RESUMEN
The adoptive transfer of CD19-specific chimeric antigen receptor engineered T cells (CAR T cells) resulted in encouraging clinical trials in indolent B-cell malignancies. However, they also show the limitations of this fascinating technology: CAR T cells can lead to even life-threatening off-tumor, on-target side effects if CAR T cells crossreact with healthy tissues. Here, we describe a novel modular universal CAR platform technology termed UniCAR that reduces the risk of on-target side effects by a rapid and reversible control of CAR T-cell reactivity. The UniCAR system consists of two components: (1) a CAR for an inert manipulation of T cells and (2) specific targeting modules (TMs) for redirecting UniCAR T cells in an individualized time- and target-dependent manner. UniCAR T cells can be armed against different tumor targets simply by replacement of the respective TM for (1) targeting more than one antigen simultaneously or subsequently to enhance efficacy and (2) reducing the risk for development of antigen-loss tumor variants under treatment. Here we provide 'proof of concept' for retargeting of UniCAR T cells to CD33- and/or CD123-positive acute myeloid leukemia blasts in vitro and in vivo.
Asunto(s)
Regulación de la Expresión Génica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Citocinas/metabolismo , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Humanos , Inmunoterapia Adoptiva/métodos , Subunidad alfa del Receptor de Interleucina-3/genética , Subunidad alfa del Receptor de Interleucina-3/inmunología , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Lentivirus/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Transducción Genética , Carga TumoralRESUMEN
Ibrutinib, a recently approved inhibitor of Bruton's tyrosine kinase (BTK), has shown great efficacy in patients with high-risk CLL. Nevertheless, there are few data regarding its use in patients who relapsed after allogeneic stem cell transplantation (alloSCT). We report clinical data from five CLL patients treated with ibrutinib for relapse after first or even second allogeneic transplantation. Additionally, we performed analyses on cytokine levels and direct measuring of CD4 Th1 and CD4 Th2 cells to evaluate possible clinically relevant immunomodulatory effects of ibrutinib. All patients achieved partial responses including one minimal residual disease (MRD)-negative remission. Within 1 year of follow-up, no relapse was observed. One patient died of severe pneumonia while on ibrutinib treatment. Beside this, no unexpected adverse events were observed. Flow cytometry and analyses of T cell-mediated cytokine levels (IL10 and TNFα) did not reveal substantial changes in T-cell distribution in favor of a CD4 Th1 T-cell shift in our patients. No acute exacerbation of GvHD was reported. In conclusion, these results support further evaluation of ibrutinib in CLL patients relapsing after alloSCT.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Terapia Recuperativa/métodos , Adenina/análogos & derivados , Adulto , Anciano , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/complicaciones , Leucemia Linfocítica Crónica de Células B/terapia , Masculino , Persona de Mediana Edad , Neutropenia/inducido químicamente , Piperidinas , Pirazoles/efectos adversos , Pirimidinas/efectos adversos , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
In patients with relapsed or refractory (r/r) acute myeloid leukemia (AML), long-term disease control can only be achieved by allogeneic hematopoietic stem cell transplantation (HSCT). We studied the safety and efficacy of clofarabine-based salvage therapy. The study was designed as phase II, multicenter, intent-to-transplant (ITT) study. A total of 84 patients with r/r AML were enrolled. All patients received at least one cycle of CLARA (clofarabine 30 mg/m(2) and cytarabine 1 g/m(2), days 1-5). Chemo-responsive patients with a donor received HSCT in aplasia after first CLARA. Generally, HSCT was performed as soon as possible. The conditioning regimen consisted of clofarabine (4 × 30 mg/m(2)) and melphalan (140 mg/m(2)). The median patient age was 61 years (range 40-75). On day 15 after start of CLARA, 26% of patients were in a morphologically leukemia-free state and 79% exposed a reduction in bone marrow blasts. Overall, 67% of the patients received HSCT within the trial. The primary end point, defined as complete remission after HSCT, was achieved by 60% of the patients. According to the ITT, overall survival at 2 years was 43% (95% confidence interval (CI), 32-54%). The 2-year disease-free survival for transplanted patients was 52% (95% CI, 40-69%). Clofarabine-based salvage therapy combined with allogeneic HSCT in aplasia shows promising results in patients with r/r AML.
Asunto(s)
Nucleótidos de Adenina/uso terapéutico , Antimetabolitos Antineoplásicos/uso terapéutico , Arabinonucleósidos/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda/terapia , Terapia Recuperativa , Adulto , Anciano , Clofarabina , Femenino , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Recurrencia , Trasplante HomólogoRESUMEN
Bispecific antibodies (bsAbs) engaging T cells are emerging as a promising immunotherapeutic tool for the treatment of hematologic malignancies. Because their low molecular mass, bsAbs have short half-lives. To achieve clinical responses, they have to be infused into patients continously, for a long period of time. As a valid alternative we examined the use of mesenchymal stromal cells (MSCs) as autonomous cellular machines for the constant production of a recently described, fully humanized anti-CD33-anti-CD3 bsAb, which is capable of redirecting human T cells against CD33-expressing leukemic cells. The immortalized human MSC line SCP-1 was genetically modified into expressing bsAb at sufficient amounts to redirect T cells efficiently against CD33 presenting target cells, both in vitro and in an immunodeficient mouse model. Moreover, T cells of patients suffering from acute myeloid leukemia (AML) in blast crisis eliminated autologous leukemic cells in the presence of the bsAb secreting MSCs over time. The immune response against AML cells could be enhanced further by providing T cells an additional co-stimulus via the CD137-CD137 ligand axis through CD137L expression on MSCs. This study demonstrates that MSCs have the potential to be used as cellular production machines for bsAb-based tumor immunotherapy in the future.
Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Inmunoterapia/métodos , Leucemia Mieloide Aguda/terapia , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/inmunología , Animales , Anticuerpos Biespecíficos/uso terapéutico , Complejo CD3/inmunología , Línea Celular Tumoral , Citometría de Flujo , Humanos , Leucemia Mieloide Aguda/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Internal tandem duplications (ITD) in the Fms-related tyrosine kinase 3 receptor (FLT3) are associated with a dismal prognosis in acute myeloid leukemia (AML). FLT3 inhibitors such as sorafenib may improve outcome, but only few patients display long-term responses, prompting the search for underlying resistance mechanisms and therapeutic strategies to overcome them. Here we identified that the nuclear factor of activated T cells, NFATc1, is frequently overexpressed in FLT3-ITD-positive (FLT3-ITD+) AML. NFATc1 knockdown using inducible short hairpin RNA or pharmacological NFAT inhibition with cyclosporine A (CsA) or VIVIT significantly augmented sorafenib-induced apoptosis of FLT3-ITD+ cells. CsA also potently overcame sorafenib resistance in FLT3-ITD+ cell lines and primary AML. Vice versa, de novo expression of a constitutively nuclear NFATc1-mutant mediated instant and robust sorafenib resistance in vitro. Intriguingly, FLT3-ITD+ AML patients (n=26) who received CsA as part of their rescue chemotherapy displayed a superior outcome when compared with wild-type FLT3 (FLT3-WT) AML patients. Our data unveil NFATc1 as a novel mediator of sorafenib resistance in FLT3-ITD+ AML. CsA counteracts sorafenib resistance and may improve treatment outcome in AML by means of inhibiting NFAT.
Asunto(s)
Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Factores de Transcripción NFATC/metabolismo , Recurrencia Local de Neoplasia/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Secuencias Repetidas en Tándem/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Ciclosporina/farmacología , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Inmunosupresores/farmacología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Mutación/genética , Factores de Transcripción NFATC/antagonistas & inhibidores , Factores de Transcripción NFATC/genética , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Niacinamida/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sorafenib , Tasa de Supervivencia , Células Tumorales Cultivadas , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Owing to the more recent positive results with the anti-CD33 immunotoxin gemtuzumab ozogamicin, therapy against acute myeloid leukemias (AMLs) targeting CD33 holds many promises. Here, CD33 and CD123 expression on AML blasts was studied by flow cytometry in a cohort of 319 patients with detailed information on French-American-British/World Health Organization (FAB/WHO) classification, cytogenetics and molecular aberrations. AMLs of 87.8% express CD33 and would therefore be targetable with anti-CD33 therapies. Additionally, 9.4% of AMLs express CD123 without concomitant CD33 expression. Thus, nearly all AMLs could be either targeted via CD33 or CD123. Simultaneous presence of both antigens was observed in 69.5% of patients. Most importantly, even AMLs with adverse cytogenetics express CD33 and CD123 levels comparable to those with favorable and intermediate subtypes. Some patient groups with unfavorable alterations, such as FMS-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations, high FLT3-ITD mutant/wild-type ratios and monosomy 5 are even characterized by high expression of CD33 and CD123. In addition, blasts of patients with mutant nucleophosmin (NPM1) revealed significantly higher CD33 and CD123 expression pointing toward the possibility of minimal residual disease-guided interventions in mutated NPM1-positive AMLs. These results stimulate the development of novel concepts to redirect immune effector cells toward CD33- and CD123-expressing blasts using bi-specific antibodies or engineered T cells expressing chimeric antigen receptors.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-3/biosíntesis , Leucemia Mieloide Aguda/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/biosíntesis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Citometría de Flujo , Humanos , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Nucleofosmina , Factores de Riesgo , Adulto Joven , Tirosina Quinasa 3 Similar a fms/sangre , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Owing to their clinical success, there is growing interest in novel bispecific antibodies (bsAbs) for retargeting of T cells to tumor cells including for the treatment of acute myeloid leukemia (AML). One potential target for retargeting of T cells to AML blasts is the surface molecule CD33. Here we describe a novel modular targeting platform that consists of a universal effector module (EM) and individual target modules (TMs). Both modules can form an immune complex via a peptide epitope. The resulting targeting complex can functionally replace a conventional bsAb. By fusion of a costimulatory domain (for example, the extracellular CD137 ligand domain) to the TM, the targeting complex can even provide a costimulatory signal to the redirected T cells at their side of interaction with the tumor cell. Furthermore, we observed that an efficient killing of tumor cells expressing low levels of the tumor target CD33 becomes critical at low effector-to-target cell ratios but can be improved by costimulation via CD137 using our novel targeting system.
Asunto(s)
Leucemia Mieloide Aguda/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Linfocitos T/inmunología , Animales , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Humanos , Leucemia Mieloide Aguda/patología , Activación de Linfocitos , MasculinoAsunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Complejo CD3/inmunología , Células Madre Hematopoyéticas/inmunología , Leucemia Mieloide Aguda/terapia , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Linfocitos T/inmunología , Citometría de Flujo , Humanos , Leucemia Mieloide Aguda/inmunologíaAsunto(s)
Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Complejo CD3/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Complejo CD3/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/patología , Subfamilia K de Receptores Similares a Lectina de Células NK/efectos de los fármacos , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
Mesenchymal stem cells have been shown to mediate immunomodulatory effects. They have been used in patients with steroid-refractory acute GVHD (aGVHD), but their relevance as a therapeutic agent targeting aGVHD has still to be defined. In this case series, we report 13 patients with steroid-refractory aGVHD who received BM-derived MSC expanded in platelet lysate-containing medium from unrelated HLA disparate donors. MSC were characterized by their morphological, phenotypical and functional properties. All tested preparations suppressed the proliferation of in vitro activated CD4+ T cells. MSC were transfused at a median dosage of 0.9 x 10(6)/kg (range 0.6-1.1). The median number of MSC applications was 2 (range 1-5). Only two patients (15%) responded and did not require any further escalation of immunosuppressive therapy. Eleven patients received additional salvage immunosuppressive therapy concomitant to further MSC transfusions, and after 28 days, five of them (45%) showed a response. Four patients (31%) are alive after a median follow-up of 257 days, including one patient who initially responded to MSC treatment. In our patient cohort, response to MSC transfusion was lower than in the series reported earlier. However, our experience supports the potential efficacy of MSC in the treatment of steroid-refractory aGVHD.
Asunto(s)
Plaquetas/fisiología , Enfermedad Injerto contra Huésped/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Enfermedad Aguda , Adulto , Anciano , Plaquetas/química , Linfocitos T CD4-Positivos/inmunología , Femenino , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas , Humanos , Inmunosupresores/uso terapéutico , Activación de Linfocitos , Masculino , Células Madre Mesenquimatosas/inmunología , Persona de Mediana Edad , Resultado del TratamientoAsunto(s)
Antineoplásicos/uso terapéutico , Cromosomas Humanos Par 5 , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Trasplante de Células Madre/métodos , Talidomida/análogos & derivados , Trasplante Homólogo/métodos , Anciano , Proliferación Celular , Citogenética , Eliminación de Gen , Humanos , Lenalidomida , Masculino , Inducción de Remisión , Talidomida/uso terapéutico , Factores de TiempoRESUMEN
STI-571 (Imatinib/Glivec) has been shown to have synergism with various chemotherapeutic agents including cytosine arabinoside (Ara-C) in BCR/ABL positive leukemia cells. The antiproliferative and proapopotic effects of STI-571 in these experiments are mainly explained by its ability to specifically block the fusion-protein BCR/ABL which has a constitutively active tyrosine kinase activity. We investigated the effects of STI-571 in combination with Ara-C on BCR/ABL negative leukemia cell lines and CD34+ hematopoietic progenitor cells in-vitro. Raji, HL-60, K562, Kasumi and KG1a leukemia cells and CD34+ cells from healthy donors were incubated with 5-20 microg/ml Ara-C for 5 h alone or in combination with 10 microg/ml STI-571. Intracellular levels of Ara-CTP measured by HPLC were increased 1.5-3 fold in leukemia cells with most promiment effects in HL-60, Kasumi and Raji cells. In HL-60 cells a linear correlation between the concentration of STI-571 (1-10 microg/ml) and the subsequent levels of Ara-CTP was observed. A linear increase of Ara-CTP could be induced by increasing the incubation time with STI-571 from 2-6 h with a ceiling effect after 8 h. In contrast coincubation of mononuclear cells or purified CD34+ cells with STI-571 at therapeutic concentrations lead to decreased intracellular levels of Ara-CTP. The synergism between Ara-C and STI-571 was even more pronounced in Raji and HL-60 cells when 300 ng/ml G-CSF were added at the beginning of the culture period. Intracellular measurements of STI-571 revealed no decreased or increased levels of the compound when increasing Ara-C concentrations were used. Our findings indicate that STI-571 can have significant impact on nucleoside metabolism in malignant and non-malignant hematopoietic cells. Further investigations will have to show whether theses effects can lead to increased cytotoxicity in primary blasts of patients with acute leukemia.
Asunto(s)
Células de la Médula Ósea/metabolismo , Citarabina/farmacología , Genes abl/genética , Piperazinas/farmacología , Pirimidinas/farmacología , Antígenos CD/análisis , Antígenos CD34/análisis , Benzamidas , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Línea Celular Tumoral , Eliminación de Gen , Humanos , Mesilato de Imatinib , Leucemia/genéticaRESUMEN
Imatinib (Glivec), STI571) is an intracellular acting drug that demonstrates high activity against BCR-ABL-positive chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). However, many patients, especially with advanced disease, develop drug resistance. Here, we show by a novel high-performance liquid chromatography-based method that intracellular levels of imatinib decrease in P-glycoprotein (Pgp)-positive leukemic cells. In a model of K562 cells with gradually increasing Pgp expression, a Pgp-dependent decline of intracellular imatinib levels was observed. Decreased imatinib levels were associated with a retained phosphorylation pattern of the Bcr-Abl target Crkl and loss of effect of imatinib on cellular proliferation and apoptosis. The modulation of Pgp by cyclosporin A (CSA) readily restored imatinib cytotoxicity in these cells. Finally, we provide first data showing a biological effect of Pgp modulation in the imatinib treatment of a patient with BCR-ABL-positive ALL. MDR1 overexpression must therefore be considered as an important clinical mechanism in the diversity of resistance development to imatinib treatment.