Yvonne Kienast

Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling

Angiopoietin-2 (ANG-2) is a key regulator of angiogenesis that exerts context-dependent effects on ECs. ANG-2 binds the endothelial-specific receptor tyrosine kinase 2 (TIE2) and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling the responsiveness of ECs to exogenous cytokines. Recent data from tumors indicate that under certain conditions ANG-2 can also promote angiogenesis. However, the molecular mechanisms of dual ANG-2 functions are poorly understood. Here, we identify a model for the opposing roles of ANG-2 in angiogenesis. We found that angiogenesis-activated endothelium harbored a subpopulation of TIE2-negative ECs (TIE2lo). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2lo ECs, subsequently inducing, in a TIE2-independent manner, phosphorylation of the integrin adaptor protein FAK, resulting in RAC1 activation, migration, and sprouting angiogenesis. Correspondingly, in vivo ANG-2 blockade interfered with integrin signaling and inhibited FAK phosphorylation and sprouting angiogenesis of TIE2lo ECs. These data establish a contextual model whereby differential TIE2 and integrin expression, binding, and activation control the role of ANG-2 in angiogenesis. The results of this study have immediate translational implications for the therapeutic exploitation of angiopoietin signaling.

Ang-2-VEGF-A CrossMab, a Novel Bispecific Human IgG1 Antibody Blocking VEGF-A and Ang-2 Functions Simultaneously, Mediates Potent Antitumor, Antiangiogenic, and Antimetastatic Efficacy

Purpose: VEGF-A blockade has been clinically validated as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of tumor angiogenesis and metastasis. Experimental Design: We have applied the recently developed CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on bevacizumab (Avastin), and the other arm recognizing Ang-2 based on LC06, an Ang-2 selective human IgG1 antibody. The potency of Ang-2-VEGF CrossMab was evaluated alone and in combination with chemotherapy using orthotopic and subcutaneous xenotransplantations, along with metastasis analysis by quantitative real-time Alu-PCR and ex vivo evaluation of vessels, hypoxia, proliferation, and apoptosis. The mechanism of action was further elucidated using Western blotting and ELISA assays. Results: Ang-2-VEGF-A CrossMab showed potent tumor growth inhibition in a panel of orthotopic and subcutaneous syngeneic mouse tumors and patient or cell line-derived human tumor xenografts, especially at later stages of tumor development. Ang-2-VEGF-A CrossMab treatment led to a strong inhibition of angiogenesis and an enhanced vessel maturation phenotype. Neoadjuvant combination with chemotherapy resulted in complete tumor regression in primary tumor-bearing Ang-2-VEGF-A CrossMab-treated mice. In contrast to Ang-1 inhibition, anti-Ang-2-VEGF-A treatment did not aggravate the adverse effect of anti-VEGF treatment on physiologic vessels. Moreover, treatment with Ang-2-VEGF-A CrossMab resulted in inhibition of hematogenous spread of tumor cells to other organs and reduced micrometastatic growth in the adjuvant setting. Conclusion: These data establish Ang-2-VEGF-A CrossMab as a promising antitumor, antiangiogenic, and antimetastatic agent for the treatment of cancer. Clin Cancer Res; 19(24); 6730–40. ©2013 AACR.

Dual angiopoietin-2 and VEGFA inhibition elicits antitumor immunity that is enhanced by PD-1 checkpoint blockade

Combination of antiangiogenic treatment and immune checkpoint inhibition improves antitumor immunity. Antitumor attack on two fronts The use of immune checkpoint inhibitors and other immunotherapies for the treatment of cancer is continuing to expand as these drugs demonstrate effectiveness in progressively more cancer types and therapeutic contexts. At the same time, the drugs are not perfect, and not all patients respond to them, so a key subject of research in this field is determining optimal ways to combine immune checkpoint therapies with other cancer treatments. Schmittnaegel et al. and Allen et al. focused their studies on the combination of antiangiogenic treatments with checkpoint inhibitors. The authors demonstrated how inhibition of tumor angiogenesis can facilitate the access of cytotoxic T cells to tumors, while the checkpoint inhibitors protect these T cells from exhaustion, enhancing their antitumor effects. Pathological angiogenesis is a hallmark of cancer and a therapeutic target. Vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2; also known as ANG2) are proangiogenic cytokines that sustain tumor angiogenesis and limit antitumor immunity. We show that combined ANGPT2 and VEGFA blockade by a bispecific antibody (A2V) provided superior therapeutic benefits, as compared to the single agents, in both genetically engineered and transplant tumor models, including metastatic breast cancer (MMTV-PyMT), pancreatic neuroendocrine tumor (RIP1-Tag2), and melanoma. Mechanistically, A2V promoted vascular regression, tumor necrosis, and antigen presentation by intratumoral phagocytes. A2V also normalized the remaining blood vessels and facilitated the extravasation and perivascular accumulation of activated, interferon-γ (IFNγ)–expressing CD8+ cytotoxic T lymphocytes (CTLs). Whereas the antitumoral activity of A2V was, at least partly, CTL-dependent, perivascular T cells concurrently up-regulated the expression of the immune checkpoint ligand programmed cell death ligand 1 (PD-L1) in tumor endothelial cells. IFNγ neutralization blunted this adaptive response, and PD-1 blockade improved tumor control by A2V in different cancer models. These findings position immune cells as key effectors of antiangiogenic therapy and support the rationale for cotargeting angiogenesis and immune checkpoints in cancer therapy.

A Novel Angiopoietin-2 Selective Fully Human Antibody with Potent Anti-Tumoral and Anti-Angiogenic Efficacy and Superior Side Effect Profile Compared to Pan-Angiopoietin-1/-2 Inhibitors

There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.

Rapid activation of tumor-associated macrophages boosts preexisting tumor immunity

Depletion of immunosuppressive tumor-associated macrophages (TAMs) or reprogramming toward a proinflammatory activation state represent different strategies to therapeutically target this abundant myeloid population. In this study, we report that inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling sensitizes TAMs to profound and rapid reprogramming in the presence of a CD40 agonist before their depletion. Despite the short-lived nature of macrophage hyperactivation, combined CSF-1R+CD40 stimulation of macrophages is sufficient to create a proinflammatory tumor milieu that reinvigorates an effective T cell response in transplanted tumors that are either responsive or insensitive to immune checkpoint blockade. The central role of macrophages in regulating preexisting immunity is substantiated by depletion experiments, transcriptome analysis of ex vivo sorted TAMs, and gene expression profiling of whole tumor lysates at an early treatment time point. This approach enabled the identification of specific combination-induced changes among the pleiotropic activation spectrum of the CD40 agonist. In patients, CD40 expression on human TAMs was detected in mesothelioma and colorectal adenocarcinoma.

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.

Arid1a regulates response to anti-angiogenic therapy in advanced hepatocellular carcinoma

BACKGROUND & AIMS AT-rich interaction domain 1a (Arid1a), a component of the chromatin remodeling complex, has emerged as a tumor suppressor gene. It is frequently mutated in hepatocellular carcinoma (HCC). However, it remains unknown how Arid1a suppresses HCC development and whether Arid1a deficiency could be exploited for therapy, we aimed to explore these questions. METHODS The expression of Arid1a in human and mouse HCCs was determined by immunohistochemical (IHC) staining. Gene expression was determined by quantitative PCR, ELISA or western blotting. Arid1a knockdown HCC cell lines were established by lentiviral-based shRNA. Tumor angiogenesis was quantified based on vessel density. The regulation of angiopoietin (Ang2) expression by Arid1a was identified by chromatin immunoprecipitation (ChIP) assay. The tumor promoting function of Arid1a loss was studied with a xenograft model in nude mice and diethylnitrosamine (DEN)-induced HCC in Arid1a conditional knockout mice. The therapeutic values of Ang2 antibody and sorafenib treatment were evaluated both in vitro and in vivo. RESULTS We demonstrate that Arid1a deficiency, occurring in advanced human HCCs, is associated with increased vessel density. Mechanistically, loss of Arid1a causes aberrant histone H3K27ac deposition at the angiopoietin-2 (Ang2) enhancer and promoter, which eventually leads to ectopic expression of Ang2 and promotes HCC development. Ang2 blockade in Arid1a-deficient HCCs significantly reduces vessel density and tumor progression. Importantly, sorafenib treatment, which suppresses H3K27 acetylation and Ang2 expression, profoundly halts the progression of Arid1a-deficient HCCs. CONCLUSIONS Arid1a-deficiency activates Ang2-dependent angiogenesis and promotes HCC progression. Loss of Arid1a in HCCs confers sensitivity to Ang2 blockade and sorafenib treatment. LAY SUMMARY AT-rich interaction domain 1a (Arid1a), is a tumor suppressor gene. Arid1a-deficiency promotes Ang2-dependent angiogenesis leading to hepatocellular carcinoma progression. Arid1a-deficiency also sensitizes tumors to Ang2 blockade by sorafenib treatment.

Impact of selective anti‐BMP9 treatment on tumor cells and tumor angiogenesis

The role of bone morphogenic protein 9 (BMP9) signaling in angiogenesis has been controversial, with a number of studies showing that it acts either as a pro‐angiogenic or, conversely, as an anti‐angiogenic factor in a context‐dependent manner. Notably, BMP9 was also reported to function in both pro‐ or anti‐tumorigenic roles during tumor progression. It has therefore remained unclear, whether selective BMP9 inhibition is a useful target for antibody therapy of cancer. To shed light on these questions, we characterized BMP9 expression in plasma of patients with different cancer indications and found elevated levels of pro‐domains and precursor BMP9 with a strong response in renal cell carcinoma (RCC). These studies prompted us to evaluate the potential of selective anti‐BMP9 cancer therapy in RCC. We generated a novel monoclonal therapeutic antibody candidate, mAb BMP9‐0093, that selectively targets all different BMP9 variants but does not bind to the closest homolog BMP10. In vitro, mAb BMP9‐0093 treatment inhibited signaling, endothelin‐1 (ET‐1) production and spreading of endothelial cells and restored BMP9‐induced decrease in pericyte migration and attachment. Furthermore, BMP9‐mediated epithelial–mesenchymal transition of renal cell carcinoma cells was reversed by mAb BMP9‐0093 treatment in vitro. In vivo, mAb BMP9‐0093 showed significant anti‐tumor activity that was associated with an increase in apoptosis as well as a decrease in tumor cell proliferation and ET‐1 release. Furthermore, mAb BMP9‐0093 induced mural cell coverage of endothelial cells, which was corroborated by a reduction in vascular permeability, demonstrated by a diminished penetration of omalizumab‐Alexa 647 into tumor tissue. Our findings provide new evidence for a better understanding of BMP9 contribution in tumor progression and angiogenesis that may result in the development of effective targeted therapeutic interventions.

Abstract 1803: Combining anti-Ang-2/VEGF-A therapy with radio- and chemotherapy in glioma

Angiogenesis is a biological hallmark of malignant gliomas, but antiangiogenic strategies especially targeting the VEGF axis did not show striking antitumor activities in the majority of patients so far. Other pathways may be more relevant in selected tumor entities or patients. Further, it remains unresolved which antiangiogenic combination regimen with standard radio- and/or chemotherapy is most effective. Therefore, we compared the therapeutic effects of anti-VEGF-A, anti-Ang-2, and bispecific anti-Ang-2/VEGF-A antibodies, alone and in combination with radio- or temozolomide (TMZ) chemotherapy in a malignant glioma model using multi-parameter two-photon in vivo microscopy in mice. We demonstrate that anti-Ang-2/VEGF-A leads to strongest vascular changes including vascular normalization, both as monotherapy and when combined with chemotherapy. The latter combination regimen was accompanied by most effective chemotherapy-induced death of cancer cells independent of blood vessel proximity, indicative of a better distribution of TMZ throughout the tumor. Furthermore, the combination of anti-Ang-2/VEGF-A plus TMZ consistently resulted in decreased tumor cell motility, and decreased formation of resistance-associated tumor microtubes (TMs), which finally lead to best tumor growth inhibition. Remarkably, all these parameters were just reverted when radiotherapy was chosen as combination partner. In contrast, when anti-VEGF-A was combined with radiotherapy, vascular normalization was highest, and TM length, nuclear motility and tumor growth were concordantly reduced. In conclusion, while TMZ chemotherapy benefits most from combination with anti-Ang-2/VEGF-A, radiotherapy does from anti-VEGF-A. The findings imply that unexpected, even divergent effects can occur when a specific antiangiogenic therapy is added to chemo- or radiotherapy in glioma, and that uninformed combination regimens should be avoided. Note: This abstract was not presented at the meeting. Citation Format: Gergely Solecki, Matthias Osswald, Weber Daniel, Malte Glock, Miriam Ratliff, Hans-Joachim Muller, Oliver Krieter, Yvonne Kienast, Wolfgang Wick, Frank Winkler. Combining anti-Ang-2/VEGF-A therapy with radio- and chemotherapy in glioma [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 1803. doi:10.1158/1538-7445.AM2017-1803

Abstract B86: Ang-2-VEGF-A CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 functions simultaneously, mediates potent antitumor, antiangiogenic, and antimetastatic efficiency

Purpose: VEGF-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of tumor angiogenesis and metastasis. Experimental Design: We have recently described a novel generic method for the production of bivalent bispecific human IgG1 antibodies. Subsequently, we have applied the CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on Bevacizumab, and the other arm recognizing Ang-2 based on LC06, a highly Ang-2 selective human IgG1 antibody. Ang-2-VEGF CrossMab was evaluated in vitro and in vivo using molecular, biochemical, cellular and histological analyses to determine its pharmacologic properties, along with its mechanism of action. Results: Ang-2-VEGF-A CrossMab showed potent tumor growth inhibition in a panel of orthotopic and subcutaneous syngeneic mouse tumors and patient and cell line-derived human tumor xenografts at both early and late stages of tumor development. Furthermore, a strong inhibition of angiogenesis and an enhanced vessel normalization phenotype were observed. In contrast to Ang-1 inhibition, anti-Ang-2-VEGF-A treatment did not aggravate the adverse effect of anti-VEGF treatment on physiological vessels. Moreover, treatment with Ang-2-VEGF-A CrossMab resulted in inhibition of hematogenous spread of tumor cells to other organs and reduced micrometastatic growth in the adjuvant setting. Conclusion: Our data indicate that Ang-2-VEGF-A CrossMab mediates potent anti-tumor, anti-angiogenic, and anti-metastatic efficacy and represents a promising therapeutic agent for the therapy of cancer. Citation Format: Erica Lorenzon, Yvonne Kienast, Romi Raemsch, Werner Scheuer, Frank Herting, Dirk Bernicke, Wolfgang Schaefer, Joerg Regula, Kay Stubenrauch, Sidney Yu, Huynh Hung The, Laurent Martarello, Christian Klein, Markus Thomas. Ang-2-VEGF-A CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 functions simultaneously, mediates potent antitumor, antiangiogenic, and antimetastatic efficiency. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B86.