RESUMO
Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies. SIGNIFICANCE: We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach. See related commentary by Capparelli and Aplin, p. 1413. This article is highlighted in the In This Issue feature, p. 1397.
Assuntos
Antígeno B7-H1 , Neoplasias , Animais , Humanos , Inibidores de Checkpoint Imunológico , Ativação Linfocitária , Camundongos , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1RESUMO
Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.
Assuntos
Ligante 4-1BB/agonistas , Anticorpos Biespecíficos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Ligante 4-1BB/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos , Humanos , Inibidores de Checkpoint Imunológico/imunologia , Tolerância Imunológica/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Imunoterapia , Ativação Linfocitária/efeitos dos fármacosRESUMO
Chimeric antigen receptor (CAR)-modified T cells are endowed with novel antigen specificity and are most often administered to patients without an engineered mechanism to control the CAR T cells once infused. "Suicide switches" such as the small molecule-controlled, inducible caspase-9 (iCas9) system afford the ability to selectively eliminate engineered T cells; however, these approaches are designed for all-or-none, irreversible termination of an ongoing immune response. In order to permit reversible and adjustable modulation, we have created a CAR that is capable of on-demand downregulation by fusing the CAR to a previously developed ligand-induced degradation (LID) domain. Addition of a small molecule ligand triggers exposure of a cryptic degron within the LID domain, resulting in proteasomal degradation of the CAR-LID fusion protein and loss of CAR on the surface of T cells. This fusion construct allowed for reversible and "tunable" inhibition of CAR T cell activity in vitro. Delivery of the triggering molecule in CAR-LID-treated tumor-bearing mice temporarily reduced CAR activity through modulation of CAR surface expression. The ability to more flexibly modulate CAR T cell expression through a small molecule provides a platform for controlling possible adverse side effects, as well as preclinical investigations of CAR T cell biology.
Assuntos
Morfolinas/química , Neoplasias/terapia , Receptores de Antígenos Quiméricos/metabolismo , Proteínas Recombinantes de Fusão/química , Bibliotecas de Moléculas Pequenas/administração & dosagem , Linfócitos T/transplante , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Imunoterapia Adotiva , Ligantes , Camundongos , Transplante de Neoplasias , Neoplasias/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Proteólise , Receptores de Antígenos Quiméricos/química , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
T cell trafficking into tumors depends on a "match" between chemokine receptors on effector cells (e.g., CXCR3 and CCR5) and tumor-secreted chemokines. There is often a chemokine/chemokine receptor "mismatch", with tumors producing minute amounts of chemokines, resulting in inefficient targeting of effectors to tumors. We aimed to alter tumors to produce higher levels of CXCL11, a CXCR3 ligand, to attract more effector cells following immunotherapy. Mice bearing established subcutaneous tumors were studied. In our first approach, we used modified chimeric antigen receptor (CAR)-transduced human T cells to deliver CXCL11 (CAR/CXCL11) into tumors. In our second approach, we intravenously (iv) administered a modified oncolytic vaccinia virus (VV) engineered to produce CXCL11 (VV.CXCL11). The effect of these treatments on T cell trafficking into the tumors and anti-tumor efficacy after subsequent CAR T cell injections or anti-tumor vaccines was determined. CAR/CXCL11 and VV.CXCL11 significantly increased CXCL11 protein levels within tumors. For CAR/CXCL11, injection of a subsequent dose of CAR T cells did not result in increased intra-tumoral trafficking, and appeared to decrease the function of the injected CAR T cells. In contrast, VV.CXCL11 increased the number of total and antigen-specific T cells within tumors after CAR T cell injection or vaccination and significantly enhanced anti-tumor efficacy. Both approaches were successful in increasing CXCL11 levels within the tumors; however, only the vaccinia approach was successful in recruiting T cells and augmenting anti-tumor efficacy. VV.CXCL11 should be considered as a potential approach to augment adoptive T cell transfer or vaccine immunotherapy.
RESUMO
Neutrophils are important innate immune cells involved in microbial clearance at the sites of infection. However, their role in cancer development is unclear. We hypothesized that neutrophils mediate antitumor effects in early tumorigenesis. To test this, we first studied the cytotoxic effects of neutrophils in vitro. Neutrophils were cytotoxic against tumor cells, with neutrophils isolated from tumor-bearing mice trending to have increased cytotoxic activities. We then injected an ELR+ CXC chemokine-producing tumor cell line into C57BL/6 and Cxcr2-/- mice, the latter lacking the receptors for neutrophil chemokines. We observed increased tumor growth in Cxcr2-/- mice. As expected, tumors from Cxcr2-/- mice contained fewer neutrophils. Surprisingly, these tumors also contained fewer CD8+ T cells, but more IL-17-producing cells. Replenishment of functional neutrophils was correlated with decreased IL-17-producing cells, increased CD8+ T cells, and decreased tumor size in Cxcr2-/- mice, while depletion of neutrophils in C57BL/6 mice showed the opposite effects. Results from a non-ELR+ CXC chemokine producing tumor further supported that functional neutrophils indirectly mediate tumor control by suppressing IL-17A production. We further studied the correlation of IL-17A and CD8+ T cells in vitro. IL-17A suppressed proliferation and IFNγ production of CD8+ T cells, while CD11b+Ly6G+ neutrophils did not suppress CD8+ T cell function. Taken together, these data demonstrate that, while neutrophils could control tumor growth by direct cytotoxic effects, the primary mechanism by which neutrophils exert antitumor effects is to regulate IL-17 production, through which they indirectly promote CD8+ T cell responses.
RESUMO
Histone/protein deacetylases (HDACs) are frequently upregulated in human malignancies and have therefore become therapeutic targets in cancer therapy. However, inhibiting certain HDAC isoforms can have protolerogenic effects on the immune system, which could make it easier for tumor cells to evade the host immune system. Therefore, a better understanding of how each HDAC isoform affects immune biology is needed to develop targeted cancer therapy. Here, we studied the immune phenotype of HDAC5(-/-) mice on a C57BL/6 background. While HDAC5(-/-) mice replicate at expected Mendelian ratios and do not develop overt autoimmune disease, their T-regulatory (Treg) cells show reduced suppressive function in vitro and in vivo. Likewise, CD4(+) T-cells lacking HDAC5 convert poorly to Tregs under appropriately polarizing conditions. To test if this attenuated Treg formation and suppressive function translated into improved anticancer immunity, we inoculated HDAC5(-/-) mice and littermate controls with a lung adenocarcinoma cell line. Cumulatively, lack of HDAC5 did not lead to better anticancer immunity. We found that CD8(+) T cells missing HDAC5 had a reduced ability to produce the cytokine, IFN-γ, in vitro and in vivo, which may offset the benefit of weakened Treg function and formation. Taken together, targeting HDAC5 weakens suppressive function and de-novo induction of Tregs, but also reduces the ability of CD8(+) T cells to produce IFN-γ.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Linhagem Celular Tumoral , Citocinas/biossíntese , Fatores de Transcrição Forkhead/metabolismo , Deleção de Genes , Marcação de Genes , Interferon gama/biossíntese , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Malignant cells drive the generation of a desmoplastic and immunosuppressive tumor microenvironment. Cancer-associated stromal cells (CASC) are a heterogeneous population that provides both negative and positive signals for tumor cell growth and metastasis. Fibroblast activation protein (FAP) is a marker of a major subset of CASCs in virtually all carcinomas. Clinically, FAP expression serves as an independent negative prognostic factor for multiple types of human malignancies. Prior studies established that depletion of FAP(+) cells inhibits tumor growth by augmenting antitumor immunity. However, the potential for immune-independent effects on tumor growth have not been defined. Herein, we demonstrate that FAP(+) CASCs are required for maintenance of the provisional tumor stroma because depletion of these cells, by adoptive transfer of FAP-targeted chimeric antigen receptor (CAR) T cells, reduced extracellular matrix proteins and glycosaminoglycans. Adoptive transfer of FAP-CAR T cells also decreased tumor vascular density and restrained growth of desmoplastic human lung cancer xenografts and syngeneic murine pancreatic cancers in an immune-independent fashion. Adoptive transfer of FAP-CAR T cells also restrained autochthonous pancreatic cancer growth. These data distinguish the function of FAP(+) CASCs from other CASC subsets and provide support for further development of FAP(+) stromal cell-targeted therapies for the treatment of solid tumors.
Assuntos
Matriz Extracelular/patologia , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/patologia , Serina Endopeptidases/metabolismo , Células Estromais/fisiologia , Microambiente Tumoral/fisiologia , Animais , Endopeptidases , Transição Epitelial-Mesenquimal/genética , Gelatinases/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/imunologia , Serina Endopeptidases/genética , Células Estromais/metabolismo , Células Tumorais CultivadasRESUMO
Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity toward B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared with standard first- and second-generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Linfócitos B/imunologia , Linfócitos do Interstício Tumoral/imunologia , Proteínas de Membrana/genética , Mesotelioma/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva , Camundongos , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Immunotherapy using vaccines or adoptively transferred tumor-infiltrating lymphocytes (TIL) is limited by T-cell functional inactivation within the solid tumor microenvironment. The purpose of this study was to determine whether a similar tumor-induced inhibition occurred with genetically modified cytotoxic T cells expressing chimeric antigen receptors (CAR) targeting tumor-associated antigens. EXPERIMENTAL DESIGN: Human T cells expressing CAR targeting mesothelin or fibroblast activation protein and containing CD3ζ and 4-1BB cytoplasmic domains were intravenously injected into immunodeficient mice bearing large, established human mesothelin-expressing flank tumors. CAR TILs were isolated from tumors at various time points and evaluated for effector functions and status of inhibitory pathways. RESULTS: CAR T cells were able to traffic into tumors with varying efficiency and proliferate. They were able to slow tumor growth, but did not cause regressions or cures. The CAR TILs underwent rapid loss of functional activity that limited their therapeutic efficacy. This hypofunction was reversible when the T cells were isolated away from the tumor. The cause of the hypofunction seemed to be multifactorial and was associated with upregulation of intrinsic T-cell inhibitory enzymes (diacylglycerol kinase and SHP-1) and the expression of surface inhibitory receptors (PD1, LAG3, TIM3, and 2B4). CONCLUSIONS: Advanced-generation human CAR T cells are reversibly inactivated within the solid tumor microenvironment of some tumors by multiple mechanisms. The model described here will be an important tool for testing T cell-based strategies or systemic approaches to overcome this tumor-induced inhibition. Our results suggest that PD1 pathway antagonism may augment human CAR T-cell function.
Assuntos
Citotoxicidade Imunológica/imunologia , Proteínas Ligadas por GPI/imunologia , Gelatinases/imunologia , Linfócitos do Interstício Tumoral/imunologia , Proteínas de Membrana/imunologia , Mesotelioma/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Serina Endopeptidases/imunologia , Linfócitos T/imunologia , Animais , Células 3T3 BALB , Endopeptidases , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Gelatinases/genética , Gelatinases/metabolismo , Humanos , Imunoterapia Adotiva , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mesotelina , Mesotelioma/metabolismo , Mesotelioma/terapia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The majority of chimeric antigen receptor (CAR) T-cell research has focused on attacking cancer cells. Here, we show that targeting the tumor-promoting, nontransformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single-chain Fv FAP [monoclonal antibody (mAb) 73.3] with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFN-γ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAP(hi) stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8(+) T-cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T-cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective, suggesting that further clinical development of anti-human FAP-CAR is warranted.
Assuntos
Transferência Adotiva/métodos , Gelatinases/imunologia , Proteínas de Membrana/imunologia , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Serina Endopeptidases/imunologia , Linfócitos T/transplante , Transferência Adotiva/efeitos adversos , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Endopeptidases , Imunidade Celular , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Transporte Proteico , Células Estromais/imunologia , Linfócitos T/imunologia , Transdução GenéticaRESUMO
Progression of premalignant lesions is restrained by oncogene-induced senescence. Oncogenic Ras triggers senescence in many organs, including the lung, which exhibits high levels of the angiogenesis inhibitor thrombospondin-1 (TSP-1). The contribution of TSP-1 upregulation to the modulation of tumorigenesis in the lung is unclear. Using a mouse model of lung cancer, we have shown that TSP-1 plays a critical and cell-autonomous role in suppressing Kras-induced lung tumorigenesis independent of its antiangiogenic function. Overall survival was decreased in a Kras-driven mouse model of lung cancer on a Tsp-1-/- background. We found that oncogenic Kras-induced TSP-1 upregulation in a p53-dependent manner. TSP-1 functioned in a positive feedback loop to stabilize p53 by interacting directly with activated ERK. TSP-1 tethering of ERK in the cytoplasm promoted a level of MAPK signaling that was sufficient to sustain p53 expression and a senescence response. Our data identify TSP-1 as a p53 target that contributes to maintaining Ras-induced senescence in the lung.
Assuntos
Adenocarcinoma/metabolismo , Adenoma/metabolismo , Carcinogênese/metabolismo , Neoplasias Pulmonares/metabolismo , Lesões Pré-Cancerosas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Trombospondina 1/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/patologia , Animais , Carcinogênese/genética , Senescência Celular , Epitélio/metabolismo , Epitélio/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Transporte Proteico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Forkhead box P3 (Foxp3)(+) T regulatory (T(reg)) cells maintain immune homeostasis and limit autoimmunity but can also curtail host immune responses to various types of tumors. Foxp3(+) T(reg) cells are therefore considered promising targets to enhance antitumor immunity, and approaches for their therapeutic modulation are being developed. However, although studies showing that experimentally depleting Foxp3(+) T(reg) cells can enhance antitumor responses provide proof of principle, these studies lack clear translational potential and have various shortcomings. Histone/protein acetyltransferases (HATs) promote chromatin accessibility, gene transcription and the function of multiple transcription factors and nonhistone proteins. We now report that conditional deletion or pharmacologic inhibition of one HAT, p300 (also known as Ep300 or KAT3B), in Foxp3(+) T(reg) cells increased T cell receptor-induced apoptosis in T(reg) cells, impaired T(reg) cell suppressive function and peripheral T(reg) cell induction, and limited tumor growth in immunocompetent but not in immunodeficient mice. Our data thereby demonstrate that p300 is important for Foxp3(+) T(reg) cell function and homeostasis in vivo and in vitro, and identify mechanisms by which appropriate small-molecule inhibitors can diminish T(reg) cell function without overtly impairing T effector cell responses or inducing autoimmunity. Collectively, these data suggest a new approach for cancer immunotherapy.
Assuntos
Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Proliferação de Células , Cromatina , Feminino , Fatores de Transcrição Forkhead/metabolismo , Células HEK293 , Humanos , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/metabolismoRESUMO
Recent clinical trials have shown promise in the use of chimeric antigen receptor (CAR)-transduced T cells; however, augmentation of their activity may broaden their clinical use and improve their efficacy. We hypothesized that because CAR action requires proteins essential for T-cell receptor (TCR) signal transduction, deletion of negative regulators of these signaling pathways would enhance CAR signaling and effector T-cell function. We tested CAR activity and function in T cells that lacked one or both isoforms of diacylglycerol kinase (dgk) expressed highly in T cells, dgkα and dgkζ, enzymes that metabolize the second messenger diacylglycerol (DAG) and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin showed greatly enhanced cytokine production and cytotoxicity when cocultured with a murine mesothelioma line that stably expresses mesothelin. In addition, we found that dgk-deficient CAR-transduced T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of tumor. Consistent with our studies in mice, pharmacologic inhibition of dgks also augments function of primary human T cells transduced with CARs. These results suggest that deletion of negative regulators of TCR signaling enhances the activity and function of CAR-expressing T cells and identify dgks as potential targets for improving the clinical potential of CARs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diacilglicerol Quinase/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Western Blotting , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Células Cultivadas , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Diglicerídeos/imunologia , Diglicerídeos/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI/imunologia , Células HEK293 , Humanos , Imunoterapia Adotiva , Isoenzimas/genética , Isoenzimas/imunologia , Isoenzimas/metabolismo , Mesotelina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologiaRESUMO
PURPOSE: Thymidylate synthase (TS) is a potential predictor of outcome after pemetrexed (Pem) in patients with malignant pleural mesothelioma (MPM), and assays measuring TS levels are commercially marketed. The goal of this study was to further evaluate the value of TS and to study another potential biomarker of response, the enzyme, folyl-polyglutamate synthase (FPGS), which activates Pem intracellularly. METHODS: Levels of TS and FPGS were semi-quantitatively determined immunohistochemically using H-scores on tissue samples from 85 MPM patients receiving Pem as primary therapy. H-score was correlated with radiographic disease control rate (DCR), time to progression (TTP) and overall survival (OS). In addition, expression levels of TS and FPGS in MPM cell lines were determined using immunoblotting and correlated with their sensitivity to Pem-induced cell death. RESULTS: H-scores from patients with disease control versus progressive disease showed extensive overlap. There were no significant correlations of DCR, TTP, or OS to either TS levels (p = 0.73, 0.93, and 0.59, respectively), FPGS levels (p = 0.95, 0.77, and 0.43, respectively) or the ratio of FPGS/TS using the median scores of each test as cutoffs. There was no correlation between TS or FPGS expression and chemosensitivity of mesothelioma cells to Pem in vitro. CONCLUSIONS: Although previous retrospective data suggest that TS and FPGS expression might be potential markers of Pem efficacy in MPM, our data indicate these markers lack sufficient predictive value in individual patients and should not be used to guide therapeutic decisions in the absence of prospective studies.
Assuntos
Biomarcadores Tumorais/metabolismo , Glutamatos/uso terapêutico , Guanina/análogos & derivados , Mesotelioma/metabolismo , Peptídeo Sintases/metabolismo , Neoplasias Pleurais/metabolismo , Timidilato Sintase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antimetabólitos Antineoplásicos/uso terapêutico , Feminino , Seguimentos , Guanina/uso terapêutico , Humanos , Técnicas Imunoenzimáticas , Masculino , Mesotelioma/tratamento farmacológico , Mesotelioma/mortalidade , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Pemetrexede , Neoplasias Pleurais/tratamento farmacológico , Neoplasias Pleurais/mortalidade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
The receptor for advanced glycation end products (RAGE) is a multiligand pattern recognition receptor implicated in multiple disease states. Although RAGE is expressed on systemic vascular endothelium, the expression and function of RAGE on lung endothelium has not been studied. Utilizing in vitro (human) and in vivo (mouse) models, we established the presence of RAGE on lung endothelium. Because RAGE ligands can induce the expression of RAGE and stored red blood cells express the RAGE ligand N(ε)-carboxymethyl lysine, we investigated whether red blood cell (RBC) transfusion would augment RAGE expression on endothelium utilizing a syngeneic model of RBC transfusion. RBC transfusion not only increased lung endothelial RAGE expression but enhanced lung inflammation and endothelial activation, since lung high mobility group box 1 and vascular cell adhesion molecule 1 expression was elevated following transfusion. These effects were mediated by RAGE, since endothelial activation was absent in RBC-transfused RAGE knockout mice. Thus, RAGE is inducibly expressed on lung endothelium, and one functional consequence of RBC transfusion is increased RAGE expression and endothelial activation.
Assuntos
Endotélio Vascular/metabolismo , Eritrócitos/fisiologia , Pulmão/metabolismo , Receptores Imunológicos/fisiologia , Animais , Células Endoteliais/fisiologia , Transfusão de Eritrócitos , Células HEK293 , Proteína HMGB1/metabolismo , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Camundongos , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/biossíntese , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
Effector T cells become rapidly inactivated after antigen exposure due to extracellular as well as intrinsic signals. We have recently demonstrated that the deletion of diacylglycerol kinases, intrinsic inhibitors of T-cell signaling, enhances the activity of adoptively transferred T cells expressing a chimeric antigen receptor (CAR) specific for a tumor-associated antigen.
RESUMO
Each year, more than 700,000 people undergo cancer surgery in the United States. However, more than 40% of those patients develop recurrences and have a poor outcome. Traditionally, the medical community has assumed that recurrent tumors arise from selected tumor clones that are refractory to therapy. However, we found that tumor cells have few phenotypical differences after surgery. Thus, we propose an alternative explanation for the resistance of recurrent tumors. Surgery promotes inhibitory factors that allow lingering immunosuppressive cells to repopulate small pockets of residual disease quickly. Recurrent tumors and draining lymph nodes are infiltrated with M2 (CD11b(+)F4/80(hi)CD206(hi) and CD11b(+)F4/80(hi)CD124(hi)) macrophages and CD4(+)Foxp3(+) regulatory T cells. This complex network of immunosuppression in the surrounding tumor microenvironment explains the resistance of tumor recurrences to conventional cancer vaccines despite small tumor size, an intact antitumor immune response, and unaltered cancer cells. Therapeutic strategies coupling antitumor agents with inhibition of immunosuppressive cells potentially could impact the outcomes of more than 250,000 people each year.
Assuntos
Vacinas Anticâncer/imunologia , Recidiva Local de Neoplasia/imunologia , Neoplasias/imunologia , Microambiente Tumoral/imunologia , Animais , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-4/imunologia , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Estimativa de Kaplan-Meier , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Neoplasias/cirurgia , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Falha de Tratamento , Vacinação/métodosRESUMO
Mesothelin is a cell-surface glycoprotein present on mesothelial cells and elicits T cell responses in a variety of cancers including pancreatic, biliary and ovarian cancer. Breast cancer is not known to express mesothelin. We postulated that mesothelin may be a unique tumor-associated antigen in triple negative breast cancer (TNBC), a less common breast cancer subtype which may have been under-represented in prior studies that characterized mesothelin expression. Therefore, we screened 99 primary breast cancer samples by immunohistochemistry analysis using formalin-fixed paraffin-embedded archival tumor tissues and confirmed that mesothelin was overexpressed in the majority of TNBC (67 %) but only rarely in <5 % ER(+) or Her2-neu(+) breast cancer, respectively. To determine whether mesothelin may be exploited as a novel immunotherapy target in breast cancer, an in vitro cell killing assay was performed to compare the ability of genetically modified T cells expressing a chimeric antibody receptor (CAR) specific for mesothelin (mesoCAR T cells) or non-transduced T cells to kill mesothelin-expressing primary breast cancer cells. A significantly higher anti-tumor cytotoxicity by mesoCAR T cells was observed (31.7 vs. 8.7 %, p < 0.001). Our results suggest that mesothelin has promise as a novel immunotherapy target for TNBC for which effective targeted therapy is lacking to date.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Proteínas Ligadas por GPI/imunologia , Imunoterapia , Transferência Adotiva , Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Estudos de Coortes , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Mesotelina , Receptor ErbB-2/deficiência , Receptores de Estrogênio/deficiência , Receptores de Progesterona/deficiência , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução GenéticaRESUMO
Since previous work using a nonreplicating adenovirus-expressing mouse interferon-ß (Ad.mIFNß) showed promising preclinical activity, we postulated that a vector-expressing IFNß at high levels that could also replicate would be even more beneficial. Accordingly a replication competent, recombinant vaccinia viral vector-expressing mIFNß (VV.mIFNß) was tested. VV.mIFNß-induced antitumor responses in two syngeneic mouse flank models of lung cancer. Although VV.mIFNß had equivalent in vivo efficacy in both murine tumor models, the mechanisms of tumor killing were completely different. In LKRM2 tumors, viral replication was minimal and the tumor killing mechanism was due to activation of immune responses through induction of a local inflammatory response and production of antitumor CD8 T-cells. In contrast, in TC-1 tumors, the vector replicated well, induced an innate immune response, but antitumor activity was primarily due to a direct oncolytic effect. However, the VV.mIFNß vector was able to augment the efficacy of an antitumor vaccine in the TC-1 tumor model in association with increased numbers of infiltrating CD8 T-cells. These data show the complex relationships between oncolytic viruses and the immune system which, if understood and harnessed correctly, could potentially be used to enhance the efficacy of immunotherapy.