RESUMEN
CD47 belongs to the immunoglobulin superfamily and is associated with ß-integrins. Recently it was reported that CD47 ligation rapidly induces apoptosis in B-chronic lymphocytic leukemia (CLL) cells. Chronic lymphocytic leukemia is still an incurable hematological malignancy even with the novel therapeutic agents; therefore, new and effective agents for the treatment of CLL in clinical settings are urgently needed. We generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Subsequently, we created a disulfide-stabilized dimer of a single-chain antibody fragment of MABL (S-S diabody) to get rid of the adverse effect of MABL such as hemagglutination. In this study, we analyzed the effects of this new antibody on cellular proliferation, and the molecular mechanism of CD47-mediated apoptosis in human lymphoid malignant cells. Treatment with S-S diabody alone induced apoptosis of CD47-positive primary B-CLL and leukemic cells (MOLT-4 and JOK-1). In addition, administration of S-S diabody significantly prolonged the survival of severe combined immunodeficiency (SCID) mice inoculated with JOK-1 cells. In gene expression profiling of the S-S diabody-treated MOLT-4 cells, hypoxia inducible factor (HIF)-1α downstream genes (RTP801 and BNIP3) were upregulated, and the mRNA expression levels of HIF-1α, RTP801 and BNIP3 were increased. Knockdown of HIF-1α by siRNA repressed S-S diabody-induced apoptosis in MOLT4 cells. In conclusion, CD47 will be a molecular target for the treatment of lymphoid malignancies, and S-S diabody might have potential as a novel therapeutic agent for B-CLL.
Asunto(s)
Apoptosis , Antígeno CD47/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Leucemia Linfocítica Crónica de Células B/terapia , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/genética , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Potenciales de la Membrana , Proteínas de la Membrana/genética , Ratones , Ratones SCID , Microscopía Electrónica , Proteínas Mitocondriales/genética , Multimerización de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Tamoxifen, a selective estrogen receptor modulator, and fulvestrant, a selective estrogen receptor down-regulator (SERD), are now available for estrogen receptor-positive breast cancer patients. However, these patients acquire drug-resistance during the treatments. We identified a new orally active nonsteroidal SERD, CH4986399, which is structurally unrelated to fulvestrant and tamoxifen. MATERIALS AND METHODS: We examined the oral antitumor activity and down-regulation of ER by CH4986399 in human breast cancer Br-10 and ZR-75-1 xenografts. RESULTS: In the Br-10 xenografts, CH4986399 (100 mg/kg p.o.) as well as fulvestrant (3 mg/body s.c.) strongly reduced tumor weight. In the ZR-75-1 xenografts, CH4986399 (100 mg/kg p.o.) strongly reduced tumor weight and ER content without agonistic activity. In contrast, tamoxifen (100 mg/kg p.o.) showed only moderate antitumor activity and no ER down-regulation. CONCLUSION: With a chemical structure different from both fulvestrant and tamoxifen, CH4986399, may help overcome drug resistance from the endocrine treatment sequence for breast cancer patients.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Desnudos , Receptores de Estrógenos/biosíntesis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos , Carcinoma Hepatocelular/tratamiento farmacológico , Glipicanos/antagonistas & inhibidores , Neoplasias Hepáticas/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Células CHO , Carcinoma Hepatocelular/inmunología , Línea Celular Tumoral , Cricetinae , Cricetulus , Glipicanos/inmunología , Humanos , Epítopos Inmunodominantes/inmunología , Neoplasias Hepáticas/inmunología , Ratones , Proteínas de Neoplasias/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human glypican 3 (GPC3) is preferentially expressed in the tumor tissues of liver cancer patients. In this study, we obtained a monoclonal antibody (mAb) against the COOH-terminal part of GPC3, which induced antibody-dependent cellular cytotoxicity (ADCC). The mAb, designated GC33, exhibited marked tumor growth inhibition of s.c. transplanted Hep G2 and HuH-7 xenografts that expressed GPC3 but did not inhibit growth of the SK-HEP-1 that was negative for GPC3. GC33 was efficacious even in an orthotopic model; it markedly reduced the blood alpha-fetoprotein levels of mice intrahepatically transplanted with Hep G2 cells. Humanized GC33 (hGC33) was as efficacious as GC33 against the Hep G2 xenograft, but hGC33 lacking carbohydrate moieties caused neither ADCC nor tumor growth inhibition. Depletion of CD56+ cells from human peripheral blood mononuclear cells markedly abrogated the ADCC caused by hGC33. The results show that the antitumor activity of hGC33 is mainly attributable to ADCC, and in human, natural killer cell-mediated ADCC is one possible mechanism of the antitumor effects by GC33. hGC33 will provide a novel treatment option for liver cancer patients with GPC3-positive tumors.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Glipicanos/inmunología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/terapia , Animales , Anticuerpos Monoclonales/administración & dosificación , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos de Neoplasias/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Bencenosulfonatos/administración & dosificación , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/inmunología , Línea Celular Tumoral , Terapia Combinada , Doxorrubicina/administración & dosificación , Glipicanos/biosíntesis , Glipicanos/genética , Humanos , Células Asesinas Naturales/inmunología , Neoplasias Hepáticas/genética , Masculino , Ratones , Ratones SCID , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Piridinas/administración & dosificación , Sorafenib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Baculoviridae/genética , Moléculas de Adhesión Celular/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/inmunología , Biblioteca de Péptidos , Proteínas del Envoltorio Viral/genética , Proteínas Virales/genética , Animales , Baculoviridae/metabolismo , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Inmunización , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Transportador de Péptidos 1 , Receptores CCR2 , Receptores de Quimiocina/inmunología , Simportadores/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
In order to develop orally active pure antiestrogens, we incorporated the carboxy-containing side chains into the 7alpha-position of the steroid scaffold and found that 17-keto derivative CH4893237 (12b) functioned as a pure antiestrogen with its oral activity much superior to clinically used pure antiestrogen, ICI182,780. Results from the pharmacokinetic evaluation indicated that the potent antiestrogen activity at oral dosing in mice attributed to both improved absorption from the intestinal wall and metabolic stability in liver.
Asunto(s)
Moduladores de los Receptores de Estrógeno/química , Moduladores de los Receptores de Estrógeno/farmacología , Administración Oral , Animales , Moduladores de los Receptores de Estrógeno/administración & dosificación , Moduladores de los Receptores de Estrógeno/síntesis química , Haplorrinos , Ratones , Estructura Molecular , Ratas , Esteroides/química , Esteroides/farmacología , Relación Estructura-ActividadRESUMEN
In order to search for alternatives to the sulfoxide moiety in the long side chain of pure antiestrogens, several molecules that may interact with water in a fashion similar to ICI164,384 were designed and it was found that compounds with the carboxy, the sulfamide, or the sulfonamide instead of the sulfoxide moiety also functioned as pure antiestrogens. Interestingly, the compound possessing the carboxy moiety showed superior antiestrogen activity compared to ICI182,780 when dosed orally. Results of the pharmacokinetic evaluation indicated that the potent antiestrogen activity at oral dosing attributed to both the improved absorption from the intestinal wall and the metabolic stability of the compound in liver.
Asunto(s)
Cromanos/farmacología , Moduladores de los Receptores de Estrógeno/química , Administración Oral , Área Bajo la Curva , Cromanos/química , Cromanos/farmacocinética , Moduladores de los Receptores de Estrógeno/administración & dosificación , Moduladores de los Receptores de Estrógeno/farmacocinética , Moduladores de los Receptores de Estrógeno/farmacologíaRESUMEN
In order to develop pure antiestrogens, a series of 7-hydroxy-3-(4-hydroxyphenyl)-3-methylchroman and 7-hydroxy-3-(4-hydroxyphenyl)-3-methylthiochroman derivatives with sulfoxide containing side chains at the 4-position were designed, synthesized, and evaluated. Among them, compounds 14b and 24b functioned as pure antiestrogens with the ability to downregulate ER, and their in vitro and in vivo antiestrogen activities were similar to those of ICI182,780. In addition, the structure-activity relationship indicated that the (3RS,4RS)-configuration between the 3- and 4-position, the methyl group at the 3-position, the 9-methylene chain between the scaffold and the sulfoxide moiety, and the terminal perfluoroalkyl moiety play an important role in increasing estrogen receptor binding and oral antiestrogen activities.
Asunto(s)
Cromanos/química , Cromanos/farmacología , Moduladores de los Receptores de Estrógeno/química , Moduladores de los Receptores de Estrógeno/farmacología , Animales , Unión Competitiva , Cromanos/síntesis química , Evaluación Preclínica de Medicamentos , Estradiol/análogos & derivados , Estradiol/farmacología , Moduladores de los Receptores de Estrógeno/síntesis química , Femenino , Ratones , Ratones Endogámicos ICR , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Tamaño de los Órganos/efectos de los fármacos , Ovariectomía , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Útero/anatomía & histología , Útero/efectos de los fármacosRESUMEN
Transporters play a critical role in many physiological and pathological states and expression of the functional transporter protein is essential in exploring its kinetics and developing effective drugs. We describe here the recovery of functional transporter protein in the baculovirus fraction. We introduced a gene encoding human peptide transporter PepT1, important for the absorption of protein hydrolytic products or peptide-mimetic drugs, into a baculovirus vector. After infection, a large amount of PepT1 appeared in the budded virus fraction compared with Sf9 cells. Uptake of [14C]glycylsarcosine was markedly increased in an acidic condition and showed a clear overshoot in PepT1-expressing virus fraction. The apparent Michaelis constant for [14C]glycylsarcosine was 0.55 +/- 0.06 mM. [14C]Glycylsarcosine uptake was inhibited by di- and tripeptides and orally active beta-lactam antibiotics. These results suggest that functional PepT1 recovers efficiently in a budded virus fraction, and, thus, this expression system will be a useful tool for characterization and screening of peptide-mimetic drugs in drug discovery.
Asunto(s)
Baculoviridae/genética , Simportadores/genética , Animales , Línea Celular , Centrifugación por Gradiente de Densidad , Clonación Molecular , Humanos , Cinética , Microscopía Electrónica , Transportador de Péptidos 1 , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/aislamiento & purificación , Mapeo Restrictivo , Spodoptera , Simportadores/aislamiento & purificaciónRESUMEN
For detection of hepatocellular carcinoma (HCC) in patients with liver cirrhosis, serum alpha-fetoprotein has been widely used, but its sensitivity has not been satisfactory, especially in small, well-differentiated HCC, and complementary serum marker has been clinically required. Glypican-3 (GPC3), a heparan sulfate proteoglycan anchored to the plasma membrane, is a good candidate marker of HCC because it is an oncofetal protein overexpressed in HCC at both the mRNA and protein levels. In this study, we demonstrated that its NH(2)-terminal portion [soluble GPC3 (sGPC3)] is cleaved between Arg(358) and Ser(359) of GPC3 and that sGPC3 can be specifically detected in the sera of patients with HCC. Serum levels of sGPC3 were 4.84 +/- 8.91 ng/ml in HCC, significantly higher than the levels seen in liver cirrhosis (1.09 +/- 0.74 ng/ml; P < 0.01) and healthy controls (0.65 +/- 0.32 ng/ml; P < 0.001). In well- or moderately-differentiated HCC, sGPC3 was superior to alpha-fetoprotein in sensitivity, and a combination measurement of both markers improved overall sensitivity from 50% to 72%. These results indicate that sGPC3 is a novel serological marker essential for the early detection of HCC.