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1.
Neurotherapeutics ; 18(3): 1980-1994, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33829411

RESUMO

Numerous therapies aimed at driving an effective anti-glioma response have been employed over the last decade; nevertheless, survival outcomes for patients remain dismal. This may be due to the expression of immune-checkpoint ligands such as PD-L1 by glioblastoma (GBM) cells which interact with their respective receptors on tumor-infiltrating effector T cells curtailing the activation of anti-GBM CD8+ T cell-mediated responses. Therefore, a combinatorial regimen to abolish immunosuppression would provide a powerful therapeutic approach against GBM. We developed a peptide ligand (CD200AR-L) that binds an uncharacterized CD200 immune-checkpoint activation receptor (CD200AR). We sought to test the hypothesis that CD200AR-L/CD200AR binding signals via he DAP10&12 pathways through in vitro studies by analyzing transcription, protein, and phosphorylation, and in vivo loss of function studies using inhibitors to select signaling molecules. We report that CD200AR-L/CD200AR binding induces an initial activation of the DAP10&12 pathways followed by a decrease in activity within 30 min, followed by reactivation via a positive feedback loop. Further in vivo studies using DAP10&12KO mice revealed that DAP10, but not DAP12, is required for tumor control. When we combined CD200AR-L with an immune-stimulatory gene therapy, in an intracranial GBM model in vivo, we observed increased median survival, and long-term survivors. These studies are the first to characterize the signaling pathway used by the CD200AR, demonstrating a novel strategy for modulating immune checkpoints for immunotherapy currently being analyzed in a phase I adult trial.


Assuntos
Antígenos CD/metabolismo , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Inibidores de Checkpoint Imunológico/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/deficiência , Sequência de Aminoácidos , Animais , Antígenos CD/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Feminino , Terapia Genética/métodos , Glioma/tratamento farmacológico , Glioma/genética , Inibidores de Checkpoint Imunológico/administração & dosagem , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Terciária de Proteína , Receptores Imunológicos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologia
3.
Cancer Immunol Res ; 2(9): 839-45, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24852944

RESUMO

Cytotoxic T cells typically are expanded ex vivo in culture with IL2 for adoptive immunotherapy. This culture period leads to a differentiated phenotype and acquisition of effector function, as well as a loss of in vivo proliferative capability and antitumor efficacy. Here, we report antigen-specific and polyclonal expansion of cytotoxic T cells in a cocktail of cytokines and small molecules that leads to a memory-like phenotype in mouse and human cells even during extended culture, leading to enhanced in vivo expansion and tumor control in mice.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Memória Imunológica , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/imunologia , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Citocinas/imunologia , Epitopos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/terapia
4.
J Immunol ; 190(12): 6259-68, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23686484

RESUMO

Overall, cancer vaccines have had a record of failure as an adjuvant therapy for malignancies that are treated with alkylating chemotherapy, and the contribution of standard treatment to that failure remains unclear. Vaccines aim to harness the proliferative potential of the immune system by expanding a small number of tumor-specific lymphocytes into a large number of antitumor effectors. Clinical trials are often conducted after treatment with alkylating chemotherapy, given either as standard therapy or for immunomodulatory effect. There is mounting evidence for synergy between chemotherapy and adoptive immunotherapy or vaccination against self-Ags; however, the impact of chemotherapy on lymphocytes primed against tumor neoantigens remains poorly defined. We report that clinically relevant dosages of standard alkylating chemotherapies, such as temozolomide and cyclophosphamide, significantly inhibit the proliferative abilities of lymphocytes in mice. This proliferative impairment was long-lasting and led to quantitative and qualitative defects in B and T cell responses to neoantigen vaccines. High-affinity responder lymphocytes receiving the strongest proliferative signals from vaccines experienced the greatest DNA damage responses, skewing the response toward lower-affinity responders with inferior functional characteristics. Together, these defects lead to inferior efficacy and overall survival in murine tumor models treated by neoantigen vaccines. These results suggest that clinical protocols for cancer vaccines should be designed to avoid exposing responder lymphocytes to alkylating chemotherapy.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Antineoplásicos Alquilantes/toxicidade , Vacinas Anticâncer/administração & dosagem , Neoplasias Experimentais/terapia , Imunidade Adaptativa/imunologia , Animais , Antineoplásicos Alquilantes/administração & dosagem , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Terapia Combinada , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
5.
Drug Deliv Transl Res ; 3(2): 143-51, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25787982

RESUMO

CD133, also known as Prominin-1, is expressed on stem cells present in many tissues and tumors. In this work, we have identified and characterized a single-chain variable fragment (scFv) for the efficient and specific recognition of CD133. Phage display was used to develop the scFv from a previously reported anti-CD133 hybridoma clone 7, which was capable of recognizing both glycosylated and non-glycosylated forms of human CD133. The scFv immunostained CD133(+) Caco-2 cells, but not CD133(-/low) U87 cells. Significantly, it immunostained CD133(-) cells transiently transfected with the mouse CD133 gene as well as CD133(+) mouse cells. Co-immunostaining studies in mouse bone marrow cells, using anti-CD133 scFv-FITC and anti-mouse CD133-PE (clone 13A4) commercial antibody, indicated that the epitopes recognized by these reagents partially overlap. Taken together, these results suggest that the scFv can recognize mouse CD133 protein in addition to recognizing human CD133. This new scFv is expected to be valuable both as a molecular diagnostic reagent for identifying CD133(+) cells and as a ligand for targeting therapeutics to CD133(+) tumor-initiating cells.

6.
Drug Deliv Transl Res ; 3(2): 195-204, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25787984

RESUMO

CD133 expression enriches for tumor-initiating cells and is a negative prognostic factor in numerous cancers. We previously developed an immunotoxin against CD133 by fusing a gene fragment encoding the scFv portion of an anti-CD133 antibody to a gene fragment encoding deimmunized PE38KDEL. The resulting fusion protein, dCD133KDEL, demonstrated potent antitumor activity following intratumoral delivery into head neck cell carcinoma xenografts. However, the efficacy against other tumors and the tolerability of systemic administration remained unclear. The purpose of this study was to evaluate the tolerability and efficacy of dCD133KDEL in a systemic human breast carcinoma model. Time course viability studies showed that dCD133KDEL selectively inhibited MDA-MB-231 ductal breast carcinoma cells that contained a minority CD133(+) subpopulation, implicating CD133(+) cells as a source for self-renewal within this cell line. Furthermore, systemic administration of dCD133KDEL caused regression or inhibition of tumor growth in mice bearing an intrasplenic MDA-MB-231 tumor challenge as a model for metastatic disease. In the same model, combined therapy with dCD133KDEL and another immunotoxin designed to target the bulk tumor mass was the most effective therapy, supporting the idea that such combination therapies might better address tumor heterogeneity. dCD133KDEL shows promise as a therapeutic agent and as a biologic tool to study cancer stem cells.

7.
Mol Cancer Ther ; 11(10): 2183-92, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22891038

RESUMO

The importance of the blood-brain barrier in preventing effective pharmacotherapy of glioblastoma has been controversial. The controversy stems from the fact that vascular endothelial cell tight junctions are disrupted in the tumor, allowing some systemic drug delivery. P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) efflux drugs from brain capillary endothelial cells into the blood. We tested the hypothesis that although the tight junctions are "leaky" in the core of glioblastomas, active efflux limits drug delivery to tumor-infiltrated normal brain and consequently, treatment efficacy. Malignant gliomas were induced by oncogene transfer into wild-type (WT) mice or mice deficient for Pgp and BCRP (knockout, KO). Glioma-bearing mice were orally dosed with dasatinib, a kinase inhibitor and dual BCRP/PgP substrate that is being currently tested in clinical trials. KO mice treated with dasatinib survived for twice as long as WT mice. Microdissection of the tumor core, invasive rim, and normal brain revealed 2- to 3-fold enhancement in dasatinib brain concentrations in KO mice relative to WT. Analysis of signaling showed that poor drug delivery correlated with the lack of inhibition of a dasatinib target, especially in normal brain. A majority of human glioma xenograft lines tested expressed BCRP or PgP and were sensitized to dasatinib by a dual BCRP/Pgp inhibitor, illustrating a second barrier to drug delivery intrinsic to the tumor itself. These data show that active efflux is a relevant obstacle to treating glioblastoma and provide a plausible mechanistic basis for the clinical failure of numerous drugs that are BCRP/Pgp substrates.


Assuntos
Encéfalo/metabolismo , Glioblastoma/tratamento farmacológico , Terapia de Alvo Molecular , Pirimidinas/metabolismo , Pirimidinas/uso terapêutico , Tiazóis/metabolismo , Tiazóis/uso terapêutico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acridinas/química , Acridinas/farmacologia , Acridinas/uso terapêutico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Dasatinibe , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Knockout , Oncogenes , Permeabilidade/efeitos dos fármacos , Pirimidinas/química , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/farmacologia , Tetra-Hidroisoquinolinas/uso terapêutico , Tiazóis/química , Tiazóis/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Distribuição Tecidual/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Clin Cancer Res ; 16(19): 4800-8, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20858839

RESUMO

PURPOSE: Atmospheric oxygen (∼20% O(2)) has been the universal condition employed to culture tumor cells used as vaccine antigen. We tested the hypothesis that reducing oxygen tension would increase the efficacy of tumor cell lysate vaccines. EXPERIMENTAL DESIGN: GL261 glioma cells and EMT6 breast carcinoma cells were grown in 5% or 20% O(2). Syngeneic tumor-bearing mice were vaccinated with these tumor cell lysates mixed with CpG oligodeoxynucleotides as an adjuvant. Tumor infiltrating T cells and apoptotic GL261 cells were quantified by immunohistochemistry. Tumor-reactive immunoglobulin was detected by Western blot. Ovalbumin and gp100-derived peptides were mixed with GL261 lysates as marker antigens to detect changes in presentation of exogenous antigen on MHC class I in vitro, and in vivo following adoptive transfer of gp100-specific CD8(+) T cells. RESULTS: Mice bearing orthotopic glioma and breast carcinoma survived significantly longer when vaccinated with 5% O(2) lysates. Antigen-specific CTL activation was significantly enhanced following stimulation with lysates derived from GL261 cells grown in 5% O(2) versus 20% O(2) through a mechanism that involved enhanced cross-presentation of exogenous antigen on MHC I. Vaccination with 5% O(2) GL261 cell lysates caused a significant increase in CTL proliferation, tumoricidal function, and trafficking into brain tumor sites, whereas 20% O(2) lysate vaccines predominantly evoked an antibody response. CONCLUSIONS: Tissue culture oxygen functions as an "immunologic switch" by dictating the cellular and humoral immune responses elicited by tumor cell lysates. These results have profound implications for cancer vaccines that utilize tumor cells as the source of antigen.


Assuntos
Vacinas Anticâncer/biossíntese , Vacinas Anticâncer/imunologia , Extratos Celulares/imunologia , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Oxigênio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/prevenção & controle , Vacinas Anticâncer/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Glioma/imunologia , Glioma/metabolismo , Glioma/patologia , Glioma/prevenção & controle , Imuno-Histoquímica , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Taxa de Sobrevida , Células Tumorais Cultivadas
9.
J Gerontol Nurs ; 33(4): 5-12, 2007 04.
Artigo em Inglês | MEDLINE | ID: mdl-17436864

RESUMO

The purpose of this study was to explore staff perceptions and concerns about the medication use process in the nursing home setting. A total of 76 staff members from 5 nursing homes in 3 Midwestern states participated in key informant interviews and focus groups. Common themes included issues related to communication, competing demands, and the challenges of a paper-based medication administration record. Concerns frequently were associated with the timeliness and accuracy of the medication administration process. Recognition of staff concerns are an important first step in improving the nursing home medication use process. Staff insight provided clarification related to impediments to safe medication practices. This study provides insight into how technology can improve the nursing home medication use process.


Assuntos
Atitude do Pessoal de Saúde , Enfermagem Geriátrica/organização & administração , Casas de Saúde , Preparações Farmacêuticas/administração & dosagem , Idoso , Documentação , Grupos Focais , Humanos
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