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1.
Clin Cancer Res ; 22(12): 2874-84, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-26787751

RESUMO

PURPOSE: This first-in-human phase I trial assessed the safety, tolerability, and preliminary antitumor activity of apitolisib (GDC-0980), a dual inhibitor of class I PI3K, and mTOR kinases. EXPERIMENTAL DESIGN: Once-daily oral apitolisib was administered to patients with solid tumors for days 1 to 21 or 1 to 28 of 28-day cycles. Pharmacokinetic and pharmacodynamic parameters were assessed. RESULTS: Overall, 120 patients were treated at doses between 2 and 70 mg. The commonest ≥G3 toxicities related to apitolisib at the recommended phase 2 dose (RP2D) at 40 mg once daily included hyperglycemia (18%), rash (14%), liver dysfunction (12%), diarrhea (10%), pneumonitis (8%), mucosal inflammation (6%), and fatigue (4%). Dose-limiting toxicities (1 patient each) were G4 fasting hyperglycemia at 40 mg (21/28 schedule) and G3 maculopapular rash and G3 fasting hyperglycemia at 70 mg (21/28 schedule). The pharmacokinetic profile was dose-proportional. Phosphorylated serine-473 AKT levels were suppressed by ≥90% in platelet-rich plasma within 4 hours at the MTD (50 mg). Pharmacodynamic decreases in fluorodeoxyglucose positron emission tomography uptake of >25% occurred in 66% (21/32) of patients dosed at 40 mg once daily. Evidence of single-agent activity included 10 RECIST partial responses (PR; confirmed for peritoneal mesothelioma, PIK3CA mutant head-and-neck cancer, and three pleural mesotheliomas). CONCLUSIONS: Apitolisib exhibited dose-proportional pharmacokinetics with target modulation at doses ≥16 mg. The RP2D was 40 mg once-daily 28/28 schedule; severe on-target toxicities were apparent at ≥40 mg, particularly pneumonitis. Apitolisib was reasonably tolerated at 30 mg, the selected dose for pleural mesothelioma patients given limited respiratory reserve. Modest but durable antitumor activity was demonstrated. Clin Cancer Res; 22(12); 2874-84. ©2016 AACR.


Assuntos
Antineoplásicos/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/efeitos adversos , Pirimidinas/farmacocinética
2.
Hum Gene Ther ; 16(7): 881-92, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16000069

RESUMO

Genetic vaccinations, gene therapy, and manufacturing of therapeutic proteins would benefit from promoter sequences that provide improved or prolonged expression levels. The cytomegalovirus (CMV) promoter is one of the most potent promoters known to date, and no previous examples of improved activity of this promoter by sequence mutagenesis have been reported. This study describes directed molecular evolution of CMV promoters derived from two human and two nonhuman primate strains of CMV by DNA shuffling and screening. Libraries of chimeric promoters were screened and analyzed for expression levels and immune responses, using plasmid DNA vectors encoding luciferase and beta-galactosidase. The results indicate that high functional diversity among CMV promoters can be generated, and the chimeric promoters selected after two rounds of DNA shuffling and particularly designed screening assays provided approximately 2-fold increased luciferase reporter gene expression and anti-beta-galactoside antibody response in vivo when compared with wild-type promoters. Sequence analysis of the shuffled promoters identified several mutations potentially contributing to the observed enhanced or reduced promoter activities and identified a 42-nucleotide region that appears obsolete for the functioning of the CMV promoter. Taken together, these data demonstrate the feasibility of generating diverse promoter sequences by DNA shuffling and screening methods, and provide novel structure- function information about CMV promoters. DNA shuffling and screening technologies provide a new approach to promoter optimization and development of optimal expression vectors for genetic vaccinations, gene therapy, and protein expression.


Assuntos
Citomegalovirus/genética , Evolução Molecular Direcionada/métodos , Vetores Genéticos/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Animais , Anticorpos Antivirais/biossíntese , Sequência de Bases , Linhagem Celular , Células Cultivadas , Embaralhamento de DNA , Regulação da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Terapia Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico
3.
Expert Opin Biol Ther ; 4(4): 589-97, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15102607

RESUMO

Viral, bacterial and parasitic pathogens have evolved multiple strategies to evade the immune response, facilitate transmission and establish chronic infections. One of the underlying strategies that pathogens have evolved is antigenic variation of immune response targets that reduce the affinity of antigen binding to antibodies and major histocompatability complex class I and II receptors. Vaccine candidates generally target a limited number of these antigen variants or combine antigens from several variants to include in multivalent vaccine formulations. DNA shuffling and screening technologies, also known as MolecularBreeding (Maxygen, Inc.) directed molecular evolution, have been successfully used to identify and develop novel and chimaeric vaccine candidates capable of inducing immune responses that recognise and control multiple antigenic variants. DNA shuffling and screening strategies also select vaccine candidates with improved immunogenicity, increased expression as recombinant polypeptides and improved growth of whole viruses in cell culture. As DNA shuffling and screening strategies can be applied to many pathogens, there remain numerous applications of DNA shuffling to solve challenging problems in vaccine process development and manufacture.


Assuntos
Variação Antigênica , Embaralhamento de DNA , Evolução Molecular Direcionada , Programas de Rastreamento , Vacinas de DNA/genética , Vacinas Virais/genética , Animais , Biologia Computacional , Engenharia Genética/métodos , Terapia Genética , Humanos , Proteínas Recombinantes de Fusão/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Vírus/genética , Vírus/imunologia
4.
J Immunother ; 31(7): 644-55, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18600180

RESUMO

Immunization against tumor-associated antigens is a promising approach to cancer therapy and prevention, but it faces several challenges and limitations, such as tolerance mechanisms associated with self-antigens expressed by the tumor cells. Costimulatory molecules B7.1 (CD80) and B7.2 (CD86) have improved the efficacy of gene-based and cell-based vaccines in animal models and are under investigation in clinical trials. However, their efficacy as vaccine adjuvants is likely limited by the fact that they mediate both stimulatory and inhibitory signals to T cells via CD28 and CTLA-4, respectively. To overcome these limitations, we have generated a B7.1-like, chimeric costimulatory molecule with preferential binding to CD28, named CD28-binding protein (CD28BP), which we combined with a modified, nonself tumor antigen variant of epithelial cell adhesion molecule (EpCAM), named TAg25. TAg25 induced a cross-reactive immune response against human wild-type EpCAM upon DNA vaccination in cynomolgus monkeys. However, TAg25 DNA immunization alone or in combination with human (h) B7.1 induced no detectable antigen-specific T cells in the peripheral blood of the animals. In contrast, TAg25 combined with CD28BP induced both CD4 and CD8 T cells specific for EpCAM. Moreover, TAg25 combined with CD28BP induced significantly higher levels of EpCAM-specific antibodies than TAg25 plus hB7.1. These improved adjuvant properties of CD28BP, when compared with hB7.1, illustrate the importance of CD28 costimulation in vaccine responses in nonhuman primates and warrant further studies on the potential of CD28BP in improving the efficacy of cancer vaccines.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer , Proteínas de Transporte/imunologia , Moléculas de Adesão Celular/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/imunologia , Animais , Antígenos de Neoplasias/genética , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Moléculas de Adesão Celular/genética , Proliferação de Células , Reações Cruzadas , Sinergismo Farmacológico , Molécula de Adesão da Célula Epitelial , Humanos , Imunização Secundária , Interferon gama/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Macaca fascicularis , Neoplasias/terapia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Tolerância a Antígenos Próprios/efeitos dos fármacos , Tolerância a Antígenos Próprios/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
5.
Vaccine ; 24(3): 335-44, 2006 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-16125280

RESUMO

We employed DNA shuffling and screening technologies to develop a single recombinant dengue envelope (E) antigen capable of inducing neutralizing antibodies against all four antigenically distinct dengue serotypes. By DNA shuffling of codon-optimized dengue 1-4 E genes, we created a panel of novel chimeric clones expressing C-terminal truncated E antigens that combined epitopes from all four dengue serotypes. DNA vaccines encoding these novel chimeras induced multivalent T cell and neutralizing antibody responses against all four dengue serotypes in mice. By contrast, a mixture of four unshuffled, parental DNA vaccines failed to produce tetravalent neutralizing antibodies in mice. The neutralizing antibody titers for some of these antigens could be further improved by extending the sequences to express full-length pre-membrane and envelope proteins. The chimeric antigens also protected mice against a lethal dengue-2 virus challenge. These data demonstrate that DNA shuffling and associated screening can lead to the selection of multi-epitope antigens against closely related dengue virus serotypes and suggest a broad utility for these technologies in optimizing vaccine antigens.


Assuntos
Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Vírus da Dengue/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Western Blotting , Linhagem Celular , Dengue/imunologia , Dengue/prevenção & controle , Dengue/virologia , Evolução Molecular Direcionada , Citometria de Fluxo , Biblioteca Gênica , Interferon gama , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Plasmídeos/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Transfecção , Vacinas de DNA/imunologia , Vacinas Sintéticas/imunologia
6.
Virology ; 353(1): 166-73, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16814355

RESUMO

DNA shuffling and screening technologies were used to produce chimeric DNA constructs expressing antigens that shared epitopes from all four dengue serotypes. Three shuffled constructs (sA, sB and sC) were evaluated in the rhesus macaque model. Constructs sA and sC expressed pre-membrane and envelope genes, whereas construct sB expressed only the ectodomain of envelope protein. Five of six, and four of six animals vaccinated with sA and sC, respectively, developed antibodies that neutralized all 4 dengue serotypes in vitro. Four of six animals vaccinated with construct sB developed neutralizing antibodies against 3 serotypes (den-1, -2 and -3). When challenged with live dengue-1 or dengue-2 virus, partial protection against dengue-1 was observed. These results demonstrate the utility of DNA shuffling as an attractive tool to create tetravalent chimeric dengue DNA vaccine constructs, as well as a need to find ways to improve the immune responses elicited by DNA vaccines in general.


Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Embaralhamento de DNA , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/genética , Evolução Molecular Direcionada , Epitopos , Humanos , Macaca mulatta , Masculino , Testes de Neutralização , Proteínas Recombinantes de Fusão/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/classificação
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