RESUMEN
The use of libraries of phage-displayed human single-chain antibody fragments (scFv) has become a new, powerful tool in rapidly obtaining therapeutically useful antibodies. Here, we describe the generation of human scFv and F(ab')2 directed against the gamma-carboxyglutamic acid (Gla) domain of coagulation factor IX. A large library of human scFv, displayed either on M13 phage or expressed as soluble proteins, was screened for binding to human Gla-domain peptide (Tyr1-Lys43). Among a panel of scFv that bound to the factor IX-Gla domain, six scFv clones recognized full-length factor IX and exhibited strong inhibitory activity of factor IX in vitro. After reformatting as F(ab')2, the affinity for factor IX of three selected clones was determined: 10C12 Kd = 1.6 nmol/l, 13D1 Kd = 2.9 nmol/l, and 13H6 Kd = 0.46 nmol/l. The antibodies specifically bound to factor IX and not to other coagulation factors, as assessed by enzyme-linked immunosorbent-type and human plasma clotting assays. The complementarity determining region amino acid sequences of clones 10C12 and 13D1 only differed at a single residue, whereas 13H6 showed little homology, suggesting that 13H6 binds to a different epitope within the factor IX-Gla domain. Despite the slightly lower affinity of 10C12 F(ab')2 versus 13H6 F(ab')2, 10C12 was consistently more potent than 13H6 in prolonging the activated partial thromboplastin time (APTT), in inhibiting platelet-mediated plasma clotting, and in inhibiting factor X activation by the intrinsic Xase complex. Finally, 10C12 F(ab')2 also recognized and neutralized factor IX/factor IXa of different species, as demonstrated by the specific APTT prolongation of dog, mouse, baboon and rabbit plasma. In summary, the results validate the usefulness of scFv phage-displayed libraries to rapidly generate fully human antibodies as potential new therapeutics for thrombotic disorders.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Factor IXa/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Sitios de Unión de Anticuerpos , Unión Competitiva , Coagulación Sanguínea/efectos de los fármacos , Factores de Coagulación Sanguínea/metabolismo , Plaquetas/fisiología , Calcio/farmacología , Bovinos , Clonación Molecular , Endotelio Vascular/citología , Ensayo de Inmunoadsorción Enzimática , Epítopos/metabolismo , Factor IXa/antagonistas & inhibidores , Factor IXa/fisiología , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fab de Inmunoglobulinas/farmacología , Región Variable de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/farmacología , Magnesio/farmacología , Datos de Secuencia Molecular , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
The cytokine thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells. TPO exerts its effect through activation of the c-Mpl receptor and of multiple downstream signal transduction pathways. While the membrane-proximal half of the cytoplasmic domain appears to be required for the activation of signaling molecules that drive proliferation, the distal half and activation of the mitogen-activated protein kinase pathway have been implicated in mediating megakaryocyte maturation in vitro. To investigate the contribution of these two regions of c-Mpl and the signaling pathways they direct in mediating the function of TPO in vivo, we used a knock-in (KI) approach to delete the carboxy-terminal 60 amino acids of the c-Mpl receptor intracellular domain. Mice lacking the C-terminal 60 amino acids of c-Mpl (Delta60 mice) have normal platelet and megakaryocyte counts compared to wild-type mice. Furthermore, platelets in the KI mice are functionally normal, indicating that activation of signaling pathways connected to the C-terminal half of the receptor is not required for megakaryocyte differentiation or platelet production. However, Delta60 mice have an impaired response to exogenous TPO stimulation and display slower recovery from myelosuppressive treatment, suggesting that combinatorial signaling by both ends of the receptor intracellular domain is necessary for an appropriate acute response to TPO.
Asunto(s)
Plaquetas/citología , Hematopoyesis , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Trombopoyetina/farmacología , Animales , Recuento de Células Sanguíneas , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Exones/genética , Fibrinógeno/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Ratones , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Activación Plaquetaria , Ploidias , Proteínas Proto-Oncogénicas/genética , Receptores de Citocinas/química , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Receptores de Trombopoyetina , Eliminación de Secuencia/genética , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
Thrombopoietin (TPO) has been established as the major regulator of megakaryocyte and platelet production. In vitro and in vivo studies have demonstrated that TPO affects both megakaryocyte proliferation and maturation. In vitro, TPO has been reported to be essential for full development of megakaryocytes and platelets. These studies are in contrast to results observed in vivo in mice deficient in the TPO or c-mpl gene (TPO-/- and c-mpl-/-). Both TPO-/- and c-mpl-/- mice exhibit a 90% reduction in megakaryocyte and platelet levels. But even with this small number of circulating platelets, these mice do not have any excessive bleeding. Ultrastructural analysis indicates that platelets and megakaryocytes present in the knockout mice are morphologically normal. Characterization of platelet function shows that platelets from knockout mice are functionally identical to the wild-type platelets as measured by upregulation of 125I-fibrinogen binding to platelets in response to adenosine diphosphate (ADP) stimulation and by platelet attachment to the immobilized extracellular matrix proteins, collagen and von Willebrand factor (vWF). These results demonstrate that in vivo, TPO is required for the control of megakaryocyte and platelet number but not for their maturation. Other factors with megakaryocytopoietic activity may be able to compensate for the maturational role of TPO and lead to the formation of normal megakaryocytes and platelets in TPO-/- and c-mpl-/- mice.
Asunto(s)
Plaquetas/citología , Megacariocitos/citología , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas/deficiencia , Receptores de Citocinas , Trombopoyetina/deficiencia , Animales , Plaquetas/fisiología , Diferenciación Celular , Megacariocitos/fisiología , Ratones , Ratones Noqueados , Microscopía Electrónica , Recuento de Plaquetas , Receptores de TrombopoyetinaRESUMEN
TGF-alpha and EGF are structurally related factors that bind to and induce tyrosine autophosphorylation of a common receptor. Proteolytic cleavage of the transmembrane TGF-alpha precursor's external domain releases several TGF-alpha species. However, membrane-bound TGF-alpha forms remain on the surface of TGF-alpha-expressing cell lines. To evaluate the biological activity of these forms, we modified two cleavage sites in the TGF-alpha precursor coding sequence, making processing into the 50 amino acid TGF-alpha impossible. Overexpression of this cDNA in a receptor-negative cell line, partial purification, and N-terminal sequence analysis indicate the existence of two transmembrane TGF-alpha forms. These solubilized precursors induce tyrosine autophosphorylation of the EGF/TGF-alpha receptor in intact receptor-overexpressing cells, and anchorage-independent growth of NRK fibroblasts. Cell-cell contact between TGF-alpha precursor-overexpressing cells and cells expressing high numbers of receptors also resulted in receptor activation. These findings suggest a role for transmembrane TGF-alpha forms in intercellular interactions in proliferating tissues.
Asunto(s)
Receptores ErbB/fisiología , Glicoproteínas de Membrana/fisiología , Procesamiento Proteico-Postraduccional , Factores de Crecimiento Transformadores/fisiología , Animales , Línea Celular , Cricetinae , Análisis Mutacional de ADN , Técnica del Anticuerpo Fluorescente , Fosforilación , Precursores de Proteínas/fisiología , Proteínas Tirosina Quinasas/fisiología , Relación Estructura-ActividadRESUMEN
We have previously shown that a mutant human insulin receptor with a COOH-terminal 43-amino acid deletion (HIR delta CT), when expressed in Rat 1 fibroblasts, binds insulin normally, autophosphorylates, and undergoes endocytosis after insulin binding in a manner comparable to the normal human insulin receptor (HIRc). In this paper we have examined the biologic activity of the truncated and normal insulin receptors. In vitro, the HIR delta CT receptors caused a 1.8-fold greater phosphorylation of a Glu4/Tyr1 polypeptide than did the HIRc receptors, but the two receptor types were nearly equivalent in their ability to phosphorylate a src-derived peptide. Furthermore, insulin preactivation of HIRc and HIR delta CT receptors in intact cells led to equivalent stimulation of tyrosine kinase activity as subsequently determined for histone in vitro. Expression of HIRc receptors in cells led to enhanced sensitivity to insulin of 2-deoxy-D-glucose uptake and glycogen synthase activation. This increased sensitivity was proportional to receptor number at low (Ro = 6400) but not at high (Ro = 1.25 X 10(6] levels of receptor expression. However, expression of HIR delta CT receptors (Ro = 2.5 X 10(5] led to little, if any, increase in insulin sensitivity of either 2-deoxy-D-glucose uptake or glycogen synthase activation. Furthermore, compared with HIRc cells, HIR delta CT cells respond poorly to an agonistic monoclonal antibody specific for the human insulin receptor. In conclusion, the HIR delta CT receptor retains intact protein kinase activity in vitro. Despite this, however, the receptor displays low activity in mediating the metabolic effects of insulin.