RESUMEN
OBJECTIVE: Chemokine pathways are co-opted by pancreatic adenocarcinoma (PDAC) to facilitate myeloid cell recruitment from the bone marrow to establish an immunosuppressive tumour microenvironment (TME). Targeting tumour-associated CXCR2+neutrophils (TAN) or tumour-associated CCR2+ macrophages (TAM) alone improves antitumour immunity in preclinical models. However, a compensatory influx of an alternative myeloid subset may result in a persistent immunosuppressive TME and promote therapeutic resistance. Here, we show CCR2 and CXCR2 combined blockade reduces total tumour-infiltrating myeloids, promoting a more robust antitumour immune response in PDAC compared with either strategy alone. METHODS: Blood, bone marrow and tumours were analysed from PDAC patients and controls. Treatment response and correlative studies were performed in mice with established orthotopic PDAC tumours treated with a small molecule CCR2 inhibitor (CCR2i) and CXCR2 inhibitor (CXCR2i), alone and in combination with chemotherapy. RESULTS: A systemic increase in CXCR2+ TAN correlates with poor prognosis in PDAC, and patients receiving CCR2i showed increased tumour-infiltrating CXCR2+ TAN following treatment. In an orthotopic PDAC model, CXCR2 blockade prevented neutrophil mobilisation from the circulation and augmented chemotherapeutic efficacy. However, depletion of either CXCR2+ TAN or CCR2+ TAM resulted in a compensatory response of the alternative myeloid subset, recapitulating human disease. This was overcome by combined CCR2i and CXCR2i, which augmented antitumour immunity and improved response to FOLFIRINOX chemotherapy. CONCLUSION: Dual targeting of CCR2+ TAM and CXCR2+ TAN improves antitumour immunity and chemotherapeutic response in PDAC compared with either strategy alone.
Asunto(s)
Carcinoma Ductal Pancreático/inmunología , Macrófagos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neoplasias Pancreáticas/inmunología , Microambiente Tumoral/inmunología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Bases de Datos Factuales , Citometría de Flujo , Humanos , Inmunohistoquímica , Inmunomodulación , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CCR2/antagonistas & inhibidores , Receptores CCR2/metabolismo , Receptores de Interleucina-8B/antagonistas & inhibidores , Receptores de Interleucina-8B/metabolismo , Análisis de Matrices Tisulares , Microambiente Tumoral/efectos de los fármacosRESUMEN
Activation of naïve cluster of differentiation (CD)8(+) cytotoxic T lymphocytes (CTLs) is a tightly regulated process, and specific dendritic cell (DC) subsets are typically required to activate naive CTLs. Potential pathways for antigen presentation leading to CD8(+) T-cell priming include direct presentation, cross-presentation, and cross-dressing. To distinguish between these pathways, we designed single-chain trimer (SCT) peptide-MHC class I complexes that can be recognized as intact molecules but cannot deliver antigen to MHC through conventional antigen processing. We demonstrate that cross-dressing is a robust pathway of antigen presentation following vaccination, capable of efficiently activating both naïve and memory CD8(+) T cells and requires CD8α(+)/CD103(+) DCs. Significantly, immune responses induced exclusively by cross-dressing were as strong as those induced exclusively through cross-presentation. Thus, cross-dressing is an important pathway of antigen presentation, with important implications for the study of CD8(+) T-cell responses to viral infection, tumors, and vaccines.
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Presentación de Antígeno , Antígenos CD/inmunología , Antígenos CD8/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Cadenas alfa de Integrinas/inmunología , Vacunación , Animales , Antígenos CD/genética , Antígenos CD8/genética , Linfocitos T CD8-positivos/citología , Células Cultivadas , Células Dendríticas/citología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Cadenas alfa de Integrinas/genética , Ratones , Ratones Noqueados , Péptidos/genética , Péptidos/inmunologíaRESUMEN
BACKGROUND: The targeted delivery of cancer therapeutics represents an ongoing challenge in the field of drug development. TRAIL is a promising cancer drug but its activity profile could benefit from a cancer-selective delivery mechanism, which would reduce potential side effects and increase treatment efficiencies. We recently developed the novel TRAIL-based drug platform TR3, a genetically fused trimer with the capacity for further molecular modifications such as the addition of tumor-directed targeting moieties. MUC16 (CA125) is a well characterized biomarker in several human malignancies including ovarian, pancreatic and breast cancer. Mesothelin is known to interact with MUC16 with high affinity. In order to deliver TR3 selectively to MUC16-expressing cancers, we investigated the possibility of targeted TR3 delivery employing the high affinity mesothelin/MUC16 ligand/receptor interaction. METHODS: Using genetic engineering, we designed the novel cancer drug Meso-TR3, a fusion protein between native mesothelin and TR3. The recombinant proteins were produced with mammalian HEK293T cells. Meso-TR3 was characterized for binding selectivity and killing efficacy against MUC16-positive cancer cells and controls that lack MUC16 expression. Drug efficacy experiments were performed in vitro and in vivo employing an intraperitoneal xenograft mouse model of ovarian cancer. RESULTS: Similar to soluble mesothelin itself, the strong MUC16 binding property was retained in the Meso-TR3 fusion protein. The high affinity ligand/receptor interaction was associated with a selective accumulation of the cancer drug on MUC16-expressing cancer targets and directly correlated with increased killing activity in vitro and in a xenograft mouse model of ovarian cancer. The relevance of the mesothelin/MUC16 interaction for attaching Meso-TR3 to the cancer cells was verified by competitive blocking experiments using soluble mesothelin. Mechanistic studies using soluble DR5-Fc and caspase blocking assays confirmed engagement of the extrinsic death receptor pathway. Compared to non-targeted TR3, Meso-TR3 displayed a much reduced killing potency on cells that lack MUC16. CONCLUSIONS: Soluble Meso-TR3 targets the cancer biomarker MUC16 in vitro and in vivo. Following attachment to the tumor via surface bound MUC16, Meso-TR3 acquires full activation with superior killing profiles compared to non-targeted TR3, while its bioactivity is substantially reduced on cells that lack the tumor marker. This prodrug phenomenon represents a highly desirable property because it has the potential to enhance cancer killing with fewer side-effects than non-targeted TRAIL-based therapeutics. Thus, further exploration of this novel fusion protein is warranted as a possible therapeutic for patients with MUC16-positive malignancies.
Asunto(s)
Antineoplásicos/farmacología , Antígeno Ca-125/metabolismo , Portadores de Fármacos , Proteínas Ligadas a GPI/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Antineoplásicos/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Ligandos , Mesotelina , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Vaccine strategies aimed at generating CD8(+) T cell memory responses are likely to show augmented efficacy against chronic challenges like tumor. The abundance in variety of memory CD8(+) T cells behooves development of vaccine strategies that generate distinct memory responses and evaluate them for tumor efficacy. In this study, we demonstrate the ability of a variety of rapamycin treatment regimens to regulate virus vaccination-induced CD8(+) T cell memory responses and tumor efficacy. Strikingly, a short course of high-dose, but not low-dose, rapamycin treatment transiently blocks viral vaccination-induced mammalian target of rapamycin activity in CD8(+) T cells favoring persistence and Ag-recall responses over type 1 effector maturation; however, prolonged high-dose rapamycin administration abrogated memory responses. Furthermore, a short course of high-dose rapamycin treatment generated CD8(+) T cell memory responses that were independent of IL-15 and IL-7 and were programmed early for sustenance and greater tumor efficacy. These results demonstrate the impact a regimen of rapamycin treatment has on vaccine-induced CD8(+) T cell responses and indicates that judicious application of rapamycin can augment vaccine efficacy for chronic challenges.
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Adyuvantes Inmunológicos/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Memoria Inmunológica/efectos de los fármacos , Proteínas/inmunología , Sirolimus/uso terapéutico , Timoma/inmunología , Neoplasias del Timo/inmunología , Vacunación , Adyuvantes Inmunológicos/farmacología , Traslado Adoptivo , Animales , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/trasplante , Virus de la Viruela de los Canarios/inmunología , Vacunas contra el Cáncer/administración & dosificación , Selección Clonal Mediada por Antígenos , Interleucina-15/deficiencia , Interleucina-15/inmunología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Complejos Multiproteicos , Trasplante de Neoplasias , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Sirolimus/farmacología , Especificidad del Receptor de Antígeno de Linfocitos T , Serina-Treonina Quinasas TOR , Timoma/terapia , Neoplasias del Timo/terapiaRESUMEN
Mammaglobin-A (Mam-A) is a 10 kDa secretory protein that is overexpressed in 80 % of primary and metastatic human breast cancers. Previous studies from our laboratory demonstrated that Mam-A cDNA vaccine can induce Mam-A-specific CD8 T cell responses and mediate regression of human breast cancer xenografts in NOD/SCID mice. In this article, we present our results on a phase I clinical trial of a Mam-A cDNA vaccination in breast cancer patients with stage-IV metastatic disease, including the impact of vaccination on the expression of the inducible co-stimulator molecule (ICOS) on CD4 T cells. Specimens from seven patients with stage-IV metastatic cancer were available for these analyses. Patients were vaccinated with a Mam-A cDNA vaccine on days 0, 28, and 56, and immune responses were assessed at serial time points following vaccination. At 6 months following the first vaccination, flow cytometric analysis demonstrated a significant increase in the frequency of CD4+ICOS(hi) T cells from 5 ± 2 % pre-vaccination to 23 ± 4 % (p < 0.001), with a concomitant decrease in the frequency of CD4+FoxP3+ T cells (regulatory T cells [Treg]) from 19 ± 6 to 10 ± 5 % (p < 0.05). ELISpot analysis of CD4+ICOS(hi) sorted T cells demonstrated that following vaccination the cytokines produced by Mam-A-specific T cells switched from IL-10 (78 ± 21 spm pre-vaccination to 32 ± 14 spm 5 months post-vaccine p < 0.001) to IFN-γ (12 ± 6 spm pre-vaccination to 124 ± 31 spm 5 months post-vaccine p < 0.001). The ratio of CD4+ICOS(hi) T cells to CD4+FoxP3+ T cells increased from 0.37 ± 0.12 before vaccination to 2.3 ± 0.72 (p = 0.021) following vaccination. Further, these activated CD4+ICOS(hi) T cells induced preferential lysis of human breast cancer cells expressing Mam-A protein. We conclude that Mam-A cDNA vaccination is associated with specific expansion and activation of CD4+ICOS(hi) T cells, with a concomitant decrease in Treg frequency. These encouraging results strongly suggest that Mam-A cDNA vaccination can induce antitumor immunity in breast cancer patients.
Asunto(s)
Neoplasias de la Mama/inmunología , Linfocitos T CD4-Positivos/inmunología , Vacunas contra el Cáncer/inmunología , Mamoglobina A/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/inmunología , Neoplasias de la Mama/terapia , Linfocitos T CD4-Positivos/metabolismo , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , ADN Complementario , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Recuento de Linfocitos , Mamoglobina A/genética , Linfocitos T Citotóxicos/metabolismo , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
PURPOSE: This phase I study was conducted to determine the safety profile and maximum tolerated dose (MTD) of IMP321, a soluble lymphocyte activation gene-3 (LAG-3) Ig fusion protein and MHC Class II agonist, combined with gemcitabine in patients with advanced pancreatic adenocarcinoma. PATIENTS AND METHODS: Patients with advanced pancreatic adenocarcinoma were treated with gemcitabine (1,000 mg/m(2))(level 1), gemcitabine (1,000 mg/m(2)) plus IMP 321 at 0.5 mg (level 2) and 2.0 mg (level 3), respectively. Safety, toxicity, and immunological markers at baseline and post treatment were assessed. RESULTS: A total of 18 patients were enrolled to the study, and 17 were evaluable for toxicity. None of the 6 patients who received 0.5 mg IMP321 experienced IMP321-related adverse events. Of the 5 patients who received IMP321 at the 2 mg dose level, 1 experienced rash, 1 reported hot flashes and 2 had mild pain at the injection sites. No severe adverse events previously attributed to IMP321 were observed. No significant differences were observed when comparing pre- and post-treatment levels of monocytes (CD11b+CD14+), conventional dendritic cells (CD11c+) or T cell subsets (CD4, CD8). CONCLUSIONS: IMP321 in combination with gemcitabine is a well-tolerated regimen. IMP321 did not result in any severe adverse events. No incremental activity observed for the additional IMP 321 to gemcitabine at the dose levels evaluated, likely due to sub-optimal dosing. Immunological markers suggested that higher dose levels of IMP321 are needed for future clinical studies.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/administración & dosificación , Antimetabolitos Antineoplásicos/administración & dosificación , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Femenino , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Gemcitabina , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
PURPOSE: Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immunosuppressive cells that are upregulated in cancer. Little is known about the prevalence and importance of MDSC in pancreas adenocarcinoma (PA). EXPERIMENTAL DESIGN: Peripheral blood, bone marrow, and tumor samples were collected from pancreatic cancer patients, analyzed for MDSC (CD15(+)CD11b(+)) by flow cytometry and compared to cancer-free controls. The suppressive capacity of MDSC (CD11b(+)Gr-1(+)) and the effectiveness of MDSC depletion were assessed in C57BL/6 mice inoculated with Pan02, a murine PA, and treated with placebo or zoledronic acid, a potent aminobisphosphonate previously shown to target MDSC. The tumor microenvironment was analyzed for MDSC (Gr1(+)CD11b(+)), effector T cells, and tumor cytokine levels. RESULTS: Patients with PA demonstrated increased frequency of MDSC in the bone marrow and peripheral circulation which correlated with disease stage. Normal pancreas tissue showed no MDSC infiltrate, while human tumors avidly recruited MDSC. Murine tumors similarly recruited MDSC that suppressed CD8(+) T cells in vitro and accelerated tumor growth in vivo. Treatment with zoledronic acid impaired intratumoral MDSC accumulation resulting in delayed tumor growth rate, prolonged median survival, and increased recruitment of T cells to the tumor. This was associated with a more robust type 1 response with increased levels of IFN-γ and decreased levels of IL-10. CONCLUSIONS: MDSC are important mediators of tumor-induced immunosuppression in pancreatic cancer. Inhibiting MDSC accumulation with zoledronic acid improves the host anti-tumor response in animal studies suggesting that efforts to block MDSC may represent a novel treatment strategy for pancreatic cancer.
Asunto(s)
Adenocarcinoma/patología , Células de la Médula Ósea/patología , Células Mieloides/patología , Neoplasias Pancreáticas/patología , Adenocarcinoma/inmunología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Difosfonatos/farmacología , Modelos Animales de Enfermedad , Humanos , Imidazoles/farmacología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Células Mieloides/inmunología , Neoplasias Pancreáticas/inmunología , Microambiente Tumoral , Regulación hacia Arriba , Ácido ZoledrónicoRESUMEN
An important mechanism by which pancreatic cancer avoids antitumor immunity is by recruiting regulatory T cells (Tregs) to the tumor microenvironment. Recent studies suggest that suppressor Tregs and effector Th17 cells share a common lineage and differentiate based on the presence of certain cytokines in the microenvironment. Because IL-6 in the presence of TGF-ß has been shown to inhibit Treg development and induce Th17 cells, we hypothesized that altering the tumor cytokine environment could induce Th17 and reverse tumor-associated immune suppression. Pan02 murine pancreatic tumor cells that secrete TGF-ß were transduced with the gene encoding IL-6. C57BL/6 mice were injected s.c. with wild-type (WT), empty vector (EV), or IL-6-transduced Pan02 cells (IL-6 Pan02) to investigate the impact of IL-6 secretion in the tumor microenvironment. Mice bearing IL-6 Pan02 tumors demonstrated significant delay in tumor growth and better overall median survival compared with mice bearing WT or EV Pan02 tumors. Immunohistochemical analysis demonstrated an increase in Th17 cells (CD4(+)IL-23R(+) cells and CD4(+)IL-17(+) cells) in tumors of the IL-6 Pan02 group compared with WT or EV Pan02 tumors. The upregulation of IL-17-secreting CD4(+) tumor-infiltrating lymphocytes was substantiated at the cellular level by flow cytometry and ELISPOT assay and mRNA level for retinoic acid-related orphan receptor γt and IL-23R by RT-PCR. Thus, the addition of IL-6 to the tumor microenvironment skews the balance toward Th17 cells in a murine model of pancreatic cancer. The delayed tumor growth and improved survival suggests that induction of Th17 in the tumor microenvironment produces an antitumor effect.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Interleucina-17/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Pancreáticas/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Separación Celular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunohistoquímica , Interleucina-17/biosíntesis , Interleucina-6/inmunología , Interleucina-6/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/metabolismo , Transducción GenéticaRESUMEN
Tumors evade immune destruction by actively inducing immune tolerance through the recruitment of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg). We have previously described increased prevalence of these cells in pancreatic adenocarcinoma, but it remains unclear what mechanisms are involved in recruiting Tregs into the tumor microenvironment. Here, we postulated that chemokines might direct Treg homing to tumor. We show, in both human pancreatic adenocarcinoma and a murine pancreatic tumor model (Pan02), that tumor cells produce increased levels of ligands for the CCR5 chemokine receptor and, reciprocally, that CD4(+) Foxp3(+) Tregs, compared with CD4(+) Foxp3(-) effector T cells, preferentially express CCR5. When CCR5/CCL5 signaling is disrupted, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor (N,N-dimethyl-N-{{4-{[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl}amino}}benzyl]-N,N-dimethyl-N- {{{4-{{{[2-(4-methylphenyl)-6,7-dihydro-5H-benzocycloheptan-8-yl]carbonyl}amino}}benzyl}}}tetrahydro-2H-pyran-4-aminiumchloride; TAK-779), Treg migration to tumors is reduced and tumors are smaller than in control mice. Thus, this study demonstrates the importance of Tregs in immune evasion by tumors, how blockade of Treg migration might inhibit tumor growth, and, specifically in pancreatic adenocarcinoma, the role of CCR5 in the homing of tumor-associated Tregs. Selective targeting of CCR5/CCL5 signaling may represent a novel immunomodulatory strategy for the treatment of cancer.
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Adenocarcinoma/patología , Adenocarcinoma/prevención & control , Antagonistas de los Receptores CCR5 , Inhibición de Migración Celular/genética , Quimiotaxis de Leucocito/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/prevención & control , Linfocitos T Reguladores/inmunología , Adenocarcinoma/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Quimiotaxis de Leucocito/inmunología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Pancreáticas/metabolismo , Receptores CCR5/biosíntesis , Receptores CCR5/genética , Receptores CCR5/fisiología , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
Ovarian cancer is the leading cause of death among women with gynecological cancer, with an overall 5-year survival rate below 50% due to a lack of specific symptoms, late stage at time of diagnosis and a high rate of recurrence after standard therapy. A better understanding of heterogeneity, genetic mutations, biological behavior and immunosuppression in the tumor microenvironment have allowed the development of more effective therapies based on anti-angiogenic treatments, PARP and immune checkpoint inhibitors, adoptive cell therapies and oncolytic vectors. Oncolytic adenoviruses are commonly used platforms in cancer gene therapy that selectively replicate in tumor cells and at the same time are able to stimulate the immune system. In addition, they can be genetically modified to enhance their potency and overcome physical and immunological barriers. In this review we highlight the challenges of adenovirus-based oncolytic therapies targeting ovarian cancer and outline recent advances to improve their potential in combination with immunotherapies.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos/administración & dosificación , Viroterapia Oncolítica/normas , Neoplasias Ováricas/terapia , Microambiente Tumoral , Animales , Femenino , Vectores Genéticos/genética , Humanos , Neoplasias Ováricas/patologíaRESUMEN
BACKGROUND: Sigma-2 receptors are over-expressed in proliferating cancer cells, making an attractive target for the targeted treatment of pancreatic cancer. In this study, we investigated the role of the novel sigma-2 receptor ligand SW43 to induce apoptosis and augment standard chemotherapy. RESULTS: The binding affinity for sigma-2 ligands is high in pancreas cancer, and they induce apoptosis with a rank order of SV119 < SW43 < SRM in vitro. Combining these compounds with gemcitabine further increased apoptosis and decreased viability. Our in vivo model showed that sigma-2 ligand treatment decreased tumor volume to the same extent as gemcitabine. However, SW43 combination treatment with gemcitabine was superior to the other compounds and resulted in stabilization of tumor volume during treatment, with minimal toxicities. CONCLUSIONS: This study shows that the sigma-2 ligand SW43 has the greatest capacity to augment gemcitabine in a pre-clinical model of pancreas cancer and has provided us with the rationale to move this compound forward with clinical investigations for patients with pancreatic cancer.
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Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores sigma/metabolismo , Animales , Antineoplásicos/farmacocinética , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Femenino , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos C57BL , Receptores sigma/agonistas , GemcitabinaRESUMEN
Pancreatic adenocarcinoma carries an ominous prognosis and has little effective treatment. Several studies have demonstrated that the potently antiapoptotic phosphatidyl inositol 3'-kinase (PI3K)-protein kinase B/AKT pathway is active in pancreas cancer. A recent study identified an endogenous AKT antagonist, carboxyl terminal modulator protein (CTMP). CTMP inhibits the phosphorylation of AKT, preventing full activation of the kinase. We screened several cell permeable peptides from the N-terminal domain of CTMP (termed TAT-CTMP1-4) in vitro and found one that caused significant apoptosis in pancreatic adenocarcinoma cell lines. An inactive variant of this peptide was synthesized and used as a negative control. In all cell lines tested, TAT-CTMP4 induced a dose-dependent increase in apoptosis as detected by %-TUNEL positive cells and %-active caspase-3 (% active caspase-3 ranged from 31.2 to 61.9 at the highest dose tested (10 microM). A screening of various cell and tissue types revealed that the proapoptotic activity was highest in pancreatic adenocarcinoma. TAT-CTMP induced similar levels of active caspase-3 as several other known inducers of apoptosis: gemcitabine, radiation therapy, wortmannin and recombinant tumor necrosis factor (TNF)-alpha. No apoptosis was observed in donor human peripheral blood mononuclear cells (PBMC, p < 0.01). We further showed that TAT-CTMP4 could augment either gemcitabine chemotherapy or radiation therapy, standard therapies for pancreas cancer. Pancreatic adenocarcinoma xenografts treated with a single dose of TAT-CTMP4 demonstrated a marked increase in caspase-3 positive tumor cells when compared with untreated controls. Additionally, pancreatic adenocarcinoma allografts treated with intratumoral TAT-CTMP and systemic gemcitabine displayed a significantly smaller tumor burden while undergoing treatment than mice in control groups (p < 0.001). These data indicate that inhibiting AKT with CTMP may be of therapeutic benefit in the treatment of pancreatic adenocarcinoma and, when combined with established therapies, may result in an increase in tumor cell death.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/farmacología , Adenocarcinoma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Proteínas de la Membrana/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Antimetabolitos Antineoplásicos/farmacología , Western Blotting , Caspasas/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Tolerancia a Radiación/efectos de los fármacos , Tioléster Hidrolasas , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , GemcitabinaRESUMEN
BACKGROUND: We have previously reported that the sigma-2 receptor is highly expressed in pancreas cancer. Furthermore, we have demonstrated that sigma-2 receptor specific ligands induce apoptosis in a dose-dependent fashion. Here, we examined whether sigma-2 receptor ligands potentiate conventional chemotherapies such as gemcitabine and paclitaxel. METHODS: Mouse (Panc-02) and human (CFPAC-1, Panc-1, AsPC-1) pancreas cancer cell lines were used in this study. Apoptosis was determined by FACS or immunohistochemical analysis after TUNEL and Caspase-3 staining. Combination therapy with the sigma-2 ligand SV119 and the conventional chemotherapies gemcitabine and paclitaxel was evaluated in an allogenic animal model of pancreas cancer. RESULTS: SV119, gemcitabine, and paclitaxel induced apoptosis in a dose-dependent fashion in all pancreas cancer cell lines tested. Combinations demonstrated increases in apoptosis. Mice were treated with SV119 (1 mg/day) which was administered in combination with paclitaxel (300 microg/day) over 7 days to mice with established tumors. A survival benefit was observed with combination therapy (p = 0.0002). Every other day treatment of SV119 (1 mg/day) in combination with weekly treatment of gemcitabine (1.5 mg/week) for 2 weeks also showed a survival benefit (p = 0.046). Animals tolerated the combination therapy and no gross toxicity was noted in serum biochemistry data or on necropsy. CONCLUSION: SV119 augments tumoricidal activity of paclitaxel and gemcitabine without major side effects. These results highlight the potential utility of the sigma-2 ligand as an adjuvant treatment in pancreas cancer.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Receptores sigma/metabolismo , Adenocarcinoma/patología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Humanos , Interfase/efectos de los fármacos , Ligandos , Ratones , Ratones Endogámicos C57BL , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Neoplasias Pancreáticas/patología , Análisis de Supervivencia , GemcitabinaRESUMEN
Most human ovarian carcinomas express mesothelin, which is shed as a diagnostically useful biomarker. We applied an ELISA to measure antibodies to native mesothelin in serum from a series of patients with divergent clinical outcomes. The level of anti-mesothelin antibodies determined as OD(450 nm) and referred to as absorption units (AU) for 1:20 diluted serum was higher in patients who remained disease-free after therapy [no evidence of disease (NED); n = 14] than in patients whose disease recurred [clinical evidence of disease (CED); n = 21; P < 0.01]. Applying AU > or = 0.5 at a serum dilution of 1:20 as cutoff, 10 of 14 (71%) ovarian carcinoma patients with NED and 9 of 21 (43%) patients with CED had antibodies to mesothelin compared with 6 of 23 (26%) healthy women (P < 0.008) and 5 of 24 (21%) women with other benign gynecologic diseases (P < 0.003), whereas 7 of 9 (78%) of women with pelvic inflammatory disease were positive. Three of the 14 (21%) NED patients had circulating mesothelin detected as an AU > or = 0.2 at a serum dilution of 1:40 (P < 0.005) compared with 15 of 21 (71%) CED patients, and 9 of 14 (64%) NED patients (P < 0.0002) were positive for antibodies and negative for antigen compared with 1 of 21 (5%) CED patients. Although our data indicate that an antibody response to mesothelin is an important correlate of ovarian carcinoma, prospective studies are needed to show whether the measurement of such antibodies (alone or together with antigen) aids the diagnosis and monitoring of patients.
Asunto(s)
Anticuerpos Antineoplásicos/sangre , Glicoproteínas de Membrana/sangre , Neoplasias Ováricas/sangre , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Proteínas Ligadas a GPI , Humanos , Mesotelina , Persona de Mediana Edad , Neoplasias Ováricas/patologíaRESUMEN
BACKGROUND: Resistance to modern adjuvant treatment is in part due to the failure of programmed cell death. Therefore the molecules that execute the apoptotic program are potential targets for the development of anti-cancer therapeutics. The sigma-2 receptor has been found to be over-expressed in some types of malignant tumors, and, recently, small molecule ligands to the sigma-2 receptor were found to induce cancer cell apoptosis. RESULTS: The sigma-2 receptor was expressed at high levels in both human and murine pancreas cancer cell lines, with minimal or limited expression in normal tissues, including: brain, kidney, liver, lung, pancreas and spleen. Micro-PET imaging was used to demonstrate that the sigma-2 receptor was preferentially expressed in tumor as opposed to normal tissues in pancreas tumor allograft-bearing mice. Two structurally distinct sigma-2 receptor ligands, SV119 and WC26, were found to induce apoptosis to mice and human pancreatic cancer cells in vitro and in vivo. Sigma-2 receptor ligands induced apoptosis in a dose dependent fashion in all pancreatic cell lines tested. At the highest dose tested (10 muM), all sigma-2 receptor ligands induced 10-20% apoptosis in all pancreatic cancer cell lines tested (p < 0.05). In pancreas tumor allograft-bearing mice, a single bolus dose of WC26 caused approximately 50% apoptosis in the tumor compared to no appreciable apoptosis in tumor-bearing, vehicle-injected control animals (p < 0.0001). WC26 significantly slowed tumor growth after a 5 day treatment compared to vehicle-injected control animals (p < 0.0001) and blood chemistry panels suggested that there is minimal peripheral toxicity. CONCLUSION: We demonstrate a novel therapeutic strategy that induces a significant increase in pancreas cancer cell death. This strategy highlights a new potential target for the treatment of pancreas cancer, which has little in the way of effective treatments.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Compuestos de Azabiciclo/farmacología , Carbamatos/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores sigma/agonistas , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Compuestos de Azabiciclo/metabolismo , Compuestos de Azabiciclo/uso terapéutico , Unión Competitiva , Encéfalo/citología , Encéfalo/metabolismo , Carbamatos/metabolismo , Carbamatos/uso terapéutico , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Riñón/citología , Riñón/metabolismo , Ligandos , Pulmón/citología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Tomografía de Emisión de Positrones , Ensayo de Unión Radioligante , Receptores sigma/metabolismo , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , TritioRESUMEN
We recently completed a phase 1 clinical trial demonstrating the safety of a mammaglobin-A DNA vaccine in patients with metastatic breast cancer. We are currently enrolling patients with early stage breast cancer in a phase 1b clinical trial. The mammaglobin-A DNA vaccine will be administered concurrently with neoadjuvant endocrine therapy, providing a unique opportunity to examine the impact of vaccination in the tumor microenvironment.
RESUMEN
Regulatory T-cells (Treg) protect the host from autoimmune disease by suppressing self-reactive immune cells. As such, Treg may also block antitumor immune responses. Recent observations by us and others showed that the prevalence of Treg is increased in cancer patients, particularly in the tumor environment. Our studies in a mouse pancreas cancer model suggest that the tumor actively promotes the accrual of Treg through several mechanisms involving activation of naturally occurring Treg as well as conversion of non-Treg into Treg. Our studies focus on further defining these mechanisms with the ultimate goal of designing strategies that block Treg-mediated suppression in cancer patients.
Asunto(s)
Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Biomarcadores de Tumor/inmunología , Neoplasias de la Mama/inmunología , Antígenos CD4/metabolismo , Vacunas contra el Cáncer/uso terapéutico , Carcinoma Ductal de Mama/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Femenino , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Humanos , Tolerancia Inmunológica , Depleción Linfocítica , Ratones , Ratones Noqueados , Neoplasias/patología , Neoplasias/terapia , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/terapia , Fenotipo , Receptores de Interleucina-2/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Mammaglobin is a gene that is expressed almost exclusively in the normal breast epithelium and human breast cancer. It is a member of the secretoglobin gene family and forms a heterodimer with lipophilin B. We have focused on the tissue-specificity of mammaglobin as a potential mechanism for the specific killing of breast cancer cells. By elucidating the promoter region of mammaglobin, we hope to utilize this site as a method for turning on the apoptosis inducer gene, Bax, in breast cancer cells. The Bax gene will only be expressed at levels necessary to induce apoptosis in mammaglobin positive cells. This would include > 80% of all breast cancers and some normal breast epithelium. This type of targeted killing could be conceptualized as a biochemical mastectomy, that is, genetic ablation of breast tumor cells and perhaps non-malignant breast epithelium while preserving the adipose and stromal components of the breast. Work is also being done to address the binding specificity of the secreted mamaglobin protein. There is early evidence that the mammaglobin heterodimer may in fact bind to breast and breast cancer cells. If this finding is validated, this creates the possibility that mammaglobin can be tagged with radioisotope or toxin, so that binding of the tagged-mammaglobin complex results in the specific killing of that breast cancer cell. Finally, mammaglobin is being explored as a target for immune-based interventions. In vitro studies have demonstrated that T cell-mediated immune responses can be induced against mammaglobin-derived peptides expressed by MHC molecules on tumor cells and antigen-presenting cells.
Asunto(s)
Biomarcadores de Tumor/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Proteínas de Neoplasias/fisiología , Uteroglobina/fisiología , Animales , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Humanos , Mamoglobina A , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Uteroglobina/biosíntesis , Uteroglobina/genéticaRESUMEN
BACKGROUND: Mammaglobin-A is an attractive target for immune-based therapy for patients with breast cancer because of its exclusive expression in breast cancer. In this study, we attempted to identify immunogenic T cell epitopes restricted by human leukocyte antigen (HLA)-A2 in mammaglobin-A protein. METHODS: To identify HLA-A2-restricted immunogenic epitopes from mammaglobin-A, 7 candidate peptides were synthesized and tested for immunogenicity. Each peptide was tested for binding to HLA-A2 in a HLA-A2 stabilization assay. Furthermore, T lymphocytes from 7 healthy donors and 1 patient with breast cancer received 3 weekly stimulations with autologous peptide-pulsed dendritic cells. Stimulated T cells were tested for specific recognition of peptide and tumor cells by interferon-gamma enzyme-linked immunosorbent assay. RESULTS: HLA-A2 binding assays showed that all designed peptides could bind to HLA-A2. Two of the 7 peptides (MAM3 and MAM7) successfully induced peptide-specific T cells. However, only MAM3-specific T cells recognized the mammaglobin overexpressing breast cancer cell line, MDA415 transfected with HLA-A2. In contrast, MAM3-specific T cell did not recognize wild type MDA415 or MDA415 transfected with HLA-A24, or the mammaglobin negative, HLA-A2 positive breast cancer cell line, MCF-7. CONCLUSIONS: Mammaglobin-A-derived peptide, MAM3, can induce mammaglobin-A-specific immunity and could be useful for vaccine strategies for patients with breast cancer.
Asunto(s)
Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/inmunología , Proteínas de Neoplasias/inmunología , Uteroglobina/inmunología , Antígenos de Neoplasias/metabolismo , Células Cultivadas , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Citometría de Flujo , Antígeno HLA-A2/metabolismo , Humanos , Inmunoterapia , Mamoglobina A , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Linfocitos T/inmunología , Uteroglobina/metabolismoRESUMEN
BACKGROUND: The need for new therapeutic regimens for cancer has led to the identification of tumor antigens in several tumors. Yet, potent antigen-presenting cells (APC) are being identified and characterized in order to find those most able to induce T-cell-mediated immunity. To facilitate the generation of cytotoxic T-lymphocytes (CTL) specific for known antigens, various sources of antigen-presenting cells (APC) were compared for their efficiency. MATERIALS AND METHODS: Allogeneic dendritic cells (DC), T2 cells and two Epstein Barr virus-transformed B cell lines, JY and TK-6, were used as antigen-presenting cells (APC). T-cells from normal donors were stimulated with solid-phase anti-CD3 antibody and reactivated with peptide-pulsed APC three times at weekly intervals. To minimize any potential alloreactivity, eighteen random combinations of JY, TK6 and T2 were used for three peptide stimulations. The APC were pulsed with the HLA-A2-binding HER2/neu-derived tumor antigen p654-662 (GP2). RESULTS: In addition to either T2 or DC alone, most combinations of APC induced significant recognition of GP-2-pulsed target cells to a varying degree. The stimulation index varied from 0.5 to 8.1 using combinations of APC. The level of peptide recognition correlated with recognition of HLA-A2+, HER2/neu+ pancreatic cancer cells; in contrast, HLA-A2-negative pancreatic cancer cells were not recognized. The recognition of tumor cells was HLA-restricted as evidenced by blocking studies with anti-HLA class I and anti-HLA-A2 antibodies. CONCLUSION: Tumor peptide-specific CTL can be generated using allogeneic B cell lines, which may provide a useful alternative to T2 or dendritic cells.