP2 levels correlated with a reduction in tumor size and a corresponding increase in tumor associated macrophages. Given that tumors with lower levels of EMP2 grow poorly, we hypothesized that targeting EMP2 may provide a therapeutic benefit. Recently, we have developed a panel of novel IgG1 monoclonal antibodies to EMP2 and tested their ability to treat both xenograft and syngeneic mouse models. Our results show that anti-EMP2 IgG1 significantly reduces tumor load in both models in part through the recruitment of M1 macrophages. To explore this observation more fully, experiments using murine syngeneic models were employed. Anti-EMP2 therapy elicits a robust ADCC response, producing an immune infiltrate rich in macrophages, NK cells, and lymphocytes. In addition, we show that CD8 cells show reduced exhaustion in the presence of anti-EMP2 antibodies. Conclusions: EMP2 is a protein upregulated in a number of cancers in women including breast, ovarian, and endometrial tumors. Our work collectively show that EMP2 is a novel immune regulator where its expression creates an immunosuppressive tumor microenvironment. We thus predict that targeting EMP2 may improve therapeutic outcomes for cancers in women. Citation Format: Jessica Tsui, Negin Ashki, Yuling Chang, Deven Patel, Jasmine Sjarif, Madhuri Wadehra. Epithelial membrane protein-2 is a novel regulator of immune editing in breast cancer [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 3965. doi:10.1158/1538-7445.AM2017-3965

Human Embryonic Stem Cell-Derived Mesenchymal Stromal Cells Decrease the Development of Severe Experimental Autoimmune Uveitis in B10.RIII Mice

ABSTRACT Purpose: We investigated the effect of exogenously administered human embryonic stem cell-derived mesenchymal stromal cells (hESC-MSCs) in experimental autoimmune uveitis (EAU) in B10.RIII mice, a murine model of severe uveitis. Methods: B10.RIII mice were immunized with an uveitogenic peptide, and intraperitoneal injections of 5 million hESC-MSCs per animal were given on the same day. Behavioral light sensitivity assays, histological evaluation, cytokine production, and regulatory T cells were analyzed at the peak of the disease. Results: Histological and behavioral evidence demonstrated that early systemic treatment with hESC-MSCs decreases the development of severe EAU in B10.RIII mice. hESC-MSCs suppress Th17 and upregulate Th1 and Th2 responses as well as IL-2 and GM-CSF in splenocytes from hESC-MSC-treated mice. Conclusions: MSCs that originate from hESC decrease the development of severe EAU in B10.RIII mice, likely through systemic immune modulation. Further investigation is needed to determine any potential effect on active EAU.

Abstract 1478: Anti-EMP2 IgG1 combined with anti-PD1/PDL1 antibodies synergistically reduce tumor load in animal models of breast cancer

Purpose: Despite significant advances in detection and medicine, the incidence and mortality due to breast cancer world-wide is still unacceptably high. Our recent work reveals that tetraspan protein epithelial membrane protein-2 (EMP2) is up-regulated in up to 70% of patients with invasive breast cancer and its expression is further augmented in metastatic lesions. New data suggests that EMP2 expression correlate with an increase in tumor associated PDL1 expression. We thus hypothesized that EMP2 expression regulated tumor immunogenicity through either direct control of PDL1 expression or MHC Class I processing. Recently, to determine the therapeutic potential of EMP2, our group created a panel of anti-EMP2 antibodies and new data shows that tumors treated with anti-EMP2 IgG1 show an increase in immune infiltrates. We thus proposed that EMP2 positive tumors will be amendable to anti-PD1/PDL1 therapy. Methods:A panel of breast cancer cell lines, both TNBC as well as hormone positive cell lines, were transfected with a vector to overexpress EMP2 or to reduce its levels. To determine if EMP2 expression influenced the expression of immune associated genes, RNAseq was performed. In addition, PDL1 protein expression was measured using standard immunohistochemistry or western blot analysis. Immune cell populations in various xenografts models were quantitated using standard immunohistochemistry. To determine the efficacy of combination anti-EMP2 and anti-PD1 therapy, syngeneic 4T1/firefly luciferase mouse models were created in BALB/c mice. Briefly, cells were injected into the mammary fat pad of female BALB/c mice, and once they approached 100 mm3, were injected IP with a)10 mg/kg dose twice a week of anti-EMP2 antibody, b) 5mg/kg twice a week with anti-PD-1 antibody (BioXCell), c) 10mg/kg ant-EMP2 and 5mg/kg of anti-PD-1 antibody twice a week, or d) saline control. Results: In multiple breast cell lines, EMP2 levels correlated with an increase in PDL1 expression, and treatment with anti-EMP2 IgG1 increased the number of infiltrating leukocytes. Using syngeneic mouse models, tumors treated with a combination of anti-EMP2 and anti-PD-1 antibodies grew slowly and had the smallest total volume compared to all other treatment groups. Anti-EMP2 IgG1 treatment was superior in reducing overall tumor volume compared to anti-PD-1 antibody treatment alone. Conclusions: Our preliminary data strongly supports the synergistic efficacy of anti-EMP2 IgG1 and anti-PD-1 antibody in reducing tumor load, suggesting that using anti-EMP2 treatment with an anti-PD1/PDL1 antibody may improve survival without inducing systemic toxicity as seen with chemotherapy. We predict that this is the direct result of EMP2 expression altering the immunogenicity of the tumor and in support of this, RNAseq data and western blot data suggest that EMP2 levels regulate PDL1 and MHC associated gene expression. Citation Format: Negin Ashki, Jessica Tsui, Madhuri Wadehra. Anti-EMP2 IgG1 combined with anti-PD1/PDL1 antibodies synergistically reduce tumor load in animal models of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1478.