ABSTRACT
By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-ß 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.
Subject(s)
Activin Receptors, Type II/metabolism , Antigens, CD/metabolism , Bone Morphogenetic Protein Receptors, Type II/metabolism , Growth Differentiation Factors/metabolism , Models, Molecular , Receptors, Cell Surface/metabolism , Activin Receptors, Type II/chemistry , Activin Receptors, Type II/genetics , Animals , Antigens, CD/chemistry , Antigens, CD/genetics , Bone Morphogenetic Protein Receptors, Type II/chemistry , Bone Morphogenetic Protein Receptors, Type II/genetics , Cells, Cultured , Dimerization , Endoglin , Female , Growth Differentiation Factor 2/blood , Growth Differentiation Factor 2/isolation & purification , Growth Differentiation Factor 2/metabolism , Growth Differentiation Factors/blood , Growth Differentiation Factors/chemistry , Growth Differentiation Factors/genetics , HEK293 Cells , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice, Inbred BALB C , Peptide Fragments/agonists , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Protein Precursors/blood , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Precursors/metabolism , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Signal Transduction , Specific Pathogen-Free OrganismsABSTRACT
As density functional calculations suggest, Cr(CO)3 -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)3 derivatives 1 to afford alkylated products 3 in good yields and with a high degree of stereochemical retention.
ABSTRACT
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.