RESUMO
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.
Assuntos
Regulação da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Modelos Animais de Doenças , Vetores Genéticos/genética , Humanos , Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-3/genética , Subunidade alfa de Receptor de Interleucina-3/imunologia , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Lentivirus/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução Genética , Carga TumoralRESUMO
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.
Assuntos
Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Terapia de Salvação/métodos , Adenina/análogos & derivados , Adulto , Idoso , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/complicações , Leucemia Linfocítica Crônica de Células B/terapia , Masculino , Pessoa de Meia-Idade , Neutropenia/induzido quimicamente , Piperidinas , Pirazóis/efeitos adversos , Pirimidinas/efeitos adversos , Estudos Retrospectivos , Resultado do TratamentoRESUMO
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.
Assuntos
Nucleotídeos de Adenina/uso terapêutico , Antimetabólitos Antineoplásicos/uso terapêutico , Arabinonucleosídeos/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda/terapia , Terapia de Salvação , Adulto , Idoso , Clofarabina , Feminino , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Recidiva , Transplante HomólogoRESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos/metabolismo , Resistencia a Medicamentos Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Piperazinas/metabolismo , Pirimidinas/metabolismo , Rodaminas/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Benzamidas , Divisão Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Resistência a Múltiplos Medicamentos , Inibidores Enzimáticos/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/fisiologia , Regulação Leucêmica da Expressão Gênica , Humanos , Mesilato de Imatinib , Imunossupressores/farmacologia , Células K562/efeitos dos fármacos , Células K562/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Nucleares/metabolismo , Piperazinas/uso terapêutico , Pirimidinas/uso terapêuticoRESUMO
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.
Assuntos
Anticorpos Biespecíficos/biossíntese , Imunoterapia/métodos , Leucemia Mieloide Aguda/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/imunologia , Animais , Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/imunologia , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Leucemia Mieloide Aguda/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Fatores de Transcrição NFATC/metabolismo , Recidiva Local de Neoplasia/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Sequências de Repetição em Tandem/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Ciclosporina/farmacologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Imunossupressores/farmacologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Mutação/genética , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Niacinamida/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sorafenibe , Taxa de Sobrevida , Células Tumorais Cultivadas , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
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.
Assuntos
Subunidade alfa de Receptor de Interleucina-3/biossíntese , Leucemia Mieloide Aguda/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Citometria de Fluxo , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Fatores de Risco , Adulto Jovem , Tirosina Quinase 3 Semelhante a fms/sangue , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
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.
Assuntos
Leucemia Mieloide Aguda/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Leucemia Mieloide Aguda/patologia , Ativação Linfocitária , MasculinoAssuntos
Antineoplásicos/uso terapêutico , Cromossomos Humanos Par 5 , Proteína 1 de Resposta de Crescimento Precoce/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Transplante de Células-Tronco/métodos , Talidomida/análogos & derivados , Transplante Homólogo/métodos , Idoso , Proliferação de Células , Citogenética , Deleção de Genes , Humanos , Lenalidomida , Masculino , Indução de Remissão , Talidomida/uso terapêutico , Fatores de TempoRESUMO
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.
Assuntos
Plaquetas/fisiologia , Doença Enxerto-Hospedeiro/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Doença Aguda , Adulto , Idoso , Plaquetas/química , Linfócitos T CD4-Positivos/imunologia , Feminino , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunossupressores/uso terapêutico , Ativação Linfocitária , Masculino , Células-Tronco Mesenquimais/imunologia , Pessoa de Meia-Idade , Resultado do TratamentoAssuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Complexo CD3/imunologia , Células-Tronco Hematopoéticas/imunologia , Leucemia Mieloide Aguda/terapia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Linfócitos T/imunologia , Citometria de Fluxo , Humanos , Leucemia Mieloide Aguda/imunologiaAssuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Complexo CD3/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Anticorpos Biespecíficos/uso terapêutico , Complexo CD3/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/patologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/efeitos dos fármacos , Lectina 3 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
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.