RESUMO
An elevated number of Gr-1+CD11b+ myeloid cells has been described in mice bearing transplantable tumors, and has been associated with immune suppression. We examined the role of such myeloid suppressor cells in mice bearing the spontaneously transformed syngeneic mouse ovarian surface epithelial cell line, 1D8. We observed high levels of CD80 expression by Gr-1+CD11b+ cells from spleen, ascites, and tumor tissue of mice bearing 1D8 ovarian carcinoma, whereas CD40 and CD86 were absent. CD80 expression was not detected on Gr-1+CD11b+ cells from naïve mice. However, the expression of CD80 by Gr-1+CD11b+ cells from naïve mice was promoted by coculture with 1D8 cells. Because irradiated 1D8 cells, but not 1D8-conditioned medium, up-regulate CD80 expression by Gr-1+CD11b+ cells, this phenomenon likely requires direct interaction. Gr-1+CD11b+ cells derived from 1D8 tumor-bearing mice provided significant suppression of antigen-specific immune responses, but Gr-1+CD11b+ cells from naïve mice did not. Both short interfering RNA-mediated knockdown and genetic knockout of CD80 expression by Gr-1+CD11b+ cells of 1D8 tumor-bearing mice alleviated the suppression of antigen-specific immune responses. Suppression via CD80 on Gr-1+CD11b+ myeloid cells was mediated by CD4+CD25+ T regulatory cells and required CD152. CD80 knockout or antibody blockade of either CD80 or CD152 retarded the growth of 1D8 tumor in mice, suggesting that expression of CD80 on Gr-1+CD11b+ myeloid cells triggered by 1D8 ovarian carcinoma suppresses antigen-specific immunity via CD152 signaling and CD4+CD25+ T regulatory cells. Thus, CD80-dependent responses to myeloid suppressor cells may contribute to tumor tolerance and the progression of ovarian carcinoma.
Assuntos
Antígeno B7-1/imunologia , Antígeno CD11b/imunologia , Neoplasias Ovarianas/imunologia , Receptores de Quimiocinas/imunologia , Animais , Antígeno B7-1/biossíntese , Antígeno B7-1/genética , Epitopos , Feminino , Tolerância Imunológica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Neoplasias Ovarianas/patologia , RNA Interferente Pequeno/genética , Linfócitos T Reguladores/imunologiaRESUMO
Purpose Our preclinical work identified depletion of ATR as a top candidate for topoisomerase 1 (TOP1) inhibitor synthetic lethality and showed that ATR inhibition sensitizes tumors to TOP1 inhibitors. We hypothesized that a combination of selective ATR inhibitor M6620 (previously VX-970) and topotecan, a selective TOP1 inhibitor, would be tolerable and active, particularly in tumors with high replicative stress. Patients and Methods This phase I study tested the combination of M6620 and topotecan in 3-week cycles using 3 + 3 dose escalation. The primary end point was the identification of the maximum tolerated dose of the combination. Efficacy and pharmacodynamics were secondary end points. Results Between September 2016 and February 2017, 21 patients enrolled. The combination was well tolerated, which allowed for dose escalation to the highest planned dose level (topotecan 1.25 mg/m2, days 1 to 5; M6620 210 mg/m2, days 2 and 5). One of six patients at this dose level experienced grade 4 thrombocytopenia that required transfusion, a dose-limiting toxicity. Most common treatment-related grade 3 or 4 toxicities were anemia, leukopenia, and neutropenia (19% each); lymphopenia (14%); and thrombocytopenia (10%). Two partial responses (≥ 18 months, ≥ 7 months) and seven stable disease responses ≥ 3 months (median, 9 months; range, 3 to 12 months) were seen. Three of five patients with small-cell lung cancer, all of whom had platinum-refractory disease, had a partial response or prolonged stable disease (10, ≥ 6, and ≥ 7 months). Pharmacodynamic studies showed preliminary evidence of ATR inhibition and enhanced DNA double-stranded breaks in response to the combination. Conclusion To our knowledge, this report is the first of an ATR inhibitor-chemotherapy combination. The maximum dose of topotecan plus M6620 is tolerable. The combination seems particularly active in platinum-refractory small-cell lung cancer, which tends not to respond to topotecan alone. Phase II studies with biomarker evaluation are ongoing.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Isoxazóis/administração & dosagem , Pirazinas/administração & dosagem , Topotecan/administração & dosagem , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Humanos , Isoxazóis/efeitos adversos , Masculino , Pessoa de Meia-Idade , Pirazinas/efeitos adversos , Topotecan/efeitos adversos , Resultado do TratamentoRESUMO
PURPOSE: Persistent infection with oncogenic human papillomaviruses (HPV) plays a central etiologic role in the development of squamous carcinomas of the cervix and their precursor lesions, cervical intraepithelial neoplasias (CIN). We carried out a prospective observational cohort study evaluating known, quantifiable prognostic variables of clinical behavior in women with high-grade cervical lesions. EXPERIMENTAL DESIGN: Our study cohort included healthy women with high-grade cervical lesions (CIN2/3) with residual visible lesions after colposcopically directed biopsy. We prospectively followed 100 women over 15 weeks before standard resection. HPV typing was done using PCR and a reverse line blot detection method. RESULTS: The rate of spontaneous histologic regression, defined as (CIN1 or less at resection) was 28%. The overall rate of HPV infection was 100%. HPV16 was identified in 68% of the lesions. Women with HPV16 only were significantly less likely to regress, compared with women with HPV types other than HPV16 (odds ratio, 0.342; 95% confidence interval, 0.117-0.997; P = 0.049). In the cohort with HPV16 only, patients who had an HLA*A201 allele had similar outcomes to those who did not carry A201. However, among patients with HPV types other than HPV16, the HLA*A201 allele interaction was significant; patients with HLA*A201 were the least likely to resolve. CONCLUSIONS: CIN2/3 lesions associated with HPV16 alone are significantly less likely to resolve spontaneously than those caused by other types. Interactions among HPV type, HLA type, and regression rate support a role for HLA-restricted HPV-specific immune responses in determining disease outcome.
Assuntos
Infecções por Papillomavirus/patologia , Displasia do Colo do Útero/patologia , Adolescente , Adulto , Idoso , Alelos , Linhagem Celular Tumoral , Estudos de Coortes , DNA Viral/análise , DNA Viral/genética , Feminino , Genótipo , Antígenos HLA/genética , Antígeno HLA-A2/genética , Humanos , Células K562 , Pessoa de Meia-Idade , Papillomaviridae/genética , Papillomaviridae/crescimento & desenvolvimento , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Estudos Prospectivos , Remissão Espontânea , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/virologiaRESUMO
Robust pharmacodynamic assay results are valuable for informing go/no-go decisions about continued development of new anti-cancer agents and for identifying combinations of targeted agents, but often pharmacodynamic results are too incomplete or variable to fulfill this role. Our experience suggests that variable reagent and specimen quality are two major contributors to this problem. Minimizing all potential sources of variability in procedures for specimen collection, processing, and assay measurements is essential for meaningful comparison of pharmacodynamic biomarkers across sample time points. This is especially true in the evaluation of pre- and post-dose tumor biopsies, which suffer from high levels of tumor insufficiency due to variations in biopsy collection techniques and significant specimen heterogeneity within and across patients. Developing methods to assess heterogeneous biopsies is necessary in order to evaluate a majority of tumor biopsies collected for pharmacodynamic biomarker studies. Improved collection devices and standardization of methods are being sought in order to improve the tumor content and quality of tumor biopsies. In terms of reagent variability, we have found that stringent initial reagent qualification and quality control of R&D-grade reagents is critical to minimize lot-to-lot variability and prevent assay failures, especially for clinical pharmacodynamic questions, which often demand assay performance that meets or exceeds clinical diagnostic assay standards. Rigorous reagent specifications and use of appropriate assay quality control methodologies help to ensure consistency between assay runs, laboratories and trials to provide much needed pharmacodynamic insights into the activity of investigational agents.
Assuntos
Antineoplásicos/farmacocinética , Biomarcadores Tumorais/análise , Manejo de Espécimes/métodos , Biópsia , Humanos , Indicadores e Reagentes , Neoplasias/patologia , Reprodutibilidade dos Testes , Manejo de Espécimes/normasRESUMO
PURPOSE: Indenoisoquinolines are non-camptothecin topoisomerase I (TopI) inhibitors that overcome the limitations of camptothecins: chemical instability and camptothecin resistance. Two dosing schedules of the novel indenoisoquinoline, indotecan (LMP400), were evaluated in patients with advanced solid tumors. METHODS: The maximum tolerated dose (MTD), toxicities, and pharmacokinetics of two indotecan drug administration schedules (daily for 5 days or weekly) were investigated. Modulation of TopI and the phosphorylation of histone H2AX (γH2AX) were assayed in tumor biopsies; γH2AX levels were also evaluated in circulating tumor cells (CTCs) and hair follicles to assess DNA damage response. RESULTS: An MTD of 60 mg/m(2)/day was established for the daily regimen, compared to 90 mg/m(2) for the weekly regimen. The TopI response to drug showed target engagement in a subset of tumor biopsies. Pharmacokinetics profiles demonstrated a prolonged terminal half-life and tissue accumulation compared to topotecan. Dose-dependent decreases in total CTCs were measured in seven patients. Formation of γH2AX-positive foci in CTCs (day 3) and hair follicles (4-6 h) was observed following treatment. CONCLUSIONS: We established the MTD of two dosing schedules for a novel TopI inhibitor, indotecan. Target engagement was demonstrated as Top1 downregulation and γH2AX response. No objective responses were observed on either schedule in this small patient cohort. The principal toxicity of both schedules was myelosuppression; no significant gastrointestinal problems were observed. Increased DNA damage response was observed in CTCs, hair follicles, and a subset of tumor biopsies.
Assuntos
Benzodioxóis/administração & dosagem , DNA Topoisomerases Tipo I/metabolismo , Histonas/metabolismo , Isoquinolinas/administração & dosagem , Neoplasias/tratamento farmacológico , Inibidores da Topoisomerase I/administração & dosagem , Adulto , Idoso , Benzodioxóis/efeitos adversos , Benzodioxóis/farmacocinética , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Esquema de Medicação , Feminino , Folículo Piloso/metabolismo , Meia-Vida , Humanos , Isoquinolinas/efeitos adversos , Isoquinolinas/farmacocinética , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/patologia , Fatores de Tempo , Distribuição Tecidual , Inibidores da Topoisomerase I/efeitos adversos , Inibidores da Topoisomerase I/farmacocinética , Topotecan/farmacocinética , Adulto JovemRESUMO
Vaccination with human papillomavirus type 16 (HPV16) L1 virus-like particles (VLP) induces both high titer neutralizing IgG and protective immunity. Because protection from experimental infection by papillomavirus is mediated by neutralizing IgG, we sought the mechanisms that trigger humoral immunity to HPV16 L1 VLP. We find that HPV16 L1 VLP bind to murine B lymphocytes thereby inducing activation-induced cytidine deaminase expression and Ig class switch recombination to cause the generation of IgG. HPV16 L1 VLP also activate production of proinflammatory factors IFN-alpha, IL-6, MIP-1alpha, RANTES, and KC, up-regulate the expression of costimulatory molecules by naive B cells, and increase the B1 B cell subpopulation. These B cell responses to HPV16 L1 VLP are dependent upon MyD88. Although MyD88(-/-) B cells produce only mu transcript after exposure to HPV16 L1 VLP, MyD88(+/+) B cells express alpha, gamma, and mu Ig H chain and activation-induced cytidine deaminase transcripts. Notably, TLR4 mutant C3H/HeJ mice exhibited significantly reduced HPV16 VLP-specific IgG1, IgG2a, IgG2b, and IgG3 titers after vaccination as compared with the control C3H/HeOuJ mice. HPV16 L1 VLP directly activated class switch recombination and costimulatory molecule expression by B cells of C3H/HeOuJ mice but not C3H/HeJ mice. Thus HPV16 L1 VLP directly activate B cells to induce CD4(+) T cell independent humoral immune responses via TLR4- and MyD88-dependent signaling.
Assuntos
Antígenos de Diferenciação/fisiologia , Linfócitos B/imunologia , Switching de Imunoglobulina , Ativação Linfocitária/imunologia , Proteínas Oncogênicas Virais/imunologia , Receptores Imunológicos/fisiologia , Vírion/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Diferenciação/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfócitos B/virologia , Ligante de CD40/fisiologia , Proteínas do Capsídeo , Switching de Imunoglobulina/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/fisiologia , Interleucina-4/fisiologia , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide , Proteínas Oncogênicas Virais/administração & dosagem , Papillomaviridae/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/virologia , Receptor 4 Toll-Like , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vírion/genéticaRESUMO
Viruses that replicate in the nucleus, including the primary causative agent of cervical cancer, human papillomavirus type 16 (HPV16), must first cross the cytoplasm. We compared the uptake of HPV16 virus-like particles (VLPs) either with or without the minor capsid protein L2. Whereas VLPs containing only the major capsid protein L1 were diffusely distributed within the cytoplasm even 6 h post-infection, VLPs comprising both L1 and L2 exhibited a radial distribution in the cytoplasm and accumulated in the perinuclear region of BPHE-1 cells within 2 h. L2 of HPV16 or bovine papillomavirus was shown to bind to a 43-kDa cellular protein that was subsequently identified as beta-actin by matrix-assisted laser desorption ionization time-of-flight analysis. A conserved domain comprising residues 25-45 of HPV16 L2 was sufficient for interaction with beta-actin. HPV16 L2 residues 25-45 fused to green fluorescent protein, but not green fluorescent protein alone, colocalized with actin and caused cell retraction and disruption of the microfilament network. Finally, wild-type L2, but not L2 with residues 25-45 deleted, facilitated HPV16 pseudovirion infection. Thus, binding of beta-actin by L2 residues 25-45 facilitates transport of HPV16 across the cytoplasm during infection, and blockade of this novel interaction may be useful for prophylaxis.
Assuntos
Proteínas do Capsídeo , Capsídeo/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/metabolismo , Infecções por Papillomavirus/virologia , Infecções Tumorais por Vírus/virologia , Actinas/química , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Papillomavirus Bovino 1/metabolismo , Bovinos , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Vírion/metabolismo , Vírion/ultraestruturaRESUMO
During the papillomavirus (PV) life cycle, the L2 minor capsid protein enters the nucleus twice: in the initial phase after entry of virions into cells and in the productive phase to mediate encapsidation of the newly replicated viral genome. Therefore, we investigated the interactions of the L2 protein of bovine PV type 1 (BPV1) with the nuclear import machinery and the viral DNA. We found that BPV1 L2 bound to the karyopherin alpha2 (Kap alpha2) adapter and formed a complex with Kap alpha2beta1 heterodimers. Previous data have shown that the positively charged termini of BPV1 L2 are required for BPV1 infection after the binding of the virions to the cell surface. We determined that these BPV1 L2 termini function as nuclear localization signals (NLSs). Both the N-terminal NLS (nNLS) and the C-terminal NLS (cNLS) interacted with Kap alpha2, formed a complex with Kap alpha2beta1 heterodimers, and mediated nuclear import via a Kap alpha2beta1 pathway. Interestingly, the cNLS was also the major DNA binding site of BPV1 L2. Consistent with the promiscuous DNA encapsidation by BPV1 pseudovirions, this DNA binding occurred without nucleotide sequence specificity. Moreover, an L2 mutant encoding a scrambled version of the cNLS, which supports production of virions, rescued the DNA binding but not the Kap alpha2 interaction. These data support a model in which BPV1 L2 functions as an adapter between the viral DNA via the cNLS and the Kaps via the nNLS and facilitates nuclear import of the DNA during infection.
Assuntos
Papillomavirus Bovino 1/patogenicidade , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Sítios de Ligação , Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/metabolismo , Proteínas do Capsídeo/genética , Bovinos , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Human papillomavirus type 16 (HPV16) is the primary etiologic agent of cervical carcinoma, whereas bovine papillomavirus type 1 (BPV1) causes benign fibropapillomas. However, the capsid proteins, L1 and L2, of these divergent papillomaviruses exhibit functional conservation. A peptide comprising residues 1 to 88 of BPV1 L2 binds to a variety of cell lines, but not to the monocyte-derived cell line D32, and blocks BPV1 infection of mouse C127 cells. Residues 13 to 31 of HPV16 L2 and BPV1 L2 residues 1 to 88 compete for binding to the cell surface, and their binding, unlike that of HPV16 L1/L2 virus-like particles, is unaffected by heparinase or trypsin pretreatment of HeLa cells. A fusion of HPV16 L2 peptide 13-31 and GFP binds (K(d), approximately 1 nM) to approximately 45,000 receptors per HeLa cell. Furthermore, mutation of L2 residues 18 and 19 or 21 and 22 significantly reduces both the ability of the HPV16 L2 13-31-GFP fusion protein to bind to SiHa cells and the infectivity of HPV16 pseudovirions. Antibody to BPV1 L2 peptides comprising residues 115 to 135 binds to intact BPV1 virions, but fails to neutralize at a 1:10 dilution. However, deletion of residues 91 to 129 from L2 abolishes the infectivity of BPV1, but not their binding to the cell surface. In summary, L2 residues 91 to 129 contain epitopes displayed on the virion surface and are required for infection, but not virion binding to the cell surface. Upon the binding of papillomavirus to the cell surface, residues 13 to 31 of L2 interact with a widely expressed, trypsin- and heparinase-resistant cell surface molecule and facilitate infection.
Assuntos
Motivos de Aminoácidos , Papillomavirus Bovino 1/patogenicidade , Proteínas do Capsídeo , Capsídeo/química , Proteínas Oncogênicas Virais/química , Papillomaviridae/patogenicidade , Receptores Virais/metabolismo , Sequência de Aminoácidos , Animais , Papillomavirus Bovino 1/metabolismo , Capsídeo/metabolismo , Bovinos , Linhagem Celular , Heparina Liase/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Tripsina/metabolismo , Vírion/química , Vírion/metabolismo , Vírion/patogenicidadeRESUMO
Human papillomavirus type-16 (HPV16) L1 virus-like particles (VLPs) activate dendritic cells (DCs) and induce protective immunity. In this study, we demonstrate, using global gene expression analysis, that HPV16 VLPs produce quite distinct innate responses in murine splenic DC subpopulations. While HPV16 VLPs increase transcription of IFN-gamma and numerous Th1-related cytokines and chemokines in CD8alpha(+)CD11c(+) DCs, CD4(+)CD11c(+) DCs up-regulate only type I IFN and a different set of Th2-associated cytokines and chemokines. Type I IFN, but not IFN-gamma, potentiates humoral immunity, notably production of VLP-specific IgG2a. However, HPV16 VLP-stimulated IL-12 production by CD8alpha(+)CD11c(+) DCs is augmented by autocrine IFN-gamma signaling. Thus, before adaptive immunity, HPV16 VLPs signal complementary defense responses in key DC subpopulations, indicating specialized DC lineages with predetermined polarization.