PS-targeting Antibodies Research Articles

Combination of a T cell activating therapy and anti-phosphatidylserine enhances anti-tumour immune responses in a\xa0HPV16 E7-expressing C3 tumour model

DPX is a novel delivery platform that generates targeted CD8 + T cells and drives antigen-specific cytotoxic T cells into tumours. Cancer cells upregulate phosphatidylserine (PS) on the cell surface as a mechanism to induce an immunosuppressive microenvironment. Development of anti-PS targeting antibodies have highlighted the ability of a PS-blockade to enhance tumour control by T cells by releasing immunosuppression. Here, C57BL/6 mice were implanted with HPV16 E7 target-expressing C3 tumours and subjected to low dose intermittent cyclophosphamide (CPA) in combination with DPX-R9F treatment targeting an E7 antigen with and without anti-PS and/or anti-PD-1 targeting antibodies. Immune responses were assessed via IFN-γ ELISPOT assay and the tumour microenvironment was further analyzed using RT-qPCR. We show that the combination of DPX-R9F and PS-targeting antibodies with and without anti-PD-1 demonstrated increased efficacy compared to untreated controls. All treatments containing DPX-R9F led to T cell activation as assessed by IFN-γ ELISPOT. Furthermore, DPX-R9F/anti-PS treatment significantly elevated cytotoxic T cells, macrophages and dendritic cells based on RT-qPCR analysis. Overall, our data indicates that anti-tumour responses are driven through a variety of immune cells within this model and highlights the need to investigate combination therapies which increase tumour immune infiltration, such as anti-phosphotidylserine.

Targeting Phosphatidylserine Enhances the Anti-Tumor Response to Tumor-Directed Radiation Therapy in a Preclinical Model of Melanoma

Phosphatidylserine (PS) is a phospholipid that is exposed on surface of apoptotic cells and has been shown to promote immunosuppressive signals in the tumor microenvironment (TME). Antibodies that block PS interaction with its receptors have been shown to repolarize tumor associated macrophages (TAMs) into a pro-inflammatory (M1) state, reduce the number of myeloid derived suppressor cells (MDSCs) in tumors and promote maturation of dendritic cells (DCs). Radiation therapy (RT) is an effective focal treatment of isolated solid tumors but is less effective in controlling metastatic cancers as a monotherapy. Tumor-directed RT is known to induce immunogenic cell death and enhance tumor-specific T cell infiltration into treated tumors. We found that a single dose of 15 Gy of tumor-directed RT on mouse B16 melanoma causes an increase in expression of PS on the surface of viable immune infiltrates. We hypothesize that expression of PS on immune cells may act similar to immune checkpoint molecules and provide a negative feedback mechanism to immune cells. Therefore, we posit that blocking PS may enhance the efficacy of tumor-directed RT. Indeed, we found that treatment with an antibody that targets PS (mch1N11) enhanced the anti-tumor efficacy of tumor-directed RT and improved overall survival. This combination led to an increase in M1 TAMs and a corresponding decrease in M2 TAMs which translated to a more pro-inflammatory TME. The addition of anti-PD-1 to RT and mch1N11 led to even greater anti-tumor efficacy and overall survival. Anti-PD-1 exerted its effects primarily by enhancing CD8+ T cell infiltration, activation, and effector function in the triple combination. Consistent with the mouse studies, we found increased PS expression on several immune subsets in the blood in patients with metastatic melanoma after receiving tumor-directed RT. These findings highlight the potential of combining PS targeting with RT and PD-1 pathway blockade to improve outcomes in patients with advanced-stage cancers and may inform the design of clinical studies combining PS-targeting antibodies with RT and/or checkpoint blockade.

Abstract 530: Phosphatidylserine-targeting antibody combined with chemoradiation enhances systemtic anti-tumor immunity

Abstract The discovery of immune checkpoint blockades have resulted in significant clinical benefits for patients with metastatic cancers including non-small cell lung cancer (NSCLC). Nevertheless, a large proportion of patients do not respond to such therapy. Strategies to enhance the therapeutic efficacy of cancer immunotherapy among a larger group of patients are urgently needed. Increasing the recognition of cancer cells by immune system may enhance response to immunotherapies. Tumor-associated macrophage (TAM) is the most abundant immune cell in most solid tumors, and is critical in bridging innate and adaptive immune system functions. Cancer cells overexpress multiple immune suppressive molecules including phosphatidylserine (PS) to inhibit immunological functions of TAMs. PS expression can be enhanced by cancer therapeutics such as radiation to induce immune suppression. Our results show that radiation increased expression of PS on lung cancer cells. The combination of PS blocking antibody with radiation decreased immune-suppressive phagocytosis and enhanced antigen presentation of macrophages. In murine lung cancer models, combination treatment of PS blockade and chemoradiation more effectively controlled tumor growth and prolonged survival than each treatment used alone. In a bilateral lung cancer tumor model, addition of PS blockade to chemoradiation promoted cytotoxic T cell infiltration and induced tumor growth inhibition of the non-irradiated tumors. Together, our results demonstrate that PS-targeting antibodies may be combined with chemoradiation to enhance intrinsic tumor immunogenicity and activate systemic anti-tumor immune responses. This approach may provide new routes for improving the efficacy of modern immunotherapeutics. Citation Format: Yifan Wang, Zhigang Liu, Weiye Deng, Betty Y. Kim, Steven H. Lin, Wen Jiang. Phosphatidylserine-targeting antibody combined with chemoradiation enhances systemtic anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 530.

Chemoradiation Combined with Phosphatidylserine-Targeting Antibody Enhances Systemic Anti-tumor Immune Responses

Recent advances in cancer immunotherapy have sparked significant interest in harnessing the body’s immune system to fight cancer. However, the response rates of cancer immunotherapies, including immune checkpoint blockade, remain low. Strategies to enhance anti-tumor immune responses by targeting different steps along the immune activation cascade that can complement T cell-based immunotherapies are clearly needed. Here, we examine whether the addition of an antibody that targets extracellular phosphatidylserine (PS), a molecule that is recognized by myeloid derived cells, can enhance anti-tumor immune responses of chemoradiation for non-small cell lung cancer (NSCLC). Chemoradiation (CRT) was combined with a murine monoclonal antibody (mch1N11). PS is highly expressed in both orthotopically and ectopically implanted 393P murine NSCLC models. Radiation dose was 2 Gy/day, given for 5 days. Chemotherapy consists of carboplatin and paclitaxel at a dose of 30 mg/kg and 10 mg/kg, respectively. PS-targeting antibody (mch1N11) was given at 3mg/kg for 2 weekly doses. For ectopic tumor models, bilateral tumors were established in the legs. Radiation was directed to the R leg and tumor on the L side was shielded. Tumor growth was measured either with CT imaging or digital caliper. Tumor infiltrating immune cell profiles were analyzed using immunohistochemistry. Survival analyses were performed using Kaplan-Meier method and compared using Log-rank test. CRT + mch1N11 treatment significantly inhibited growth and improved survival in mice implanted with orthotopic 393P tumors as compared to CRT alone (median: 21 vs. 15 days, HR: 2.77, P = 0.006). Tissue analyses showed that CRT significantly increased the expression of PD-L1 within the tumor and drastically reduced the number of tumor infiltrating CD8 T cells. For the CRT + mch1N11 group, a similar up-regulation of PD-L1 expression was noted. However, the addition of mch1N11 re-populated the infiltrating CD8 T cells within the tumor. In the bilateral tumor model, the addition of mch1N11 antibody to CRT resulted in tumor regression in ∼40% of the non-irradiated tumors in the contralateral side as compared to 0% in the CRT alone or chemotherapy + mch1N11 groups. We showed that PS-targeting combined with standard CRT can significantly prolong survival in preclinical models of NSCLC and generate enhanced systemic anti-tumor immunity against lesions outside of the irradiated field. CRT up-regulated PD-L1 expression within the tumor and depleted tumor infiltrating cytotoxic CD8 T cells. The addition of mch1N11 antibody to CRT, however, was able to re-populate this critical immune effector cell population. Together, our results demonstrate that PS-targeting antibodies may be combined with CRT to enhance intrinsic tumor immunogenicity, activate systemic anti-tumor immune responses, and act as a priming strategy to sensitize the tumor to immune checkpoint inhibition with PD-1 or PD-L1 antibodies.

Abstract 3657: Phosphatidylserine-targeting antibodies enhance anti-tumor activity of a tumor vaccine in a HPV-induced tumor model

Abstract Antibodies targeting phosphatidylserine (PS) have been shown to induce anti-tumor responses by induction of tumor-specific T cells. Based on this observation, we evaluated the responses of PS and PD-1 targeting antibody therapy to enhance anti-tumor responses of a HPV16 peptide vaccine formulated in DepoVax™ (DPX) in mice bearing HPV-transformed C3 mouse tumors. The addition of PS-targeting antibody (mch1N11) to DPX/metronomic cyclophosphamide (mCPA) immunotherapy prolonged survival in comparison to mice receiving an isotype control in combination with DPX/mCPA. When anti-PD-1 was added to mch1N11 + mCPA, there was no increase in survival. The addition of mch1N11 to DPX/mCPA immunotherapy had no effect on tumor growth or survival in the aggressive B16-F10 model. TIL analysis revealed an increase in CD8+ T cells, antigen specific CD8+ T cells and PD-1+ T cells in the tumor with mch1N11 treatment. The expression of surface markers for macrophages (CD68high, F4/80) and dendritic cells (CD11c) were also increased in the tumors of mice treated with mch1N11. RT-qPCR analysis of the tumor confirmed higher mRNA expression of T cells markers (CD8, Granzyme B, PD-1) and antigen presenting cell markers (F4/80, CD74). In the spleen, expression of cell surface markers for monocytes (CD11b) and PD-1+ T cells (CD8) were elevated in groups treated with mch1N11 in combination with anti-PD-1. Combined, these findings indicate that in this model, PS-targeting antibodies can enhance the activity of phagocytic cells involved in antigen presentation. We have found that PD-1 expression increases as anti-tumor activity increases, therefore these results also provide an indication that antibodies targeting PS enhance the anti-tumor immune response induced by DPX/mCPA therapy. The observations suggest that PS-targeting antibodies may enhance therapeutic vaccines for the treatment of cancer. Citation Format: Genevieve Weir, Tara Quinton, Jeff T. Hutchins, Bruce D. Freimark, Marianne Stanford. Phosphatidylserine-targeting antibodies enhance anti-tumor activity of a tumor vaccine in a HPV-induced tumor model [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 3657. doi:10.1158/1538-7445.AM2017-3657

Abstract 574: Phosphatidylserine targeting antibody in combination with tumor radiation and immune checkpoint blockade promotes anti-tumor activity in mouse B16 melanoma

Abstract Phosphatidylserine (PS) is a phospholipid that is exposed on surface of apoptotic cells, tumor cells and tumor endothelium. PS has been shown to promote immunosuppressive signals in the tumor microenvironment. Antibodies that target PS have been shown to reactivate anti-tumor immunity by polarizing tumor associated macrophages into a pro-inflammatory M1 phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs. In a mouse B16 melanoma model, targeting PS in combination with immune checkpoint blockade promoted greater anti-tumor activity than either agent alone. This combination was shown to enhance CD4+ and CD8+ T cell infiltration and activation in the tumors of treated animals. Radiation therapy (RT) is an effective focal treatment of primary solid tumors, but is less effective in treating metastatic solid tumors as a monotherapy. There is evidence that RT induces immunogenic tumor cell death and enhances tumor-specific T cell infiltration in treated tumors. The abscopal effect, a phenomenon in which tumor regression occurs outside the site of RT, has been observed in both preclinical and clinical trials when RT is combined with immunotherapy. In this study, we show that irradiation treatment of B16 melanoma causes an increase in PS expression on the surface of viable tumor and immune infiltrates. We subsequently examined the effects of combining RT with an antibody that targets PS (mch1N11) and immune checkpoint blockade (anti-PD-1) in B16 melanoma. We found that treatment with mch1N11 synergizes with RT to improve anti-tumor activity and overall survival in tumor bearing mice. In addition, the triple combination of mch1N11, RT and anti-PD-1 treatment displayed even greater anti-tumor and survival benefit. Analysis of local immune responses in the tumors of treated animals revealed an increase in tumor-associated macrophages with a shift towards a pro-inflammatory M1 phenotype after treatment with RT and mch1N11. In addition, analysis of the systemic immune responses in the spleen and tumor draining lymph nodes revealed an increase in CD8 T cell activation, effector cytokine production and differentiation into effector memory cells in the triple combination. This finding highlights the potential of combining these three agents to improve outcome in patients with advanced-stage melanoma and other cancers and may inform the design of clinical studies combining PS-targeting antibodies with RT and/or checkpoint blockade. Citation Format: Sadna Budhu, Olivier De Henau, Roberta Zappasodi, Rachel Giese, Luis F. Campesato, Christopher Barker, Bruce Freimark, Jeff Hutchins, Jedd D. Wolchok, Taha Merghoub. Phosphatidylserine targeting antibody in combination with tumor radiation and immune checkpoint blockade promotes anti-tumor activity in mouse B16 melanoma [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 574. doi:10.1158/1538-7445.AM2017-574

Highlights of This Issue

Resistance to anti-androgen therapy in prostate cancer (PCa) can occur through increased intratumoral/adrenal production of androgens, overexpression of the androgen receptor (AR), and/or AR mutants that exhibit altered ligand specificity or activate transcription in a ligand-independent manner. Given the diversity of mechanisms that can lead to endocrine therapy resistance, it has been difficult to develop drugs that directly and effectively inhibit AR in advanced disease. However, taking the alternative approach of targeting critical processes downstream of AR, Stice and colleagues demonstrate that inhibition of the AR-regulated cyclin D1-CDK 4/6 signaling axis results in substantial tumor inhibition in the most treatment recalcitrant animal models of PCa. These results suggest that CDK4/6 inhibitors (CDK4/6i) are a viable alternative to taxanes, the current standard of care in endocrine therapy-resistant prostate cancer.Circulating microvesicles (MVs) have been described as important players in cell-to-cell communication and tumor-derived MVs can include potentially oncogenic information in cancers. In plasma of leukemia patients, circulating MVs were shown to carry leukemia specific fusion transcripts. Here, using gene expression profiling it is demonstrated that MVs cargo of leukemic cells encloses oncogenic fusion transcripts and mRNA related to basic functions of leukemic cells. In co-culture with mesenchymal stem cells, MVs transferred to target cells enhanced their proliferation. As carriers of oncogenic protein coding messages, MVs potentially jeopardize cell-directed therapy and facilitate the spread to other compartments of the body.Hypoxia-induced changes in composition and stiffness of the extracellular matrix (ECM) in the tumor microenvironment (TME) are well-established. Ju and colleagues now define the role of hypoxia in the regulation of ECM-binding receptors by analyzing the expression of individual integrin subunits within a large clinical cohort of breast cancer (BCa) patients and a panel of 20 BCa cell lines. Furthermore, it was demonstrated that integrin α5 is required for BCa metastasis to lymph nodes and lungs. In clinical specimens, increased integrin α5 expression correlates with poor prognosis, suggesting that targeting integrin α5 has therapeutic benefit for patients with hypoxic tumors.Tyro3, Axl and Mertk (TAM receptors) have been implicated in human cancers, whereby aberrantly elevated expression and signaling is associated with cancer progression, metastasis, and resistance to targeted therapies. Here, Kasikara and colleagues explore the function of TAMs as phosphatidylserine (PS) sensing receptors and reveal that activation of TAMs by apoptotic cells and Gas6 promotes epithelial cell efferocytosis, AKT-mediated chemoresistance, and up-regulation of PD-L1. TAM-mediated up-regulation of PD-L1 could be partially blocked by PS-targeting antibodies or Annexin V; thus, providing a rationale that PS-targeting, anti-TAMs, and anti-PD1 based therapeutics have merit as combinatorial checkpoint inhibitors.

Abstract B36: Modulating the tumor microenvironment to enhance cancer immunotherapy by inducing phosphatidylserine expression on the tumor surface

Abstract Background and Hypothesis: While immune checkpoint blockade, in particular PD-1 or PD-L1 inhibition, has revolutionized cancer immunotherapy, a significant number of patients with solid tumors fail to respond to therapy. An emerging consensus is the need for a local infiltration of immune effector T cells, in combination with PD-1 or PD-L1, for anticancer responses. We are exploring an improved strategy to modulate the tumor microenvironment by targeting phosphatidylserine (PS), which is a phospholipid membrane component maintained on the cytosolic side of the cell membrane in viable cells. When a cell undergoes apoptosis or stress induced by the microenvironment, PS is no longer restricted to the cytosolic domain and gets transposed to the extracellular surface where it serves a variety of functions, including as a ligand for another immune checkpoint, TIM-3. In fact, PS-targeting antibodies induce immunity against weakly immunogenic tumor cells (He at al, 2009), and may serve to enhance anti-cancer immunotherapy. Chemotherapy and radiation therapy are often used to treat cancers. In addition, photodynamic therapy using a photosensitizer conjugated with a target specific molecule has been recognized as an emerging therapeutic strategy. Therefore, we sought to determine the expression of PS following treatment with chemotherapy, radiation, or photodynamic therapy, and to determine if the addition of a PS-targeting antibody could augment antitumor response, especially in combination with PD-1 inhibition. Methods and Results: We first confirmed the upregulation of PS on malignant human cells following treatment with chemotherapy by FACS analysis using PS-targeting antibody mch1N11. PS expression was increased (50.2% positive compared to 5.0% in untreated cells) after 18 h of incubation with etoposide (25µM). We then tested PS expression on murine triple negative breast cancer cell lines (E0771-OVA, 4T1) at days 1, 2, and 4 after a single dose of X-ray (30, 40, or 50 Gy). E0771-OVA cells showed increased PS expression at day 2, while 4T1 cell lines increased PS expression at day 4. E0771-OVA cells were also irradiated using a more conventional schedule, specifically daily administration (6 Gy/day 5 days), leading to increase PS expression. Additionally, we tested PS expression on E0771-OVA cells after photodynamic therapy using HS201, which is a synthesized photosensitizer compound constructed of verteporfin and HSP 90 inhibitor. PS expression increased at day 1 when the cells were treated with a single dose of laser (10 or 60J) after 30 minutes conjugation of HS201 (1 or 10 μM). After confirming upregulation of PS by chemotherapy, radiotherapy, and photodynamic therapy, we next tested the combination of PS-targeting mAb mch1N11 with chemotherapy in vivo. We treated C57BL/6 mice bearing E0771-OVA tumors with paclitaxel (PTX), PS-targeting mAb mch1N11, anti-PD-L1 Ab, or combination. As single agents, both mch1N11 and anti-PD-L1 mAbs showed tumor growth suppression; however, combination of those two and 3 combination showed stronger effect. On the other hand, PTX didn't seem to add any additional effect. Conclusions: PS expression is upregulated by chemotherapy, radiotherapy, and photodynamic therapy in vitro. A combination therapy blocking PS by PS-targeting mAb and immune checkpoint blockade with anti-PD-L1 mAb appears superior to single agent therapy. Our future plan is to investigate the effect of local irradiation or photodynamic therapy combined with PS-targeting mAb and anti-PD-L1 Ab. Citation Format: Kensuke Kaneko, Takuya Osada, Bruce D. Freimark, Herbert Kim Lyerly. Modulating the tumor microenvironment to enhance cancer immunotherapy by inducing phosphatidylserine expression on the tumor surface. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B36.

Phosphatidylserine-Targeted Nanotheranostics for Brain Tumor Imaging and Therapeutic Potential.

Phosphatidylserine (PS), the most abundant anionic phospholipid in cell membrane, is strictly confined to the inner leaflet in normal cells. However, this PS asymmetry is found disruptive in many tumor vascular endothelial cells. We discuss the underlying mechanisms for PS asymmetry maintenance in normal cells and its loss in tumor cells. The specificity of PS exposure in tumor vasculature but not normal blood vessels may establish it a useful biomarker for cancer molecular imaging. Indeed, utilizing PS-targeting antibodies, multiple imaging probes have been developed and multimodal imaging data have shown their high tumor-selective targeting in various cancers. There is a critical need for improved diagnosis and therapy for brain tumors. We have recently established PS-targeted nanoplatforms, aiming to enhance delivery of imaging contrast agents across the blood–brain barrier to facilitate imaging of brain tumors. Advantages of using the nanodelivery system, in particular, lipid-based nanocarriers, are discussed here. We also describe our recent research interest in developing PS-targeted nanotheranostics for potential image-guided drug delivery to treat brain tumors.

Anti-Tumor Responses By Ibrutinib and Anti-PD-1 Blockade Is Enhanced By Phosphatidylserine-Targeting Antibody Therapy

Introduction: Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane that becomes exposed on vascular endothelial cells and tumor cells in the tumor microenvironment, particularly in response to chemotherapy and irradiation. Binding of antibodies targeting PS on the tumor endothelial cells and tumors induces the recruitment of immune cells and engages the immune system to destroy tumor and associated vasculature and by blocking the immunosuppressive action of PS. Recent studies have demonstrated that PS-targeting antibodies enhance the anti-tumor activity of immune checkpoint antibody blockade to CTLA-4 and PD-1 in mouse breast and melanoma tumor models (Freimark et al. Cancer Immunol. Res. 2016; Gray et al. Breast Cancer Res 2016). Ibrutinib is an approved anticancer drug targeting B-cell malignancies that is a selective, covalent inhibitor of the enzyme Bruton's tyrosine kinase(BTK) in B-cell tumors. Data from recent mouse tumor studies demonstrate that ibrutinib in combination with anti-PD-1 antibody blockade inhibits growth of solid tumors (lacking BTK expression) suggesting that ibrutinib may inhibit kinases of the immune system such as interleukin-2 inducible T-cell kinase (ITK), to enhance specific anti-tumor responses (Sagiv-Barfli et al. PNAS 20 2015).Methods: The present study was conducted to evaluate the anti-tumor effects of combination therapy including PS-targeting antibody mouse chimeric 1N11 (mch1N11), ibrutinib (32765) and anti-PD-1 antibody using C57BL/6 mice bearing triple negative E0771 breast tumors. Tumors were staged to an initial volume of ~100mm3and randomized to treatment groups (N=10) with mch1N11 or isotype at 10 mg/kg qw, anti-PD-1 at 2.5 mg/kg qw or ibrunitib 6 mg/kg or vehicle qd x 8. Tumor volumes were measured twice per week to determine tumor growth inhibition (TGI) relative to control treated animals until a maximum volume of 1500-2000mm3. The in vitro sensitivity of E0771 tumor cells to ibrutinib was compared to drug sensitive Jeko-1 lymphoma cells in a 72 hour growth and viability assay.Results: The E0771 cell line is resistant in vitroto 10 mM ibrutinib compared to the drug-sensitive Jeko-1 cell line (Figure 1). Mice bearing E0771 tumors treated with mch1N11, ibrutinib and anti-PD-1 alone had 22.2%, 23.5% and 32.6% TGI respectively. Combination of two agents increased the TGI for mch1N11 and ibrutinib to 30.5%, ibrutinib and anti-PD-1 to 34.5%, mch1N11 and anti-PD-1 to 36.1%. A triple combination therapy had statistically greater TGI compared to control treated mice (59.9%, p = 0.0084) and was greater than single and double combination therapies.Conclusion:Treatment of solid tumors with a combination of inhibitors that target PS, ITK and the PD-1/PD-L1 axis in the tumor microenvironment provides a novel treatment for solid tumors, including triple negative breast cancer. [Display omitted] [Display omitted] DisclosuresGong:Peregrine Pharmaceuticals, Inc.: Employment. Gray:Peregrine Pharmaceuticals, Inc.: Employment. Hutchins:Peregrine Pharmaceuticals, Inc.: Employment. Freimark:Peregrine Pharmaceuticals, Inc.: Employment.

Abstract B019: LAG3 is an immunotherapeutic target in murine triple negative breast cancers whose activity is significantly enhanced in combination with phosphatidylserine targeting antibodies

Abstract Previous pre-clinical modeling demonstrated that phosphatidylserine (PS) targeting antibodies are capable of significantly enhancing the effectiveness of anti- programmed cell death ligand 1 (PD-1) therapy in murine breast cancers (BC). These results prompted our hypothesis that PS blocking antibodies should also be capable of enhancing the activity of additional immunotherapies in BC, including triple negative breast cancer (TNBC). PS normally resides in the inner leaflet of the plasma membrane in most cell types. Conditions that promote cellular stress in the tumor microenvironment, such as reactive oxygen species (ROS), hypoxia, and irradiation, cause PS externalization and exposure on tumor associated endothelial cells and tumor cells, where it is recognized by specific receptors, including members of the TIM and TAM family. This PS recognition promotes an innate system driven immunosuppressive condition by promoting an influx of myeloid derived suppressor cells (MDSCs), an M2-macrophage shift, and suppression of dendritic cell maturation, while inducing anti-inflammatory cytokines. To identify additional immuno-therapeutic targets that may be potential targets for combinational treatment with PS-targeting antibodies, we employed gene expression analysis on tumors from murine TNBC treated with single and combinations of PS and PD-1 targeting antibodies. Of the genes assayed, expression of the lymphocyte activation gene 3 (LAG3) increased in each treatment arm, suggesting it may be a potential target for combinational therapy in breast cancers. To test our hypothesis, C57BL/6 mice hosting the murine TNBC line E0771 were treated with a PS targeting antibody or a LAG3 targeting antibody alone or in combination. Our data demonstrate that while PS-blocking and anti-LAG3 therapies each have moderate efficacy as single agents, combinational treatment significantly impeded the growth of TNBC, and was capable of increasing tumor infiltrating lymphocytes (TILs), including CD8 + and CD3 + T-cells, and reducing the percentage of MDSCs over that observed by single treatments. Combined, our data demonstrate that LAG3 targeting also represents a viable option for the treatment of TNBC, and that the addition of PS targeting antibodies to LAG3 therapy can effectively increase the anti-tumor and immune-activating effects mediated by multiple checkpoint therapies. Citation Format: Michael J. Gray, Jian Gong, Jeff Hutchins, Bruce Freimark. LAG3 is an immunotherapeutic target in murine triple negative breast cancers whose activity is significantly enhanced in combination with phosphatidylserine targeting antibodies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B019.

Abstract PR09: Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms

Abstract Background: The expression of phosphatidylserine (PS) on cell surfaces drives immunosuppressive mechanisms associated with tolerogenic cell death. In the tumor microenvironment, PS is exposed on tumor cells and tumor vascular endothelial cells treated with conventional therapies. PS signals through multiple immune cell signaling receptors where it drives the expansion of myeloid-derived suppressor cells (MDSCs), regulatory T cells, M2 macrophages, and stimulates their production of immunosuppressive cytokines (e.g., TGFβ and IL-10), rendering the tumor microenvironment non-responsive to immune activation. PS targeting antibodies have significant anti-tumor effects in multiple tumor xenografts, syngeneic tumors, and genetically engineered preclinical tumor models through mechanisms that re-program the tumor microenvironment from immunosuppressive to immune potentiating. Methods: The combination of PS-targeting and anti-PD1 antibodies was compared to single agent therapy in multiple pre-clinical mouse models, including syngeneic melanoma and breast tumors, and a genetically engineered mouse model (GEMM) of pancreatic cancer. Mice were treated weekly IP at 5 mg/kg with a PS targeting antibody, anti-PD1, or the combination for the duration of each experiment; tumor and spleen tissue microenvironments were immune profiled by FACS, ELISPOT, and immunohistochemistry. Results: In all tumor models examined, the anti-tumor effect of combination therapy was significantly superior to single agent therapy. In B16 and K1735 melanoma tumors, the combination more than doubled the anti-tumor effect of anti-PD-1 alone. Combination therapy significantly inhibited tumor growth by over 90% in E077.1 breast tumors and prolonged animal survival by 30% (n>25 each group) in the GEMM of pancreatic cancer despite the fact that anti-PD1 therapy alone was ineffective in each of these models. Analysis of immune cell subsets in the tumor microenvironment indicated that the combination of antibody-mediated blockade of PS and PD-1 significantly enhanced the effector function of tumor infiltrating CD8+ T cells and increased the ratio of T effector to T regulatory cells, as demonstrated by significant increases in TILs producing IFNγ, TNFα, IL-2, granzyme B, and Ki-67. Combination therapy re-conditioned the tumor microenvironment to favor immune potentiation, as demonstrated by significant decreases in the frequency of MDSC, the ratio of M2 to M1 macrophages, the expression of surface PD-L1, and reductions in TGFβ and IL-10. Furthermore, combination treatment induced systemic tumor specific CD8 T cell immunity, as mice demonstrating complete responses (in the K1735 and E077.1 models) rejected tumor cells upon re-challenge. Splenocytes from these mice had significantly higher numbers of tumor-specific IFNγ-producing cells in ELISPOT assay. Studies were completed without toxicity in any setting. Conclusions: Antibody-mediated PS blockade inhibits PS-mediated immune suppression and stimulates FcRγ activation. This results in immune activation that enables an otherwise non-responsive tumor microenvironment to respond to checkpoint inhibition. This abstract is also presented as Poster B08. Citation Format: Xianming Huang, Jian Gong, Michael Gray, Van Nguyen, Ryan Parks, Chris Hughes, Jeff Hutchins, Rolf Brekken, Bruce Freimark. Antibody-mediated blockade of phosphatidylserine synergizes with immune checkpoint blockade by inhibiting multiple immune suppressive mechanisms. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR09.

Antibody-Mediated Phosphatidylserine Blockade Enhances the Antitumor Responses to CTLA-4 and PD-1 Antibodies in Melanoma.

In tumor-bearing animals, the membrane phospholipid phosphatidylserine (PS) suppresses immune responses, suggesting that PS signaling could counteract the antitumor effect of antibody-driven immune checkpoint blockade. Here, we show that treating melanoma-bearing mice with a PS-targeting antibody enhances the antitumor activity of downstream checkpoint inhibition. Combining PS-targeting antibodies with CTLA-4 or PD-1 blockade resulted in significantly greater inhibition of tumor growth than did single-agent therapy. Moreover, combination therapy enhanced CD4(+) and CD8(+) tumor-infiltrating lymphocyte numbers; elevated the fraction of cells expressing the proinflammatory cytokines IL2, IFNγ, and TNFα; and increased the ratio of CD8 T cells to myeloid-derived suppressor cells and regulatory T cells in tumors. Similar changes in immune cell profiles were observed in splenocytes. Taken together, these data show that antibody-mediated PS blockade enhances the antitumor efficacy of immune checkpoint inhibition. Cancer Immunol Res; 4(6); 531-40. ©2016 AACR.

Abstract P4-04-03: Targeting of phosphatidylserine by monoclonal antibodies augments the activity of immune checkpoint inhibitor PD-1/PD-L1 therapy in murine breast tumors

Abstract Phosphatidylserine (PS) is a phospholipid that typically resides in the inner leaflet of the plasma membrane in many types of cells, including both tumor and tumor associated endothelial cells. Conditions that cause cellular stress, including those that occur from oxygen radicals, hypoxia, irradiation, and chemotherapy, cause a dramatic shift in PS localization. This change in localization results in PS shifting to the outer plasma membrane, allowing its recognition by components of the tumor microenvironment. Recognition of PS promotes an immunosuppressive environment that encourages tumor growth, in part by promoting the recruitment of myeloid derived suppressor cells, immature dendritic cells, and M2-like macrophages, in addition to inducing production of anti-inflammatory cytokines. Currently the chimeric PS-targeting antibody, bavituximab, is being used in combination with chemotherapies to treat patients with solid tumors in multiple late-stage clinical trials, where it is believe to help augment the efficacy of chemotherapeutics by blockade of PS-mediated immunosuppression and triggering an Fc-FcR mediated pro-inflammatory response in the tumor microenvironment. While the results with PS targeting therapies with chemotherapeutics are encouraging, the effectiveness of PS targeting therapies in conjunction with therapies towards immune checkpoint regulators remains largely unknown. To better understand the role of PS-targeting in breast cancers, and its effectiveness when used in combination with checkpoint inhibitors, immune competent mice harboring either EMT-6 or E0771 breast tumors were utilized. Treatments comprised PS targeting antibodies and an anti-PD1 antibody (to interrupt the PD-1/PD-L1 signaling axis) either alone or in combination with each other, and the effect on tumor growth and immune suppression determined. In both models, which showed differential sensitivity to therapy, the inclusion of PS targeting antibodies with the checkpoint blocker antibody had a significantly greater anti-tumor response than either single agent alone. Citation Format: Gray M, Gong J, Nguyen V, Hutchins J, Freimark B. Targeting of phosphatidylserine by monoclonal antibodies augments the activity of immune checkpoint inhibitor PD-1/PD-L1 therapy in murine breast tumors. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-03.

Phosphatidylserine (PS) Is Exposed in Choroidal Neovascular Endothelium: PS-Targeting Antibodies Inhibit Choroidal Angiogenesis In Vivo and Ex Vivo.

Choroidal neovascularization (CNV) accounts for 90% of cases of severe vision loss in patients with advanced age-related macular degeneration. Identifying new therapeutic targets for CNV may lead to novel combination therapies to improve outcomes and reduce treatment burden. Our goal was to test whether phosphatidylserine (PS) becomes exposed in the outer membrane of choroidal neovascular endothelium, and whether this could provide a new therapeutic target for CNV. Choroidal neovascularization was induced in C57BL/6J mice using laser photocoagulation. Choroidal neovascularization lesions costained for exposed PS and for intercellular adhesion molecule 2 (or isolectin B4) were imaged in flat mounts and in cross sections. The laser CNV model and a choroidal sprouting assay were used to test the effect of PS-targeting antibodies on choroidal angiogenesis. Choroidal neovascularization lesion size was determined by intercellular adhesion molecule 2 (ICAM-2) staining of flat mounts. We found that PS was exposed in CNV lesions and colocalized with vascular endothelial staining. Treatment with PS-targeting antibodies led to a 40% to 80% reduction in CNV lesion area when compared to treatment with a control antibody. The effect was the same as that seen using an equal dose of an anti-VEGF antibody. Results were confirmed using the choroid sprouting assay, an ex vivo model of choroidal angiogenesis. We demonstrated that PS is exposed in choroidal neovascular endothelium. Furthermore, targeting this exposed PS with antibodies may be of therapeutic value in CNV.

Abstract 4289: Targeting of phosphatidylserine by monoclonal antibodies enhances the activity of immune checkpoint inhibitors in breast tumors

Abstract Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane that becomes exposed on tumor vascular endothelial cells (ECs) and tumor cells. PS exposure becomes enhanced in response to chemotherapy, irradiation, and oxidative stresses in the tumor microenvironment. PS exposure in tumors promotes an immunosuppressive microenvironment which includes the recruitment of myeloid derived suppressor cells (MDSCs), immature dendritic cells and M2-like macrophages, as well as, the production of anti-inflammatory cytokines. Binding of PS targeting antibodies on tumor endothelial cells, tumor cells and their secreted microparticles triggers an Fc-FcR mediated pro-inflammatory cellular and cytokine response that reverses the immunosuppressive PS meditated checkpoint, thereby enhancing anti-tumor immunity. A chimeric PS-targeting antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in multiple late-stage clinical trials. Using breast tumors in immune competent mice, we demonstrate PS targeting antibodies enhance the anti-tumor activity of combination therapies including anti-PD-1 antibodies. Tumor growth inhibition correlates with statistically significant increases in the infiltration of CD8+ T cells and a reduction of myeloid-derived suppressor cells (MDSCs). The combination of these mechanisms promotes strong, localized, anti-tumor responses without the side-effects of systemic immune activation. Citation Format: Jian Gong, Shen Yin, Van Nguyen, Jeff Hutchins, Bruce D. Freimark. Targeting of phosphatidylserine by monoclonal antibodies enhances the activity of immune checkpoint inhibitors in breast tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4289. doi:10.1158/1538-7445.AM2015-4289

Abstract P5-04-12: Targeting of phosphatidylserine by monoclonal antibodies enhances activity of immune checkpoint inhibitors in breast tumors

Abstract Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane and becomes exposed on tumor vascular endothelial cells (ECs) and tumor cells. PS exposure becomes enhanced in response to chemotherapy, irradiation, and oxidative stresses in the tumor microenvironment. PS exposure in tumors promotes an immunosuppressive microenvironment which includes the recruitment of myeloid derived suppressor cells (MDSCs), immature dendritic cells, and M2-like macrophages as well as the production of anti-inflammatory cytokines. Binding of PS targeting antibodies on tumor endothelial cells, tumor cells and their secreted microparticles triggers an Fc-FcR mediated pro-inflammatory cellular and cytokine response that reverses the immunosuppressive PS meditated checkpoint, thereby enhancing anti-tumor immunity. A chimeric PS-targeting antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in multiple late-stage clinical trials. Using breast tumors in immune competent mice, we demonstrate PS targeting antibodies enhance the anti-tumor activity of anti-CTLA-4 and anti-PD-1 antibodies in the presence and absence of conventional chemotharapy. Tumor growth inhibition correlates with infiltration of immune cells in tumors and induction of adaptive immunity. The combination of these mechanisms promotes strong, localized, anti-tumor responses without the side-effects of systemic immune activation. Citation Format: Bruce Freimark, Jian Gong, Van Nguyen, Shen Yin, Rich Archer, Jeff Hutchins. Targeting of phosphatidylserine by monoclonal antibodies enhances activity of immune checkpoint inhibitors in breast tumors [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-12.

Abstract 4978: Targeting of phosphatidylserine by monoclonal antibodies enhances activity of immune checkpoint inhibitors in tumors

Abstract Phosphatidylserine (PS) is a phospholipid normally residing in the inner leaflet of the plasma membrane and becomes exposed on tumor vascular endothelial cells (ECs) and tumor cells. PS exposure becomesenhanced in response to chemotherapy, irradiation, and oxidative stresses in the tumor microenvironment. PS exposure in tumors promotes an immunosuppressive microenvironment which includes the recruitment of myeloid derived suppressor cells (MDSCs), immature dendritic cells, and M2-like macrophages as well as the production of anti-inflammatory cytokines. Binding of PS targeting antibodies on tumor endothelial cells, tumor cells and their secreted microparticles triggers an Fc-FcR mediated pro-inflammatory cellular and cytokine response that reverses the immunosuppressive PS meditated checkpoint, thereby enhancing anti-tumor immunity. A chimeric PS-targeting antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in multiple late-stage clinical trials. Using multiple syngeneic tumors in immune competent mice, we demonstrate PS targeting antibodies enhance the anti-tumor activity of anti-CTLA-4 and anti-PD-1 antibodies. Tumor growth inhibition correlates with infiltration of immune cells in tumors and induction of adaptive immunity. The combination of these mechanisms promotes strong, localized, anti-tumor responses without the side-effects of systemic immune activation. Citation Format: Jian Gong, Van Nguyen, Shen Yin, Rich Archer, Jeff Hutchins, Bruce Freimark. Targeting of phosphatidylserine by monoclonal antibodies enhances activity of immune checkpoint inhibitors in tumors. [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 4978. doi:10.1158/1538-7445.AM2014-4978

Abstract 1244: Phosphatidylserine-targeting antibody reactivates tumor immunity and destroys tumor vasculature in mice.

Abstract The immunosuppressive lipid, phosphatidylserine (PS), becomes exposed on tumor blood vessels and tumor cells responding to therapy. The exposed PS contributes to the immunosuppressed tumor microenvironment. Bavituximab is a phosphatidylserine (PS)-targeting antibody that is being combined with chemotherapy in clinical trials in cancer patients. Here, we tested the hypothesis that innate immunity against cancer can be elicited by combining chemotherapy with a monoclonal antibody that indirectly binds exposed phosphatidylserine (PS). We used a murine version of bavituximab, 2aG4, plus docetaxel to treat prostate tumors in mice. Combination treatment markedly retarded tumor growth, prolonged survival times and delayed progression to androgen-independent disease. Antibody treatment reduced the presence of myeloid-derived suppressor cells (MDSCs) in the tumors and caused macrophages to repolarize from the immunosuppressive, proangiogenic M2-like state into a tumoricidal M1-like state. The reactivated macrophages destroyed antibody-coated tumor vasculature and tumor cells. Addition of 2aG4 to MDSCs in vitro caused them to differentiate into M1-like macrophages and DCs. Thus, PS-targeting antibodies appear to reactivate innate immunity in tumors resulting in tumor growth inhibition. Citation Format: Yi Yin, Xianming Huang, Dan Ye, Philip Thorpe. Phosphatidylserine-targeting antibody reactivates tumor immunity and destroys tumor vasculature in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1244. doi:10.1158/1538-7445.AM2013-1244

Abstract 2510: Increased Fc-FcR interaction of human phosphatidylserine targeting antibody enhances pro-inflammatory and ADCC anti-tumor mechanisms

Abstract Anionic phospholipids, principally phosphatidylserine (PS), become exposed on the external surface of vascular endothelial cells in tumors, providing an excellent marker for tumor vascular targeting. Binding of antibodies targeting PS on the tumor endothelial cells and tumors recruits immune cells and engages the immune system to destroy tumor vasculature. The antibodies also enhance anti-tumor immunity by blocking the immunosuppressive action of PS. A chimeric anti-PS antibody, bavituximab, is being used in combination with chemotherapy to treat patients with solid tumors in Phase II trials. We have engineered mutations in the Fc region of fully human PS-targeting antibody PGN635 that enhance the interaction with FcRgammaIIIa and have characterized their ability to exert anti-tumor effects through various mechanisms, including ADCC and cytokine induction. Mutations that enhance Fc-FcR interaction of PS targeting antibodies enhance production of pro-inflammatory chemokines such as MCP-1 (CCL2), MIP-1alpha (CCL3) and MIP-1beta (CCL4). In addition, the same Fc mutations enhance ADCC of PS expressing tumor targets. Our data indicate that improvement of multiple immune effector functions via enhanced interaction of Fc-FcR interaction of PS-targeting antibodies may improve the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2510. doi:1538-7445.AM2012-2510