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1.
Cancer Gene Ther ; 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31155611

RESUMO

Effective chimeric antigen receptor-modified T-cell (CAR-T) therapy for liver metastases (LM) will require innovative solutions to ensure efficient delivery and minimization of systemic toxicity. We previously demonstrated the safety of CAR-T hepatic artery infusions (HAI). We subsequently conducted the phase 1b HITM-SIR trial, in which six patients (pts) with CEA+ LM received anti-CEA CAR-T HAIs and selective internal radiation therapy (SIRT). The primary endpoint was safety with secondary assessments of biologic activity. Enrolled pts had a mean LM size of 6.4 cm, 4 pts had >10 LM, and pts received an average of two lines of prior systemic therapy. No grade 4 or 5 toxicities were observed, and there were no instances of severe cytokine-release syndrome (CRS) or neurotoxicity. The mean transduction efficiency was 60.4%. Following CAR-T HAI, reduced levels of GM-CSF-R, IDO, and PD-L1 were detected in LM, and serum CEA levels were stable or decreased in all subjects. Median survival time was 8 months (mean 11, range 4-31). Anti-CEA CAR-T HAI with subsequent SIRT was well tolerated, and biologic responses were demonstrated following failure of conventional therapy. HAI of CAR-T was once again confirmed not to be associated with severe CRS or neurotoxicity.

2.
iScience ; 2: 105-122, 2018 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-30428369

RESUMO

T cells infiltrate affected organs in chronic infections and malignancy, but they may fail to eradicate virus-infected cells or tumor because of exhaustion. This report describes a Yin Yang-1 (YY1)-centered mechanism for diverse components that have been correlated with exhaustion. Utilizing an in vitro reconstruction of chronic T cell activation, YY1 is shown to positively regulate the checkpoint receptors PD1, Lag3, and Tim3 and to negatively regulate the type I cytokines interleukin-2 (IL-2) (in collaboration with Ezh2 histone methyltransferase) and interferon gamma (IFN-?). Other tests suggest that IL-2 failure drives a large component of cytotoxic functional decline rather than solely checkpoint receptor-ligand interactions that have been the focus of current anti-exhaustion therapies. Clinical evaluations confirm elevated YY1 and Ezh2 in melanoma tumor-infiltrating lymphocytes and in PD1+ T cells in patients with HIV. Exhaustion is revealed to be an active process as the culmination of repetitive two-signal stimulation in a feedback loop via CD3/CD28?p38MAPK/JNK?YY1? exhaustion.

3.
Mol Ther Methods Clin Dev ; 10: 371-378, 2018 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-30211249

RESUMO

Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of vector products used in the clinical trials were generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral vector product generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value.

4.
Oncogene ; 2018 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158673

RESUMO

Immunosuppressive myeloid-derived suppressor cells (MDSC) subvert antitumor immunity and limit the efficacy of chimeric antigen receptor T cells (CAR-T). Previously, we reported that the GM-CSF/JAK2/STAT3 axis drives liver-associated MDSC (L-MDSC) proliferation and blockade of this axis rescued antitumor immunity. We extended these findings in our murine liver metastasis (LM) model, by treating tumor-bearing mice with STAT3 inhibitors (STATTIC or BBI608) to further our understanding of how STAT3 drives L-MDSC suppressive function. STAT3 inhibition caused significant reduction of tumor burden as well as L-MDSC frequencies due to decrease in pSTAT3 levels. L-MDSC isolated from STATTIC or BBI608-treated mice had significantly reduced suppressive function. STAT3 inhibition of L-MDSC was associated with enhanced antitumor activity of CAR-T. Further investigation demonstrated activation of apoptotic signaling pathways in L-MDSC following STAT3 inhibition as evidenced by an upregulation of the pro-apoptotic proteins Bax, cleaved caspase-3, and downregulation of the anti-apoptotic protein Bcl-2. Accordingly, there was also a decrease of pro-survival markers, pErk and pAkt, and an increase in pro-death marker, Fas, with activation of downstream JNK and p38 MAPK. These findings represent a previously unrecognized link between STAT3 inhibition and Fas-induced apoptosis of MDSCs. Our findings suggest that inhibiting STAT3 has potential clinical application for enhancing the efficacy of CAR-T cells in LM through modulation of L-MDSC.

5.
Sci Rep ; 8(1): 3846, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29497107

RESUMO

Axl is a tyrosine kinase receptor that is commonly overexpressed in many cancers. As such, Axl represents an attractive therapeutic target. The transfer of engineered T cell expressing chimeric antigen receptor (CAR) is an exciting cancer therapeutic approach that shows high efficacy against cancers in clinical trials, especially for B cell malignancies. Furthermore, recently developed synthetic Notch (synNotch) receptor has demonstrated potential in enhancing the specificity of CAR T cell therapy and delivering therapeutic payloads to tumors in an antigen-dependent manner. Therefore, a CAR or synNotch against Axl could be a valuable therapeutic reagent against many cancers. Here, we develop a single-chain variable fragment from a humanized monoclonal antibody against Axl. The scFv is attached to CD3ζ, CD28, and 4-1BB signaling domains to generate an anti-Axl CAR. When introduced into human primary T cells, the anti-Axl CAR can lead to cytokine production and cell killing in response to tumor cells expressing Axl. Moreover, an anti-Axl synNotch generated using the same scFv can be activated with Axl expressing tumor cells. Given the fact that Axl is an important cancer therapeutic target, these receptors could be valuable reagents for developing anti-Axl therapies.

6.
J Arthroplasty ; 32(10): 2990-2994, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28757131

RESUMO

BACKGROUND: Tranexamic acid (TXA) reduces intraoperative blood loss and transfusions in patients undergoing total knee arthroplasty. Although numerous studies demonstrate the efficacy of intravenous and topical TXA in these patients, few demonstrate the effectiveness and appropriate dosing recommendations of oral formulations. METHODS: A retrospective cohort study was performed to evaluate differences in transfusion requirements in patients undergoing primary unilateral total knee arthroplasty with either no TXA (n = 866), a single-dose of oral TXA (n = 157), or both preoperative and postoperative oral TXA (n = 1049). Secondary outcomes included postoperative hemoglobin drop, total units transfused, length of stay, drain output, and cell salvage volume. RESULTS: Transfusion rates decreased from 15.4% in the no-oral tranexamic acid (OTA) group to 9.6% in the single-dose OTA group (P < .001) and 7% in the 2-dose group (P < .001), with no difference in transfusion rates between the single- and 2-dose groups (P = .390). In addition, postoperative hemoglobin drop was reduced from 4.2 g/dL in the no-OTA group to 3.5 g/dL in the single-dose group (P < .01) and to 3.4 g/dL in the 2-dose group (P < .01), without a difference between the single- and 2-dose groups (P = .233). CONCLUSION: OTA reduces transfusions, with greater ease of administration and improved cost-effectiveness relative to other forms of delivery.


Assuntos
Antifibrinolíticos/administração & dosagem , Artroplastia do Joelho/efeitos adversos , Perda Sanguínea Cirúrgica/prevenção & controle , Transfusão de Sangue/estatística & dados numéricos , Ácido Tranexâmico/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Antifibrinolíticos/economia , Análise Custo-Benefício , Drenagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Ácido Tranexâmico/economia
7.
J Leukoc Biol ; 102(2): 201-208, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28546503

RESUMO

Chimeric antigen receptor expressing T cells (CAR-T) are a promising form of immunotherapy, but the influence of age-related immune changes on CAR-T production remains poorly understood. We showed that CAR-T cells from geriatric donors (gCAR-T) are functionally impaired relative to CAR-T from younger donors (yCAR-T). Higher transduction efficiencies and improved cell expansion were observed in yCAR-T cells compared with gCAR-T. yCAR-T demonstrated significantly increased levels of proliferation and signaling activation of phosphorylated (p)Erk, pAkt, pStat3, and pStat5. Furthermore, yCAR-T contained higher proportions of CD4 and CD8 effector memory (EM) cells, which are known to have enhanced cytolytic capabilities. Accordingly, yCAR-T demonstrated higher levels of tumor antigen-specific cytotoxicity compared with gCAR-T. Enhanced tumor killing by yCAR-T correlated with increased levels of perforin and granzyme B. yCAR-T had increased α5ß1 integrin expression, a known mediator of retroviral transduction. We found that treatment with M-CSF or TGF-ß1 rescued the impaired transduction efficiency of the gCAR-T by increasing the α5ß1 integrin expression. Neutralization of α5ß1 confirmed that this integrin was indispensable for CAR expression. Our study suggests that the increase of α5ß1 integrin expression levels enhances CAR expression and thereby improves tumor killing by gCAR-T.


Assuntos
Envelhecimento/imunologia , Imunoterapia/métodos , Integrina alfa5beta1/biossíntese , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Western Blotting , Quimera , Citotoxicidade Imunológica/imunologia , Feminino , Citometria de Fluxo , Humanos , Integrina alfa5beta1/imunologia , Ativação Linfocitária/imunologia , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Adulto Jovem
9.
Prostate ; 76(14): 1257-70, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27324746

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR)-modified "designer" T cells (dTc, CAR-T) against PSMA selectively target antigen-expressing cells in vitro and eliminate tumors in vivo. Interleukin 2 (IL2), widely used in adoptive therapies, was proven essential in animal models for dTc to eradicate established solid tumors. METHODS: Patients under-went chemotherapy condi-tion-ing, followed by dTc dosing under a Phase I escalation with continuous infusion low dose IL2 (LDI). A target of dTc escalation was to achieve ≥20% engraftment of infused activated T cells. RESULTS: Six patients enrolled with doses prepared of whom five were treated. Patients received 10(9) or 10(10) autologous T cells, achieving expansions of 20-560-fold over 2 weeks and engraftments of 5-56%. Pharmacokinetic and pharmacodynamic analyses established the impact of conditioning to promote expansion and engraftment of the infused T cells. Unexpectedly, administered IL2 was depleted up to 20-fold with high engraftments of activated T cells (aTc) in an inverse correlation (P < 0.01). Clinically, no anti-PSMA toxicities were noted, and no anti-CAR reactivities were detected post-treatment. Two-of-five patients achieved clinical partial responses (PR), with PSA declines of 50% and 70% and PSA delays of 78 and 150 days, plus a minor response in a third patient. Responses were unrelated to dose size (P = 0.6), instead correlating inversely with engraftment (P = 0.06) and directly with plasma IL2 (P = 0.03), suggesting insufficient IL2 with our LDI protocol to support dTc anti-tumor activity under optimal (high) dTc engraftments. CONCLUSIONS: Under a Phase I dose escalation in prostate cancer, a 20% engraftment target was met or exceeded in three subjects with adequate safety, leading to study conclusion. Clinical responses were obtained but were suggested to be restrained by low plasma IL2 when depleted by high levels of engrafted activated T cells. This report presents a unique example of how the pharmaco-dynamics of "drug-drug" interactions may have a critical impact on the efficacy of their co-application. A new Pilot/Phase II trial is planned to test moderate dose IL2 (MDI) together with high dTc engraftments for anticipated improved therapeutic efficacy. Prostate 76:1257-1270, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Glutamato Carboxipeptidase II/antagonistas & inibidores , Interleucina-2/administração & dosagem , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/terapia , Receptores de Antígenos de Linfócitos T/administração & dosagem , Linfócitos T/transplante , Idoso , Antígenos de Superfície/sangue , Glutamato Carboxipeptidase II/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/sangue , Transplante Autólogo/métodos , Resultado do Tratamento
10.
Proc Natl Acad Sci U S A ; 112(42): 13045-50, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26438866

RESUMO

Despite significant advances in the treatment of Hodgkin's lymphoma (HL), a significant proportion of patients will not respond or will subsequently relapse. We identified CD25, the IL-2 receptor alpha subunit, as a favorable target for systemic radioimmunotherapy of HL. The scientific basis for the clinical trial was that, although most normal cells with exception of Treg cells do not express CD25, it is expressed by a minority of Reed-Sternberg cells and by most polyclonal T cells rosetting around Reed-Sternberg cells. Forty-six patients with refractory and relapsed HL were evaluated with up to seven i.v. infusions of the radiolabeled anti-CD25 antibody (90)Y-daclizumab. (90)Y provides strong ß emissions that kill tumor cells at a distance by a crossfire effect. In 46 evaluable HL patients treated with (90)Y-daclizumab there were 14 complete responses and nine partial responses; 14 patients had stable disease, and nine progressed. Responses were observed both in patients whose Reed-Sternberg cells expressed CD25 and in those whose neoplastic cells were CD25(-) provided that associated rosetting T cells expressed CD25. As assessed using phosphorylated H2AX (γ-H2AX) as a bioindicator of the effects of radiation exposure, predominantly nonmalignant cells in the tumor microenvironment manifested DNA damage, as reflected by increased expression of γ-H2AX. Toxicities were transient bone-marrow suppression and myelodysplastic syndrome in six patients who had not been evaluated with bone-marrow karyotype analyses before therapy. In conclusion, repeated (90)Y-daclizumab infusions directed predominantly toward nonmalignant T cells rosetting around Reed-Sternberg cells provided meaningful therapy for select HL patients.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Doença de Hodgkin/tratamento farmacológico , Imunoglobulina G/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/imunologia , Radioisótopos de Ítrio/química , Adulto , Idoso , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Daclizumabe , Feminino , Doença de Hodgkin/imunologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Fosforilação , Recidiva , Adulto Jovem
11.
Cancer Immunol Immunother ; 64(7): 817-29, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25850344

RESUMO

Chimeric antigen receptor-modified T cell (CAR-T) technology, a promising immunotherapeutic tool, has not been applied specifically to treat liver metastases (LM). While CAR-T delivery to LM can be optimized by regional intrahepatic infusion, we propose that liver CD11b+Gr-1+ myeloid-derived suppressor cells (L-MDSC) will inhibit the efficacy of CAR-T in the intrahepatic space. We studied anti-CEA CAR-T in a murine model of CEA+ LM and identified mechanisms through which L-MDSC expand and inhibit CAR-T function. We established CEA+ LM in mice and studied purified L-MDSC and responses to treatment with intrahepatic anti-CEA CAR-T infusions. L-MDSC expanded threefold in response to LM, and their expansion was dependent on GM-CSF, which was produced by tumor cells. L-MDSC utilized PD-L1 to suppress anti-tumor responses through engagement of PD-1 on CAR-T. GM-CSF, in cooperation with STAT3, promoted L-MDSC PD-L1 expression. CAR-T efficacy was rescued when mice received CAR-T in combination with MDSC depletion, GM-CSF neutralization to prevent MDSC expansion, or PD-L1 blockade. As L-MDSC suppressed anti-CEA CAR-T, infusion of anti-CEA CAR-T in tandem with agents targeting L-MDSC is a rational strategy for future clinical trials.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno Carcinoembrionário/imunologia , Neoplasias Hepáticas/patologia , Células Mieloides/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fígado/citologia , Fígado/patologia , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Fator de Transcrição STAT3/metabolismo , Carga Tumoral
12.
Clin Cancer Res ; 21(14): 3149-59, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25850950

RESUMO

PURPOSE: Chimeric antigen receptor-modified T cells (CAR-T) have demonstrated encouraging results in early-phase clinical trials. Successful adaptation of CAR-T technology for CEA-expressing adenocarcinoma liver metastases, a major cause of death in patients with gastrointestinal cancers, has yet to be achieved. We sought to test intrahepatic delivery of anti-CEA CAR-T through percutaneous hepatic artery infusions (HAIs). EXPERIMENTAL DESIGN: We conducted a phase I trial to test HAI of CAR-T in patients with CEA(+) liver metastases. Six patients completed the protocol, and 3 received anti-CEA CAR-T HAIs alone in dose-escalation fashion (10(8), 10(9), and 10(10) cells). We treated an additional 3 patients with the maximum planned CAR-T HAI dose (10(10) cells × 3) along with systemic IL2 support. RESULTS: Four patients had more than 10 liver metastases, and patients received a mean of 2.5 lines of conventional systemic therapy before enrollment. No patient suffered a grade 3 or 4 adverse event related to the CAR-T HAIs. One patient remains alive with stable disease at 23 months following CAR-T HAI, and 5 patients died of progressive disease. Among the patients in the cohort that received systemic IL2 support, CEA levels decreased 37% (range, 19%-48%) from baseline. Biopsies demonstrated an increase in liver metastasis necrosis or fibrosis in 4 of 6 patients. Elevated serum IFNγ levels correlated with IL2 administration and CEA decreases. CONCLUSIONS: We have demonstrated the safety of anti-CEA CAR-T HAIs with encouraging signals of clinical activity in a heavily pretreated population with large tumor burdens. Further clinical testing of CAR-T HAIs for liver metastases is warranted.


Assuntos
Adenocarcinoma/terapia , Quimioterapia do Câncer por Perfusão Regional/métodos , Imunoterapia/métodos , Neoplasias Hepáticas/terapia , Receptores de Antígenos de Linfócitos T/administração & dosagem , Linfócitos T/transplante , Adenocarcinoma/secundário , Idoso , Feminino , Neoplasias Gastrointestinais/patologia , Neoplasias Gastrointestinais/terapia , Humanos , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade
13.
Cytokine ; 71(2): 339-47, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25516298

RESUMO

T cells reactive to tumor antigens and viral antigens lose their reactivity when exposed to the antigen-rich environment of a larger tumor bed or viral load. Such non-responsive T cells are termed exhausted. T cell exhaustion affects both CD8+ and CD4+ T cells. T cell exhaustion is attributed to the functional impairment of T cells to produce cytokines, of which the most important may be Interleukin 2 (IL2). IL2 performs functions critical for the elimination of cancer cells and virus infected cells. In one such function, IL2 promotes CD8+ T cell and natural killer (NK) cell cytolytic activities. Other functions include regulating naïve T cell differentiation into Th1 and Th2 subsets upon exposure to antigens. Thus, the signaling pathways contributing to T cell exhaustion could be linked to the signaling pathways contributing to IL2 loss. This review will discuss the process of T cell exhaustion and the signaling pathways that could be contributing to T cell exhaustion.


Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Regulação para Baixo , Interleucina-2/metabolismo , Regiões 3' não Traduzidas , Animais , Apoptose , Infecções Bacterianas/imunologia , Diferenciação Celular , Cromatina/metabolismo , Biologia Computacional , Epigênese Genética , Humanos , Imunoterapia , Células Matadoras Naturais/citologia , Doenças Parasitárias/imunologia , RNA Mensageiro/metabolismo , Transdução de Sinais
14.
Mol Ther Methods Clin Dev ; 1: 14022, 2014 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-25419532

RESUMO

Transduction and expression procedures in gene therapy protocols may optimally transfer more than a single gene to correct a defect and/or transmit new functions to recipient cells or organisms. This may be accomplished by transduction with two (or more) vectors, or, more efficiently, in a single vector. Occasionally, it may be useful to coexpress homologous genes or chimeric proteins with regions of shared homology. Retroviridae include the dominant vector systems for gene transfer (e.g., gamma-retro and lentiviruses) and are capable of such multigene expression. However, these same viruses are known for efficient recombination-deletion when domains are duplicated within the viral genome. This problem can be averted by resorting to two-vector strategies (two-chain two-vector), but at a penalty to cost, convenience, and efficiency. Employing a chimeric antigen receptor system as an example, we confirm that coexpression of two genes with homologous domains in a single gamma-retroviral vector (two-chain single-vector) leads to recombination-deletion between repeated sequences, excising the equivalent of one of the chimeric antigen receptors. Here, we show that a degenerate codon substitution strategy in the two-chain single-vector format efficiently suppressed intravector deletional loss with rescue of balanced gene coexpression by minimizing sequence homology between repeated domains and preserving the final protein sequence.

15.
J Med Primatol ; 43(5): 341-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25138734

RESUMO

BACKGROUND: To increase the immunosurveillance in HIV infection, we used retroviral vectors expressing CD4-chimeric antigen receptors (CARs) to genetically modify autologous T cells and redirect CTL toward HIV. The CD4 extracellular domain targets envelope and the intracellular signaling domains activate T cells. The maC46 fusion inhibitor binds HIV and blocks viral replication. METHODS: We stimulated rhesus PBMCs with antibodies to CD3/CD28 and cotransduced T cells with CD4-CAR and maC46 vectors. CD4-CAR-transduced T cells were added to Env(+) 293T cells at E:T of 1:1. Killing of target cells was measured as reduced impedance. RESULTS: We observed gene expression in 60-70% of rhesus CD3(+) CD8(+) T cells with the individual vectors and in 35% of the cells with both vectors. CD4-CAR-transduced populations specifically killed Env(+) cells. CONCLUSIONS: In these studies, we showed that designer T cells were redirected to kill Env(+) cells. Control of viremia without HAART would revolutionize treatment for HIV patients.


Assuntos
Condutividade Elétrica , Infecções por HIV/imunologia , HIV-1/fisiologia , Linfócitos T Citotóxicos/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Impedância Elétrica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Humanos , Imunoterapia , Macaca mulatta , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
16.
Prostate ; 74(3): 286-96, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24174378

RESUMO

BACKGROUND: Adoptive immunotherapy by infusion of designer T cells (dTc) engineered with chimeric antigen receptors (CARs) for tumoricidal activity represents a potentially highly specific modality for the treatment of cancer. In this study, 2nd generation (gen) anti-prostate specific membrane antigen (PSMA) dTc were developed for improving the efficacy of previously developed 1st gen dTc for prostate cancer immunotherapy. The 1st gen dTc are modified with chimeric immunoglobulin-T cell receptor (IgTCR) while the 2nd gen dTc are engineered with an immunoglobulin-CD28-T cell receptor (IgCD28TCR), which incorporates a CD28 costimulatory signal for optimal T cell activation. METHODS: A 2nd gen anti-PSMA IgCD28TCR CAR was constructed by inserting the CD28 signal domain into the 1st gen CAR. 1st and 2nd gen anti-PSMA dTc were created by transducing human T cells with anti-PSMA CARs and their antitumor efficacy was compared for specific activation on PSMA-expressing tumor contact, cytotoxicity against PSMA-expressing tumor cells in vitro, and suppression of tumor growth in an animal model. RESULTS: The 2nd gen dTc can be optimally activated to secrete larger amounts of cytokines such as IL2 and IFNγ than 1st gen and to proliferate more vigorously on PSMA-expressing tumor contact. More importantly, the 2nd gen dTc preserve the PSMA-specific cytotoxicity in vitro and suppress tumor growth in animal models with significant higher potency. CONCLUSIONS: Our results demonstrate that 2nd gen anti-PSMA designer T cells exhibit superior antitumor functions versus 1st gen, providing a rationale for advancing this improved agent toward clinical application in prostate cancer immunotherapy.


Assuntos
Imunoterapia Adotiva/métodos , Neoplasias da Próstata/terapia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais , Antígenos CD28/imunologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Citotoxicidade Imunológica , Vetores Genéticos/genética , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Células Jurkat , Ativação Linfocitária , Masculino , Camundongos , Camundongos Nus , Próstata/imunologia , Receptores de Antígenos/genética , Receptores de Antígenos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão , Retroviridae/genética , Transdução Genética , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Transpl Immunol ; 30(1): 40-5, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24262132

RESUMO

Impairing dendritic cell (DC) function to prevent graft versus host disease (GvHD) is an appealing concept. DC antigen presentation is NF-κB pathway-dependent and bortezomib might therefore play a role in preventing alloreactivity. We obtained DC from the blood of patients enrolled in a phase I study using post-transplant cyclophosphamide and bortezomib for prevention of GvHD. Control samples were obtained from patients receiving standard GvHD prevention regimen. Pre-treatment samples were also collected from enrolled patients. DC isolated on days +1, +4, and +7 showed progressive decrease in the expression of maturation markers in comparison to control. In a DC-CD4+ mixed lymphocyte reaction (MLR) where DC isolated from the recipient blood before graft infusion were the stimulator cells, T cell proliferation measured by bromodeoxyuridine (BrdU) integration was decreased in samples obtained on days +14 and +21 in comparison to control group. Finally, measured by real-time PCR, the expression of IκB progressively increased while the expression of NF-κB decreased in DC on days +1, +4, and +7, in comparison to pre-treatment paired controls. We conclude that our data further justify exploring the role of bortezomib in GvHD prevention and propose a novel mechanism of action of bortezomib in DC.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Proteínas I-kappa B/metabolismo , NF-kappa B/metabolismo , Apresentação do Antígeno/efeitos dos fármacos , Apresentação do Antígeno/imunologia , Ácidos Borônicos/administração & dosagem , Bortezomib , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Ciclofosfamida/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Doença Enxerto-Hospedeiro/imunologia , Humanos , Proteínas I-kappa B/genética , Imunossupressores/administração & dosagem , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , NF-kappa B/genética , Cuidados Pós-Operatórios , Pirazinas/administração & dosagem
18.
Virology ; 446(1-2): 268-75, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24074590

RESUMO

The current antiretroviral therapy (ART) can effectively reduce plasma HIV loads to undetectable levels, but cannot eliminate latently infected resting memory CD4 T cells that persist for the lifetime of infected patients. Therefore, designing new therapeutic approaches to eliminate these latently infected cells or the cells that produce HIV upon reactivation from latency is a priority in the ART era in order to progress to a cure of HIV. Here, we show that "designer" T cells expressing chimeric antigen receptor (CAR), CD4-CD28-CD3ζ, can target and kill HIV Env-expressing cells. Further, they secrete effector cytokines upon contact with HIV Env+ target cells that can reactivate latent HIV in a cell line model, thereby exposing those cells to recognition and killing by anti-HIV CAR+ T cells. Taken to the limit, this process could form the basis for an eventual functional or sterilizing cure for HIV in patients.


Assuntos
Antígenos CD28/biossíntese , Complexo CD3/biossíntese , Antígenos CD4/biossíntese , HIV/imunologia , Receptores de Antígenos/biossíntese , Linfócitos T/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Antígenos CD28/genética , Complexo CD3/genética , Antígenos CD4/genética , Linhagem Celular , Citotoxicidade Imunológica , Infecções por HIV/terapia , Humanos , Imunoterapia/métodos , Receptores de Antígenos/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/biossíntese
19.
J Leukoc Biol ; 94(4): 813-23, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23883516

RESUMO

Biliary obstruction is a common clinical problem that is associated with intrahepatic inflammation and impaired immunity. PD-1 is well known to mediate T cell dysfunction but has been reported to promote and attenuate acute inflammation in various injury models. With the use of a well-established murine model of BDL, we studied the effects of intrahepatic PD-1 expression on LTC function, inflammation, and cholestasis. Following BDL, PD-1 expression increased significantly among LTCs. Increased PD-1 expression following BDL was associated with decreased LTC proliferation and less IFN-γ production. Elimination of PD-1 expression resulted in significantly improved proliferative capacity among LTC following BDL, in addition to a more immunostimulatory cytokine profile. Not only was LTC function rescued in PD-1(-/-) mice, but also, the degrees of biliary cell injury, cholestasis, and inflammation were diminished significantly compared with WT animals following BDL. PD-1-mediated acute inflammation following BDL was associated with expansions of intrahepatic neutrophil and Th17 cell populations, with the latter dependent on IL-6. PD-1 blockade represents an attractive strategy for reversing intrahepatic immunosuppression while limiting inflammatory liver damage.


Assuntos
Colestase/imunologia , Colestase/fisiopatologia , Inflamação/imunologia , Fígado/patologia , Neutrófilos/patologia , Receptor de Morte Celular Programada 1/metabolismo , Células Th17/patologia , Animais , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Proliferação de Células , Colestase/complicações , Colestase/patologia , Inflamação/complicações , Inflamação/patologia , Interleucina-6/metabolismo , Icterícia/complicações , Icterícia/imunologia , Icterícia/patologia , Icterícia/fisiopatologia , Ligadura , Fígado/imunologia , Fígado/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Regulação para Cima
20.
J Transl Med ; 11: 46, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23433424

RESUMO

BACKGROUND: Imatinib mesylate is an effective treatment for metastatic gastrointestinal stromal tumor (GIST). However, most patients eventually develop resistance and there are few other treatment options. Immunotherapy using genetically modified or designer T cells (dTc) has gained increased attention for several malignancies in recent years. The aims of this study were to develop and test novel anti-KIT dTc engineered to target GIST cells. METHODS: Human anti-KIT dTc were created by retroviral transduction with novel chimeric immune receptors (CIR). The gene for stem cell factor (SCF), the natural ligand for KIT, was cloned into 1st generation (SCF-CD3ζ, 1st gen) and 2nd generation (SCF-CD28-CD3ζ, 2nd gen) CIR constructs. In vitro dTc proliferation and tumoricidal capacity in the presence of KIT+ tumor cells were measured. In vivo assessment of dTc anti-tumor efficacy was performed by treating immunodeficient mice harboring subcutaneous GIST xenografts with dTc tail vein infusions. RESULTS: We successfully produced the 1st and 2nd gen anti-KIT CIR and transduced murine and human T cells. Average transduction efficiencies for human 1st and 2nd gen dTc were 50% and 42%. When co-cultured with KIT+ tumor cells, both 1st and 2nd gen dTc proliferated and produced IFNγ. Human anti-KIT dTc were efficient at lysing GIST in vitro compared to untransduced T cells. In mice with established GIST xenografts, treatment with either 1st or 2nd gen human anti-KIT dTc led to significant reductions in tumor growth rates. CONCLUSIONS: We have constructed a novel anti-KIT CIR for production of dTc that possess specific activity against KIT+ GIST in vitro and in vivo. Further studies are warranted to evaluate the therapeutic potential and safety of anti-KIT dTc.


Assuntos
Tumores do Estroma Gastrointestinal/terapia , Proteínas Proto-Oncogênicas c-kit/imunologia , Linfócitos T/citologia , Animais , Sequência de Bases , Proliferação de Células , Primers do DNA , Tumores do Estroma Gastrointestinal/patologia , Masculino , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase , Linfócitos T/imunologia
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