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.

The stilbene phytochemicals resveratrol and piceatannol have been reported to possess substantial antitumorigenic and antileukemic activities, respectively. Although recent experimental data revealed the proapoptotic potency of resveratrol, the molecular mechanisms underlying the antileukemic activity have not yet been studied in detail. In the present study, we show that resveratrol, as well as the hydroxylated analog piceatannol, are potent inducers of apoptotic cell death in BJAB Burkittlike lymphoma cells with an ED 50 concentration of 25 M. Further experiments revealed that treatment of BJAB cells with both substances led to a concentration-dependent activation of caspase-3 and mitochondrial permeability transition. Using BJAB cells overexpressing a dominant-negative mutant of the Fas-associated death domain (FADD) adaptor protein to block death receptor-mediated apoptosis, we demonstrate that resveratrol-and piceatannol-induced cell death in these cells is independent of the CD95/Fas signaling pathway. To explore the antileukemic properties of both compounds in more detail, we extended our study to primary, leukemic lymphoblasts. Interestingly, piceatannol but not resveratrol is a very efficient inducer of apoptosis in this ex vivo assay with leukemic lymphoblasts of 21 patients suffering from childhood lymphoblastic leukemia (ALL). Leukemia (2001Leukemia ( ) 15, 1735Leukemia ( -1742

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-␤ 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 BMP9⅐pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using realtime 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, coreceptor 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, BMP9⅐pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and prodomains 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.Bone morphogenic protein 9 (BMP9 2 ; also known as growth and differentiation factor 2 (GDF2)), is a member of the transforming growth factor ␤ (TGF␤) superfamily. BMP9 is constitutively expressed in liver and secreted into the circulation at active concentrations (1). Circulating BMP9 is a potent biological effector signaling through type I receptor activin-receptorlike kinase 1 (ALK1) in endothelial cells, thereby maintaining vascular homeostasis (2, 3). BMP9 and ALK1 are required for properly organized blood and lymphatic vascular development (4 -6). Human mutations in ALK1 lead to a genetic vascular disorder known as hereditary hemorrhagic telangiectasia (7). Recently, mutations in BMP9 have been identified in individuals with a vascular disorder phenotypically overlapping with hereditary hemorrhagic telangiectasia (8). BMP9 was also discovered to function as a neurotropic factor, potently inducing and maintaining the cholinergic phenotype in the central nervous system (9), and is also the most potent BMP for inducing osteogenic, and to a lesser extent adipogenic and chondrogenic differentiation (10, 11). Osteogenic signaling requires both ALK1 and the low affinity type I receptor ALK2 (12). Type II receptors activin receptor IIA and IIB (ActRIIA and ActRIIB) and BMP receptor II (BMPRII) have also been implicated in ALK1/BMP9 signaling (13). Moreover, endoglin (ENG) has been identified as a co-receptor that can increase BMP9/ALK1 signaling (3,14). This is reflected in a model where ENG and ALK1 act together to bind...

As density functional calculations suggest, Cr(CO) -complexed benzylic radicals (such as 2) exhibit a significant degree of configurational stablility. This was exploited in an efficient method for the electron transfer mediated transformations of readily available 1-arylalkanol-Cr(CO) derivatives 1 to afford alkylated products 3 in good yields and with a high degree of stereochemical retention.

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