RESUMO
We previously reported that radioimmunotherapy (RIT) using 90Y-labeled anti-ROBO1 IgG (90Y-B5209B) achieved significant anti-tumor effects against small-cell lung cancer (SCLC) xenografts. However, subsequent tumor regrowth suggested the necessity for more effective therapy. Here, we evaluated the efficacy of combination 90Y-B5209B and cisplatin therapy in NCI-H69 SCLC xenograft mice. Mice were divided into four therapeutic groups: saline, cisplatin only, RIT only, or combination therapy. Either saline or cisplatin was administered by injection one day prior to the administration of either saline or 90Y-B5209B. Tumor volume, body weight, and blood cell counts were monitored. The pathological analysis was performed on day seven post injection of 90Y-B5209B. The survival duration of the combination therapy group was significantly longer than that of the group treated with RIT alone. No significant survival benefit was observed following the isolated administration of cisplatin (relative to saline). Pathological changes following combination therapy were more significant than those following the isolated administration of RIT. Although combination therapy was associated with an increase of several adverse effects such as weight loss and pancytopenia, these were transient. Thus, cisplatin pre-treatment can potentially enhance the efficacy of 90Y-B5209B, making it a promising therapeutic strategy for SCLC.
Assuntos
Cisplatino/farmacologia , Neoplasias/terapia , Radioimunoterapia , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/patologia , Resultado do TratamentoRESUMO
OBJECTIVE: We previously reported In-labeled anti-cadherin17 (CDH17) IgG visualized CDH17-positive gastric cancer xenografts. Unfortunately, a long waiting time was required to obtain high-contrast images due to long blood retention (blood half-life: 26 h). To accelerate blood clearance, we have developed anti-CDH17 minibody (D2101 minibody) and evaluated the pharmacokinetics in gastric cancer mouse models. METHODS: Two different single chain Fvs (scFvs), D2101 mutant and D2111, were developed from each parental IgG. The binding ability to CDH17 and stability in plasma were evaluated. D2101 minibody, constructed based on D2101 mutant scFv, was labeled with Cu (Cu-D2101 minibody), and the in-vitro and in-vivo properties were evaluated by cell ELISA, biodistribution experiments, and PET imaging in mice bearing CDH17-positive AGS and CDH17-negative MKN74 tumors. RESULTS: D2101 mutant and D2111 scFvs showed similar affinities to CDH17. D2101 mutant scFv was more stable than D2111 scFv in plasma. No loss of binding affinity of the D2101 minibody by chelate conjugation and radiolabeling procedures was observed. The biodistribution of Cu-D2101 minibody showed high uptake in AGS tumors and low uptake in MKN74. The blood half-life of Cu-D2101 minibody was 6.5 h. Improved blood clearance of Cu-D2101 minibody provided high tumor-to-blood ratios compared with the previous results of parental IgG in AGS xenograft mice. PET studies showed consistent results with biodistribution studies. CONCLUSIONS: Cu-D2101 minibody exhibited higher tumor-to-blood ratios at earlier time points than those of the radiolabeled parental IgG. Cu-D2101 minibody has potential as an immunoimaging agent for CDH17-positive tumors.
Assuntos
Transformação Celular Neoplásica , Radioisótopos de Cobre , Fragmentos de Imunoglobulinas/química , Neoplasias Gástricas/diagnóstico por imagem , Neoplasias Gástricas/patologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Humanos , Marcação por Isótopo , Camundongos , Tomografia por Emissão de Pósitrons , Fatores de Tempo , Distribuição TecidualRESUMO
Biparatopic fragment antibodies can overcome deficiencies in avidity of conventional antibody fragments. Here, we describe a technology for generating biparatopic antibodies through two-step targeting using a pair of polypeptides, SpyTag and SpyCatcher, that spontaneously react to form a covalent bond between antibody fragments. In this method, two antibody fragments, each targeting different epitopes of the antigen, are fused to SpyTag and to SpyCatcher. When the two polypeptides are serially added to the antigen, their proximity on the antigen results in covalent bond formation and generation of a biparatopic antibody. We validated the system with purified recombinant antigen. Results in antigen-overexpressing cells were promising although further optimization will be required. Because this strategy results in high-affinity targeting with a bipartite molecule that has considerably lower molecular weight than an antibody, this technology is potentially useful for diverse applications.
RESUMO
OBJECTIVE: Cadherin-17 (CDH17) is a transmembrane protein that mediates cell-cell adhesion and is frequently expressed in adenocarcinomas, including gastric cancer. CDH17 may be an effective diagnostic marker for the staging of gastric cancer. Here, we developed an 111In-labeled anti-CDH17 monoclonal antibody (Mab) as an imaging tracer and performed biodistribution and single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies using mice with CDH17-positive gastric cancer xenografts. CDH17 expression in gastric cancer specimens was also analyzed. METHODS: The cross-reactivity and affinity of our anti-CDH17 Mab D2101 was evaluated by surface plasmon resonance analysis and cell enzyme-linked immunosorbent assay, respectively. Biodistribution and SPECT/CT studies of 111In-labeled D2101 (111In-D2101) were performed. CDH17 expression in gastric cancer specimens was evaluated by immunohistochemistry. RESULTS: Surface plasmon resonance analysis revealed that D2101 specifically recognizes human CDH17, but not murine CDH17. The affinity of D2101 slightly decreased as a result of the radiolabeling procedures. The biodistribution study revealed high uptake of 111In-D2101 in tumors (maximum, 39.2 ± 9.5% ID/g at 96 h postinjection), but low uptake in normal organs, including the stomach. Temporal SPECT/CT imaging with 111In-D2101 visualized tumors with a high degree of tumor-to-nontumor contrast. Immunohistochemical analysis revealed that, compared with HER2, which is a potential marker of N-stage, CDH17 had a higher frequency of positivity in specimens of primary and metastatic gastric cancer. CONCLUSION: Our 111In-anti-CDH17 Mab D2101 depicted CDH17-positive gastric cancer xenografts in vivo and has the potential to be an imaging probe for the diagnosis of primary lesions and lymph-node metastasis in gastric cancer.
Assuntos
Caderinas/imunologia , Imunoconjugados/química , Imunoconjugados/imunologia , Radioisótopos de Índio/química , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Neoplasias Gástricas/diagnóstico por imagem , Neoplasias Gástricas/patologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoconjugados/farmacocinética , Marcação por Isótopo , Metástase Linfática , Camundongos , Estadiamento de Neoplasias , Distribuição TecidualRESUMO
To investigate favorable single amino acid substitutions that improve antigen-antibody interactions, alanine (Ala) mutagenesis scanning of the interfacial residues of a cancer-targeted antibody, B5209B, was performed based on X-ray crystallography analysis. Two substitutions were shown to significantly enhance the binding affinity for the antigen, by up to 30-fold. One substitution improved the affinity by a gain of binding enthalpy, whereas the other substitution improved the affinity by a gain of binding entropy. Molecular dynamics simulations showed that the enthalpic improvement could be attributed to the stabilization of distant salt bridges located at the periphery of the antigen-antibody interface. The entropic improvement was due to the release of water molecules that were stably trapped in the antigen-antibody interface of the wild-type antibody. Importantly, these effects of the Ala substitutions were caused by subtle adjustments of the binding interface. These results will be helpful to design high-affinity antibodies with avoiding entropy-enthalpy compensation.
Assuntos
Alanina/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Neoplasias/imunologia , Substituição de Aminoácidos , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Neoplasias/terapia , Ligação Proteica , Conformação Proteica , Engenharia de ProteínasRESUMO
Molecular recognition is a fundamental event at the core of essentially every biological process. In particular, intermolecular H-bonds have been recognized as key stabilizing forces in antibody-antigen interactions resulting in exquisite specificity and high affinity. Although equally abundant, the role of intramolecular H-bonds is far less clear and not universally acknowledged. Herein, we have carried out a molecular-level study to dissect the contribution of intramolecular H-bonds in a flexible peptide for the recognition by an antibody. We show that intramolecular H-bonds may have a profound, multifaceted and favorable effect on the binding affinity by up to 2 kcal mol-1 of free energy. Collectively, our results suggest that antibodies are fine tuned to recognize transiently stabilized structures of flexible peptides in solution, for which intramolecular H-bonds play a key role.
Assuntos
Anticorpos/química , Peptídeos/química , Reações Antígeno-Anticorpo , Calorimetria , Ligação de Hidrogênio , Modelos Moleculares , Peptídeos/isolamento & purificação , Redobramento de ProteínaRESUMO
Cadherin-17 (CDH17) is highly expressed in gastric cancer and is thus considered to be a good target for antibody therapy. CDH17 is classified as a nonclassical cadherin, in that it is composed of seven extracellular cadherin domains. We generated anti-CDH17 monoclonal antibodies (mAbs) which recognize the extracellular domain of CDH17. Competitive assay using AGS, a gastric cancer cell line, cells revealed that five selected anti-CDH17 mAbs recognize different epitopes on CDH17. As AGS cells were shown to exhibit broad expression pattern of CDH17 by flow cytometry, we separated three clones with a low (10,000/cell), medium (50,000/cell), and high (200,000/cell) expression level, designating them as AGSlow, AGSmed, and AGShigh, respectively. The mAbs, coupled with saporin, exhibited effective cytotoxicity to AGShigh, but poor cytotoxicity to AGSlow. By contrast, the immunotoxin cocktail using the three clones D2101, D2005, and D2008, which recognize different epitopes, exhibited efficient cytotoxicity, even to the AGSlow group. The effect of the immunotoxin cocktail is synergistic, as the combination index was demonstrated to be below 1.0, as calculated by the method of Chou and Talalay using CalcuSyn software. These results suggest that the immunotoxin cocktail targeted to multiple epitopes has synergistic effects on low expression level cells, which expand the applicable range of immunotoxin therapy for cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Caderinas/imunologia , Sinergismo Farmacológico , Epitopos/imunologia , Imunotoxinas/farmacologia , Neoplasias Gástricas/patologia , Biomarcadores Tumorais/metabolismo , Caderinas/antagonistas & inibidores , Caderinas/metabolismo , Humanos , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismo , Células Tumorais CultivadasRESUMO
Accumulated evidence suggests a physiological relationship between the transcription factor NRF3 (NFE2L3) and cancers. Under physiological conditions, NRF3 is repressed by its endoplasmic reticulum (ER) sequestration. In response to unidentified signals, NRF3 enters the nucleus and modulates gene expression. However, molecular mechanisms underlying the nuclear translocation of NRF3 and its target gene in cancer cells remain poorly understood. We herein report that multiple regulation of NRF3 activities controls cell proliferation. Our analyses reveal that under physiological conditions, NRF3 is rapidly degraded by the ER-associated degradation (ERAD) ubiquitin ligase HRD1 and valosin-containing protein (VCP) in the cytoplasm. Furthermore, NRF3 is also degraded by ß-TRCP, an adaptor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase in the nucleus. The nuclear translocation of NRF3 from the ER requires the aspartic protease DNA-damage inducible 1 homolog 2 (DDI2) but does not require inhibition of its HRD1-VCP-mediated degradation. Finally, NRF3 mediates gene expression of the cell cycle regulator U2AF homology motif kinase 1 (UHMK1) for cell proliferation. Collectively, our study provides us many insights into the molecular regulation and biological function of NRF3 in cancer cells.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Ciclo Celular/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células COS , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Chlorocebus aethiops , Proteínas Culina/genética , Proteínas Culina/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Células Epiteliais/patologia , Células HCT116 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/genética , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
Bispecific antibody targeting of two different antigens is promising, but when fragment-based antibodies are used, homogeneous production is difficult. To overcome this difficulty, we developed a method using the SpyTag/SpyCatcher system in which a covalent bond is formed between the two polypeptides. Using this method, we constructed a bispecific antibody that simultaneously interacted with two different epitopes of roundabout homologue 1 (ROBO1), a membrane protein associated with cancer progression. A bispecific tetravalent antibody with an additional functional moiety was also constructed by using a dimeric biotin-binding protein. An interaction analysis of ROBO1-expressing cells and the recombinant antigen demonstrated the improved binding ability of the bispecific antibodies through spontaneous binding of the two antibody fragments to their respective epitopes. In addition, multivalency delayed dissociation, which is advantageous in therapy and diagnosis.
Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Antígenos/química , Epitopos/química , Humanos , Fragmentos de Imunoglobulinas/imunologiaRESUMO
Prostaglandin D2 (PGD2) is a lipid mediator involved in sleep regulation and inflammation. PGD2 interacts with 2 types of G protein-coupled receptors, DP1 and DP2/CRTH2 (chemoattractant receptor homologous molecule expressed on T helper type 2 cells)/GPR44 to show a variety of biological effects. DP1 activation leads to Gs-mediated elevation of the intracellular cAMP level, whereas activation of DP2 decreases this level via the Gi pathway; and it also induces G protein-independent, arrestin-mediated cellular responses. Activation of DP2 by PGD2 causes the progression of inflammation via the recruitment of lymphocytes by enhancing the production of Th2-cytokines. Here we developed monoclonal antibodies (MAbs) against the extracellular domain of mouse DP2 by immunization of DP2-null mutant mice with DP2-overexpressing BAF3, murine interleukin-3 dependent pro-B cells, to reduce the generation of antibodies against the host cells by immunization of mice. Moreover, we immunized DP2-KO mice to prevent immunological tolerance to mDP2 protein. After cell ELISA, immunocytochemical, and Western blot analyses, we successfully obtained a novel monoclonal antibody, MAb-1D8, that specifically recognized native mouse DP2, but neither human DP2 nor denatured mouse DP2, by binding to a particular 3D receptor conformation formed by the N-terminus and extracellular loop 1, 2, and 3 of DP2. This antibody inhibited the binding of 0.5 nM [3H]PGD2 to mouse DP2 (IC50 = 46.3 ± 18.6 nM), showed antagonistic activity toward 15(R)-15-methyl PGD2-induced inhibition of 300 nM forskolin-activated cAMP production (IC50 = 16.9 ± 2.6 nM), and gave positive results for immunohistochemical staining of DP2-expressing CD4+ Th2 lymphocytes that had accumulated in the kidney of unilateral ureteral obstruction model mice. This monoclonal antibody will be very useful for in vitro and in vivo studies on DP2-mediated diseases.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Receptores Imunológicos/imunologia , Receptores de Prostaglandina/imunologia , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Linfócitos T CD4-Positivos/metabolismo , Células CHO , Células COS , Cricetulus , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos , Células HEK293 , Humanos , Hibridomas/metabolismo , Imunização , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Precursoras de Linfócitos B/imunologia , Prostaglandina D2/análogos & derivados , Prostaglandina D2/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores de Prostaglandina/genética , Obstrução Ureteral/imunologia , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , beta-Arrestinas/metabolismoRESUMO
The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, â¼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface.
Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Baculoviridae/metabolismo , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Animais , Baculoviridae/genética , Células CHO , Cricetulus , Humanos , Cinética , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/genética , Proteínas RoundaboutRESUMO
Slit proteins and their receptors, the Roundabout (Robo) family, are known to have a pivotal role in the vascular system. Slit2/Robo1 regulates the migration of human umbilical vein endothelial cells (HUVECs) and tumor-associated endothelial cells. Robo4, the endothelial-specific Robo, is also considered to be involved in vascular cell migration. However, the Slit/Robo signaling pathway is still unclear. Using a Boyden chamber assay, we found that Slit2 induces the migration of HUVECs under a Robo4 knockdown condition. This effect disappeared in Robo1 knockdown cells. The co-existence of the N-terminal extracellular portion of Robo1 blocked the Slit2-evoked migration of HUVECs, while that of Robo4 caused no effect. These results show that the Slit2 signal is transduced through Robo1, while the negative regulation of Robo4 is an intracellular event. Targeted proteomics using an anti-Robo1 monoclonal antibody identified CdGAP, an adhesion-localized Rac1-and Cdc42-specific GTPase activating protein, as a candidate for Slit2/Robo1 signaling. Robo1 and CdGAP were co-immunoprecipitated from CHO cells co-transfected with Robo1 and CdGAP genes. These results suggest that Slit2/Robo1 binding exerts an effect on cell migration, which is negatively regulated by Robo4, and Robo1 may function by interacting with CdGAP in HUVECs.
Assuntos
Movimento Celular/fisiologia , Células Endoteliais/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Linhagem Celular , Células Endoteliais/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Transdução de Sinais/fisiologia , Proteínas RoundaboutRESUMO
Copper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1(-/-) mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFκB-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Isquemia/genética , Metalochaperonas/genética , NADPH Oxidases/genética , Neovascularização Patológica/genética , Proteína-Lisina 6-Oxidase/genética , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Transporte de Cátions/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Proteínas de Transporte de Cobre , ATPases Transportadoras de Cobre , Regulação da Expressão Gênica , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Isquemia/metabolismo , Isquemia/patologia , Perna (Membro)/irrigação sanguínea , Perna (Membro)/patologia , Metalochaperonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares , Monócitos/metabolismo , Monócitos/patologia , NADPH Oxidases/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteína-Lisina 6-Oxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a 'gated-pore' transport mechanism in such monosaccharide transporters.
Assuntos
Frutose/metabolismo , Transportador de Glucose Tipo 5/química , Transportador de Glucose Tipo 5/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Bovinos , Membrana Celular/metabolismo , Cristalografia por Raios X , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Frutose/química , Glucose/química , Glucose/metabolismo , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 5/genética , Modelos Moleculares , Mutação Puntual/genética , Conformação Proteica , Ratos , Sais/química , Eletricidade Estática , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Simportadores/química , Simportadores/metabolismoRESUMO
SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol.
Assuntos
Proteínas Metiltransferases/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Aminoácidos , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Ácidos Graxos Insaturados/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Histona-Lisina N-Metiltransferase , Humanos , Carioferinas/antagonistas & inibidores , Leupeptinas/farmacologia , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteínas Metiltransferases/genética , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Frações Subcelulares/metabolismo , Proteína Exportina 1RESUMO
INTRODUCTION: ROBO1 is a membrane protein that contributes to tumor metastasis and angiogenesis. We previously reported that 90Y-labeled anti-ROBO1 monoclonal antibody (90Y-anti-ROBO1 IgG) showed an antitumor effect against ROBO1-positive tumors. In this study, we performed a biodistribution study and radioimmunotherapy (RIT) against ROBO1-positive small cell lung cancer (SCLC) models. METHODS: For the biodistribution study, 111In-labeled anti-ROBO1 monoclonal antibody (111In-anti-ROBO1 IgG) was injected into ROBO1-positive SCLC xenograft mice via the tail vein. To evaluate antitumor effects, an RIT study was performed, and SCLC xenograft mice were treated with 90Y-anti-ROBO1 IgG. Tumor volume and body weight were periodically measured throughout the experiments. The tumors and organs of mice were then collected, and a pathological analysis was carried out. RESULTS: As a result of the biodistribution study, we observed tumor uptake of 111In-anti-ROBO1 IgG. The liver, kidney, spleen, and lung showed comparably high accumulation of 111In-labeled anti-ROBO1. In the RIT study, 90Y-anti-ROBO1 IgG significantly reduced tumor volume compared with baseline. Pathological analyses of tumors revealed coagulation necrosis and fatal degeneration of tumor cells, significant reduction in the number of Ki-67-positive cells, and an increase in the number of apoptotic cells. A transient reduction of hematopoietic cells was observed in the spleen, sternum, and femur. CONCLUSIONS: These results suggest that RIT with 90Y-anti-ROBO1 IgG is a promising treatment for ROBO1-positive SCLC.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas do Tecido Nervoso/imunologia , Receptores Imunológicos/imunologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Radioisótopos de Ítrio/uso terapêutico , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Radioimunoterapia/métodos , Receptores Imunológicos/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/radioterapia , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Radioisótopos de Ítrio/química , Radioisótopos de Ítrio/farmacocinética , Proteínas RoundaboutRESUMO
ROBO1, fibronectin Type-III domain (Fn)-containing protein, is a novel immunotherapeutic target for hepatocellular carcinoma in humans. The crystal structure of the antigen-binding fragment (Fab) of B2212A, the monoclonal antibody against the third Fn domain (Fn3) of ROBO1, was determined in pursuit of antibody drug for hepatocellular carcinoma. This effort was conducted in the presence or absence of the antigen, with the chemical features being investigated by determining the affinity of the antibody using molecular dynamics (MD) and thermodynamics. The structural comparison of B2212A Fab between the complex and the free form revealed that the interfacial Tyr(L) 50 (superscripts L, H, and F stand for the residues in the light chain, heavy chain, and Fn3, respectively) played important roles in Fn3 recognition. That is, the aromatic ring of Tyr(L) 50 pivoted toward Phe(F) 68, forming a CH/π interaction and a new hydrogen bond with the carbonyl O atom of Phe(F) 68. MD simulations predicted that the Tyr(L) 50-Phe(F) 68 interaction almost entirely dominated Fab-Fn3 binding, and Ala-substitution of Tyr(L) 50 led to a reduced binding of the resultant complex. On the contrary, isothermal titration calorimetry experiments underscored that Ala-substitution of Tyr(L) 50 caused an increase of the binding enthalpy between B2212A and Fn3, but importantly, it induced an increase of the binding entropy, resulting in a suppression of loss in the Gibbs free energy in total. These results suggest that mutation analysis considering the binding entropy as well as the binding enthalpy will aid in the development of novel antibody drugs for hepatocellular carcinoma.
Assuntos
Fibronectinas/química , Proteínas do Tecido Nervoso/química , Receptores Imunológicos/química , Tirosina/química , Fibronectinas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/metabolismo , Estrutura Terciária de Proteína , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termodinâmica , Tirosina/metabolismo , Proteínas RoundaboutRESUMO
The monoclonal antibody has become an important therapeutic in the treatment of both hematological malignancies and solid tumors. The recent success of antibody-drug conjugates (ADCs) has broadened the extent of the potential target molecules in cancer immunotherapy. As a result, even molecules of low abundance have become targets for cytotoxic reagents. The multi-pass membrane proteins are an emerging target for the next generation antibody therapeutics. One outstanding challenge is the difficulty in preparing a sufficient amount of these membrane proteins so as to be able to generate the functional antibody. We have pursued the expression of various membrane proteins on the baculovirus particle and the utilization of displayed protein for immunization. The strong antigenicity of the virus acts either as a friend or foe in the making of an efficient antibody against an immunologically tolerant antigen. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.
RESUMO
BACKGROUND: ROBO1 is a membrane protein that functions in axon guidance. ROBO1 contributes to tumour metastasis and angiogenesis and may have potential as a target protein of immunotherapy because ROBO1 is specifically expressed at high levels in hepatocellular carcinoma. In this study, we examined biodistribution and radioimmunotherapy (RIT) using a radioisotope-labelled anti-ROBO1 monoclonal antibody (MAb) against hepatocellular carcinoma models. METHODS: ROBO1-positive HepG2 human hepatocellular carcinoma xenograft nude mice were used in this study. We conjugated anti-ROBO1 MAb with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and the conjugates were labelled with (111)In and (90)Y. To study biodistribution, the (111)In-DOTA-anti-ROBO1 MAb was injected into HepG2 xenograft mice via the tail vein. To evaluate any antitumour effect, a RIT study was performed, and the (90)Y-DOTA-anti-ROBO1 MAb was injected via the tail vein. Tumour volume, mouse weight, and blood cell count were periodically measured throughout the experiments. The tumours and organs of mice were collected, and a histopathological analysis was carried out. RESULTS: The tumour uptake of (111)In-anti-ROBO1 MAb in HepG2 xenograft mice was 15.0% ± 0.69% injected dose per gram at 48 h after injection. Immunotherapy with cold-anti-ROBO1 MAb (70 µg) did not cause a significant antitumour effect. RIT with 6.7 MBq of (90)Y-anti-ROBO1 MAb caused significant tumour growth suppression. Transient body weight loss and bone-marrow suppression were observed. Histopathological analyses of tumours revealed the fatal degeneration of tumour cells, significant reduction of the Ki-67 index, and an increase of the apoptosis index. Normal organs showed no significant injury, but a transient reduction of hematopoietic cells was observed in the spleen and in the sternal bone marrow. CONCLUSIONS: These results suggest that RIT with (90)Y-anti-ROBO1 MAb is a promising treatment for ROBO1-positive hepatocellular carcinoma.
RESUMO
DJ-1, the product of a causative gene of a familial form of Parkinson disease, undergoes preferential oxidation of Cys106 (cysteine residue at position 106) under oxidative stress. Using specific monoclonal antibodies against Cys106 oxidized DJ-1 (oxDJ-1), we examined oxDJ-1 immunoreactivity in brain sections from DJ-1 knockout and wild-type mice and in human brain sections from cases classified into different Lewy body stages of Parkinson disease and Parkinson disease with dementia. Oxidized DJ-1 immunoreactivity was prominently observed in neuromelanin-containing neurons and neuron processes of the substantia nigra; Lewy bodies also showed oxDJ-1 immunoreactivity. Oxidized DJ-1 was also detected in astrocytes in the striatum, in neurons and glia in the red nucleus, and in the inferior olivary nucleus, all of which are related to regulation of movement. These observations suggest the relevance of DJ-1 oxidation to homeostasis in multiple brain regions, including neuromelanin-containing neurons of the substantia nigra, and raise the possibility that oxDJ-1 levels might change during the progression of Lewy body-associated neurodegenerative diseases.