Despite substantial progress in lung cancer immunotherapy, the overall response rate in patients with KRAS-mutant lung adenocarcinoma (LUAD) remains low. Combining standard immunotherapy with adjuvant approaches that enhance adaptive immune responsessuch as epigenetic modulation of antitumor immunity-is therefore an attractive strategy. To identify epigenetic regulators of tumor immunity, we constructed an epigenetic-focused single guide RNA library and performed an in vivo CRISPR screen in a Kras G12D / Trp53 −/− LUAD model. Our data showed that loss of the histone chaperone Asf1a in tumor cells sensitizes tumors to anti-PD-1 treatment. Mechanistic studies revealed that tumor cell-intrinsic Asf1a defi ciency induced immunogenic macrophage differentiation in the tumor microenvironment by upregulating GM-CSF expression and potentiated T-cell activation in combination with anti-PD-1. Our results provide a rationale for a novel combination therapy consisting of ASF1A inhibition and anti-PD-1 immunotherapy. SIGNIFICANCE: Using an in vivo epigenetic CRISPR screen, we identifi ed Asf1a as a critical regulator of LUAD sensitivity to anti-PD-1 therapy. Asf1a defi ciency synergized with anti-PD-1 immunotherapy by promoting M1-like macrophage polarization and T-cell activation. Thus, we provide a new immunotherapeutic strategy for this subtype of patients with LUAD.

Purpose: The vascular targeting antibody bavituximab is being combined with chemotherapy in clinical trials in cancer patients. Bavituximab targets the membrane phospholipid, phosphatidylserine, complexed with β2-glycoprotein I. Phosphatidylserine is normally intracellular but becomes exposed on the luminal surface of vascular endothelium in tumors. Phosphatidylserine exposure on tumor vessels is increased by chemotherapy and irradiation. Here, we determined whether treatment with the murine equivalent of bavituximab, 2aG4, combined with irradiation can suppress tumor growth in a rat model of glioblastoma. Experimental Design: F98 glioma cells were injected into the brains of syngeneic rats where they grow initially as a solid tumor and then infiltrate throughout the brain. Rats with established tumors were treated with 10 Gy whole brain irradiation and 2aG4. Results: Combination treatment doubled the median survival time of the rats, and 13% of animals were rendered disease free. Neither treatment given individually was as effective. We identified two mechanisms. First, irradiation induced phosphatidylserine exposure on tumor blood vessels and enhanced antibody-mediated destruction of tumor vasculature by monocytes/macrophages. Second, the antibody treatment induced immunity to F98 tumor cells, which are normally weakly immunogenic. Surviving rats were immune to rechallenge with F98 tumor cells. In vitro, 2aG4 enhanced the ability of dendritic cells (DCs) to generate F98-specific cytotoxic T cells. Phosphatidylserine exposure, which is induced on tumor cells by irradiation, likely suppresses tumor antigen presentation, and 2aG4 blocks this tolerogenic effect. Conclusion: Bavituximab combined with radiotherapy holds promise as a vascular targeting and immune enhancement strategy for the treatment of human glioblastoma. (Clin Cancer Res 2009;15(22):6871-80)

Purpose: New treatment strategies aimed at damaging tumor vasculature could potentially improve tumor response to radiation therapy. We recently showed that anionic phospholipids, principally phosphatidylserine, are specifically exposed on the luminal surface of tumor blood vessels. Here we tested the hypothesis that radiation therapy can increase phosphatidylserine exposure on lung tumor vasculature, thereby enhancing the antitumor properties of the antiphosphatidylserine antibody 2aG4. Experimental Design: The therapeutic efficacy of radiation therapy plus 2aG4 was tested in nude mice bearing radiation-resistant A549 human lung tumors. Radiation-induced phosphatidylserine exposure on endothelial cells and A549 tumor cells was analyzed by immunofluoresence staining. The mechanism of the enhanced antitumor effect was examined by histology and antibody-dependent cell-mediated cytotoxicity experiments. Results: Focal irradiation of A549 human lung cancer xenografts increased the percentage of tumor vessels with exposed phosphatidylserine from 4% to 26%. Treatment of mice bearing A549 tumors with 2aG4 plus focal radiation therapy inhibited tumor growth by 80% and was superior to radiation therapy or 2aG4 alone (P < 0.01). Combination therapy reduced blood vessel density and enhanced monocyte infiltration into the tumor mass beyond that observed with individual treatments. In vitro, 2aG4 enhanced the ability of macrophages to kill endothelial cells with exposed phosphatidylserine in an Fc ¶-dependent manner. Conclusion: These results suggest that 2aG4 enhances the antitumor effects of radiation therapy by increasing antibody-dependent cell-mediated cytotoxicity toward tumor vessels with externalized phosphatidylserine. Bavituximab, a chimeric version of 2aG4 in clinical trials, has the potential to enhance the therapeutic efficacy of radiation therapy in lung cancer patients.

Pancreatic cancer continues to have a dismal prognosis and novel therapy is needed. In this study, we evaluate a promising new target for therapy, phosphatidylserine (PS). PS is an anionic phospholipid located normally on the inner leaflet of the plasma membrane in mammalian cells. In the tumor microenvironment, PS becomes externalized on vascular endothelium. The monoclonal antibody 3G4 binds PS and promotes an inflammatory response against tumor blood vessels, resulting in reduction of tumor growth. Mice with orthotopic pancreatic tumors were treated with 3G4, gemcitabine or a combination of both drugs. Tumor burden including pancreas weight and metastatic lesions (liver, lymph node and peritoneal) were reduced 3-to 5-fold by the combination therapy as compared with 1.5-to 2-fold with 3G4 and gemcitabine alone, respectively. Treatment of tumor-bearing animals with the combination therapy increased macrophage infiltration into the tumor mass 10-fold and reduced microvessel density in the tumor by 2.5-fold compared with tumors from untreated animals. Gemcitabine alone and 3G4 alone were less effective than the combination of the 2 agents together. The additive therapeutic effect of both agents appears to be because chemotherapy increases PS exposure on tumor vascular endothelium and amplifies the target for attack by 3G4. In conclusion, 3G4 enhanced the anti-tumor and anti-metastatic activity of gemcitabine without contributing to toxicity. ' 2005 Wiley-Liss, Inc.

Ongoing clinical trials will provide important information regarding the future development of TRAIL-R agonists.

A promising target on tumor vasculature is phosphatidylserine (PS), an anionic phospholipid that resides exclusively on the inner leaflet of the plasma membrane of resting mammalian cells. We have shown previously that PS becomes exposed on the surface of endothelial cells (EC) in solid tumors. To target PS on tumor vasculature, the murine monoclonal antibody 3G4 was developed. 3G4 localizes to tumor vasculature, inhibits tumor growth, and enhances anti-tumor chemotherapies without toxicity in mice. A chimeric version of 3G4 is in clinical trials. In this study, we investigated the basis for the interaction between 3G4 and EC with surface-exposed PS. We demonstrate that antibody binding to PS is dependent on plasma protein ␤-2-glycoprotein 1 (␤2GP1). ␤2GP1 is a 50-kDa glycoprotein that binds weakly to anionic phospholipids under physiological conditions. We show that 3G4 enhances binding of ␤2GP1 to EC induced to expose PS. We also show that divalent 3G4-␤2GP1 complexes are required for enhanced binding, since 3G4 Fab fragments do not bind EC with exposed PS. Finally, we demonstrate that an artificial dimeric ␤2GP1 construct binds to EC with exposed PS in the absence of 3G4, confirming that antibody binding is mediated by dimerization of ␤2GP1. Together, these data indicate that 3G4 targets tumor EC by increasing the avidity of ␤2GP1 for anionic phospholipids through formation of multivalent 3G4-␤2GP1 complexes.We recently reported the development of a monoclonal antibody, 3G4, that targets anionic phospholipids exposed on the surface of tumor vascular endothelial cells (EC) 2 (1, 2). Phosphatidylserine (PS) is the most abundant anionic phospholipid of the plasma membrane and is considered the primary target of 3G4. It is well established that PS is actively confined to the internal leaflet of the plasma membrane under normal conditions in most cell types (3). PS asymmetry is maintained by an ATP-dependent aminophospholipid translocase that catalyzes the transport of aminophospholipids from the external leaflet to the internal leaflet of the plasma membrane (4). Loss of PS asymmetry results from outward movement of aminophospholipids in response to increased Ca 2ϩ fluxes. This leads to inhibition of the translocase (5) and/or activation of an exporter of PS that transports PS to the outer membrane surface (6). Loss of asymmetry is observed under several physiological and pathological conditions, including apoptosis (7), cell activation (8), cell injury (9), and malignant transformation (10).Conditions in the tumor microenvironment contain a number of factors that may activate and/or injure tumor EC as follows: (a) tumor-derived interleukin-1 and tumor necrosis factor-␣ activate the endothelium and induce expression of cell adhesion molecules (11, 12); (b) reactive oxygen species (ROS) generated by leukocytes that adhere to the tumor endothelium (12); and (c) ROS generated by tumor cells as a by-product of metabolism (11, 13) or as a result of exposure to hypoxia followed by reoxygenation (14). In this regard, we...

We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8+ T cells specifically recognizing the model antigen in a H-2b–restricted manner. The assay was screened with a small molecule library to identify compounds that inhibit or enhance T cell–mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8+ cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting EGFR that sensitized tumor cells to T cell–mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.

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