RESUMO
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.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Antígenos CD/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Modelos Moleculares , Receptores de Superfície Celular/metabolismo , Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/genética , Animais , Antígenos CD/química , Antígenos CD/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Células Cultivadas , Dimerização , Endoglina , Feminino , Fator 2 de Diferenciação de Crescimento/sangue , Fator 2 de Diferenciação de Crescimento/isolamento & purificação , Fator 2 de Diferenciação de Crescimento/metabolismo , Fatores de Diferenciação de Crescimento/sangue , Fatores de Diferenciação de Crescimento/química , Fatores de Diferenciação de Crescimento/genética , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/sangue , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Organismos Livres de Patógenos EspecíficosRESUMO
Despite remarkable progress in applied Surface Plasmon Resonance (SPR)-based methods, concise monitoring of kinetic properties for native biomarkers from patient samples is still lacking. Not only are low concentrations of native targets in patient samples, often in the pM range, a limiting and challenging factor, but body fluids as complex matrices furthermore complicate measurements. The here-described method enables the determination of kinetic constants and resulting affinities for native antigens from patients' cerebrospinal fluid (CSF) and sera binding to antibodies. Using a significantly extended target-enrichment step, we modified a common sandwich-assay protocol, based on a primary and secondary antibody. We successfully analyze antibody kinetics of native targets from a variety of origins, with consistent results, independent of their source. Moreover, native neurofilament light chain (NFL) was investigated as an exemplary biomarker. Obtained data reveal antibodies recognizing recombinant NFL with high affinities, while showing no, or only significantly weakened binding to native NFL. The indicated differences for recombinant vs. native material demonstrate another beneficial application. Our assay is highly suitable for gaining valuable insights into characteristics of native biomarkers, thus impacting on the binder development of diagnostic reagents or pharmaceutical drugs.
Assuntos
Antígenos , Ressonância de Plasmônio de Superfície , Humanos , Ressonância de Plasmônio de Superfície/métodos , Anticorpos , BiomarcadoresRESUMO
Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development.
Assuntos
Terapia de Alvo Molecular/métodos , Receptores Proteína Tirosina Quinases/imunologia , Anticorpos de Cadeia Única/imunologia , Especificidade de Anticorpos , Apoptose/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Ativação Enzimática , Humanos , Engenharia de Proteínas , Receptores Proteína Tirosina Quinases/metabolismo , Anticorpos de Cadeia Única/genéticaRESUMO
The mutation of well behaved enzymes in order to simulate less manageable cognates is the obvious approach to study specific features of the recalcitrant target. Accordingly, the prototypical protein kinase PKA serves as a model for many kinases, including the closely related PKB, an AGC family protein kinase now implicated as oncogenic in several cancers. Two residues that differ between the alpha isoforms of PKA and PKB at the adenine-binding site generate differing shapes of the binding surface and are likely to play a role in ligand selectivity. As the corresponding mutations in PKA, V123A would enlarge the adenine pocket, while L173M would alter both the shape and its electronic character of the adenine-binding surface. We have determined the structures of the corresponding double mutant (PKAB2: PKAalpha V123A, L173M) in apo and MgATP-bound states, and observed structural alterations of a residue not previously involved in ATP-binding interactions: the side-chain of Q181, which in native PKA points away from the ATP-binding site, adopts in apo double mutant protein a new rotamer conformation, which places the polar groups at the hinge region in the ATP pocket. MgATP binding forces Q181 back to the position seen in native PKA. The crystal structure shows that ATP binding geometry is identical with that in native PKA but in this case was determined under conditions with only a single Mg ion ligand. Surface plasmon resonance spectroscopy studies show that significant energy is required for this ligand-induced transition. An additional PKA/PKB mutation, Q181K, corrects the defect, as shown both by the crystal structure of triple mutant PKAB3 (PKAalpha V123A, L173M, Q181K) and by surface plasmon resonance spectroscopy binding studies with ATP and three isoquinoline inhibitors. Thus, the triple mutant serves well as an easily crystallizable model for PKB inhibitor interactions. Further, the phenomenon of Q181 shows how crystallographic analysis should accompany mutant studies to monitor possible spurious structural effects.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Técnicas Biossensoriais , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/química , Magnésio/metabolismo , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Mutação , Conformação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Homologia de Sequência de Aminoácidos , Enxofre/químicaRESUMO
Macrophage infiltration has been identified as an independent poor prognostic factor in several cancer types. The major survival factor for these macrophages is macrophage colony-stimulating factor 1 (CSF-1). We generated a monoclonal antibody (RG7155) that inhibits CSF-1 receptor (CSF-1R) activation. In vitro RG7155 treatment results in cell death of CSF-1-differentiated macrophages. In animal models, CSF-1R inhibition strongly reduces F4/80(+) tumor-associated macrophages accompanied by an increase of the CD8(+)/CD4(+) T cell ratio. Administration of RG7155 to patients led to striking reductions of CSF-1R(+)CD163(+) macrophages in tumor tissues, which translated into clinical objective responses in diffuse-type giant cell tumor (Dt-GCT) patients.