Phosphatidylserine-targeting Antibody Research Articles

The phosphatidylserine targeting antibody bavituximab plus pembrolizumab in unresectable hepatocellular carcinoma: a phase 2 trial

Immune checkpoint inhibitors targeting PD-1/L1 have modest efficacy in hepatocellular carcinoma as single agents. Targeting membranous phosphatidylserine may induce pro-inflammatory and -immune stimulating effects that enhance immunotherapy activity. This hypothesis was tested in a single-arm phase 2 trial evaluating frontline bavituximab, a phosphatidylserine targeting antibody, plus pembrolizumab (anti-PD-1) in patients with unresectable hepatocellular carcinoma (NCT03519997). The primary endpoint was investigator-assessed objective response rate among evaluable patients, and secondary end points included progression-free survival, incidence of adverse events, overall survival, and duration of response. Among 28 evaluable patients, the confirmed response rate was 32.1%, which met the pre-specified endpoint, and the median progression-free survival was 6.3 months (95% CI, 1.3–11.3 months). Treatment related-adverse events of any grade occurred in 45.7% of patients, with grade 3 or greater adverse events in 14.3% of patients. Adverse events of any cause were observed in 33 patients (94.3%), with grade 3 or greater adverse events in 11 patients (31.4%). Prespecified exploratory analyses of baseline tumor specimens showed that a depletion of B cells, and the presence of fibrotic tissue and expression of immune checkpoints in stroma was associated with tumor response. These results suggest that targeting phosphatidylserine may lead to synergistic effects with PD-1 blockade without increasing toxicity rates, and future studies on this therapeutic strategy may be guided by biomarkers characterizing the pre-treatment tumor microenvironment.

Abstract 1272: Aberrantly exposed phosphatidylserine as a drug delivery target in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) has risen to become the 3rd leading cause of cancer related deaths in the United States with a 5-year survival rate of 11% and a median survival of just 4-6 months. PDA is notoriously chemo-resistant and has shown little to no sensitivity to the current immune checkpoint blockade therapy. A novel marker for the tumor microenvironment is phosphatidylserine, first characterized as a selective marker of tumor vasculature. Phosphatidylserine (PS) is a phospholipid exposed on the outer leaflet of the plasma membrane of tumor associated endothelial cells, apoptotic tumor cells, and some viable tumor cells, where it functions to suppress the immune response. Exposed PS drives immunosuppression by binding to PS receptors expressed on tumor-infiltrating macrophages and dendritic cells. To this point, PS has been targeted with antibodies, such as Bavituximab, that have shown excellent specificity for tumor vasculature and an immune stimulatory environment evidence by polarization of macrophages to a pro-inflammatory M1-like phenotype, a reduction in myeloid-derived suppressor cells, enhanced maturation of dendritic cells and increased primed T cell activity. We have advanced this concept by developing the next generation of PS targeting agent, a fusion protein (betabody) that exploits the PS-binding domain of β2-glycoprotein 1 (β2GP1), an abundant serum protein that binds PS. Betabodies were constructed by fusing domain V of β2GP1 to the Fc of an IgG2a. Betabodies are smaller than full length antibodies (~85 kDa vs 150 kDa), have good affinity for PS (~1 nM) and bind directly to PS. We have demonstrated that betabodies bind specifically to externalized PS through ELISA, ICC and flow cytometry. We established that betabodies localize robustly and specifically to the tumor microenvironment through in vivo localization experiments in tumor-bearing mice. Further studies will exploit the specificity of betabodies to deliver novel microtubule-disrupting agents to exposed PS in the microenvironment of PDA. Because betabodies are internalized inefficiently, therapeutic payloads will be linked to betabodies via enzyme cleavable linkers. This first-in-class tumor targeting therapeutic has potential to provide efficacy in therapy-resistant pancreatic tumors. In addition, similarly to PS targeting antibodies, preliminary data in vitro has shown that treatment of both bone marrow derived macrophages and Raw 264.7 with the unconjugated betabodies repolarize M2 macrophages to a M1 phenotype. The mechanism of action has not yet been fully elucidated; however, it may involve the interaction between the β2GP1 domains and endogenous LPS. This first-in-class tumor targeting therapeutic has potential to provide efficacy in therapy-resistant pancreatic tumors via targeted drug delivery as well as modulation of the immuno-microenvironment like its PS targeting predecessors. Citation Format: Natalie Zara Phinney, Xianming Huang, Mary Lynn Trawick, Kevin Pinney, Rolf Brekken. Aberrantly exposed phosphatidylserine as a drug delivery target in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1272.

Inhibiting Growth of Human Triple-Negative Breast Cancer Cells by Activating Mutant p53 Protein Alone or in Combination With a Phosphatidylserine-Targeting Antibody

Triple-negative human breast cancers (TNBC) lack three proteins commonly targeted by chemotherapy; estrogen and progesterone receptors, and her-2-neu. Most current protocols used to treat TNBC are largely ineffective and aggressive tumors frequently re-emerge, leading to metastasis and patient death. Thus, new therapies for TNBC are needed. Recent studies show that around 80% of TNBC express mutant p53 (mtp53), a functionally defective form of the p53 tumor suppressor protein. If mtp53 is converted into the active wild-type protein (wtp53), tumor suppressor functions are recovered. Most p53 mutations occur in the DNA-binding domain, causing normal regulation of p53 target genes involved in apoptosis, cell-cycle arrest, and angiogenesis to be blocked. This promotes metastasis and renders tumors resistant to chemotherapy. APR-246 is a small-molecule drug that re-activates mtp53 by covalent modification of the DNA-binding core domain of the mutant protein through alkylation of thiol groups and has been shown to reactivate mtp53 and restore p53 function. We examined whether APR-246 could inhibit TNBC growth, both in vitro and in vivo. Cell viability assays and FACS were used to measure in vitro TNBC cell growth and apoptosis respectively, in MDA-MB-231 and MDA-MB-468 cells, with MCF-7 cells (which express wtp53) as controls. Analysis of TNBC growth in vivo was assessed in a mouse model of MDA-MB-231 derived xenografts. Nuclear extracts of APR-246-treated TNBC cells exhibited significantly increased p53 DNA binding compared with untreated cells, indicating that APR-246 converts mtp53 to wtp53 in these cells. APR-246 significantly reduced TNBC cell viability in vitro, but had no effect on normal mammary cells or wtp53-expressing MCF-7 cells. Pro-apoptotic proteins, Bax, p21 and caspase-3 were elevated in APR-246 treated cells, while the cell survival protein Bcl-2 was suppressed. In the xenograft model, animals were given an intravenous (iv) tail vein injection of APR-246 alone (100 mg/kg/day) once tumors reached 100 mm3. A second group received an intraperitoneal (ip) injection of 2aG4 antibody (100 µg/mouse/day), which targets phosphatidylserine and disrupts tumor blood vessel formation. A third group was given both APR-246 and 2aG4 using the same doses above. A control group received antibody C44 (100 µg/mouse/day, ip) and/or PBS (0.1 mL/day, iv). A total of 18 treatments were used. Administration of APR-246 alone or in combination with 2aG4, significantly reduced TNBC tumor growth, as well as two markers of angiogenesis (vascular endothelial growth factor expression and blood-vessel density). APR-246 in combination with 2aG4 completely eradicated almost 20% of the TNBC tumors. We conclude that TNBC is inhibited by APR-246 and 2aG4. Such treatment could represent an effective and innovative means of combating these particularly aggressive and deadly types of cancer.

Phosphatidylserine targeting antibody enhances anti-tumor activity of adoptive cell therapies in a mouse melanoma model

Abstract Adoptive cell therapy has emerged as a viable strategy to treat cancer. T cells that recognize tumor antigens can be reinvigorated ex-vivo or autologous T cells can be genetically modified to express anti-tumor T cell receptors (TCRs) or chimeric antigen receptors (CARs). However, once re-infused into patients, these tumor specific T cells are subjected to immunosuppressive signals within the tumor. A critical immune checkpoint within tumors is phosphatidylserine (PS), a phospholipid that is exposed on apoptotic cells and tumor cells. Innate cells exposed to PS secrete suppressive cytokines that can significantly impair the function of tumor specific T cells. Antibodies that target PS can reactivate anti-tumor immunity by reducing the number of MDSCs in tumors and promoting the maturation of functional APCs. Our lab has shown that the mouse chimeric version of PS Targeting monoclonal antibody Bavituximab (1N11), in combination with transgenic CD4+ T cells that recognize melanoma antigen Trp1, can regress advanced melanoma tumors in mice. Here, we demonstrate a 2nd generation CAR T cell, that binds Trp1 on the surface of B16 melanoma, in combination with 1N11 can improve anti-tumor activity and survival in B16 tumor bearing mice. Flow cytometry analysis of immune responses in the tumor of mice treated with tumor specific T cells and 1N11 shows a decrease in M2 macrophages and FoxP3+ regulatory T cells. These findings highlight that diminishing suppressive mechanisms locally with PS targeting can enhance the efficacy of transgenic TCR and CAR T cells to improve the outcome in patients with advanced-stage melanoma. Our studies may inform the design of clinical trials combining PS Targeting antibodies with CAR T cell therapy in solid tumors.

A phase II clinical trial of the phosphatidylserine targeting antibody, bavituximab, in combination with pembrolizumab in patients with advanced hepatocellular carcinoma.

TPS599 Background: Phosphatidylserine (PS) is an immunosuppressive lipid segregated to the inner leaflet of the plasma membrane of normal cells. PS is externalized to the outer leaflet of the plasma membrane on cells that line tumor blood vessels, tumor cells and exosomes in the tumor microenvironment creating a specific target for anticancer treatments. Bavituximab is a PS targeting antibody that binds to PS via β2 glycoprotein-1 (β2GP1), an abundant serum glycoprotein. Bavituximab can modulate the tumor immune microenvironment to promote anti-tumor immune activity. This clinical trial is evaluating the effect of combining bavituxumab and the checkpoint inhibitor pembrolizumab in patients with advanced hepatocellular carcinoma (HCC). Methods: This is a single arm phase 2 clinical trial for patients with histologically confirmed HCC which is not eligible for curative and/or loco-regional therapy. No prior systemic therapy for HCC is allowed. Patients should be > 18 years, have Child-Pugh Score A, ECOG 0-1 and meet the following laboratory criteria: total bilirubin ≤ 2.0, INR ≤ 1.7; Hgb ≥ 8.5 g/dl; AST, ALT ≤5 times ULN; platelet ≥ 50,000/mm3; and albumin ≥ 2.5 g/dl. Major exclusion criteria include thromboembolic event within the last 6 months, clinically evident ascites, GI bleeding within 2 months, uncontrolled hypertension, HIV and autoimmune disease (except DM1, childhood allergies and thyroid abnormalities if adequately controlled. Dosing is as follows: Bavituximab: 3 mg/kg IV weekly, pembrolizumab: 200 mg IV every 3 weeks. The primary objective is to determine the overall response rate (ORR) of therapy. Secondary objectives are to determine the safety, tolerability and other measures of clinical efficacy (overall survival, 6 month PFS, and duration of response). Tumor and blood samples are being collected to assess for pharmacodynamic biomarkers. A minimax two-stage method will be used to analyze ORR after the first 15 patients are accrued. If 3 or more of the first 15 patients have either a complete or partial response, 13 additional patients will be enrolled. ORR will be analyzed by RECIST 1.1 criteria. Clinical trial information: NCT03519997.

Combined treatment with p53-activating drug APR-246 and a phosphatidylserine-targeting antibody, 2aG4, inhibits growth of human triple-negative breast cancer xenografts

Combined treatment with p53-activating drug APR-246 and a phosphatidylserine-targeting antibody, 2aG4, inhibits growth of human triple-negative breast cancer xenografts

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.

APR-246 alone and in combination with a phosphatidylserine-targeting antibody inhibits lung metastasis of human triple-negative breast cancer cells in nude mice.

BackgroundApproximately 15–20% of all human breast cancers are classified as triple-negative because they lack estrogen and progesterone receptors and Her-2-neu, which are commonly targeted by chemotherapeutic drugs. New treatment strategies are therefore urgently needed to combat triple-negative breast cancers (TNBCs). Almost 80% of the triple-negative tumors express mutant p53 (mtp5), a functionally defective tumor suppressor protein. Whereas wild-type p53 (wtp53) promotes cell-cycle arrest and apoptosis and inhibits vascular endothelial growth factor-dependent angiogenesis, mtp53 fails to regulate these functions, resulting in tumor vascularization, growth, resistance to chemotherapy, and metastasis. Restoration of p53 function is therefore a promising drug-targeted strategy for suppressing TNBC metastasis.MethodsAPR-246 is a small-molecule drug that reactivates mtp53, thereby restoring p53 function. In this study, we sought to determine whether administration of APR-246, either alone or in combination with 2aG4, an antibody that targets phosphatidylserine residues on tumor blood vessels and disrupts tumor vasculature, effectively inhibits stem cell-like characteristics of tumor cells and migration in vitro, and metastasis of human mtp53-expressing TNBC cells to the lungs in mouse models.ResultsAPR-246 reduced both the stem cell-like properties and migration of TNBC cells in vitro. In mouse models, administration of either APR-246 or 2aG4 reduced metastasis of TNBC cells to the lungs; a combination of the two diminished lung metastasis to the same extent as either agent alone. Combination treatment significantly reduced the incidence of lung metastasis compared either single agent alone.ConclusionMetastasis of human mtp53-expressing TNBC cells to the lungs of nude mice is inhibited by the treatment that combines activation of mtp53 with targeting of phosphatidylserine residues on tumor blood vessels. We contend therefore that our findings strongly support the use of combination treatment involving mtp53 activation and immunotherapy in patients with TNBC.

Abstract 3568: Phosphatidylserine targeting antibody enhances antitumor activity of CAR T cells in mouse melanoma

Abstract A viable strategy to treat advanced cancers includes transferring of tumor-specific T cells. T cells that recognize tumor antigens can be expanded and reinvigorated ex-vivo. Furthermore, autologous T cells can be genetically modified to express antitumor T cell receptors or chimeric antigen receptors (CARs). Although the potency and specificity of tumor-specific T cells can be manipulated ex vivo, once reinfused into patients, the T cells are subjected to immunosuppressive mechanisms established by the tumor. An important immune checkpoint regulator within tumors is phosphatidylserine (PS), a phospholipid that is exposed on apoptotic cells, tumor cells and tumor endothelium. Innate immune cells exposed to PS secrete suppressive cytokines and chemokines that can significantly impair the function and activation of antitumor T cells. Antibodies that target PS have been shown to reactivate antitumor immunity by polarizing tumor-associated macrophages into a proinflammatory M1 phenotype, reducing the number of MDSCs in tumors and promoting the maturation of dendritic cells into functional APCs. Our lab has previously shown that a PS targeting monoclonal antibody (mch1N11), in combination with transgenic CD4+ T cells that recognize the melanoma antigen Trp1, can regress very advanced melanomas in all treated mice. Here, we further those studies with data showing that a 2nd-generation CAR T cell that binds Trp1 on the surface of B16 melanoma, in combination with mch1N11 can improve antitumor activity and overall survival in B16 tumor-bearing mice. Additionally, in vitro killing assays with antigen-specific T cells sorted from the tumor reveal that mch1N11 enhances the cytolytic function of these T cells against B16 melanoma. Flow cytometry analysis of local immune responses in the tumors of animals treated with tumor-specific T cells and mch1N11 showed a decrease in anti-inflammatory (M2) macrophages and FoxP3+ regulatory T cells. These findings highlight that diminishing suppressive mechanisms locally with mch1N11 can enhance the efficacy of transgenic TCR and CAR T cells to improve the outcome in patients with advanced-stage melanoma. Our studies may inform the design of clinical trials combining PS targeting antibodies with CAR T cell therapy in solid tumors. Citation Format: Sara Schad, Daniel Hirschhorn-Cymerman, Sadna Budhu, Hong Zhong, Xia Yang, Joseph Shan, Steven King, Taha Merghoub, Jedd Wolchok. Phosphatidylserine targeting antibody enhances antitumor activity of CAR T cells in mouse melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3568.

Abstract 2767: Phosphatidylserine targeting and radiation improves survival in a mouse tumor model resistant to checkpoint blockade

Abstract Background: Despite the excitement surrounding checkpoint blockade, many tumors remain resistant thus there is a need to sensitize tumors to immunotherapy and augment tumor rejection. Phosphatidylserine (PS) is a phospholipid expressed on the outer surface of apoptotic cells and a variety of immune cell types. PS signaling polarizes macrophages to the M2 phenotype, inhibits TLR signaling and is upregulated on the surface of tumor cells by radiotherapy (RT). RT also promotes infiltration of CD8 T-cells to the tumor microenvironment. Accordingly, RT may improve CD8 T-cell mediated tumor rejection in the context of checkpoint blockade and sensitize tumors to PS antagonism. We hypothesize PS-targeting antibody, mch1n11, in combination with local RT, may be an effective adjuvant or alternative treatment in patients with tumors resistant to checkpoint blockade such as anti-PD-1 (aPD-1). Furthermore, we hypothesize the combination of aPD-1 with mch1n11 and RT will synergize to improve the effector function of CD8 T-cells and enhance tumor elimination. Methods: B16-F10 murine melanoma was injected into the hind limb of C57BL/6 mice. One cohort received triple combination therapy comprised of aPD-1, mch1n11, and RT given simultaneously at an early time point. Another cohort that showed continued tumor growth after therapy with aPD-1 was used to model checkpoint blockade resistance. These mice received delayed treatment with intra-peritoneal mch1n11 in combination with a one-time 15 Gy RT dose targeted to the tumor-bearing hind limb. The aPD-1 resistant cohort was subdivided into two groups: in one cohort aPD-1 was administered prior to and continued with mch1n11+RT therapy. In the other cohort, aPD-1 was discontinued prior to mch1n11+RT administration. Untreated mice, mice treated with aPD-1 alone, mice treated with isotype antibodies, and mice treated without initial aPD-1 therapy that received delayed mch1n11+RT therapy served as controls. Results: Early triple combination therapy results in almost complete tumor elimination and leads to statistically significant prolonged survival. In the aPD-1 resistant cohort, both prior and continuous aPD-1 treatment with mch1n11+RT improved survival compared to all four controls. There was no significant difference in tumor size or survival between the adjuvant and alternative experimental groups. Experiments are ongoing to characterize immune infiltrates of tumors and the individual effects of aPD-1, RT and mch1n11. Conclusion: This preclinical model suggests a phosphatidylserine targeting antibody combined with single dose RT presents an alternative or adjuvant therapy for tumors resistant to checkpoint blockade. The data from this preclinical model will be used to develop a clinical trial for patients with tumors resistant to checkpoint blockade. Citation Format: Rachel Giese, Sadna Budhu, Christopher Barker, Aditi Gupta, Steve King, Joseph Shan, Jedd Wolchok, Taha Merghoub. Phosphatidylserine targeting and radiation improves survival in a mouse tumor model resistant to checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2767.

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, some tumor cells and tumor endothelium. It has been shown to promote immunosuppressive signals in the tumor microenvironment. 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. In this study, we show that irradiation 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 immune responses in the tumors of treated animals revealed a shift towards a pro-inflammatory M1 phenotype after treatment with RT and mch1N11. In addition, analysis of the adaptive immune response revealed an increase in antigen-specific CD8 T cell infiltration in the tumors as well as increased activation, effector function and differentiation in the triple combination therapy. This finding highlights the potential of combining these agents to improve outcome in patients with advanced-stage cancer and may inform the design of future clinical trials.

A combination of p53-activating APR-246 and phosphatidylserine-targeting antibody potently inhibits tumor development in hormone-dependent mutant p53-expressing breast cancer xenografts.

BackgroundBetween 30 and 40% of human breast cancers express a defective tumor suppressor p53 gene. Wild-type p53 tumor suppressor protein promotes cell-cycle arrest and apoptosis and inhibits vascular endothelial growth factor–dependent angiogenesis, whereas mutant p53 protein (mtp53) lacks these functions, resulting in tumor cell survival and metastasis. Restoration of p53 function is therefore a promising drug-targeted strategy for combating mtp53-expressing breast cancer.MethodsIn this study, we sought to determine whether administration of APR-246, a small-molecule drug that restores p53 function, in combination with 2aG4, an antibody that targets phosphatidylserine residues on tumor blood vessels and disrupts tumor vasculature, effectively inhibits advanced hormone-dependent breast cancer tumor growth.ResultsAPR-246 reduced cell viability in mtp53-expressing BT-474 and T47-D human breast cancer cells in vitro, and significantly induced apoptosis in a dose-dependent manner. However, APR-246 did not reduce cell viability in MCF-7 breast cancer cells, which express wild-type p53. We next examined APR-246’s anti-tumor effects in vivo using BT-474 and T47-D tumor xenografts established in female nude mice. Tumor-bearing mice were treated with APR-246 and/or 2aG4 and tumor volume followed over time. Tumor growth was more effectively suppressed by combination treatment than by either agent alone, and combination therapy completely eradicated some tumors. Immunohistochemistry analysis of tumor tissue sections demonstrated that combination therapy more effectively induced apoptosis and reduced cell proliferation in tumor xenografts than either agent alone. Importantly, combination therapy dramatically reduced the density of blood vessels, which serve as the major route for tumor metastasis, in tumor xenografts compared with either agent alone.ConclusionBased on our findings, we contend that breast tumor growth might effectively be controlled by simultaneous targeting of mtp53 protein and tumor blood vessels in mtp53-expressing cancers.

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.

Phosphatidylserine: A cancer cell targeting biomarker.

Cancer is a leading cause of mortality and morbidity globally. Many prominent cancer-associated molecules have been identified over the recent years which include EGFR, CD44, TGFbRII, HER2, miR-497, NMP22, BTA, Fibrin/FDP etc. These biomarkers are often used for screening, detection, diagnosis, prognosis, prediction and monitoring of cancer development. Phosphatidylserine (PS) is an essential component in all human cells which is present on the inner leaflet of the cell membrane. The oxidative stress causes exposure of PS on the surface of the vascular endothelium in the cancer cells (lung, breast, pancreatic, bladder, skin, brain metastasis, rectal adenocarcinoma etc.) but not on the normal cells. The external PS is regulated by calcium-dependent flippase activity. Cancer cell lines with high surface PS have low flippase activity and high intracellular calcium content. Human Annexin-V, PS targeting antibodies (PGN635 and bavituximab and mch1N11), lysosomal protein, phospholipid Saposin C dioleoylphosphatidylserine (SapC-DOPS), peptide-peptoid hybrid PPS1, PS-binding 14-mer peptide (PSBP-6) and hexapeptide (E3) have been reported to target PS present on cancer cell surface. High expression of CD47 inhibits tumor cell phagocytosis by macrophages. The PS cancer biomarker has also been used to target the drugs to cancer cells specifically without affecting other healthy cells. Currently, the fusion protein (FP) consisting of L-methionase linked to human Annexin-V has been reported to target the cancer cells. The FP catalyzes the conversion of non-toxic prodrug selenomethionine into toxic methyl selenol which thus also prevents the methionine (essential amino acid) supplementation to the cancer cells.

Abstract 3652: Combinational activity of LAG3 and PD-1 targeted therapies is significantly enhanced by the addition of phosphatidylserine targeting antibodies and establishes an anti-tumor memory response in murine triple negative breast cancer

Abstract Previous studies utilizing NanoString immune profile analysis demonstrated that intratumoral levels of LAG3 (lymphocyte activation gene 3) mRNA increased in response to phosphatidylserine (PS) and PD-1 targeting antibodies in murine triple negative breast cancers (TNBC). This suggests LAG3 acts to attenuate immune system activation during I/O therapies - and that PD-1 and LAG3 function cooperatively in suppressing immune system activation. Here we show that adding PS targeting antibodies can further enhance the effectiveness of antibodies targeting LAG3 and/or LAG3+PD-1. We first examined expression of LAG3 and PD-1 in the murine TNBC model E0771 and found that tumor associated T-cells (CD4+ and CD8+) have expression of both markers. Mice implanted with TNBC tumors were next treated with antibodies targeting PS, PD-1, and LAG3 alone and in combination with each other. Interestingly, the addition of PS targeting antibodies not only increased the effectiveness anti-PD-1 effectiveness as previously observed, but also enhanced anti-LAG3 treatment, showing that PS targeting antibodies are capable of augmenting additional I/O therapeutic regimens. Comparison of anti-PD-1+LAG3 combination vs. single anti-PD-1 or anti-LAG3 treatments showed moderately more anti-tumor activity than single treatments, however the addition of PS targeting antibodies to either checkpoint inhibitor was as equally effective in inhibiting tumor growth as observed in the anti-LAG3+PD-1 treatment. Further comparison of antibody treatments targeting PD-1+LAG3 vs. PS+PD-1+LAG3 demonstrated that the addition of PS targeting antibodies resulted in a significant decrease in tumor growth with complete tumor regression in 80% of the animals (along with the ability to completely reject secondary TNBC challenge) compared to 0% in the anti-PD-1+LAG3 treatment group. Immunoprofiling showed that the addition of PS targeting antibodies to these checkpoint therapies, including the combination of anti-PD-1+LAG3, resulted in a phenotype associated with enhanced immune system activation and immune-surveillance including increased tumor infiltrating lymphocytes (TILs) with upregulation of T-cell associated activation pathways, increased Th1 to Th2 profile, and enhanced antigen presentation processing /presentation mechanisms along with cytokines associated with immune system activation. Overall our data demonstrate that adding PS targeting antibodies to clinically relevant therapies, including PD-1 and LAG3, may significantly enhance their ability to activate and redirect the host immune system into recognition and elimination of tumor cells compared to single and combinational treatments that lack PS targeting antibodies. Citation Format: Michael J. Gray, Jian Gong, Jeff T. Hutchins, Bruce D. Freimark. Combinational activity of LAG3 and PD-1 targeted therapies is significantly enhanced by the addition of phosphatidylserine targeting antibodies and establishes an anti-tumor memory response in murine triple negative 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 3652. doi:10.1158/1538-7445.AM2017-3652

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

Preclinical Evaluation of Sequential Combination of Oncolytic Adenovirus Delta-24-RGD and Phosphatidylserine-Targeting Antibody in Pancreatic Ductal Adenocarcinoma.

Delta-24-RGD (DNX-2401) is a conditional replication-competent oncolytic virus engineered to preferentially replicate in and lyse tumor cells with abnormality of p16/RB/E2F pathway. In a phase I clinical trial, Delta-24-RGD has shown favorable safety profile and promising clinical efficacy in brain tumor, which prompted us to evaluate its anticancer activity in pancreatic ductal adenocarcinoma (PDAC), which also has high frequency of homozygous deletion and promoter methylation of CDKN2A encoding the p16 protein. Our results demonstrate that Delta-24-RGD can induce dramatic cytotoxicity in a subset of PDAC cell lines with high cyclin D1 expression. Induction of autophagy and apoptosis by Delta-24-RGD in sensitive PDAC cells was confirmed with LC3B-GFP autophagy reporter and acridine orange staining as well as Western blotting analysis of LC3B-II expression. Notably, we found that Delta-24-RGD induced phosphatidylserine exposure in infected cells independent of cells' sensitivity to Delta-24-RGD, which renders a rationale for combination of Delta-24-RGD viral therapy and phosphatidylserine targeting antibody for PDAC. In a mouse PDAC model derived from a liver metastatic pancreatic cancer cell line, Delta-24-RGD significantly inhibited tumor growth compared with control (P < 0.001), and combination of phosphatidylserine targeting antibody 1N11 further enhanced its anticancer activity (P < 0.01) possibly through inducing synergistic anticancer immune responses. Given that these 2 agents are currently in clinical evaluation, our study warrants further clinical evaluation of this novel combination strategy in pancreatic cancer therapy. Mol Cancer Ther; 16(4); 662-70. ©2016 AACR.

Abstract P2-04-12: Targeting of phosphatidylserine by monoclonal antibodies enhances the activity of immune checkpoint lag3 targeting antibodies in murine breast tumors

Abstract Previous preclinical modeling has demonstrated that antibodies targeting the programmed cell death ligand 1 (PD-1) have activity in murine breast cancers (BC), and this activity is significantly enhanced when combined with phosphatidylserine (PS) targeting antibodies. This suggested that the addition of PS targeting antibodies could be capable of augmenting anti-tumorigenic properties of other checkpoint inhibitors and alternative immune activating therapies in BC. The ability of PS targeting antibodies to enhance the activity of anti-PD-1 therapy occurs in part through increasing tumor infiltrating lymphocytes (TILs), boosting the Th1 immuno-profile, and the suppression of tumor promoting cytokines induced by anti-PD-1 therapy. PS normally resides in the inner leaflet of the plasma membrane in many types of cells. Conditions that incite cellular stress in the tumor microenvironment, such as ROS, hypoxia, and irradiation, promote PS externalization and exposure on tumor associated endothelial cells and tumor cells, where it is recognized by specific receptors, including members of TIM and TAM family. This PS recognition promotes an innate system driven immunosuppressive condition in part by promoting the recruitment of myeloid derived suppressor cells (MDSCs), M2-like macrophages, and suppressing dendritic cells maturation, while inducing anti-inflammatory cytokines. To identify additional immuno-therapeutic targets that may have activity when used in combination with PS targeting antibodies, we employed bioinformatics analysis on tumors from murine triple negative breast cancer (TNBC) treated with PS and PD-1 targeting antibodies, alone or in combination. Interestingly, 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 this hypothesis, immune competent mice harboring the murine TNBC line E0771 were treated with a PS targeting antibody or a LAG3 targeting antibody as single agent or in combination. Our data demonstrate that while PS-blocking and anti-LAG3 therapies each have efficacy in E0771 as single agents, combinational treatment significantly improved growth inhibition and was capable of increasing TILs, including CD8 + and CD3 + T-cells, while reducing the population of MDSCs. Overall, our data suggest that LAG3 targeting may also represent a viable option for the treatment of breast cancer and that the addition of PS targeting antibodies to LAG3 therapy can effectively increase the anti-tumor and immune-activating effects mediated by additional T-cell checkpoint therapies. Citation Format: Gray MJ, Gong J, Hutchins JT, Freimark BD. Targeting of phosphatidylserine by monoclonal antibodies enhances the activity of immune checkpoint lag3 targeting antibodies in murine breast tumors [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-12.

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.