Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy

Edwin L. Steele Laboratory of Tumor Biology, Department of Radiation Oncology and Vaccine and Immunotherapy Center, Infectious Diseases Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 10/2012; 109(43):17561-17566. DOI: 10.1073/pnas.1215397109


The recent approval of a prostate cancer vaccine has renewed hope for anticancer immunotherapies. However, the immunosuppressive
tumor microenvironment may limit the effectiveness of current immunotherapies. Antiangiogenic agents have the potential to
modulate the tumor microenvironment and improve immunotherapy, but they often are used at high doses in the clinic to prune
tumor vessels and paradoxically may compromise various therapies. Here, we demonstrate that targeting tumor vasculature with
lower vascular-normalizing doses, but not high antivascular/antiangiogenic doses, of an anti-VEGF receptor 2 (VEGFR2) antibody
results in a more homogeneous distribution of functional tumor vessels. Furthermore, lower doses are superior to the high
doses in polarizing tumor-associated macrophages from an immune inhibitory M2-like phenotype toward an immune stimulatory
M1-like phenotype and in facilitating CD4+ and CD8+ T-cell tumor infiltration. Based on this mechanism, scheduling lower-dose anti-VEGFR2 therapy with T-cell activation induced
by a whole cancer cell vaccine therapy enhanced anticancer efficacy in a CD8+ T-cell–dependent manner in both immune-tolerant and immunogenic murine breast cancer models. These findings indicate that
vascular-normalizing lower doses of anti-VEGFR2 antibody can reprogram the tumor microenvironment away from immunosuppression
toward potentiation of cancer vaccine therapies. Given that the combinations of high doses of bevacizumab with chemotherapy
have not improved overall survival of breast cancer patients, our study suggests a strategy to use antiangiogenic agents in
breast cancer more effectively with active immunotherapy and potentially other anticancer therapies.

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    • "Given that hypoxia and reoxygenation cycles are strong stimuli for neoangiogenesis that drive therapeutic resistance and metastasis ( Dewhirst et al , 2008 ; De Bock et al , 2011 ) , our finding of increased cell death at the expense of tumour hypoxia suggests that dual inhibition could offer a therapeutic strategy to reduce these risks . Equally , the increased stability and perfusion of the residual tumour vessels in the surviving and well - oxygenated tumour areas should not only benefit the penetration of cytostatic drugs , but also lower the frequency of metastatic dissemination ( Holopainen et al , 2012 ) , and create a more supportive tumour microenvironment for immuno - or chemother - apy ( Huang et al , 2012 ) . When endothelial cells regress after VEGF inhibition , they leave empty vascular BMS that are durable and not degraded by macrophages and persist long after the blood vessels have disappeared ( Inai et al , 2004 ) . "
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    ABSTRACT: Background: Effective vascular normalisation following vascular endothelial growth factor (VEGF) inhibition is associated with endothelial cell regression leaving empty basement membrane sleeves (BMS). These long-lived BMS permit the rapid regrowth of tumour vasculature upon treatment cessation and promote resistance to VEGF-targeting drugs. Previous attempts at removing BMS have failed. Angiopoietin-2 (Ang2) is a vascular destabilizing factor that antagonises normalisation. We hypothesised that Ang2 inhibition could permit vascular normalisation at significantly reduced doses of VEGF inhibition, avoiding excessive vessel regression and the formation of empty BMS. Methods: Mice xenografted with human colorectal cancer cells (LS174T) were treated with low (0.5 mg kg(-1)) or high (5 mg kg(-1)) doses of the VEGF-targeting antibody bevacizumab with or without an Ang2 blocking peptibody L1-10. Tumour growth, BMS formation and normalisation parameters were examined including vessel density, pericyte coverage, adherence junctions, leakiness, perfusion, hypoxia and proliferation. Results: Dual targeting of VEGF and Ang2 achieved effective normalisation at only one-tenth of the dose required with bevacizumab alone. Pericyte coverage, vascular integrity, adherence junctions and perfusion as prerequisites for improved access of chemotherapy were improved without inducing empty BMS that facilitate rapid vascular regrowth. Conclusions: Dual targeting of VEGF and Ang2 can potentiate the effectiveness of VEGF inhibitors and avoid the formation of empty BMS.
    British Journal of Cancer 01/2015; 112(3). DOI:10.1038/bjc.2014.629 · 4.84 Impact Factor
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    • "Administration of low-doses of anti-VEGFR2 antibody results in a transient vascular normalization and improves the CD4+ and CD8+ tumor infiltration. Association of low-doses of anti-VEGFR2 with whole cancer cell vaccine induces anti-tumor efficacy (94). "
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    ABSTRACT: The progressive conversion of normal cells into cancer cells is characterized by the acquisition of eight hallmarks. Among these criteria, the capability of the cancer cell to avoid the immune destruction has been noted. Thus, tumors develop mechanisms to become invisible to the immune system, such as the induction of immunosuppressive cells, which are able to inhibit the development of an efficient immune response. Molecules produced in the tumor microenvironment are involved in the occurrence of an immunosuppressive microenvironment. Recently, it has been shown that vascular endothelial growth factor A (VEGF-A) exhibits immunosuppressive properties in addition to its pro-angiogenic activities. VEGF-A can induce the accumulation of immature dendritic cells, myeloid-derived suppressor cells, regulatory T cells, and inhibit the migration of T lymphocytes to the tumor. Other pro-angiogenic factors such as placental growth factor (PlGF) could also participate in tumor-induced immunosuppression, but only few works have been performed on this point. Here, we review the impact of pro-angiogenic factors (especially VEGF-A) on immune cells. Anti-angiogenic molecules, which target VEGF-A/VEGFR axis, have been developed in the last decades and are commonly used to treat cancer patients. These drugs have anti-angiogenic properties but can also counteract the tumor-induced immunosuppression. Based on these immunomodulatory properties, anti-angiogenic molecules could be efficiently associated with immunotherapeutic strategies in preclinical models. These combinations are currently under investigation in cancer patients.
    Frontiers in Oncology 04/2014; 4:70. DOI:10.3389/fonc.2014.00070
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    • "Single cell suspensions were prepared from spleens. Cells were plated in round-bottomed 96-well plates, pulsed with a validated CD8+ T-cell Her2/neu peptide (PDSLRDLSVF, 1 μg/ml; EZBiolab) [25,43], an in-house designed H2d-restricted MSLN Ld1 peptide (IPLSYLCDF, 1 μg/ml; EZBiolab) that did not induce ovarian cancer specific T-cell response in H-2q FVB mice, or medium alone for 72 hours when Golgi Plug (BD Bioscience) was added for the last 5 hours as previously described [44], and then stained with fluorophore-conjugated anti-CD3, anti-CD4, anti-CD8, anti-IFNγ (BD Pharmingen), and anti-Granzyme B (eBioscience) antibodies. Cells were then analyzed on a LSRII 4 laser (BD Biosciences). "
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    ABSTRACT: Although dendritic cell (DC) vaccines are considered to be promising treatments for advanced cancer, their production and administration is costly and labor-intensive. We developed a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting mesothelin (MSLN), which is overexpressed on ovarian cancer and mesothelioma cells, to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70), which is a potent immune activator that stimulates monocytes and DCs, enhances DC aggregation and maturation and improves cross-priming of T cells mediated by DCs. Binding of this fusion protein with MSLN on the surface of tumor cells was measured by flow cytometry and fluorescence microscopy. The therapeutic efficacy of this fusion protein was evaluated in syngeneic and orthotopic mouse models of papillary ovarian cancer and malignant mesothelioma. Mice received 4 intraperitoneal (ip) treatments with experimental or control proteins post ip injection of tumor cells. Ascites-free and overall survival time was measured. For the investigation of anti-tumor T-cell responses, a time-matched study was performed. Splenocytes were stimulated with peptides, and IFNgamma- or Granzyme B- generating CD3+CD8+ T cells were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect, we performed in vivo CD8+ cell depletion. We further determined if the fusion protein increases DC maturation and improves antigen presentation as well as cross-presentation by DCs. We demonstrated in vitro that the scFvMTBHsp70 fusion protein bound to the tumor cells used in this study through the interaction of scFv with MSLN on the surface of these cells, and induced maturation of bone marrow-derived DCs Use of this bifunctional fusion protein in both mouse models significantly enhanced survival and slowed tumor growth while augmenting tumor-specific CD8+ T-cell dependent immune responses. We also demonstrated in vitro and in vivo that the fusion protein enhanced antigen presentation and cross-presentation by targeting tumor antigens towards DCs. This new cancer immunotherapy has the potential to be cost-effective and broadly applicable to tumors that overexpress mesothelin.
    Journal of Hematology & Oncology 02/2014; 7(1):15. DOI:10.1186/1756-8722-7-15 · 4.81 Impact Factor
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