RESUMO
Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART®) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (Tm1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.
RESUMO
LOX-1 is a scavenger receptor that functions as the primary receptor for oxidized low-density lipoprotein (OxLDL) in endothelial cells. The binding of OxLDL to LOX-1 is believed to lead to endothelial activation, dysfunction, and injury, which constitute early atherogenic events. Because of its potential pathological role in atherosclerosis, LOX-1 has been proposed as a therapeutic target for the treatment of this disease. In order to antagonize the ligand-binding function of cell surface LOX-1, we generated a series of recombinant human LOX-1-crystallizable fragment (Fc) fusion proteins and subsequently characterized their biochemical properties and ligand-binding activities in vitro. Consistent with the notion that oligomerization of cell surface LOX-1 is required for high-avidity binding of ligands, we found that LOX-1-Fc fusion protein containing four ligand-binding domains per Fc dimer, but not the one containing two ligand-binding domains, exhibited ligand-binding activity. Optimal ligand-binding activity could be achieved via crosslinking of LOX-1-Fc fusion proteins with a polyclonal antibody against Fc. The crosslinked LOX-1-Fc protein also effectively inhibited the binding and internalization of OxLDL by cell surface LOX-1. These findings demonstrate that functional oligomerization is required for recombinant LOX-1-Fc to function as an effective antagonist.
Assuntos
Membrana Celular/metabolismo , Lipoproteínas LDL/metabolismo , Modelos Moleculares , Proteínas Recombinantes de Fusão/farmacologia , Receptores Depuradores Classe E/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/química , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Reagentes de Ligações Cruzadas/química , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Receptores Depuradores Classe E/antagonistas & inibidores , Receptores Depuradores Classe E/genéticaRESUMO
Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and sensitivity to ionizing radiation (IR). We have previously shown that a herpes simplex virus type 1 (HSV-1) amplicon vector carrying the human ataxia-telangiectasia mutated (ATM) complementary DNA (cDNA) is able to correct aspects of the cellular phenotype of human A-T cells in culture, and is also able to transfer the ATM cDNA to the Atm(-/-) mouse cerebellum. In order to achieve stable gene replacement, we have generated an HSV/adeno-associated virus (AAV) hybrid amplicon vector carrying the expression cassettes for the ATM cDNA [(9.2 kilobases (kb)] and enhanced green fluorescent protein (EGFP), flanked by AAV inverted terminal repeats (ITRs). This hybrid vector, in the presence of AAV Rep proteins, mediates site-specific integration into the AAVS1 site on chromosome 19 in human cells and in Atm(-/-) mice carrying that human locus. The functional activity of the vector-derived ATM was confirmed in vitro and in vivo by ATM autophosphorylation at Ser-1981 after IR. This proof-of-principle study establishes the ability of HSV/AAV hybrid amplicon vectors to mediate functional targeted integration of the ATM cDNA into A-T cells in culture and in Atm(-/-) mice in vivo, thus laying a foundation for possible gene therapy approaches in the treatment of A-T patients.
Assuntos
Proteínas de Ciclo Celular/genética , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/genética , Dependovirus/genética , Marcação de Genes , Vetores Genéticos/genética , Genoma Humano , Proteínas Serina-Treonina Quinases/genética , Simplexvirus/genética , Proteínas Supressoras de Tumor/genética , Animais , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Linhagem Celular Transformada , Chlorocebus aethiops , Proteínas de Ligação a DNA/deficiência , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Supressoras de Tumor/deficiência , Células VeroRESUMO
The distribution of lamprey gonadotropin-releasing hormone (GnRH)-I and -III has been extensively characterized by immunocytochemistry in the forebrain of the sea lamprey, Petromyzon marinus. However, the cellular location of lamprey GnRH-III mRNA expression by in situ hybridization in the lamprey brain has not been determined. We show for the first time the location of expression of lamprey GnRH-III, as well as provide a more comprehensive in situ study of lamprey GnRH-I and glutamic acid decarboxylase (GAD; GABA-synthesizing enzyme) mRNA expression in the brain of the lamprey in different reproductive life stages. Colorimetric and dual-label fluorescent amplification methods of in situ hybridization were used on brain tissue sections of adult, juvenile, and larval sea lamprey. In each life stage of the lamprey, expression of lamprey GnRH-I was shown in the preoptic area (POA) and the hypothalamus forming the characteristic arc-like cell population extending from the preoptic nucleus (NPO) to the neurohypophysis. Lamprey GnRH-III expression was also seen in the POA of each life stage in close proximity to lamprey GnRH-I mRNA containing neurons. GAD expression was shown in distinct cell clusters in and around the POA, in the olfactory bulb, in the dorsal thalamus beneath the habenular region, and also in the ventral-medial hypothalamus stretching from the periventricular region to the anterior portion of the rhombencephalon. Using dual-label in situ hybridization, we have shown that lamprey GnRH-I and -III mRNA are colocalized in the same cells in the POA in adult lampreys. Dual-label in situ hybridization also showed close proximity of GAD mRNA containing neurons and GnRH containing neurons in the POA. These data suggest that gamma-aminobutyric acid (GABA) may directly affect GnRH release in the brain of the sea lamprey.
Assuntos
Encéfalo/metabolismo , Glutamato Descarboxilase/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/metabolismo , Petromyzon/metabolismo , Animais , Encéfalo/citologia , Feminino , Regulação da Expressão Gênica , Glutamato Descarboxilase/genética , Hormônio Liberador de Gonadotropina/genética , Hipotálamo/citologia , Hipotálamo/metabolismo , Hibridização In Situ , Masculino , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Petromyzon/anatomia & histologia , Área Pré-Óptica/citologia , Área Pré-Óptica/metabolismo , Isoformas de Proteínas/metabolismo , RNA Mensageiro/análise , Distribuição TecidualRESUMO
gamma-Aminobutyric acid (GABA) is a neurotransmitter with a demonstrated neuroregulatory role in reproduction in most representative species of vertebrate classes via the hypothalamus. The role of GABA on the hypothalamus-pituitary axis in lampreys has not been fully elucidated. Recent immunocytochemical and in situ hybridization studies suggest that there may be a neuroregulatory role of GABA on the gonadotropin-releasing hormone (GnRH) system in lampreys. To assess possible GABA-GnRH interactions, the effects of GABA and its analogs on lamprey GnRH in vitro and in vivo were studied in adult female sea lampreys (Petromyzon marinus). In vitro perfusion of GABA and its analogs at increasing concentrations (0.1-100 microM) was performed over a 3-h time course. There was a substantial increase of GnRH-I and GnRH-III following treatment of muscimol at 100 microM. In in vivo studies, GABA or muscimol injected at 200 microg/kg significantly increased lamprey GnRH concentration in the brain 0.5 h after treatment compared to controls in female sea lampreys. No significant change in lamprey GnRH-I or GnRH-III was observed following treatment with bicuculline. These data provide novel physiological data supporting the hypothesis that GABA may influence GnRH in the brain of sea lamprey.