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1.
Proc Natl Acad Sci U S A ; 111(5): 1766-71, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24443552

RESUMO

Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low-antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.


Assuntos
Anticorpos/metabolismo , Imunoconjugados/metabolismo , Preparações Farmacêuticas/síntese química , Engenharia de Proteínas/métodos , Animais , Anticorpos/sangue , Anticorpos/química , Anticorpos/toxicidade , Técnicas de Cultura Celular por Lotes , Células CHO , Morte Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Cricetulus , Cisteína/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Imunoconjugados/toxicidade , Preparações Farmacêuticas/sangue , Preparações Farmacêuticas/química , Estabilidade Proteica/efeitos dos fármacos , Ratos
2.
Proc Natl Acad Sci U S A ; 110(44): 17796-801, 2013 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-24127589

RESUMO

Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ~ 100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.


Assuntos
Descoberta de Drogas/métodos , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Animais , Complexo CD3/imunologia , Xenoenxertos/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoterapia/métodos , Leucócitos Mononucleares , Masculino , Camundongos , Neoplasias da Próstata/imunologia , Engenharia de Proteínas
3.
Proc Natl Acad Sci U S A ; 109(40): 16101-6, 2012 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-22988081

RESUMO

Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2(+) cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.


Assuntos
Aminoácidos/química , Anticorpos Monoclonais Humanizados/química , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/química , Engenharia de Proteínas/métodos , Aminobenzoatos/química , Animais , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Feminino , Humanos , Imunoconjugados/farmacocinética , Imunoconjugados/uso terapêutico , Imunoglobulina G/química , Camundongos , Camundongos SCID , Oligopeptídeos/química , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Trastuzumab
5.
Cancer Res ; 65(7): 2846-53, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15805286

RESUMO

Radiotherapy is an effective approach for the treatment of local prostate cancer. However, once prostate cancer metastasizes, radiotherapy cannot be used due to the distribution of multiple metastases to lymph nodes and bones. In contrast, radioimmunotherapy should still be efficacious in metastatic prostate cancer as radioisotopes are brought to tumor cells by targeting antibodies. Here we identify and validate a prostate-expressed molecule, tomoregulin, as a target for radioimmunotherapy of prostate cancer. Tomoregulin is a transmembrane protein selectively expressed in the brain, prostate, and prostate cancer, but not expressed in other normal tissues. Immunohistochemical studies of tomoregulin protein in clinical samples show its location in the luminal epithelium of normal prostate, benign prostatic hyperplasia, and prostatic intraepithelial neoplasia. More importantly, the tomoregulin protein is expressed in primary prostate tumors and in their lymph node and bone metastases. The nature of tomoregulin as a transmembrane protein and its tissue-specific expression make tomoregulin an attractive target for radioimmunotherapy, in which tomoregulin-specific antibodies will deliver a radioisotope to prostate tumor cells and metastases. Indeed, biodistribution studies using a prostate tumor xenograft model showed that the (111)In-labeled anti-tomoregulin antibody 2H8 specifically recognizes tomoregulin protein in vivo, leading to a strong tumor-specific accumulation of the antibody. In efficacy studies, a single i.p. dose of 150 microCi (163 microg) (90)Y-labeled 2H8 substantially inhibits the growth rate of established LNCaP human prostate tumor xenograft in nude mice but produces no overt toxicity despite cross-reactivity of 2H8 with mouse tomoregulin. Our data clearly validate tomoregulin as a target for radioimmunotherapy of prostate cancer.


Assuntos
Imunotoxinas/uso terapêutico , Radioisótopos de Índio/uso terapêutico , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/radioterapia , Radioisótopos/uso terapêutico , Itérbio/uso terapêutico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imunotoxinas/imunologia , Imunotoxinas/farmacocinética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Nus , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Próstata/metabolismo , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Radioimunoterapia , Compostos Radiofarmacêuticos/imunologia , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/uso terapêutico , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Mol Cancer Ther ; 5(9): 2158-64, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16985048

RESUMO

The ability of cancer cells to undergo invasion and migration is a prerequisite for tumor metastasis. Rho, a Ras-related small GTPase, and the Rho-associated coiled coil-containing protein kinases (Rho kinases, ROCK1 and ROCK2) are key regulators of focal adhesion, actomyosin contraction, and thus cell motility. Inhibitors of this pathway have been shown to inhibit tumor cell motility and metastasis. Here, we show that fasudil [1-(5-isoquinolinesulfonyl)-homopiperazine], an orally available inhibitor of Rho kinases, and its metabolite 1-(hydroxy-5-isoquinoline sulfonyl-homopiperazine) (fasudil-OH) modify tumor cell morphology and inhibit tumor cell migration and anchorage-independent growth. In addition, we show that fasudil inhibited tumor progression in three independent animal models. In the MM1 peritoneal dissemination model, tumor burden and ascites production were reduced by > 50% (P < 0.05). In the HT1080 experimental lung metastasis model, fasudil decreased lung nodules by approximately 40% (P < 0.05). In the orthotopic breast cancer model with MDA-MB-231, there were 3-fold more tumor-free mice in the fasudil-treated group versus saline control group (P < 0.01). Fasudil has been approved for the treatment of cerebral vasospasm and associated cerebral ischemic symptoms. In patients, fasudil is well tolerated without any serious adverse reactions. Therefore, the concept of Rho kinase inhibition as an antimetastatic therapy for cancer can now be clinically explored.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Neoplasias da Mama/tratamento farmacológico , Fibrossarcoma/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Feminino , Fibrossarcoma/enzimologia , Fibrossarcoma/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Ratos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Chem Biol Drug Des ; 74(1): 43-50, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19519743

RESUMO

Heat-shock protein-90 is an attractive target for anticancer drugs, as heat-shock protein-90 blockers such as the ansamycin 17-(allylamino)-17-demethoxygeldanamycin greatly reduce the expression of many signaling molecules that are disregulated in cancer cells and are key drivers of tumor growth and metastasis. While 17-(allylamino)-17-demethoxygeldanamycin has shown promise in clinical trials, this compound class has significant template-related drawbacks. In this paper, we describe a new, potent non-ansamycin small-molecule inhibitor of heat-shock protein-90, BX-2819, containing resorcinol and triazolothione rings. Structural studies demonstrate binding of BX-2819 to the ADP/ATP-binding pocket of heat-shock protein-90. The compound blocked expression of heat-shock protein-90 client proteins in cancer cell lines and inhibited cell growth with a potency similar to 17-(allylamino)-17-demethoxygeldanamycin. In a panel of four cancer cell lines, BX-2819 blocked growth with an average IC(50) value of 32 nM (range of 7-72 nM). Efficacy studies demonstrated that treatment with BX-2819 significantly inhibited the growth of NCI-N87 and HT-29 tumors in nude mice, consistent with pharmacodynamic studies showing inhibition of heat-shock protein-90 client protein expression in tumors for greater than 16 h after dosing. These data support further studies to assess the potential of BX-2819 and related analogs for the treatment of cancer.


Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Triazóis/farmacologia , Animais , Benzoquinonas/química , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Simulação por Computador , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Choque Térmico HSP90/metabolismo , Células HT29 , Humanos , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacologia , Camundongos , Camundongos Nus , Transplante Heterólogo , Triazóis/química , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Am J Pathol ; 168(2): 608-20, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16436674

RESUMO

The mammary gland microenvironment during postlactational involution shares similarities with inflammation, including high matrix metalloproteinase activity, fibrillar collagen deposition, and release of bioactive fragments of fibronectin and laminin. Because inflammation can promote tumorigenesis, we evaluated whether the tissue microenvironment of the involuting gland is also promotional. Extracellular matrix was isolated from mammary glands of nulliparous rats or rats with mammary glands undergoing weaning-induced involution. Using these matrices as substratum, nulliparous matrix was found to promote ductal organization of normal mammary epithelial MCF-12A cells in three-dimensional culture and to suppress invasion of mammary tumor MDA-MB-231 cells in transwell filter assays. Conversely, involution matrix failed to support ductal development in normal cells and promoted invasiveness in tumor cells. To evaluate the effects of these matrices on metastasis in vivo, MDA-MB-231 cells, premixed with Matrigel, nulliparous matrix, or involution matrix, were injected into mammary fat pads of nude mice. Metastases to lung, liver, and kidney were increased in the involution matrix group, and correlated with a twofold increase in tumor vascular endothelial growth factor expression and increased angiogenesis. These data suggest that the mammary gland microenvironment becomes promotional for tumor cell dissemination during involution, thus providing a plausible mechanism to explain the high rate of metastases that occur with pregnancy-associated breast cancer.


Assuntos
Neoplasias da Mama/patologia , Neoplasias Renais/secundário , Lactação/fisiologia , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Glândulas Mamárias Animais/fisiologia , Animais , Colágeno/metabolismo , Combinação de Medicamentos , Matriz Extracelular/patologia , Feminino , Humanos , Neoplasias Renais/patologia , Laminina/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica/patologia , Neovascularização Patológica , Gravidez , Prenhez , Proteoglicanas/metabolismo , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Int J Cancer ; 114(6): 1002-9, 2005 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-15645422

RESUMO

1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) is a deoxycytidine analog that has been shown previously to have impressive anti-proliferative and cytotoxic effects in vitro and in vivo toward colorectal and gastric tumors. In our present studies, the pharmacokinetic behavior in nude mice and the effectiveness of 4'-thio-FAC against human pancreatic and ovarian tumor growth were assessed in comparison with standard chemotherapeutic agents. Potent in vitro anti-proliferative effects were observed against pancreatic (Capan-1, MIA-PaCa-2, BxPC-3) and ovarian (SK-OV-3, OVCAR-3, ES-2) cancer cell lines with IC(50) of 0.01-0.2 microM. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously (s.c.) implanted human pancreatic tumor xenografts or intraperitoneally (i.p.) disseminated human ovarian xenografted tumors. Oral daily administration of 4'-thio-FAC for 8-10 days significantly inhibited the growth of gemcitabine-resistant BxPC-3 pancreatic tumors and induced regression of gemcitabine-refractory Capan-1 tumors. 4'-Thio-FAC was also a highly effective inhibitor of ovarian peritoneal carcinomatosis. In the SK-OV-3 and ES-2 ovarian cancer models, 4'-thio-FAC prolonged survival to a greater extent than that observed with gemcitabine. Furthermore, the superiority of 4'-thio-FAC to carboplatin and paclitaxel was demonstrated in the ES-2 clear cell ovarian carcinoma model. Studies provide evidence that 4'-thio-FAC is a promising new alternative to gemcitabine and other chemotherapeutic drugs in the treatment of a variety of tumor indications, including pancreatic and ovarian carcinoma.


Assuntos
Carcinoma/tratamento farmacológico , Citarabina/análogos & derivados , Citarabina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Carcinoma/patologia , Carcinoma/veterinária , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Nus , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/veterinária , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/veterinária , Transplante Heterólogo , Células Tumorais Cultivadas
10.
J Biol Chem ; 280(20): 19867-74, 2005 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-15772071

RESUMO

The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.


Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Domínio Catalítico , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HeLa , Humanos , Técnicas In Vitro , Cinética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Melanoma Experimental/secundário , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Pirimidinas/química , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
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