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1.
Cancers (Basel) ; 16(10)2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38791979

RESUMO

The vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key regulators of blood vessel formation, including in tumors, where their deregulated function can promote the production of aberrant, leaky blood vessels, supporting tumor development. Here we investigated the VEGFR1 ligand VEGF-B, which we demonstrate to be expressed in tumor cells and in tumor stroma and vasculature across a range of tumor types. We examined the anti-VEGF-B-specific monoclonal antibody 2H10 in preclinical xenograft models of breast and colorectal cancer, in comparison with the anti-VEGF-A antibody bevacizumab. Similar to bevacizumab, 2H10 therapy was associated with changes in tumor blood vessels and intra-tumoral diffusion consistent with normalization of the tumor vasculature. Accordingly, treatment resulted in partial inhibition of tumor growth, and significantly improved the response to chemotherapy. Our studies indicate the importance of VEGF-B in tumor growth, and the potential of specific anti-VEGF-B treatment to inhibit tumor development, alone or in combination with established chemotherapies.

2.
Brain ; 134(Pt 4): 1089-100, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21436112

RESUMO

While considerable effort has focused on developing positron emission tomography ß-amyloid imaging radiotracers for the early diagnosis of Alzheimer's disease, no radiotracer is available for the non-invasive quantification of tau. In this study, we detail the characterization of (18)F-THK523 as a novel tau imaging radiotracer. In vitro binding studies demonstrated that (18)F-THK523 binds with higher affinity to a greater number of binding sites on recombinant tau (K18Δ280K) compared with ß-amyloid(1-42) fibrils. Autoradiographic and histofluorescence analysis of human hippocampal serial sections with Alzheimer's disease exhibited positive THK523 binding that co-localized with immunoreactive tau pathology, but failed to highlight ß-amyloid plaques. Micro-positron emission tomography analysis demonstrated significantly higher retention of (18)F-THK523 (48%; P < 0.007) in tau transgenic mice brains compared with their wild-type littermates or APP/PS1 mice. The preclinical examination of THK523 has demonstrated its high affinity and selectivity for tau pathology both in vitro and in vivo, indicating that (18)F-THK523 fulfils ligand criteria for human imaging trials.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Compostos de Anilina/farmacologia , Encéfalo/diagnóstico por imagem , Fluordesoxiglucose F18/farmacologia , Quinolinas/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Análise de Variância , Animais , Autorradiografia , Sítios de Ligação , Encéfalo/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Cintilografia
3.
Proc Natl Acad Sci U S A ; 106(13): 5082-7, 2009 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-19289842

RESUMO

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR(287-302) complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR(C271A/C283A) mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR(C271A/C283A). Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Receptores ErbB/imunologia , Proteínas de Neoplasias/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Complexo Antígeno-Anticorpo/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cristalografia por Raios X , Epitopos , Humanos , Camundongos , Camundongos Nus , Conformação Proteica , Desnaturação Proteica/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
FASEB J ; 19(7): 780-2, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15774576

RESUMO

Overexpression of the EGFR is commonly caused by EGFR gene amplification and is sometimes associated with expression of a variant EGFR (de2-7 EGFR or EGFRvIII) bearing an internal deletion in its extracellular domain. mAb 806 is a novel EGFR antibody with significant antitumor activity that recognizes both the de2-7 EGFR and a subset of the wild-type (wt) EGFR when overexpressed, but does not bind the EGFR expressed in normal tissues. Recently, we demonstrated that the mAb 806 epitope is restricted to a short cysteine loop of the EGFR (amino acids 287-302) that is only available for antibody binding in a transitional form of the receptor, which occurs as the receptor changes from its inactive tethered conformation to a dimeric untethered form. The truncation associated with the de2-7 EGFR mutation renders this receptor constitutively untethered, leading to increased binding of mAb 806. We now show that mAb 806 preferentially binds the immature high-mannose wt and de2-7 EGFR precursors normally located in the endoplasmic reticulum, indicating that this form of the wt EGFR is also constitutively untethered. Using the unique specificity of mAb 806, we clearly demonstrated the presence of these high-mannose EGFR precursors on the cell surface. Given that the high-mannose forms of the wt EGFR must be untethered they may contribute to the spontaneous EGFR signaling reported in cells overexpressing the receptor. These precursor forms of the EGFR thus represent novel tumor targets and contribute to the exceptional selectivity of mAb 806 for EGFR when overexpressed in cancer cells. As our observations are likely to apply to other receptors overexpressed in cancer, they suggest a strategy for developing antitumor antibodies even when the target receptor is expressed in normal tissue.


Assuntos
Anticorpos Monoclonais/imunologia , Membrana Celular/química , Receptores ErbB/genética , Receptores ErbB/imunologia , Expressão Gênica , Manose/análise , Especificidade de Anticorpos , Astrocitoma , Carcinoma de Células Escamosas , Linhagem Celular Tumoral , Dimerização , Retículo Endoplasmático/química , Epitopos/química , Epitopos/imunologia , Receptores ErbB/química , Amplificação de Genes , Glicosilação , Neoplasias de Cabeça e Pescoço , Humanos , Técnicas de Imunoadsorção , Relação Estrutura-Atividade
5.
Nucl Med Biol ; 41(5): 419-25, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24767600

RESUMO

[¹8F]FMISO is the most widely validated PET radiotracer for imaging hypoxic tissue. However, as a result of the pharmacokinetics of [¹8F]FMISO a 2h wait between tracer administration and patient scanning is required for optimal image acquisition. In order to develop hypoxia imaging agents with faster kinetics, we have synthesised and evaluated several F-18 labelled anilino sulfoxides. In this manuscript we report on the synthesis, in vitro and in vivo evaluation of a novel fluoroethyltriazolyl propargyl anilino sulfoxide. The radiolabelling of the novel tracer was achieved via 2-[¹8F]fluoroethyl azide click chemistry. Radiochemical yields were 23 ± 4% based on 2-[¹8F]fluoroethyl azide and 7 ± 2% based on K[¹8F]F. The radiotracer did not undergo metabolism or defluorination in an in vitro assay using S9 liver fractions. Imaging studies using SK-RC-52 tumors in BALB/c nude mice have indicated that the tracer may have a higher pO2 threshold than [¹8F]FMISO for uptake in hypoxic tumors. Although clearance from muscle was faster than [¹8F]FMISO, uptake in hypoxic tumors was slower. The average tumor to muscle ratio at 2h post injection in large, hypoxic tumors with a volume greater than 686 mm³ was 1.7, which was similar to the observed ratio of 1.75 for [¹8F]FMISO. Although the new tracer showed improved pharmacokinetics when compared with the previously synthesised sulfoxides, further modifications to the chemical structure need to be made in order to offer significant in vivo imaging advantages over [¹8F]FMISO.


Assuntos
Neoplasias Renais/patologia , Tomografia por Emissão de Pósitrons/métodos , Sulfóxidos , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Humanos , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo , Camundongos , Radioquímica , Sulfóxidos/química , Sulfóxidos/farmacocinética
6.
Cancer Res ; 74(16): 4470-81, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25125683

RESUMO

Eph receptor tyrosine kinases are critical for cell-cell communication during normal and oncogenic tissue patterning and tumor growth. Somatic mutation profiles of several cancer genomes suggest EphA3 as a tumor suppressor, but its oncogenic expression pattern and role in tumorigenesis remain largely undefined. Here, we report unexpected EphA3 overexpression within the microenvironment of a range of human cancers and mouse tumor xenografts where its activation inhibits tumor growth. EphA3 is found on mouse bone marrow-derived cells with mesenchymal and myeloid phenotypes, and activation of EphA3(+)/CD90(+)/Sca1(+) mesenchymal/stromal cells with an EphA3 agonist leads to cell contraction, cell-cell segregation, and apoptosis. Treatment of mice with an agonistic α-EphA3 antibody inhibits tumor growth by severely disrupting the integrity and function of newly formed tumor stroma and microvasculature. Our data define EphA3 as a novel target for selective ablation of the tumor microenvironment and demonstrate the potential of EphA3 agonists for anticancer therapy.


Assuntos
Anticorpos Monoclonais/farmacologia , Receptores Proteína Tirosina Quinases/agonistas , Receptores Proteína Tirosina Quinases/biossíntese , Receptor EphA3/agonistas , Receptor EphA3/biossíntese , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphA3/imunologia , Receptor EphA3/metabolismo , Transdução de Sinais , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Microambiente Tumoral/efeitos dos fármacos
7.
Clin Cancer Res ; 19(21): 5984-93, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24045184

RESUMO

PURPOSE: CS-1008 (tigatuzumab; phase I/II), an antihuman death receptor 5 (DR5) agonist, induces apoptosis and has cytotoxic activity against human cancer cell lines. This study reports on the preclinical validation of (111)In-labeled anti-DR5 humanized antibody CS-1008 as a diagnostic tool to study the DR5 occupancy in patients with cancer and establish dose ranges for receptor saturation kinetics in vivo. EXPERIMENTAL DESIGN: CS-1008 was radiolabeled and characterized for DR5 binding and labeling efficiency on TRAIL-sensitive DR5-positive colorectal cancer cells (COLO 205 and WiDr). Pharmacokinetic and biodistribution studies were conducted in BALB/c nu/nu mice bearing COLO 205, WiDr, or DR5-negative CT26 colon tumors. Planar gamma camera imaging and computerized tomography (CT) images were obtained to study receptor occupancy in vivo. RESULTS: Scatchard analysis showed high and specific binding affinity (Kd, 1.05 ± 0.12 nmol/L) of (111)In-labeled CS-1008. (111)In-labeled CS-1008 was specifically taken up in mice bearing COLO 205 and WiDr tumors with prolonged tumor retention (26.25 ± 2.85%ID/g vs. 12.20 ± 2.24 at 168 hours post injection; n = 5, SD), and uptake correlated both with DR5 expression on tumor cells and antitumor activity. DR5 saturation was shown in vivo via both biodistribution studies and planar gamma camera imaging/CT imaging of (111)In-labeled CS-1008. Saturation of DR5 corresponded to maximal in vivo antitumor efficacy. CONCLUSIONS: Imaging of DR5 receptor occupancy in vivo correlates with tumor concentration and in vivo efficacy, and is a novel molecular imaging technique that can be used to determine receptor occupancy and effective dose levels of DR5 agonist antibodies in the clinic.


Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Imagem Molecular , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacocinética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Radioisótopos de Índio , Marcação por Isótopo , Cinética , Camundongos , Ligação Proteica , Cintilografia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Distribuição Tecidual
8.
Nucl Med Biol ; 39(6): 871-82, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22336372

RESUMO

The significance of imaging hypoxia with the PET ligand [(18)F]FMISO has been demonstrated in a variety of cancers. However, the slow kinetics of [(18)F]FMISO require a 2-h delay between tracer administration and patient scanning. Labelled chloroethyl sulfoxides have shown faster kinetics and higher contrast than [(18)F]FMISO in a rat model of ischemic stroke. However, these nitrogen mustard analogues are unsuitable for routine production and use in humans. Here we report on the synthesis and in vitro and in vivo evaluation of two novel sulfoxides which we synthesised from a single precursor molecule via either 2-[(18)F]fluoroethyl azide click chemistry or conventional nucleophilic displacement of a chloride leaving group. The yields of the click chemistry approach were 90±5% of [(18)F]2 based on 2-[(18)F]fluoroethyl azide, and the yields for the S(N) reaction were 15±5% of [(18)F]1 based on K[(18)F]F. Both radiotracers underwent metabolism in an in vitro assay using S9 liver fractions with biological half-lives of 32.39 and 43.32 min, respectively. Imaging studies using an SK-RC-52 tumor model in BALB/c nude mice have revealed that only [(18)F]1 is retained in hypoxic tumors, whereas [(18)F]2 is cleared from those tumors at a rate similar to that of muscle tissue. [(18)F]1 has emerged as a promising new lead structure for further development of sulfoxide-based hypoxia imaging agents. In particular, the mechanism of uptake needs to be elucidated and changes to the chemical structure need to be made in order to reduce metabolism and improve radiotracer kinetics.


Assuntos
Carcinoma de Células Renais/diagnóstico por imagem , Carcinoma de Células Renais/patologia , Tomografia por Emissão de Pósitrons/métodos , Sulfóxidos/química , Animais , Carcinoma de Células Renais/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo , Camundongos , Radioquímica , Ratos , Sulfóxidos/metabolismo , Sulfóxidos/farmacocinética
9.
Proc Natl Acad Sci U S A ; 102(52): 19051-6, 2005 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-16365285

RESUMO

In this study, human T cells were provided with a reactivity against the Lewis-Y (Le(Y)) carbohydrate antigen, which is overexpressed on 70% of epithelial-derived tumors, but not normally recognized by T cells. Antitumor reactivity was achieved by transduction of T cells with a gene encoding a cell-surface chimeric receptor composed of single-chain anti-Le(Y) antibody linked to an enhanced cytoplasmic signaling domain made up of CD28 and CD3-zeta. Importantly, the single-chain antibody was humanized to try to reduce potential problems of human anti-mouse antibody responses in patients receiving chimeric receptor-modified T cells in future clinical trials. T cells expressing the chimeric receptor were demonstrated to secrete cytokines and proliferate in response to receptor ligation and lysed Le(Y+) tumors in vitro. Another aspect of this study was the finding that no activity was observed against normal tissue, as represented by autologous neutrophils that express low levels of Le(Y). Significantly, systemic delivery of anti-Le(Y) T cells dramatically inhibited established s.c. human ovarian OVCAR-3 tumors (a recognized difficult model to treat) in mice. Finally, we demonstrated that anti-Le(Y) T cells preferentially expanded or accumulated in the tumor compared with control empty vector T cells, thereby providing mechanistic insight into the specific antitumor response. This study supports the use of humanized gene-modified T cells as a potential therapy for Le(Y+) malignancies.


Assuntos
Transferência Adotiva , Imunoterapia/métodos , Antígenos do Grupo Sanguíneo de Lewis/química , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/citologia , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Autoimunidade , Antígenos CD28/biossíntese , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Ensaios Clínicos como Assunto , Citocinas/metabolismo , Citometria de Fluxo , Vetores Genéticos , Humanos , Imuno-Histoquímica , Leucócitos Mononucleares/citologia , Camundongos , Transplante de Neoplasias , Neoplasias/metabolismo , Neutrófilos/metabolismo , Proteínas Recombinantes de Fusão/química , Retroviridae/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Linfócitos T/metabolismo , Fatores de Tempo
10.
Hybrid Hybridomics ; 22(4): 219-28, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-14511567

RESUMO

The chimeric monoclonal antibody ch806 specifically targets the tumor-associated mutant epidermal growth factor receptor (de 2-7EGFR or EGFRVIII) and is currently under investigation for its potential use in cancer therapy. The humanised monoclonal antibody hu3S193 specifically targets the Lewis Y epithelial antigen and is currently in Phase I clinical trials in patients with advanced breast, colon, and ovarian carcinomas. To assist the clinical evaluation of ch806 and hu3S193, laboratory assays are required to monitor their serum pharmacokinetics and quantitate any immune responses to the antibodies. Mice immunized with ch806 or hu3S193 were used to generate hybridomas producing antibodies with specific binding to ch806 or hu3S193 and competitive for antigen binding. These anti-idiotype antibodies (designated Ludwig Melbourne Hybridomas, LMH) were investigated as reagents suitable for use as positive controls for HAHA or HACA analyses and for measuring hu3S193 or ch806 in human serum. Anti-idiotypes with the ability to concurrently bind two target antibody molecules were identified, which enabled the development of highly reproducible, sensitive, specific ELISA assays for determining serum concentrations of hu3S193 and ch806 with a 3 ng/mL limit of quantitation using LMH-3 and LMH-12, respectively. BIAcore analyses determined high apparent binding affinity for both idiotypes: LMH-3 binding immobilized hu3S193, Ka = 4.76 x 10(8) M(-1); LMH-12 binding immobilised ch806, Ka = 1.74 x 10(9) M(-1). Establishment of HAHA or HACA analysis of sera samples using BIAcore was possible using LMH-3 and LMH-12 as positive controls for quantitation of immune responses to hu3S193 or ch806 in patient sera. These anti-idiotypes could also be used to study the penetrance and binding of ch806 or hu3S193 to tumor cells through immunohistochemical analysis of tumor biopsies. The generation of anti-idiotype antibodies capable of concurrently binding a target antibody on each variable domain provides reagents with high sensitivity for the assessment of safety and pharmacokinetic profiles of target antibodies administered clinically.


Assuntos
Anticorpos Anti-Idiotípicos/biossíntese , Anticorpos Anti-Idiotípicos/uso terapêutico , Imunoterapia , Laboratórios , Animais , Afinidade de Anticorpos , Linhagem Celular Tumoral , Células Clonais/imunologia , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hibridomas/imunologia , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmocitoma/imunologia , Plasmocitoma/patologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Reprodutibilidade dos Testes , Soro/imunologia , Ressonância de Plasmônio de Superfície
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