RESUMEN
BACKGROUND: Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment. Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells. We sought to better understand the biology of IL32 in human melanoma. METHODS: We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset. We evaluated global gene expression patterns associated with IL32 expression. We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro. In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5' upstream region of the human IL32 gene into a luciferase reporter vector. RESULTS: A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF). Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, ß, γ) of IL32. Cis-acting elements within this 5' upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression. In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature. CONCLUSIONS: Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature. Constitutive and induced expression are regulated, in part, by cis-acting sequences within the 5' upstream region.
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Interleucinas/genética , Melanoma/genética , Neoplasias Cutáneas/genética , Biopsia , Desdiferenciación Celular/genética , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Melanoma/patología , Análisis por Micromatrices , Metástasis de la Neoplasia , Neoplasias Cutáneas/patología , Transcriptoma , Microambiente Tumoral/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Modern immune therapies (PD-1/PD-L1 and CTLA-4 checkpoints blockade and adoptive cell transfer) have remarkably improved the response rates of metastatic melanoma. These modalities rely on the killing potential of CTL as proximal mediator of antimelanoma responses. Mechanisms of tumor resistance to and the predominant cytotoxic pathway(s) used by melanoma-reactive CTL are important outcome determinants. We hypothesized that downmodulation of death receptors (DRs) in addition to aberrant apoptotic signaling might confer resistance to death signals delivered by CTL. To test these two hypotheses, we used an in vitro model of MART CTL-resistant melanoma sublines. TCR-transgenic and patient-derived CTLs used the TRAIL cytotoxic pathway through DR5. Furthermore, recombinant human TRAIL and drozitumab (anti-DR5 agonistic mAb) were used to explicitly verify the contribution of the DR5/TRAIL pathway in killing melanomas. CTL resistance was due to DR5 downregulation and an inverted ratio of pro- to antiapoptotic molecules, both of which were reversed by the histone deacetylase inhibitor suberoylanilide hydroxanic acid. Apoptosis negative (c-IAP-2 and Bcl-xL) and positive (DR5) regulators were potential incriminators partly regulating CTL sensitivity. These preclinical findings suggest that exposure to this chromatin remodeling drug of immune-resistant melanomas can skew toward an intracellular proapoptotic milieu, increase DR expression, and overcome acquired immune resistance.
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Apoptosis/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Melanoma/inmunología , Melanoma/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Apoptosis/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica , Humanos , Isoantígenos/genética , Isoantígenos/inmunología , Melanoma/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Linfocitos T Citotóxicos/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Transducción GenéticaRESUMEN
BACKGROUND: Alpha fetoprotein (AFP) is an oncofetal antigen over-expressed by many hepatocellular cancers (HCC). We previously demonstrated that HLA-A2-restricted epitopes derived from AFP are immunogenic in vitro and in vivo despite high circulating levels of this oncofetal antigen. In order to test a more broadly applicable, HLA-unrestricted, inexpensive, cell-free vaccine platform capable of activating tumor antigen-specific CD8+ and CD4+ T cells, we tested full length AFP in a plasmid DNA construct in combination with an AFP-expressing replication-deficient adenovirus (AdV) in a prime-boost vaccine strategy. METHODS: HCC patients who had an AFP+ tumor and previous treatment for HCC were screened and two patients received vaccination with three plasmid DNA injections followed by a single AdV injection, all delivered intramuscularly (i.m.). RESULTS: The vaccine was well tolerated and safe. Both patients showed immunologic evidence of immunization. The first patient had a weak AFP-specific T cell response, a strong AdV-specific cellular response and recurred with an AFP-expressing HCC at nine months. The second patient developed a strong AFP-specific CD8+ and CD4+ cellular response and an AdV neutralizing antibody response, and recurred at 18 months without an increase in serum AFP. CONCLUSIONS: The AFP DNA prime-AdV boost vaccine was safe and immunogenic. Circulating anti-AdV neutralizing antibodies at baseline did not prohibit the development of AFP-specific cellular immunity. The patient who developed CD8+ and CD4+ AFP-specific T cell immunity had more favorable progression-free survival. The observations with these two patients support development of this vaccine strategy in a larger clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00093548.
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Adenoviridae/inmunología , Vacunas contra el Cáncer/administración & dosificación , Carcinoma Hepatocelular/inmunología , Neoplasias Hepáticas/inmunología , Vacunas de ADN/administración & dosificación , alfa-Fetoproteínas/genética , Formación de Anticuerpos , Quimiocinas/sangre , Citocinas/sangre , Ensayo de Inmunoadsorción Enzimática , Sustancias de Crecimiento/sangre , HumanosRESUMEN
Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34(+) HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8(+) and CD4(+) T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4-5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8(+) and CD4(+) T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34(+) HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-ß genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.
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Alelos , Células Madre Hematopoyéticas/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Subgrupos de Linfocitos T/metabolismo , Animales , Antígenos CD34/metabolismo , Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Epítopos de Linfocito T/inmunología , Femenino , Orden Génico , Vectores Genéticos/genética , Supervivencia de Injerto , Trasplante de Células Madre Hematopoyéticas , Humanos , Interferón gamma/biosíntesis , Lentivirus/genética , Masculino , Melanoma/genética , Melanoma/inmunología , Melanoma/terapia , Ratones , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Timocitos/citología , Timocitos/metabolismo , Transducción Genética , Trasplante HeterólogoRESUMEN
Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFß), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFß by transduction with a TGFß dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma. Smaller numbers of DN Pmel-1 T cells effectively mediated tumor regression and retained the ability to produce interferon-γ in the tumor microenvironment. These results support efforts to incorporate this DN receptor in clinical trials of adoptive cell therapy for cancer.
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Traslado Adoptivo , Linfocitos T CD8-positivos/metabolismo , Modelos Animales , Neoplasias Experimentales/terapia , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Western Blotting , Linfocitos T CD8-positivos/citología , Citometría de Flujo , Ratones , Ratones Transgénicos , Microambiente TumoralRESUMEN
MHC class I-restricted human melanoma epitope MART-1(27-35) specific TCR-engineered CD4+CD25- T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25- T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-gamma, TNF-alpha, IL-2, and MIP1ss), lyse target cells, and "help" the expansion of the MART-1(27-35) specific CD8+ T cells when stimulated by the MART-1(27-35) peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-1(27-35) specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies.
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Linfocitos T CD4-Positivos/inmunología , Epítopos/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T CD4-Positivos/clasificación , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Ingeniería Genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoterapia Activa , Inmunoterapia Adoptiva , Técnicas In Vitro , Activación de Linfocitos , Melanoma/inmunología , Melanoma/terapia , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T Colaboradores-Inductores/clasificación , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Transducción GenéticaRESUMEN
Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4(+) T cells. Considering the difficulties in simultaneously engaging CD4(+) and CD8(+) T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4(+) T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4(+) T cells has emerged as a strategic consideration. Such TCR-engineered CD4(+) T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4(+) T cells engineered to express the alpha- and beta-chains of a high functional avidity TCR specific for the melanoma epitope, MART-1(27-35), as a prototypic human tumor Ag system. We found that unpolarized CD4(+)CD25(-) T cells engineered to express the MART-1(27-35) TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8(+) CTL. Such TCR engineered CD4(+) T cells, therefore, might be useful in clinical immunotherapy.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Epítopos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Células TH1/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Citocinas/inmunología , Epítopos/metabolismo , Vectores Genéticos , Humanos , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Células TH1/metabolismo , Transducción Genética , TransgenesRESUMEN
PURPOSE: CTL-associated antigen 4 (CTLA4)-blocking monoclonal antibodies induce long-term regression of metastatic melanoma in some patients, but the exact mechanism is unknown. In this study, biopsies of selected accessible tumor lesions from patients treated with tremelimumab were examined to further elucidate the mechanism of its antitumor activity. EXPERIMENTAL DESIGN: Fifteen tumor biopsies from 7 patients who had been treated with tremelimumab (CP-675,206) were collected. Samples were analyzed for melanoma markers, immune cell subset markers, the presence of the T regulatory-specific transcription factor FoxP3 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO). RESULTS: Clinically responding lesions had diffuse intratumoral infiltrates of CD8(+) T cells that were markedly increased in cases where comparison with a baseline biopsy was available. Nonregressing lesions had sparse, patchy CD8(+) intratumoral infiltrates. Patients with regressing lesions had an increased frequency of CD8(+) cells with or without a concomitant increase in CD4(+) cells. Two of 3 responding patients with paired samples showed a slight increase in the number of FoxP3(+) cells in the postdosing biopsies. In patients with regressing lesions who had paired samples, the intensity of IDO staining in macrophages and/or melanoma cells showed no clear pattern of change postdosing. CONCLUSIONS: Administration of tremelimumab was associated with massive intratumoral infiltrates of CD8(+) CTLs in patients with regressing tumors but had varying effects on intratumoral infiltrates of CD4(+) and FoxP3(+) cells or intratumoral expression of IDO.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/inmunología , Factores de Transcripción Forkhead/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Linfocitos T Citotóxicos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados , Biopsia , Linfocitos T CD4-Positivos/inmunología , Antígeno CTLA-4 , Femenino , Humanos , Masculino , Melanoma/inmunología , Melanoma/terapia , Persona de Mediana EdadRESUMEN
BACKGROUND: CTLA4-blocking antibodies induce tumor regression in a subset of patients with melanoma. Analysis of immune parameters in peripheral blood may help define how responses are mediated. METHODS: Peripheral blood from HLA-A*0201-positive patients with advanced melanoma receiving tremelimumab (formerly CP-675,206) at 10 mg/kg monthly was repeatedly sampled during the first 4 cycles. Samples were analyzed by 1) tetramer and ELISPOT assays for reactivity to CMV, EBV, MART1, gp100, and tyrosinase; 2) activation HLA-DR and memory CD45RO markers on CD4+/CD8+ cells; and 3) real-time quantitative PCR of mRNA for FoxP3 transcription factor, preferentially expressed by T regulatory cells. The primary endpoint was difference in MART1-specific T cells by tetramer assay. Immunological data were explored for significant trends using clustering analysis. RESULTS: Three of 12 patients eligible for immune monitoring had tumor regression lasting > 2 years without relapse. There was no significant change in percent of MART1-specific T cells by tetramer assay. Additionally, there was no generalized trend toward postdosing changes in other antigen-specific CD8+ cell populations, FoxP3 transcripts, or overall changes in surface expression of T-cell activation or memory markers. Unsupervised hierarchical clustering based on immune monitoring data segregated patients randomly. However, clustering according to T-cell activation or memory markers separated patients with clinical response and most patients with inflammatory toxicity into a common subgroup. CONCLUSION: Administration of CTLA4-blocking antibody tremelimumab to patients with advanced melanoma results in a subset of patients with long-lived tumor responses. T-cell activation and memory markers served as the only readout of the pharmacodynamic effects of this antibody in peripheral blood. CLINICAL TRIAL REGISTRATION NUMBER: NCT00086489.
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Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Melanoma/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados , Antígeno CTLA-4 , Análisis por Conglomerados , Femenino , Antígenos HLA-A/metabolismo , Antígeno HLA-A2 , Humanos , Sistema Inmunológico , Masculino , Melanoma/inmunología , Melanoma/metabolismo , Persona de Mediana Edad , Linfocitos T/metabolismoRESUMEN
A promising arsenal of targeted and immunotherapy treatments for metastatic melanoma has emerged over the last decade. With these therapies, we now face new mechanisms of tumor-acquired resistance. We report here a patient whose metastatic melanoma underwent dedifferentiation as a resistance mechanism to adoptive T-cell transfer therapy (ACT) to the MART1 antigen, a phenomenon that had been observed only in mouse studies to date. After an initial period of tumor regression, the patient presented in relapse with tumors lacking melanocytic antigens (MART1, gp100) and expressing an inflammation-induced neural crest marker (NGFR). We demonstrate using human melanoma cell lines that this resistance phenotype can be induced in vitro by treatment with MART1 T cell receptor-expressing T cells or with TNFα, and that the phenotype is reversible with withdrawal of inflammatory stimuli. This supports the hypothesis that acquired resistance to cancer immunotherapy can be mediated by inflammation-induced cancer dedifferentiation.Significance: We report a patient whose metastatic melanoma underwent inflammation-induced dedifferentiation as a resistance mechanism to ACT to the MART1 antigen. Our results suggest that future melanoma ACT protocols may benefit from the simultaneous targeting of multiple tumor antigens, modulating the inflammatory response, and inhibition of inflammatory dedifferentiation-inducing signals. Cancer Discov; 8(8); 935-43. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899.
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Resistencia a Antineoplásicos , Antígeno MART-1/inmunología , Melanoma/terapia , Proteínas del Tejido Nervioso/metabolismo , Nevo Pigmentado/terapia , Receptores de Factor de Crecimiento Nervioso/metabolismo , Desdiferenciación Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Humanos , Inmunoterapia Adoptiva , Masculino , Melanoma/inmunología , Persona de Mediana Edad , Metástasis de la Neoplasia , Nevo Pigmentado/inmunología , Receptores Quiméricos de Antígenos/metabolismo , RecurrenciaRESUMEN
Adoptive cell therapy (ACT) consisting of genetically engineered T cells expressing tumor antigen-specific T-cell receptors displays robust initial antitumor activity, followed by loss of T-cell activity/persistence and frequent disease relapse. We characterized baseline and longitudinal T-cell phenotype variations resulting from different manufacturing and administration protocols in patients who received ACT. Patients with melanoma who enrolled in the F5-MART-1 clinical trial (NCT00910650) received infusions of MART-1 T-cell receptors transgenic T cells with MART-1 peptide-pulsed dendritic cell vaccination. Patients were divided into cohorts based on several manufacturing changes in the generation and administration of the transgenic T cells: decreasing ex vivo stimulation/expansion time, increased cell dose, and receiving fresh instead of cryopreserved cells. T-cell phenotypes were analyzed by flow cytometry at baseline and longitudinally in peripheral blood. Transgenic T cells with shorter ex vivo culture/expansion periods displayed significantly increased expression of markers associated with less differentiated naive/memory populations, as well as significantly decreased expression of the inhibitory receptor programmed death 1 (PD1). Patients receiving fresh infusions of transgenic cells demonstrated expansion of central memory T cells and delayed acquisition of PD1 expression compared with patients who received cryopreserved products. Freshly infused transgenic T cells showed persistence and expansion of naive and memory T-cell populations and delayed acquisition of PD1 expression, which correlated with this cohort's superior persistence of transgenic cells and response to dendritic cell vaccines. These results may be useful in designing future ACT protocols.
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Vacunas contra el Cáncer/inmunología , Inmunoterapia Adoptiva/métodos , Melanoma/terapia , Neoplasias Cutáneas/terapia , Linfocitos T Citotóxicos/inmunología , Adulto , Anciano , Células Cultivadas , Criopreservación , Femenino , Humanos , Memoria Inmunológica , Inmunofenotipificación , Activación de Linfocitos , Antígeno MART-1/metabolismo , Masculino , Melanoma/inmunología , Persona de Mediana Edad , Fenotipo , Neoplasias Cutáneas/inmunologíaRESUMEN
PURPOSE: To determine the ocular safety of CP-675,206 (Pfizer, New York, New York, USA), a fully human anti-cytotoxic T lymphocyte-associated antigen 4 monoclonal antibody in clinical trials of immunotherapy of metastatic melanoma. DESIGN: Prospective, nonrandomized study of the eye and vision in phase I/II clinical trials of CP-675,206 in metastatic melanoma conducted at the University of California, Los Angeles. METHODS: Patients with regional or distant metastatic melanoma were enrolled in phase I/II clinical trials evaluating the safety and antitumor efficacy of CP-675,206 alone or in combination with melanoma antigen peptide-pulsed dendritic cell vaccines. Ophthalmic evaluation was performed at the onset of CP-675,206 immunotherapy (baseline evaluation), two months or more after the onset of CP-675,206 immunotherapy (end-study evaluation), and at two- to three-month intervals thereafter in patients who continued to receive CP-675,206 immunotherapy (poststudy evaluation). Baseline and end-study evaluations included comprehensive ophthalmic examination, psychophysical and electrophysiologic visual function assessment, fundus photography, fluorescein angiography, and visual function assessment. RESULTS: Twenty patients with metastatic melanoma arising from the skin, mucosa, eye, or unknown site were evaluated. Systemic toxicity attributed to CP-675,206 included dermatologic manifestations, diarrhea, and autoimmune hepatitis with panhypopituitarism. A subset of patients receiving CP-675,206 demonstrated antitumor efficacy with partial response or complete response of metastatic melanoma. Comparison of ophthalmic baseline with end-study evaluations in all 20 patients and limited-term poststudy evaluations showed no adverse effect of CP-675,206 immunotherapy on the eye or vision. CONCLUSIONS: In this study, CP-675,206 immunotherapy for metastatic melanoma did not adversely affect the eye or vision.
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Anticuerpos Bloqueadores/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Inmunoconjugados/inmunología , Inmunoterapia , Melanoma/terapia , Neoplasias/terapia , Fenómenos Fisiológicos Oculares , Visión Ocular/fisiología , Abatacept , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Bloqueadores/efectos adversos , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados , Antígenos de Neoplasias/inmunología , Neoplasias del Ano/patología , Neoplasias del Ano/terapia , Neoplasias de la Coroides/patología , Neoplasias de la Coroides/terapia , Quimioterapia Combinada , Electrooculografía , Electrorretinografía , Femenino , Angiografía con Fluoresceína , Humanos , Antígeno MART-1 , Masculino , Melanoma/secundario , Persona de Mediana Edad , Proteínas de Neoplasias/inmunología , Neoplasias/patología , Estudios Prospectivos , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Resultado del Tratamiento , Agudeza VisualRESUMEN
PURPOSE: Define an immunologic response using the tetramer and enzyme-linked immunospot (ELISPOT) assays. EXPERIMENTAL DESIGN: Ten healthy subjects and 21 patients with melanoma (all HLA-A*0201) donated a total of 121 blood samples to determine the lower limit of detection (LLD), analytic coefficient of variation (aCV), and physiologic CV (pCV) of the tetramer and ELISPOT assays. The mean, SD, and reference change value (RCV) were calculated to define changes beyond the assay imprecision, and its application was tested in the monitoring of T-cell expansion after CTLA4 blockade with ticilimumab (CP-675,206). RESULTS: The LLD for the tetramer assay was 0.038% CD8+ cells and seven spots per 10(5) peripheral blood mononuclear cells for the ELISPOT assay. The aCV of the tetramer assay was <10% and was higher for the ELISPOT (24.69-36.32%). There was marked between-subject variability on baseline homeostatic values, which was correlated to prior antigen exposure. An immunologic response was defined as an increase beyond the mean + 3 SD in antigen-specific cells for subjects with baseline levels below the LLD, or beyond the assay RCV for baseline levels above the LLD. In four patients receiving ticilimumab, expansions of antigen-specific T cells beyond the assay variability were noted for EBV and MART1 antigens. CONCLUSIONS: A combined approach of change from negative (below the LLD) to positive (above the LLD) and a percentage change beyond the assay variability using the RCV score can be computed to define which change in circulating antigen-specific T cells represents a response to immunotherapy.
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Ensayo de Inmunoadsorción Enzimática , Inmunoensayo , Complejo Mayor de Histocompatibilidad/inmunología , Melanoma/inmunología , Linfocitos T/inmunología , Adulto , Anticuerpos Monoclonales/uso terapéutico , Femenino , Humanos , Inmunoterapia , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Masculino , Melanoma/terapia , Persona de Mediana Edad , Sensibilidad y EspecificidadRESUMEN
Alpha-fetoprotein (AFP) is a self protein expressed by fetal liver at high levels, but is transcriptionally repressed at birth. AFP is up-regulated in hepatocellular carcinomas, and patients with active disease could have plasma levels as high as 1 mg/mL. We previously identified four immunodominant HLA-A*0201-restricted peptides [hAFP(137-145) (PLFQVPEPV), hAFP(158-166) (FMNKFIYEI), hAFP(325-334) (GLSPNLNRFL), and hAFP(542-550) (GVALQTMKQ)] derived from human AFP that could stimulate specific T cell responses in healthy donor peripheral blood lymphocytes in vitro. We conducted a phase I/II clinical trial in which HLA-A*0201 patients with AFP-positive hepatocellular carcinoma were immunized with three biweekly intradermal vaccinations of the four AFP peptides pulsed onto autologous dendritic cells (DC). DCs were prepared from adherent peripheral blood mononuclear cells cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 7 days. Sixteen subjects were enrolled and 10 were treated. Peripheral blood lymphocytes were isolated from these patients before, during, and after AFP peptide/DC immunization and were tested ex vivo with MHC tetramer and IFNgamma ELISPOT analysis. Six of 10 subjects expanded statistically significant levels of AFP-specific T cells postvaccine to at least one peptide by MHC tetramer. Also, 6 of 10 subjects increased IFNgamma producing AFP-specific T cell responses to at least one of the peptides postvaccination, by ELISPOT. We conclude that the human T cell repertoire is capable of responding to the AFP self antigen after the administration of AFP peptide-pulsed DC even in an environment of high circulating levels of this oncofetal antigen.
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Carcinoma Hepatocelular/terapia , Células Dendríticas/trasplante , Neoplasias Hepáticas/terapia , Fragmentos de Péptidos/uso terapéutico , alfa-Fetoproteínas/uso terapéutico , Adulto , Anciano , Secuencia de Aminoácidos , Carcinoma Hepatocelular/inmunología , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Hepáticas/inmunología , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/toxicidad , alfa-Fetoproteínas/toxicidadRESUMEN
Dendritic cell (DC)-based immunization in cancer has proven to be a promising approach. However, just as DCs are crucial accessory cells in generating immune responses, they also seem to participate in tolerance induction, especially against peripheral "self" antigens. The bulk of the evidence that DCs present peripheral self antigens to induce tolerance has, however, come mostly from studies in transgenic animal models. A tolerogenic function of DCs for peripheral self antigens in a human model has not been critically examined. In this study using the Melan-A/MART-1(27-35) peptide as a model for self but melanoma-associated antigen-against which human hosts often harbor CD8(+) CTL precursors with high frequencies-we confirm that although immature dendritic cells (iDCs) are inefficient antigen presenting cells (APCs), fully activated DCs efficiently activate melanoma epitope-specific CD8(+) CTL precursors, in vitro. We, however, show that in a direct epitope presentation schema, iDCs neither delete nor anergize epitope-specific CD8(+) T cells in primary or secondary stimulation. Interestingly, iDCs and activated DCs can delete a large fraction of the epitope-specific CTLs on tertiary stimulation. The deletion is induced in an epitope-specific manner and through apoptosis. These observations, therefore, have implications on the DC-based cancer vaccine designs and are relevant in the inquiry into the role of DCs on tolerance induction.
Asunto(s)
Células Dendríticas/inmunología , Epítopos de Linfocito T/inmunología , Melanoma/inmunología , Linfocitos T Citotóxicos/inmunología , Autoantígenos/inmunología , Epítopos/inmunología , Humanos , Tolerancia Inmunológica/inmunología , Activación de Linfocitos/inmunología , Proteínas de Neoplasias/inmunologíaRESUMEN
This article reviews the immunologic basis of clinical trials that test means of tumor antigen recognition and immune activation, with the goal to provide the clinician with a mechanistic understanding of ongoing cancer vaccine and cellular immunotherapy clinical trials. Multiple novel immunotherapy strategies have reached the stage of testing in clinical trials that were accelerated by recent advances in the characterization of tumor antigens and by a more precise knowledge of the regulation of cell-mediated immune responses. The key steps in the generation of an immune response to cancer cells include loading of tumor antigens onto antigen-presenting cells in vitro or in vivo, presenting antigen in the appropriate immune stimulatory environment, activating cytotoxic lymphocytes, and blocking autoregulatory control mechanisms. This knowledge has opened the door to antigen-specific immunization for cancer using tumor-derived proteins or RNA, or synthetically generated peptide epitopes, RNA, or DNA. The critical step of antigen presentation has been facilitated by the coadministration of powerful immunologic adjuvants, the provision of costimulatory molecules and immune stimulatory cytokines, and the ability to culture dendritic cells. Advances in the understanding of the nature of tumor antigens and their optimal presentation, and in the regulatory mechanisms that govern the immune system, have provided multiple novel immunotherapy intervention strategies that are being tested in clinical trials.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer , Inmunoterapia/tendencias , Neoplasias/inmunología , Neoplasias/terapia , Animales , Ensayos Clínicos como Asunto , HumanosRESUMEN
Tumor antigen gene-modified dendritic cells (DC) generates robust antigen-specific protective antitumor responses. Though the role of CD4 positive and CD8 positive cells in the immunological response to gene-modified DC has been well-characterized, the role of NK cells in this response has been somewhat less clear. Owing to the significant contribution of innate immunity in other model systems, we postulated that NK cells would hold a critical position in the generation of an immune response following immunization with tumor antigen-engineered DC. Immunization with MART-1 melanoma antigen-engineered DC in C57BL/6 mice resulted in the generation of antigen-specific cytotoxic T lymphocytes and in vivo protective responses to the murine B16 melanoma. These responses were dependent on the presence of functional NK cells, although NK cells alone were not sufficient in generating protective responses. Adoptive transfer of NK cells into an NK-deficient but T-cell-competent environment restored the protective response to gene-modified DC immunization. In conclusion, protective immunity after tumor antigen gene-modified DC immunization requires collaboration between CD4+ and CD8+ T cells and NK cells.