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ABSTRACT: CD19-specific chimeric antigen receptor (CAR) T cells have demonstrated impressive responses in patients with relapsed and refractory B cell malignancies. However, many patients relapse or fail to respond to CD19 CAR T cells, demonstrating the need to improve its efficacy and durability. Current protocols for generating CAR T cells involve T cell activation through CD3 stimulation to facilitate efficient CAR transfer followed by ex vivo expansion with exogenous cytokines to obtain adequate cell numbers for treatment. Both T cell activation and expansion inevitably lead to terminal differentiation and replicative senescence, which are suboptimal for therapy. Interleukin-7 (IL-7) was previously shown to allow for lentiviral transduction of T cells in the absence of activation. In these studies, we used IL-7 to generate CD19 CAR T cells without stimulating CD3. Nonactivated and IL-7 cultured (NICE) CD19 CAR T cells were enriched with the T memory stem cell population, retained novel markers of stemness, had lower expression of exhaustion markers, and increased proliferative potential. Furthermore, our findings are consistent with engraftment of NICE CD19 CAR T cells and demonstrate a superior therapeutic response in both intraperitoneal and subcutaneous in vivo B cell lymphoma models. These results suggest that NICE CD19 CAR T cells may improve outcomes for B cell malignancies and warrant clinical evaluation.
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Interleucina-7 , Recurrencia Local de Neoplasia , Receptores de Antígenos de Linfocitos T , Humanos , Linfocitos T , Células Madre , FenotipoRESUMEN
Adoptive T cell therapy with T cell receptor (TCR)-modified T cells has shown promise in treating metastatic melanoma and other malignancies. However, studies are needed to improve the efficacy and durability of responses of TCR-modified T cells. Standard protocols for generating TCR-modified T cells involve activating T cells through CD3 stimulation to allow for the efficient transfer of tumor-reactive receptors with viral vectors. T cell activation results in terminal differentiation and shortening of telomeres, which are likely suboptimal for therapy. In these studies, we demonstrate efficient T cell transduction with the melanoma-reactive TIL1383I TCR through culturing with interleukin 7 (IL-7) in the absence of CD3 activation. The TIL1383I TCR-modified T cells generated following IL-7 culture were enriched with naïve (TN) and memory stem cell populations (TSCM) while maintaining longer telomere lengths. Furthermore, we demonstrated melanoma-reactivity of TIL1383I TCR-modified cells generated following IL-7 culture using in vitro assays and a superior response in an in vivo melanoma model. These results suggest that utilizing IL-7 to generate TCR-modified T cells in the absence of activation is a feasible strategy to improve adoptive T cell therapies for melanoma and other malignancies.
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Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Melanoma/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Línea Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Femenino , Células HEK293 , Humanos , Memoria Inmunológica/inmunología , Interleucina-7/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fenotipo , Transducción Genética/métodosRESUMEN
T cells that are gene-modified with tumor-specific T cell receptors are a promising treatment for metastatic melanoma patients. In a clinical trial, we treated seven metastatic melanoma patients with autologous T cells transduced to express a tyrosinase-reactive T cell receptor (TCR) (TIL 1383I) and a truncated CD34 molecule as a selection marker. We followed transgene expression in the TCR-transduced T cells after infusion and observed that both lentiviral- and retroviral-transduced T cells lost transgene expression over time, so that by 4 weeks post-transfer, few T cells expressed either lentiviral or retroviral transgenes. Transgene expression was reactivated by stimulation with anti-CD3/anti-CD28 beads and cytokines. TCR-transduced T cell lentiviral and retroviral transgene expression was also downregulated in vitro when T cells were cultured without cytokines. Transduced T cells cultured with interleukin (IL)-15 maintained transgene expression. Culturing gene-modified T cells in the presence of histone deacetylase (HDAC) inhibitors maintained transgene expression and functional TCR-transduced T cell responses to tumor. These results implicate epigenetic processes in the loss of transgene expression in lentiviral- and retroviral-transduced T cells.
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BACKGROUND: ALK fusions are targetable drivers in non-small-cell lung cancer (NSCLC). However, patients with NSCLC harboring ALK rearrangements without a fusion partner identified in DNA have also been shown to respond to ALK inhibitors. We aimed to characterize complex ALK variants that may predict sensitivity to multiple approved ALK inhibitors. METHODS: Comprehensive genomic profiling (CGP) of DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue or blood-based circulating tumor DNA was performed for 39,159 NSCLC patients during routine clinical care. For a subset of cases, RNA sequencing was performed, and prior ALK test results and clinical treatment information were collected from treating physicians. RESULTS: We queried the Foundation Medicine NSCLC database and identified ALK internal inversions, as well as internal deletions, as the sole ALK rearrangements in 6 (0.02%) and 3 (0.01%) of cases, respectively. In cases with ALK internal inversions, RNA testing identified an EML4-ALK fusion in 2/2 cases evaluated, and 3/3 patients treated with ALK inhibitors had durable responses. A single patient with an ALK internal deletion and clinical data available responded to multiple ALK inhibitors. RNA data available for a subset of non-NSCLC cases suggest that ALK internal deletions removing a portion of the N-terminus are drivers themselves and do not result in ALK fusions. Fluorescence in situ hybridization (FISH) results were inconsistent for both classes of DNA events. CONCLUSION: Rare internal inversions of ALK appear to be indicative of ALK fusions, which can be detected in RNA, and response to ALK inhibitors in patients with NSCLC. In contrast, ALK internal deletions are not associated with ALK fusions in RNA but likely represent targetable drivers themselves. These data suggest that CGP of DNA should be supplemented with immunohistochemistry or RNA-based testing to further resolve these events and match patients to effective therapies.
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Malignant melanoma incidence has been increasing for over 30 years, and despite promising new therapies, metastatic disease remains difficult to treat. We describe preliminary results from a Phase I clinical trial (NCT01586403) of adoptive cell therapy in which three patients received autologous CD4+ and CD8+ T cells transduced with a lentivirus carrying a tyrosinase-specific TCR and a marker protein, truncated CD34 (CD34t). This unusual MHC Class I-restricted TCR produces functional responses in both CD4+ and CD8+ T cells. Parameters monitored on transduced T cells included activation (CD25, CD69), inhibitory (PD-1, TIM-3, CTLA-4), costimulatory (OX40), and memory (CCR7) markers. For the clinical trial, T cells were activated, transduced, selected for CD34t+ cells, then re-activated, and expanded in IL-2 and IL-15. After lymphodepleting chemotherapy, patients were given transduced T cells and IL-2, and were followed for clinical and biological responses. Transduced T cells were detected in the circulation of three treated patients for the duration of observation (42, 523, and 255 days). Patient 1 tolerated the infusion well but died from progressive disease after 6 weeks. Patient 2 had a partial response by RECIST criteria then progressed. After progressing, Patient 2 was given high-dose IL-2 and subsequently achieved complete remission, coinciding with the development of vitiligo. Patient 3 had a mixed response that did not meet RECIST criteria for a clinical response and developed vitiligo. In two of these three patients, adoptive transfer of tyrosinase-reactive TCR-transduced T cells into metastatic melanoma patients had clinical and/or biological activity without serious adverse events.
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Antígenos de Neoplasias/inmunología , Melanoma/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Neoplasias Cutáneas/terapia , Subgrupos de Linfocitos T/trasplante , Adulto , Anciano , Humanos , Masculino , Melanoma/inmunología , Melanoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/secundario , Subgrupos de Linfocitos T/inmunología , Trasplante AutólogoRESUMEN
The authors would like to make the following corrections to the published article.
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Serum microRNAs hold great promise as easily accessible and measurable biomarkers of disease. In prostate cancer, serum miRNA signatures have been associated with the presence of disease as well as correlated with previously validated risk models. However, it is unclear whether miRNAs can provide independent prognostic information beyond current risk models. Here, we focus on a group of low-risk prostate cancer patients who were eligible for active surveillance, but chose surgery. A major criteria for the low risk category is a Gleason score of 6 or lower based on pre-surgical biopsy. However, a third of these patients are upgraded to Gleason 7 on post surgical pathological analysis. Both in a discovery and a validation cohort, we find that pre-surgical serum levels of miR-19, miR-345 and miR-519c-5p can help identify these patients independent of their pre-surgical age, PSA, stage, and percent biopsy involvement. A combination of the three miRNAs increased the area under a receiver operator characteristics curve from 0.77 to 0.94 (p<0.01). Also, when combined with the CAPRA risk model the miRNA signature significantly enhanced prediction of patients with Gleason 7 disease. In-situ hybridizations of matching tumors showed miR-19 upregulation in transformed versus normal-appearing tumor epithelial, but independent of tumor grade suggesting an alternative source for the increase in serum miR-19a/b levels or the release of pre-existing intracellular miR-19a/b upon progression. Together, these data show that serum miRNAs can predict relatively small steps in tumor progression improving the capacity to predict disease risk and, therefore, potentially drive clinical decisions in prostate cancer patients. It will be important to validate these findings in a larger multi-institutional study as well as with independent methodologies.
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MicroARNs/sangre , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Humanos , Hibridación in Situ , MasculinoRESUMEN
OBJECTIVE: To assess the ability of current nomograms to predict disease progression at repeat biopsy or at delayed radical prostatectomy (RP) in a prospectively accrued cohort of patients managed by active surveillance (AS). MATERIALS AND METHODS: A total of 273 patients meeting low-risk criteria who were managed by AS and who underwent multiple biopsies and/or delayed RP were included in the study. The Kattan (base, medium and full), Steyerberg, Nakanishi and Chun nomograms were used to calculate the likelihood of indolent disease ('nomogram probability') as well as to predict 'biopsy progression' by grade or volume, 'surgical progression' by grade or stage, or 'any progression' on repeat biopsy or surgery. We evaluated the associations between each nomogram probability and each progression outcome using logistic regression with (area under the receiver-operating characteristic curve (AUC) values and decision curve analysis. RESULTS: The nomogram probabilities of indolent disease were lower in patients with biopsy progression (P < 0.01) and any progression on repeat biopsy or surgical pathology (P < 0.05). In regression analyses, nomograms showed a modest ability to predict biopsy progression, adjusted for total number of biopsies (AUC range 0.52-0.67) and any progression (AUC range 0.52-0.70). Decision curve analyses showed that all the nomograms, except for the Kattan base model, have similar value in predicting biopsy progression and any progression. Nomogram probabilities were not associated with surgical progression in a subgroup of 58 men who underwent delayed RP. CONCLUSIONS: Existing nomograms have only modest accuracy in predicting the outcomes of patients undergoing AS. Improvements to existing nomograms should be made before they are implemented in clinical practice and used to select patients for AS.
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Nomogramas , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Espera Vigilante , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Progresión de la Enfermedad , Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Probabilidad , Curva ROC , Medición de RiesgoRESUMEN
Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity involving FcγRIIIa (CD16) likely contributes to the clinical efficacy of rituximab. To assess the in vivo effects of CD16 polymorphisms on rituximab-induced NK activation, blood was evaluated before and 4 hours after initiation of the initial dose of rituximab in 21 lymphoma subjects. Rituximab induced NK activation and a drop in circulating NK-cell percentage in subjects with the high-affinity [158(VF/VV)] but not the low-affinity [158(FF)] CD16 polymorphism. There was no correlation between NK-cell activation or NK-cell percentage and polymorphisms in CD32A, C1q, or CH50. We conclude that NK activation occurs within 4 hours of rituximab infusion in subjects with the high-affinity CD16 polymorphism but not those with the low-affinity CD16 polymorphism. This finding may help explain the superior clinical outcome seen in the subset of high-affinity CD16 polymorphism lymphoma patients treated with single-agent rituximab.
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Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Linfoma , Receptores de IgG/metabolismo , Adulto , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Afinidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Femenino , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Humanos , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/uso terapéutico , Infusiones Intravenosas , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Linfoma/tratamiento farmacológico , Linfoma/inmunología , Linfoma/patología , Masculino , Persona de Mediana Edad , Polimorfismo Genético/inmunología , Unión Proteica , Receptores de IgG/inmunología , RituximabRESUMEN
Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the clinical response to monoclonal antibody (mAb) therapy of lymphoma. Recent in vitro analysis suggests C3b can inhibit mAb-induced natural killer (NK)-cell activation and ADCC. Further studies were conducted to assess the effect of C3 depletion on mAb-induced NK activation and therapy of lymphoma. Normal human serum inhibited the ability of rituximab-coated lymphoma cells to activate NK cells as previously reported. Serum did not inhibit NK-cell activation when it was preincubated with cobra venom factor (CVF) to deplete C3. Similar results were found when transudative pleural fluid or nonmalignant ascites was used as surrogates for extravascular fluid, suggesting the inhibitory effect of complement may be present in the extravascular compartment, in which many malignant lymphocytes reside. In vivo, C3 was depleted before mAb treatment in a syngeneic murine model of lymphoma. Survival of lymphoma-bearing mice after treatment with CVF plus mAb and with a human C3 derivative with CVF-like functions (HC3-1496) plus mAb was both superior to that of mAb alone. These studies show that complement depletion enhances NK-cell activation induced by rituximab-coated target cells and improves the efficacy of mAb therapy in a murine lymphoma model.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Complemento C3/deficiencia , Linfoma/tratamiento farmacológico , Linfoma/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , RituximabRESUMEN
The effective and practical use of mAbs in cancer therapy became a reality with the development of the chimeric anti-CD20 mAb, rituximab. Several additional mAbs have since been approved for clinical use. Despite these successes, the mechanisms by which mAbs mediate antitumor activity are still unclear. Preclinical studies indicate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) both can contribute to mAb-induced tumor cell lysis. However, evidence related to the relative clinical importance of each mechanism, and whether they are synergistic or antagonistic, is conflicting. New ways to enhance both CDC and ADCC are being developed in attempt to develop a more effective anticancer mAb. Continued research on the mechanisms of mAb therapy will be necessary if we are to take optimal advantage of the current mAbs and develop more effective mAbs in the future.
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Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos , Vía Clásica del Complemento , Citotoxicidad Inmunológica , Inmunoterapia/métodos , Neoplasias/terapia , Animales , Anticuerpos Monoclonales de Origen Murino , Antígenos CD55/efectos de los fármacos , Antígenos CD59/efectos de los fármacos , Diseño de Fármacos , Humanos , Isotipos de Inmunoglobulinas/inmunología , Isotipos de Inmunoglobulinas/uso terapéutico , Proteína Cofactora de Membrana/antagonistas & inhibidores , Ratones , Neoplasias/inmunología , Rituximab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-dependent cellular cytotoxicity (ADCC) and complement fixation both appear to play a role in mediating antitumor effects of monoclonal antibodies (mAbs), including rituximab. We evaluated the relationship between rituximab-induced complement fixation, natural killer (NK)-cell activation, and NK cell-mediated ADCC. Down-modulation of NK- cell CD16 and NK-cell activation induced by rituximab-coated target cells was blocked by human serum but not heat-inactivated serum. This inhibition was also observed in the absence of viable target cells. C1q and C3 in the serum were required for these inhibitory effects, while C5 was not. An antibody that stabilizes C3b on the target cell surface enhanced the inhibition of NK-cell activation induced by rituximab-coated target cells. Binding of NK cells to rituximab-coated plates through CD16 was inhibited by the fixation of complement. C5-depleted serum blocked NK cell-mediated ADCC. These data suggest that C3b deposition induced by rituximab-coated target cells inhibits the interaction between the rituximab Fc and NK-cell CD16, thereby limiting the ability of rituximab-coated target cells to induce NK activation and ADCC. Further studies are needed to define in more detail the impact of complement fixation on ADCC, and whether mAbs that fail to fix complement will be more effective at mediating ADCC.
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Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Complemento C3b/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Anticuerpos Monoclonales de Origen Murino , Antígenos de Protozoos/inmunología , Antígenos de Superficie/inmunología , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Complemento C1q/inmunología , Regulación hacia Abajo/efectos de los fármacos , Calor , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Antígeno de Macrófago-1/metabolismo , Unión Proteica , Receptores de IgG/metabolismo , Rituximab , Suero , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Growing evidence indicates that the affinity of monoclonal antibodies (mAbs) for CD16 (FcgammaRIII) plays a central role in the ability of the mAb to mediate antitumor activity. We evaluated how CD16 polymorphisms, and mAb with modified affinity for target antigen and CD16, affect natural killer (NK) cell phenotype when CD20(+) malignant B cells were also present. The mAb consisted of rituximab (R), anti-CD20 with enhanced affinity for CD20 (AME-B), and anti-CD20 with enhanced affinity for both CD20 and CD16 (AME-D). Higher concentrations of mAb were needed to induce CD16 modulation, CD54 up-regulation, and antibody-dependent cellular cytotoxicity (ADCC) on NK cells from subjects with the lower affinity CD16 polymorphism. The dose of mAb needed to induce NK activation was lower with AME-D irrespective of CD16 polymorphism. At saturating mAb concentrations, peak NK activation was greater for AME-D. Similar results were found with measurement of CD16 modulation, CD54 up-regulation, and ADCC. These data demonstrate that cells coated with mAb with enhanced affinity for CD16 are more effective at activating NK cells at both low and saturating mAb concentrations irrespective of CD16 polymorphism, and they provide further evidence for the clinical development of such mAbs with the goal of improving clinical response to mAb.