RESUMO
Cancer cell vascular invasion is a crucial step in the malignant progression toward metastasis. Here we used a genome-wide RNA interference screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these Krtaps have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/ß4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a Krtap can serve as a switch toward malignant progression.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Neoplasias Mamárias Experimentais/irrigação sanguínea , Animais , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Queratinas/metabolismo , Masculino , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Peixe-ZebraRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a high degree of inflammation and profound immune suppression. Here we identify Yes-associated protein (Yap) as a critical regulator of the immunosuppressive microenvironment in both mouse and human PDAC. Within Kras:p53 mutant pancreatic ductal cells, Yap drives the expression and secretion of multiple cytokines/chemokines, which in turn promote the differentiation and accumulation of myeloid-derived suppressor cells (MDSCs) both in vitro and in vivo. Pancreas-specific knockout of Yap or antibody-mediated depletion of MDSCs promoted macrophage reprogramming, reactivation of T cells, apoptosis of Kras mutant neoplastic ductal cells and pancreatic regeneration after acute pancreatitis. In primary human PDAC, YAP expression levels strongly correlate with an MDSC gene signature, and high expression of YAP or MDSC-related genes predicts decreased survival in PDAC patients. These results reveal multifaceted roles of YAP in PDAC pathogenesis and underscore its promise as a therapeutic target for this deadly disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Adenocarcinoma/imunologia , Carcinoma Ductal Pancreático/imunologia , Inflamação/imunologia , Neoplasias Pancreáticas/imunologia , Pancreatite/imunologia , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Doença Aguda , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proteínas de Ciclo Celular , Diferenciação Celular , Proliferação de Células , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Mutação/genética , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/patologia , Estadiamento de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/metabolismo , Pancreatite/patologia , Fosfoproteínas/genética , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Taxa de Sobrevida , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Fatores de Transcrição , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAP , Neoplasias PancreáticasRESUMO
Resistance to therapies targeting the estrogen pathway remains a challenge in the treatment of estrogen receptor-positive breast cancer. To address this challenge, a systems biology approach was used. A library of small interfering RNAs targeting an estrogen receptor (ER)- and aromatase-centered network identified 46 genes that are dispensable in estrogen-dependent MCF7 cells, but are selectively required for the survival of estrogen-independent MCF7-derived cells and multiple additional estrogen-independent breast cancer cell lines. Integration of this information identified a tumor suppressor gene TOB1 as a critical determinant of estrogen-independent ER-positive breast cell survival. Depletion of TOB1 selectively promoted G1 phase arrest and sensitivity to AKT and mammalian target of rapmycin (mTOR) inhibitors in estrogen-independent cells but not in estrogen-dependent cells. Phosphoproteomic profiles from reverse-phase protein array analysis supported by mRNA profiling identified a significant signaling network reprogramming by TOB1 that differed in estrogen-sensitive and estrogen-resistant cell lines. These data support a novel function for TOB1 in mediating survival of estrogen-independent breast cancers. These studies also provide evidence for combining TOB1 inhibition and AKT/mTOR inhibition as a therapeutic strategy, with potential translational significance for the management of patients with ER-positive breast cancers.
Assuntos
Neoplasias da Mama/patologia , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Estrogênios/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células MCF-7 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The therapeutic utility of antibodies and their derivatives is achieved by various means. The FDA has approved several targeted antibodies that disrupt signaling of various growth factor receptors for the treatment of a number of cancers. Rituximab, and other anti-CD20 monoclonal antibodies are active in B cell malignancies. As more experience has been gained with anti-CD20 monoclonal antibodies, the multifactorial nature of their anti-tumor mechanisms has emerged. Other targeted antibodies function to dampen inhibitory checkpoints. These checkpoint inhibitors have recently achieved dramatic results in several cancers, including melanoma. These and related antibodies continue to be investigated in the clinical and pre-clinical settings. Novel antibody structures that target two or more antigens have also made their way into clinical use. Tumor targeted antibodies can also be conjugated to chemo- or radiotherapeutic agents, or catalytic toxins, as a means to deliver toxic payloads to cancer cells. Here we provide a review of these mechanisms and a discussion of their relevance to current and future clinical applications.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoterapia/métodos , Neoplasias/terapia , Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Antígenos CD20/metabolismo , Antígeno CTLA-4/imunologia , Antígeno CTLA-4/metabolismo , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Humanos , Imunoterapia/tendências , Neoplasias/imunologia , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Resultado do TratamentoRESUMO
Met is a receptor tyrosine kinase that promotes cancer progression. In addition, Met has been implicated in resistance of tumors to various targeted therapies such as epidermal growth factor receptor inhibitors in lung cancers, and has been prioritized as a key molecular target for cancer therapy. However, the underlying mechanism of resistance to Met-targeting drugs is poorly understood. Here, we describe screening of 1310 genes to search for key regulators related to drug resistance to an anti-Met therapeutic antibody (SAIT301) by using a small interfering RNA-based synthetic lethal screening method. We found that knockdown of 69 genes in Met-amplified MKN45 cells sensitized the antitumor activity of SAIT301. Pathway analysis of these 69 genes implicated fibroblast growth factor receptor (FGFR) as a key regulator for antiproliferative effects of Met-targeting drugs. Inhibition of FGFR3 increased target cell apoptosis through the suppression of Bcl-xL expression, followed by reduced cancer cell growth in the presence of Met-targeting drugs. Treatment of cells with the FGFR inhibitors substantially restored the efficacy of SAIT301 in SAIT301-resistant cells and enhanced the efficacy in SAIT301-sensitive cells. In addition to FGFR3, integrin ß3 is another potential target for combination treatment with SAIT301. Suppression of integrin ß3 decreased AKT phosphorylation in SAIT301-resistant cells and restored SAIT301 responsiveness in HCC1954 cells, which are resistant to SAIT301. Gene expression analysis using CCLE database shows that cancer cells with high levels of FGFR and integrin ß3 are resistant to crizotinib treatment, suggesting that FGFR and integrin ß3 could be used as predictive markers for Met-targeted therapy and provide a potential therapeutic option to overcome acquired and innate resistance for the Met-targeting drugs.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genética , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Crizotinibe , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Biblioteca de Peptídeos , Pirazóis/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Increased activity of SRC family kinases promotes tumor invasion and metastasis, and overexpression of the mitotic regulator Aurora kinase A (AURKA) drives tumor aneuploidy and chromosomal instability. These functions nominate SRC and AURKA as valuable therapeutic targets for cancer, and inhibitors for SRC and Aurora kinases are now being used in the clinic. In this study, we demonstrate potent synergy between multiple inhibitors of Aurora and SRC kinases in ovarian and colorectal cancer cell lines, but not in normal ovarian epithelial cell lines. Combination of Aurora and SRC inhibitors selectively killed cells that have undergone a preceding aberrant mitosis, and was associated with a postmitotic reattachment defect, and selective removal of aneuploid cell populations. Combined inhibition of Aurora kinase and SRC potentiated dasatinib-dependent loss of activated (Y(416)-phosphorylated) SRC. SRC and AURKA share a common interaction partner, NEDD9, which serves as a scaffolding protein with activities in cell attachment and mitotic control, suggesting SRC and AURKA might interact directly. In vitro, we observed physical interaction and mutual cross-phosphorylation between SRC and AURKA that enhanced SRC kinase activity. Together, these findings suggest that combination of SRC and Aurora-targeting inhibitors in the clinic may be a productive strategy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinases da Família src/antagonistas & inibidores , Aurora Quinase A , Aurora Quinases , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dasatinibe , Feminino , Humanos , Mitose/efeitos dos fármacos , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Tiazóis/farmacologia , Quinases da Família src/fisiologiaRESUMO
A series of novel derivatives of neuropeptides with a metal-chelating moiety was synthesized and examined for various properties related to iron (Fe) chelation and neuroprotective action. All derivatives chelated Fe to form stable Fe complexes in water. Some strongly inhibited Fe-induced lipid peroxidation with an IC(50) value of about 12 microm. In PC12 cell culture, several compounds, at concentrations as low as 1 microm, attenuated serum-free stimulated cell death and improved cell survival by 20-35%. At this concentration, these analogs also protected against 6-hydroxydopamine (6-OHDA)-induced cell death, increasing cell viability by 20-30%. Electron paramagnetic resonance (EPR) studies indicated that besides being good Fe chelators, these analogs act as radical scavengers to directly scavenge hydroxyl radicals. Together, the data indicate that some of the analogs could be further developed as possible neuroprotective agents for treatment of neurodegenerative diseases such as Parkinson's, Alzheimer's, and Huntington's diseases, Friedreich's atxia, amyotrophic, and lateral sclerosis where Fe misregulation has been reported.
Assuntos
Quelantes/síntese química , Doenças Neurodegenerativas/tratamento farmacológico , Neuropeptídeos/síntese química , Animais , Quelantes/farmacologia , Sequestradores de Radicais Livres/metabolismo , Metais Pesados/metabolismo , Doenças Neurodegenerativas/metabolismo , Neuropeptídeos/metabolismo , Neuropeptídeos/farmacologia , Células PC12 , RatosRESUMO
Acetylcholinesterase (AChE) has been found to be associated with the core of senile plaques. We have shown that AChE interacts with the amyloid beta-peptide (Abeta) and promotes amyloid fibril formation by a hydrophobic environment close to the peripheral anionic binding site (PAS) of the enzyme. Here we present evidence for the structural motif of AChE involved in this interaction. First, we modeled the docking of Abeta onto the structure of Torpedo californica AChE, and identified four potential sites for AChE-Abeta complex formation. One of these, Site I, spans a major hydrophobic sequence exposed on the surface of AChE, which had been previously shown to interact with liposomes [Shin et al. (1996) Protein Sci. 5, 42-51]. Second, we examined several AChE-derived peptides and found that a synthetic 35-residue peptide corresponding to the above hydrophobic sequence was able to promote amyloid formation. We also studied the ability to promote amyloid formation of two synthetic 24-residue peptides derived from the sequence of a Omega-loop, which has been suggested as an AChE-Abeta interacting motif. Kinetic analyses indicate that only the 35-residue hydrophobic peptide mimics the effect of intact AChE on amyloid formation. Moreover, RP-HPLC analysis revealed that the 35-residue peptide was incorporated into the growing Abeta-fibrils. Finally, fluorescence binding studies showed that this peptide binds Abeta with a K(d) = 184 microM, independent of salt concentration, indicating that the interaction is primarily hydrophobic. Our results indicate that the homologous human AChE motif is capable of accelerating Abeta fibrillogenesis.
Assuntos
Acetilcolinesterase/química , Peptídeos beta-Amiloides/química , Placa Amiloide/química , Acetilcolinesterase/isolamento & purificação , Sequência de Aminoácidos , Animais , Química Encefálica , Bovinos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Placa Amiloide/ultraestrutura , Conformação Proteica , Solubilidade , TorpedoRESUMO
Antitumor monoclonal antibodies must bind to tumor antigens with high affinity to achieve durable tumor retention. This has spurred efforts to generate high affinity antibodies for use in cancer therapy. However, it has been hypothesized that very high affinity interactions between antibodies and tumor antigens may impair efficient tumor penetration of the monoclonal antibodies and thus diminish effective in vivo targeting (K. Fujimori et al., J. Nucl. Med., 31: 1191-1198, 1990). Here we show that intrinsic affinity properties regulate the quantitative delivery of antitumor single-chain Fv (scFv) molecules to solid tumors and the penetration of scFv from the vasculature into tumor masses. In biodistribution studies examining a series of radioiodinated scFv mutants with affinities ranging from 10(-7)-10(-11) M, quantitative tumor retention did not significantly increase with enhancements in affinity beyond 10(-9) M. Similar distribution patterns were observed when the scFv were evaluated in the absence of renal clearance in anephric mice, indicating that the rapid renal clearance of the scFv was not responsible for these observations. IHC and IF evaluations of tumor sections after the i.v. administration of scFv affinity mutants revealed that the lowest affinity molecule exhibited diffuse tumor staining whereas the highest affinity scFv was primarily retained in the perivascular regions of the tumor. These results indicate that antibody-based molecules with extremely high affinity have impaired tumor penetration properties that must be considered in the design of antibody-based cancer therapies.
Assuntos
Afinidade de Anticorpos , Antígenos de Neoplasias/imunologia , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/metabolismo , Neoplasias Ovarianas/metabolismo , Receptor ErbB-2/imunologia , Animais , Antígenos de Neoplasias/metabolismo , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Humanos , Rim/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos , Camundongos SCID , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/imunologia , Coelhos , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We tested the hypothesis that bispecific Abs (Bsab) with increased binding affinity for tumor Ags augment retargeted antitumor cytotoxicity. We report that an increase in the affinity of Bsab for the HER2/neu Ag correlates with an increase in the ability of the Bsab to promote retargeted cytotoxicity against HER2/neu-positive cell lines. A series of anti-HER2/neu extracellular domain-directed single-chain Fv fragments (scFv), ranging in affinity for HER2/neu from 10(-7) to 10(-11) M, were fused to the phage display-derived NM3E2 human scFV: NM3E2 associates with the extracellular domain of human FcgammaRIII (CD16). The resulting series of Bsab promoted cytotoxicity of SKOV3 human ovarian carcinoma cells overexpressing HER2/neu by human PBMC preparations containing CD16-positive NK cells. The affinity for HER2/neu clearly influenced the ability of the Bsab to promote cytotoxicity of (51)Cr-labeled SKOV3 cells. Lysis was 6.5% with an anti-HER2/neu K(D) = 1.7 x 10(-7) M, 14.5% with K(D) = 5.7 x 10(-9) M, and 21.3% with K(D) = 1.7 x 10(-10) M at 50:1 E:T ratios. These scFv-based Bsab did not cross-link receptors and induce leukocyte calcium mobilization in the absence of tumor cell engagement. Thus, these novel Bsab structures should not induce the dose-limiting cytokine release syndromes that have been observed in clinical trials with intact IgG BSAB: Additional manipulations in Bsab structure that improve selective tumor retention or facilitate the ability of Bsab to selectively cross-link tumor and effector cells at tumor sites should further improve the utility of this therapeutic strategy.
Assuntos
Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/toxicidade , Anticorpos Biespecíficos/metabolismo , Anticorpos Biespecíficos/toxicidade , Citotoxicidade Celular Dependente de Anticorpos , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Sítios de Ligação de Anticorpos , Especificidade de Anticorpos , Sinalização do Cálcio/imunologia , Testes Imunológicos de Citotoxicidade , Humanos , Região Variável de Imunoglobulina/metabolismo , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Cinética , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Células Tumorais CultivadasRESUMO
PURPOSE: To investigate mechanism-directed regimens in maximizing the efficacy of fluorouracil (5-FU) in advanced colorected cancer. PATIENTS AND METHODS: Based on promising phase II data, a randomized comparison of various methods for the biochemical modulation of 5-FU was undertaken in patients with advanced colorectal cancer. The control group received single-agent 5-FU as a 24-hour infusion weekly. Patients (N = 1,120) with no prior chemotherapy for metastatic disease were randomized to one of the following arms: arm A, 5-FU 2,600 mg/m2 by 24-hour infusion, weekly; arm B, N-phosphonoacetyl-l-aspartic acid 250 mg/m2 day l, 5-FU 2,600 mg/m2 by 24-hour infusion day 2, weekly; arm C, 5-FU 600 mg/m2 with oral leucovorin (LV) 125 mg/m2 hourly for the preceding 4 hours, weekly; arm D, 5-FU 600 mg/m2 with intravenous (IV) LV 600 mg/m2, weekly; arm E, 5-FU 750 mg/m2/d IV by continuous infusion for 5 days, then 750 mg/m2 weekly, and recombinant interferon alfa-2a 9 million units subcutaneously three times weekly. Median follow-up was 4.8 years. RESULTS: Of the 1,098 assessable patients, 57% had measurable disease. The toxicity of all the regimens was tolerable. Grade 4 or worse toxicity occurred in 11%, 11%, 30%, 24%, and 22% on each arm, respectively; diarrhea was the most common adverse effect. These toxicity patterns favored significantly (P <.001) the 24-hour infusion arms. Median survival (months) by arm was A, 14.8; B, 11.9; C, 13.5; D, 13.6; and E, 15.2. These survival durations did not differ significantly. CONCLUSION: We conclude that a weekly infusion regimen of 5-FU is significantly less toxic than and as effective as 5-FU bolus regimens modulated by either LV or interferon in patients with metastatic colorectal cancer.
Assuntos
Antineoplásicos/administração & dosagem , Ácido Aspártico/análogos & derivados , Ácido Aspártico/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/administração & dosagem , Interferon-alfa/administração & dosagem , Leucovorina/administração & dosagem , Ácido Fosfonoacéticos/análogos & derivados , Ácido Fosfonoacéticos/administração & dosagem , Administração Oral , Idoso , Neoplasias Colorretais/mortalidade , Feminino , Fluoruracila/efeitos adversos , Humanos , Infusões Intravenosas , Interferon alfa-2 , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas RecombinantesRESUMO
Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to be an effective vaccine adjuvant because it enhances antigen processing and presentation by dendritic cells. ALVAC-CEA B7.1 is a canarypox virus encoding the gene for the tumor-associated antigen carcinoembryonic antigen (CEA) and for a T-cell costimulatory molecule, B7.1. After an initial dose escalation phase, this study evaluated vaccination with 4.5 x 10(8) plaque-forming units ALVAC-CEA B7.1 alone (n = 30) or with GM-CSF (n = 30) in patients with advanced CEA-expressing tumors to determine whether the addition of the adjuvant GM-CSF enhances induction of CEA-specific T-cells. Patients were vaccinated with vaccine intradermally every other week for 8 weeks. GM-CSF was given s.c. for 5 days beginning 2 days before vaccination. Patients with stable or responding disease after four immunizations received monthly boost injections alone or with GM-CSF. Biopsies of vaccine sites were obtained 48 h after vaccination to evaluate leukocytic infiltration and CEA expression. Induction of peripheral blood CEA-specific T-cell precursors was assessed in HLA-A2 positive patients by an ELISPOT assay looking for the production of IFN-gamma. Therapy was well tolerated. All of the patients had evidence of leukocytic infiltration and CEA expression in vaccine biopsy sites. In the patients receiving GM-CSF, leukocytic infiltrates were greater in cell number but were less likely to have a predominant lymphocytic infiltrate compared with patients receiving vaccine in the absence of the cytokine adjuvant. After four vaccinations, CEA-specific T-cell precursors were statistically increased in HLA-A2 positive patients who received vaccine alone. However, the GM-CSF plus vaccine cohort of HLA-A2 positive did not demonstrate a statistically significant increase in their CEA-specific T-cell precursor frequencies compared with baseline results. The number of prior chemotherapy regimens was negatively correlated with the generation of a T-cell response, whereas there was a positive correlation between the number of months from the last chemotherapy regimen and the T-cell response. ALVAC-CEA B7.1 is safe in patients with advanced, recurrent adenocarcinomas that express CEA, is associated with the induction of a CEA-specific T-cell response in patients treated with vaccine alone but not with vaccine and GM-CSF, and can lead to disease stabilization for up to 13 months.
Assuntos
Vacinas Anticâncer/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Neoplasias/tratamento farmacológico , Vacinas Sintéticas/uso terapêutico , Adulto , Idoso , Biópsia , Vacinas Anticâncer/efeitos adversos , Quimioterapia Adjuvante , Estudos de Coortes , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos adversos , Humanos , Imunidade/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Neoplasias/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Resultado do Tratamento , Vacinas Sintéticas/efeitos adversosRESUMO
Phage display technology makes possible the direct isolation of monovalent single-chain Fv antibody fragments. For many applications, however, it is useful to restore Fc mediated antibody functions such as avidity, effector functions and a prolonged serum half-life. We have constructed vectors for the convenient, rapid expression of a single-chain antibody Fv domain (scFv) fused to the Fc portion of human IgG1 in the methylotrophic yeast Pichia pastoris. The scFv-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The increased size of the dimer (approximately 106 kDa vs. approximately 25 kDa for a scFv) results in a prolonged serum half-life in vivo, with t(1/2) of the beta phase of clearance increasing from 3.5 h for a typical scFv to 93 h for a scFv-Fc fusion in mice. The scFv-Fc fusion is capable of mediating antibody-dependent cellular cytotoxicity against tumor target cells using human peripheral blood mononuclear cells as effectors. Finally, the Fc domain is a convenient, robust affinity handle for purification and immunochemical applications, eliminating the need for proteolytically sensitive epitope and/or affinity tags on the scFv.
Assuntos
Fragmentos Fc das Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/genética , Pichia/genética , Sequência de Aminoácidos , Animais , Citotoxicidade Celular Dependente de Anticorpos , Sequência de Bases , Primers do DNA/genética , Vetores Genéticos , Humanos , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/isolamento & purificação , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Masculino , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Biblioteca de Peptídeos , Plasmídeos/genética , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
To investigate the effect of adding tamoxifen to megestrol in the hormonal therapy for advanced endometrial cancer, 66 patients were entered in this study. Initially, 41 patients were randomized to either the standard progestin therapy of megestrol or to the combination of megestrol and tamoxifen between October 1982 and October 1984. The megestrol arm was terminated because of poor accrual and 25 patients were directly assigned to the combination arm. Among the 20 eligible cases on the megestrol arm, the response rate of 20% consisted of I complete response and 3 partial responses. The response rate on the megestrol plus tamoxifen arm was 19% with 1 (2%) complete response and 7 (17%) partial responses among 42 eligible cases. The median survival times were 12.0 months and 8.6 months, respectively. Only mild and moderate toxicities were observed on megestrol compared with more toxic complications observed on the combination of megestrol and tamoxifen, including a life-threatening case of pulmonary embolism. Although we could not carry out a comparative evaluation as intended, we conclude that the combination of megestrol and tamoxifen offers no clinical advantage over megestrol alone in the treatment of advanced endometrial carcinoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Endométrio/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias do Endométrio/patologia , Feminino , Humanos , Megestrol/administração & dosagem , Megestrol/efeitos adversos , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia/patologia , Modelos de Riscos Proporcionais , Taxa de Sobrevida , Tamoxifeno/administração & dosagemAssuntos
Neoplasias/radioterapia , Radioimunoterapia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos de Neoplasias/imunologia , Antígeno Carcinoembrionário/imunologia , Desenho de Fármacos , Radioisótopos de Flúor/farmacocinética , Radioisótopos de Flúor/uso terapêutico , Humanos , Fragmentos de Imunoglobulinas/imunologia , Radioisótopos do Iodo/farmacocinética , Radioisótopos do Iodo/uso terapêutico , Camundongos , Peso Molecular , Neoplasias/diagnóstico por imagem , Neoplasias/imunologia , Radioimunodetecção , Compostos Radiofarmacêuticos/farmacocinética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
PURPOSE: To determine the safety and feasibility of delivering multiple cycles of front-line high-dose carboplatin, paclitaxel, and topotecan with peripheral-blood stem-cell (PBSC) support. PATIENTS AND METHODS: Patients were required to have a malignant solid tumor for which they had received no prior chemotherapy. Mobilization of PBSC was achieved with either filgrastim alone or in combination with cyclophosphamide and paclitaxel. Patients then received three or four cycles of high-dose carboplatin (area under the concentration-time curve [AUC] 16), paclitaxel (250 mg/m(2)), and topotecan (10-15 mg/m(2)), with the latter two agents administered as 24-hour infusions and supported with PBSC and filgrastim. Cycles were repeated every 28 days. RESULTS: Twenty patients were enrolled onto the trial and were assessable for toxicity and clinical outcome. Dose-limiting toxicities were stomatitis and prolonged hematopoietic recovery. The maximum-tolerated dose of topotecan was 12.5 mg/m(2) when given with high-dose carboplatin and paclitaxel for three cycles. Four cycles were able to be given with a dose of topotecan of 10 mg/m(2). The pharmacokinetics of each compound were not affected by the other agents. Eleven (85%) of 13 patients with assessable disease responded. CONCLUSION: Multiple cycles of high-dose carboplatin, paclitaxel, and topotecan can be safely administered with filgrastim and PBSC support. The recommended doses for phase II study are carboplatin AUC 16, paclitaxel 250 mg/m(2), and topotecan 10 mg/m(2). Trials are currently being conducted with this regimen as front-line treatment in patients with advanced ovarian cancer and extensive small-cell carcinoma. This approach remains experimental and should be used only in the context of a clinical trial.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Neoplasias/terapia , Paclitaxel/administração & dosagem , Topotecan/administração & dosagem , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Esquema de Medicação , Feminino , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Neutropenia/induzido quimicamente , Estomatite/induzido quimicamenteRESUMO
In immunodeficient mice antitumor single-chain Fv (scFv) molecules penetrate tumors rapidly and have rapid serum clearance, leading to excellent tumor:normal organ ratios. However, the absolute quantity of scFv retained in the tumor is low due to rapid serum clearance and monovalent scFv binding. We previously demonstrated that the presence of an additional binding site prolongs in vitro and in vivo association of scFv-based molecules with tumor cells expressing relevant antigen. The contribution of the intrinsic affinity of each component scFv to the association between a dimeric scFv and its target antigen is largely unknown. Here, we have constructed bivalent diabody molecules from three affinity mutants of the human anti-ErbB2 (HER2/neu) scFv molecule C6.5 by shortening the peptide linker between the heavy (VH) and light (VL) chains variable domains from 15 to 5 amino acids. The shorter linker prevents intramolecular pairing of VH and VL, resulting in intermolecular pairing and creation of a dimeric Mr 50,000 molecule with two antigen-binding sites. The scFv used to create the diabodies span a 133-fold range of affinity for the same epitope of ErbB2 [133 nM (C6G98A), 25 nM (C6.5), and 1 nM (C6ML3-9)] and differ by only one to three amino acids. Diabody binding kinetics were determined by surface plasmon resonance on the immobilized ErbB2 extracellular domain. The association rate constants obtained for each diabody molecule were similar to that of the parental (component) scFv. However, the dissociation rate constants obtained for the bivalent diabodies were up to 15-fold slower. The magnitude of the decrease in the bivalent dissociation rate constant was inversely proportional to the monovalent interaction, ranging from only 3-fold for that of the C6ML3-9 diabody to 15-fold for the C6G98A diabody. This resulted in only a 22-fold difference in bivalent affinity, compared with a 133-fold difference in affinity for the respective scFv. Equilibrium-binding constants obtained by surface plasmon resonance correlated well with the equilibrium-binding constants determined in vitro on ErbB2 overexpressing cells. Biodistribution studies were performed in scid mice bearing established SKOV3 tumors. At 24 h, 3-37-fold more diabody was retained in tumor compared with the parental scFv monomers. This likely results from a higher apparent affinity, because of bivalent binding, and a slower serum clearance. Surprisingly, the differences in affinity between diabodies did not result in differences in quantitative tumor retention or tumor to blood ratios. In fact, the diabody constructed from the lowest affinity scFv exhibited the best tumor-targeting properties. We conclude that, above a threshold affinity, other factors regulate quantitative tumor retention. In addition, straightforward dimerization of a low-affinity scFv leads to significantly greater tumor localization than does exhaustive scFv affinity maturation.
Assuntos
Anticorpos Biespecíficos/imunologia , Afinidade de Anticorpos/imunologia , Fragmentos de Imunoglobulinas/imunologia , Neoplasias Ovarianas/imunologia , Receptor ErbB-2/imunologia , Animais , Anticorpos Biespecíficos/metabolismo , Anticorpos Biespecíficos/farmacocinética , Feminino , Humanos , Fragmentos de Imunoglobulinas/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Neoplasias Ovarianas/metabolismo , Ressonância de Plasmônio de Superfície , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Intravenously administered anti-tumor single-chain Fv (scFv) and diabody molecules exhibit rapid clearance kinetics and accumulation in tumors that express their cognate antigen. In an attempt to fit the rate of isotope decay to the timing of delivery and duration of tumor retention, anti-HER2/neu CHX-A" DTPA-C6.5K-A scFv and diabody conjugates were labeled with the alpha-particle emitter (213)Bi (t(1/2) = 47 min). Radioimmunotherapy studies employing 0.64, 0.35, or 0.15 microCi of (213)Bi-labeled C6.5K-A diabody or 1.1, 0.6, or 0. 3 microCi of (213)Bi-labeled C6.5K-A scFv were performed in nude mice bearing early, established SK-OV-3 tumors. Only the 0.3 microCi dose of (213)Bi-labeled C6.5K-A scFv resulted in both acceptable toxicity and a reduction in tumor growth rate. The specificity of the anti-tumor effects was determined by comparing the efficacy of treatment with 0.3 and 0.15 microCi doses of (213)Bi-labeled C6.5K-A scFv and (213)Bi-labeled NM3E2 (an irrelevant scFv) in nude mice bearing large established tumors. The 0.3 microCi dose of (213)Bi on both the C6.5K-A and NM3E2 scFvs resulted in similar anti-tumor effects (p = 0.46) indicating that antigen-specific targeting was not a factor. This suggests that the physical half-life of (213)Bi may be too brief to be effectively paired with systemically-administered diabody or scFv molecules.
Assuntos
Partículas alfa , Bismuto/uso terapêutico , Fragmentos de Imunoglobulinas/uso terapêutico , Neoplasias Experimentais/radioterapia , Radioimunoterapia , Animais , Masculino , Camundongos , Camundongos NusRESUMO
Perillyl alcohol (POH) is a monoterpene with anticarcinogenic and antitumor activity in murine tumor models. Putative mechanisms of action include activation of the transforming growth factor beta pathway and/or inhibition of p21ras signaling, leading to differentiation or apoptosis. In this Phase I trial, 17 patients took POH p.o. three times daily for 14 days of each 28-day cycle. The starting dose of POH was 1600 mg/m2/dose, with escalations to 2100 and 2800 mg/m2/dose in subsequent cohorts. Chronic nausea and fatigue were dose-limiting toxic effects at 2800 mg/m2. Grade 1-2 hypokalemia was common at 2100 and 2800 mg/m2. Although POH could not be detected in plasma, two of its metabolites, dihydroperillic acid (DHPA) and perillic acid (PA), were measured in plasma and urine on days 1 and 15 after the first and last doses of POH, respectively. Both area under the concentration versus time curve and peak plasma concentration (Cmax) values increased with dose and exhibited high intersubject variability. Day 15 DHPA Cmax values ranged from a mean +/- SD of 22.6+/-12 microM at 1600 mg/m2/dose to 42.4+/-15.24 microM at 2800 mg/m2/dose. Corresponding mean +/- SD Cmax values for PA were 433.2+/-245.8 and 774.1+/-439.6 microM. One patient treated at the 2800 mg/m2/dose had markedly prolonged plasma levels of both PA and DHPA and developed grade 3 mucositis. POH treatment did not consistently alter the expression of p21ras, rap1, or rhoA in peripheral blood mononuclear cells obtained from patients treated at the highest dose level. The metabolites PA and DHPA did not change expression or isoprenylation of p21ras in MCF-7 breast or DU145 prostate carcinoma cells at concentrations that exceeded those achieved in patient plasma after POH treatment. We conclude that POH at 1600-2100 mg/m2 p.o. three times daily is well tolerated on a 14-day on/14-day off dosing schedule. Inhibition of p21ras function in humans is not likely to occur after POH administration at safe doses of the present oral formulation.