RESUMO
BACKGROUND AND AIMS: Janus kinase 2 (JAK2) signaling is increased in human and experimental liver fibrosis with portal hypertension. JAK2 inhibitors, such as pacritinib, are already in advanced clinical development for other indications and might also be effective in liver fibrosis. Here, we investigated the antifibrotic role of the JAK2 inhibitor pacritinib on activated hepatic stellate cells (HSCs) in vitro and in two animal models of liver fibrosis in vivo . APPROACH AND RESULTS: Transcriptome analyses of JAK2 in human livers and other targets of pacritinib have been shown to correlate with profibrotic factors. Although transcription of JAK2 correlated significantly with type I collagen expression and other profibrotic genes, no correlation was observed for interleukin-1 receptor-associated kinase and colony-stimulating factor 1 receptor. Pacritinib decreased gene expression of fibrosis markers in mouse primary and human-derived HSCs in vitro . Moreover, pacritinib decreased the proliferation, contraction, and migration of HSCs. C 57 BL/6J mice received ethanol in drinking water (16%) or Western diet in combination with carbon tetrachloride intoxication for 7 weeks to induce alcoholic or nonalcoholic fatty liver disease. Pacritinib significantly reduced liver fibrosis assessed by gene expression and Sirius red staining, as well as HSC activation assessed by alpha-smooth muscle actin immunostaining in fibrotic mice. Furthermore, pacritinib decreased the gene expression of hepatic steatosis markers in experimental alcoholic liver disease. Additionally, pacritinib protected against liver injury as assessed by aminotransferase levels. CONCLUSIONS: This study demonstrates that the JAK2 inhibitor pacritinib may be promising for the treatment of alcoholic and nonalcoholic liver fibrosis and may be therefore relevant for human pathology.
Assuntos
Janus Quinase 2 , Cirrose Hepática , Humanos , Camundongos , Animais , Janus Quinase 2/metabolismo , Cirrose Hepática/patologia , Fígado/patologia , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Fibrose , Células Estreladas do Fígado/metabolismoRESUMO
PURPOSE: Hepatic fibrosis develops as a response to chronic liver injury, resulting in the formation of fibrous scars. This process is initiated and driven by collagen-producing activated myofibroblasts which reportedly express high levels of platelet derived growth factor receptor-ß (PDGFRß). We therefore regard PDGFRß as an anchor for diagnosis and therapy. The Fibrobody® SP02SP26-ABD is a biparatopic VHH-construct targeting PDGFRß. Here, we explore its potential as a theranostic vector for liver fibrosis. METHODS: Specificity, cross-species binding, and cellular uptake of SP02SP26-ABD was assessed using human, mouse and rat PDGFRß ectodomains and PDGFRß-expressing cells. Cellular uptake by PDGFRß-expressing cells was also evaluated by equipping the Fibrobody® with auristatinF and reading out in vitro cytotoxicity. The validity of PDGFRß as a marker for active fibrosis was confirmed in human liver samples and 3 mouse models of liver fibrosis (DDC, CCl4, CDA-HFD) through immunohistochemistry and RT-PCR. After radiolabeling of DFO*-SP02SP26-ABD with 89Zr, its in vivo targeting ability was assessed in healthy mice and mice with liver fibrosis by PET-CT imaging, ex vivo biodistribution and autoradiography. RESULTS: SP02SP26-ABD shows similar nanomolar affinity for human, mouse and rat PDGFRß. Cellular uptake and hence subnanomolar cytotoxic potency of auristatinF-conjugated SP02SP26-ABD was observed in PDGFRß-expressing cell lines. Immunohistochemistry of mouse and human fibrotic livers confirmed co-localization of PDGFRß with markers of active fibrosis. In all three liver fibrosis models, PET-CT imaging and biodistribution analysis of [89Zr]Zr-SP02SP26-ABD revealed increased PDGFRß-specific uptake in fibrotic livers. In the DDC model, liver uptake was 12.15 ± 0.45, 15.07 ± 0.90, 20.23 ± 1.34, and 20.93 ± 4.35%ID/g after 1,2,3 and 4 weeks of fibrogenesis, respectively, compared to 7.56 ± 0.85%ID/g in healthy mice. Autoradiography revealed preferential uptake in the fibrotic (PDGFRß-expressing) periportal areas. CONCLUSION: The anti-PDGFRß Fibrobody® SP02SP26-ABD shows selective and high-degree targeting of activated myofibroblasts in liver fibrosis, and qualifies as a vector for diagnostic and therapeutic purposes.
RESUMO
The PtII linker [ethylenediamineplatinum(II)]2+ , coined Lx, has emerged as a novel non-conventional approach to antibody-drug conjugates (ADCs) and has shown its potential in preclinical inâ vitro and inâ vivo benchmark studies. A crucial improvement of the Lx conjugation reaction from initially <15 % to ca. 75-90 % conjugation efficiency is described, resulting from a systematic screening of all relevant reaction parameters. NaI, a strikingly simple inorganic salt additive, greatly improves the conjugation efficiency as well as the conjugation selectivity simply by exchanging the leaving chloride ligand on Cl-Lx-drug complexes (which are direct precursors for Lx-ADCs) for iodide, thus generating I-Lx-drug complexes as more reactive species. Using this iodide effect, we developed a general and highly practical conjugation procedure that is scalable: our lead Lx-ADC was produced on a 5â g scale with an outstanding conjugation efficiency of 89 %.
Assuntos
Anticorpos Monoclonais/química , Complexos de Coordenação/química , Imunoconjugados/química , Platina/química , Animais , Linhagem Celular Tumoral , Desferroxamina/química , Humanos , Imunoconjugados/sangue , Imunoconjugados/metabolismo , Imunoconjugados/uso terapêutico , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Receptor ErbB-2/imunologia , Iodeto de Sódio/química , Distribuição Tecidual , Transplante Heterólogo , Trastuzumab/química , Trastuzumab/imunologia , Trastuzumab/uso terapêuticoRESUMO
Recombinant adeno-associated virus (rAAV) has become the vector of choice for the development of novel human gene therapies. High-yield manufacturing of high-quality vectors can be achieved using the baculovirus expression vector system. However, efficient production of rAAV in this insect cell-based system requires a genetic redesign of the viral protein 1 (VP1) operon. In this study, we generated a library of rationally designed rAAV serotype 5 variants with modulations in the translation-initiation region of VP1 and investigated the potency of the resulting vectors. We found that the initiation strength at the VP1 translational start had downstream effects on the VP2/VP3 ratio. Excessive incorporation of VP3 into a vector type decreased potency, even when the VP1/VP2 ratio was in balance. Finally, we successfully generated a potent rAAV vector based on serotype 5 with a balanced VP1/VP2/VP3 stoichiometry.
Assuntos
Terapia Genética , Vetores Genéticos/genética , Parvovirinae/genética , Proteínas Virais/genética , Baculoviridae/genética , Proteínas do Capsídeo/genética , Dependovirus , Vetores Genéticos/uso terapêutico , Humanos , Óperon/genética , Sorogrupo , Proteínas Virais/uso terapêuticoRESUMO
Background & Aims: Intestine-restricted inhibitors of the apical sodium-dependent bile acid transporter (ASBT, or ileal bile acid transporter) are approved as treatment for several inheritable forms of cholestasis but are also associated with abdominal complaints and diarrhoea. Furthermore, blocking ASBT as a single therapeutic approach may be less effective in moderate to severe cholestasis. We hypothesised that interventions that lower hepatic bile salt synthesis in addition to intestinal bile salt uptake inhibition provide added therapeutic benefit in the treatment of cholestatic disorders. Here, we test combination therapies of intestinal ASBT inhibition together with obeticholic acid (OCA), cilofexor, and the non-tumorigenic fibroblast growth factor 15 (Fgf15)/fibroblast growth factor 19 (FGF19) analogue aldafermin in a mouse model of cholestasis. Methods: Wild-type male C57Bl6J/OlaHsd mice were fed a 0.05% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and received daily oral gavage with 10 mg/kg OCA, 30 mg/kg cilofexor, 10 mg/kg ASBT inhibitor (Linerixibat; ASBTi), or a combination. Alternatively, wild-type male C57Bl6J/OlaHsd mice were injected with adeno-associated virus vector serotype 8 (AAV8) to express aldafermin, to repress bile salt synthesis, or to control AAV8. During a 3-week 0.05% DDC diet, mice received daily oral gavage with 10 mg/kg ASBTi or placebo control. Results: Combination therapy of OCA, cilofexor, or aldafermin with ASBTi effectively reduced faecal bile salt excretion. Compared with ASBTi monotherapy, aldafermin + ASBTi further lowered plasma bile salt levels. Cilofexor + ASBTi and aldafermin + ASBTi treatment reduced plasma alanine transaminase and aspartate transaminase levels and fibrotic liver immunohistochemistry stainings. The reduction in inflammation and fibrogenesis in mice treated with cilofexor + ASBTi or aldafermin + ASBTi was confirmed by gene expression analysis. Conclusions: Combining pharmacological intestinal bile salt uptake inhibition with repression of bile salt synthesis may form an effective treatment strategy to reduce liver injury while dampening the ASBTi-induced colonic bile salt load. Impact and Implications: Combined treatment of intestinal ASBT inhibition with repression of bile salt synthesis by farnesoid X receptor agonism (using either obeticholic acid or cilofexor) or by expression of aldafermin ameliorates liver damage in cholestatic mice. In addition, compared with ASBT inhibitor monotherapy, combination treatments lower colonic bile salt load.
RESUMO
Alloreactive (allo)-HLA-directed T cell responses after HLA-mismatched allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion are typically considered detrimental responses mediating graft-versus-host disease (GVHD). Allo-HLA-reactive T cells with beneficial and selective graft-versus-leukemia (GVL) reactivity, however, can also be identified within an HLA-mismatched context. We investigated whether allo-HLA class II-directed T cells with beneficial GVL reactivity induced in NOD/scid mice engrafted with human chronic myelogenous leukemia in lymphoid blast crisis after treatment with donor lymphocyte infusion - mediated detrimental xenogeneic GVHD as a result of broad off-target cross-reactivity. The results demonstrate that beneficial GVL reactivity and xenogeneic GVHD are mediated by separate T cells. GVL reactivity was mediated by human T cells recognizing allo-HLA class II molecules, whereas xenoreactivity was exerted by human T cells recognizing H-2 molecules. Taken together, our data indicate a limited risk for detrimental off-target effects by allo-HLA class II-directed T cells and thereby provide a basis for the development of strategies for selecting allo-HLA-restricted T cells with selective GVL reactivity for adoptive transfer after HLA-mismatched allogeneic hematopoietic stem cell transplantation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença Enxerto-Hospedeiro/terapia , Efeito Enxerto vs Leucemia/imunologia , Transplante de Células-Tronco Hematopoéticas , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Doença Aguda , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/transplante , Modelos Animais de Doenças , Feminino , Expressão Gênica , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Teste de Histocompatibilidade , Humanos , Imunoterapia Adotiva , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante HomólogoRESUMO
In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy-that is, the genetic induction of a conditional suicide phenotype into donor T cells-allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk(+) T cells contained HA-1- and H-Y-specific CD8(+) cytotoxic T cells (CTL) precursors. Thymidine kinase-positive HA-1- and H-Y-specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Proliferação de Células , Genes Transgênicos Suicidas/imunologia , Leucemia/diagnóstico , Receptores de Interleucina-7/fisiologia , Animais , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Feminino , Expressão Gênica/fisiologia , Terapia Genética/métodos , Vetores Genéticos/imunologia , Humanos , Imunoterapia Adotiva/métodos , Leucemia/genética , Leucemia/imunologia , Leucemia/terapia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Prognóstico , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/fisiologia , Transplante HomólogoRESUMO
BACKGROUND: The Rho-kinase ROCK II plays a major role in the activation of hepatic stellate cells (HSC), which are the key profibrotic and contractile cells contributing to the development of chronic liver disease. Inhibition of ROCK II ultimately blocks the phosphorylation of the myosin light chain (MLC) and thus inhibits stress fibre assembly and cell contraction. We investigated the effects of the ROCK inhibitors Y-33075 as well as Y-27632 in murine and human hepatic stellate cells. METHODS: Primary isolated HSC from FVB/NJ mice and the immortalized human HSC line TWNT-4 were culture-activated and incubated with Y-27632 and Y-33075 (10nM to 10µM) for 24h. Protein expression levels were analyzed by Western Blots and transcriptional levels of pro-fibrotic markers and proliferative markers were evaluated using real-time qPCR. Migration was investigated by wound-healing assay. Proliferation was assessed by BrdU assay. Contraction of HSC was measured using 3D collagen matrices after incubation with Y-27632 or Y-33075 in different doses. RESULTS: Both Rho-kinase inhibitors, Y-27632 and Y-33075, reduced contraction, fibrogenesis and proliferation in activated primary mouse HSC (FVB/NJ) and human HSC line (TWNT-4) significantly. Y-33075 demonstrated a 10-times increased potency compared to Y-27632. Surprisingly, both inhibitors mediated a substantial and unexpected increase in migration of HSC in FVB/NJ. CONCLUSION: ROCK inhibition by the tested compounds decreased contraction but increased migration. Y-33075 proved more potent than Y27632 in the inhibition of contraction of HSCs and should be further evaluated in chronic liver disease.
Assuntos
Transdução de Sinais , Quinases Associadas a rho , Animais , Humanos , Camundongos , Células Cultivadas , Células Estreladas do Fígado/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
B-lineage acute lymphoblastic leukemia (ALL) may express CD52 and CD20. Alemtuzumab (ALM) and rituximab (RTX) are therapeutic antibodies directed against CD52 and CD20, respectively, but showed limited activity against ALL in clinical trials. The mechanisms for the impaired responses remained unclear. We studied expression of CD52 and CD20 on ALL cells and found that most cases coexpressed CD52 and CD20. However, distinct CD52-negative (CD52(-)) subpopulations were detected in most cases as the result of defective glycophosphatidyl-inositol anchoring. Although ALM efficiently eradicated CD52-positive (CD52(+)) cells in NOD/scid mice engrafted with primary human ALL, CD52(-) subclones escaped therapy. In the same model, RTX showed limited activity resulting from occurrence of CD20 down-modulation. However, CD52(-) cells concurrently lacked the glycophosphatidyl-inositol-anchored complement regulators CD55 and CD59 and showed increased susceptibility to RTX-mediated complement-dependent cytotoxicity in vitro. At the same time, ALM was shown to inhibit down-modulation of CD20 in response to RTX by depleting the trogocytic capacity of phagocytic cells. Probably because of these complementary mechanisms, combined administration of ALM and RTX induced complete responses in vivo. Based on these data, we propose a mechanistic rationale for combined application of RTX and ALM in ALL.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD20/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Glicosilfosfatidilinositóis/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Alemtuzumab , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Antineoplásicos/administração & dosagem , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos CD20/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Western Blotting , Antígeno CD52 , Antígenos CD59/imunologia , Antígenos CD59/metabolismo , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Citometria de Fluxo , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RituximabRESUMO
Currently, individuals with pre-existing neutralizing antibodies (NABs) against adeno-associated virus (AAV) above titer of 5 are excluded from systemic AAV-based clinical trials. In this study we explored the impact of pre-existing anti-AAV5 NABs on the efficacy of AAV5-based gene therapy. AMT-060 (AAV5-human FIX) was evaluated in 10 adults with hemophilia B who tested negative for pre-existing anti-AAV5 NABs using a GFP-based assay. In this study, using a more sensitive luciferase-based assay, we show that 3 of those 10 patients tested positive for anti-AAV5 NABs. However, no relationship was observed between the presence of pre-treatment anti-AAV5 NABs and the therapeutic efficacy of AMT-060. Further studies in non-human primates (NHPs) showed that AAV5 transduction efficacy was similar following AMT-060 treatment, irrespective of the pre-existing anti-AAV5 NABs titers. We show that therapeutic efficacy of AAV5-mediated gene therapy was achieved in humans with pre-existing anti-AAV5 NABs titers up to 340. Whereas in NHPs circulating human factor IX (hFIX) protein was achieved, at a level therapeutic in humans, with pre-existing anti-AAV5 NABs up to 1030. Based on those results, no patients were excluded from the AMT-061 (AAV5-hFIX-Padua) phase IIb clinical trial (n = 3). All three subjects presented pre-existing anti-AAV5 NABs, yet had therapeutic hFIX activity after AMT-061 administration.
RESUMO
Gene therapy for severe hemophilia B is advancing and offers sustained disease amelioration with a single treatment. We have reported the efficacy and safety of AMT-060, an investigational gene therapy comprising an adeno-associated virus serotype 5 capsid encapsidating the codon-optimized wild-type human factor IX (WT hFIX) gene with a liver-specific promoter, in patients with severe hemophilia B. Treatment with 2 × 1013 gc/kg AMT-060 showed sustained and durable FIX activity of 3%-13% and a substantial reduction in spontaneous bleeds without T cell-mediated hepatoxicity. To achieve higher FIX activity, we modified AMT-060 to encode the R338L "Padua" FIX variant that has increased specific activity (AMT-061). We report the safety and increased FIX activity of AMT-061 in non-human primates. Animals (n = 3/group) received intravenous AMT-060 (5 × 1012 gc/kg), AMT-061 (ranging from 5 × 1011 to 9 × 1013 gc/kg), or vehicle. Human FIX protein expression, FIX activity, and coagulation markers including D-dimer and thrombin-antithrombin complexes were measured. At equal doses, AMT-060 and AMT-061 resulted in similar human FIX protein expression, but FIX activity was 6.5-fold enhanced using AMT-061. Both vectors show similar safety and transduction profiles. Thus, AMT-061 holds great promise as a more potent FIX replacement gene therapy with a favorable safety profile.
RESUMO
Introduction: Compared to the antibody and drug components of an ADC, the linker part has been somewhat neglected. However, its importance for the reduction of failures in ADC approvals is increasingly recognized. Next of being a stable glue between drug and antibody, an ideal linker should improve the manufacturability and widen the therapeutic window of ADCs. Areas covered: The biopharmaceutical company LinXis started an ADC development program in which platinum(II) is the key element of the first metal-organic linker. The cationic complex [ethylenediamineplatinum(II)]2+, herein called 'Lx®', is used successfully for conjugation of drugs to antibodies. Expert opinion: Based on lessons learned from ADC development, Lx linker technology fulfills most of the desirable linker characteristics. Lx allows large-scale cost-effective manufacturing of ADCs via a straightforward two-step 'plug-and-play' process. First clinical candidate trastuzumab-Lx-auristatin F shows favorable preclinical safety as well as outstanding in vivo tumor targeting performance and therapeutic efficacy.
Assuntos
Aminobenzoatos/química , Antineoplásicos/química , Imunoconjugados/química , Oligopeptídeos/química , Compostos Organoplatínicos/química , Trastuzumab/química , Aminobenzoatos/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Oligopeptídeos/uso terapêutico , Compostos Organoplatínicos/uso terapêutico , Trastuzumab/uso terapêuticoRESUMO
PURPOSE: The poor immunogenicity of most leukemias and the lack of specificity of the donor T cells limit the in vivo effectiveness of conventional donor lymphocyte infusions in many patients suffering from persistent or recurrent leukemia after allogeneic stem cell transplantation. These limitations may be overcome by the adoptive transfer of in vitro generated leukemia-reactive T cells. Although the potential clinical efficacy of this approach has been shown previously, lack of reproducibility of the procedure and the inability to show persistence and survival of the transferred T cells hampered further clinical application. The purpose of this study was to develop a new, broadly applicable strategy for the efficient generation and isolation of leukemia-reactive T cells with a better probability to survive and expand in vivo. EXPERIMENTAL DESIGN: Myeloid and B-cell leukemias were modified into professional immunogenic antigen-presenting cells, and used to stimulate HLA-matched donor T cells. After two stimulations, responding donor T cells were isolated based on their secretion of IFN-gamma and tested for their capacity to recognize and kill the primary leukemia. RESULTS: Using one universal stimulation and isolation protocol for various forms of leukemia, T-cell populations containing high frequencies of leukemia-reactive T cells could reproducibly be generated and early isolated under mild stimulatory conditions. Isolated T cells still had high proliferative potential and their reactivity seemed to be restricted to cells of the patient's hematopoiesis. CONCLUSION: We here show a new robust procedure for the generation and isolation of leukemia-reactive T cells for adoptive transfer.
Assuntos
Transferência Adotiva , Imunoterapia Adotiva/métodos , Interferon gama/metabolismo , Linfócitos T/citologia , Sobrevivência Celular , Antígenos HLA/metabolismo , Humanos , Imunofenotipagem , Imunoterapia Adotiva/instrumentação , Leucemia/metabolismo , Leucemia de Células B/patologia , Leucemia de Células B/terapia , Leucemia Mieloide/patologia , Leucemia Mieloide/terapia , Monócitos/metabolismo , Transplante de Células-Tronco/métodos , Linfócitos T/metabolismo , Transplante HomólogoRESUMO
The experimental cytotoxic drug cyclopentenyl cytosine (CPEC) is a non-competitive inhibitor of the enzyme cytidine triphosphate (CTP) synthethase. We evaluated the in vitro and in vivo antitumor activity of CPEC on human acute lymphoblastic leukemia (ALL) cell lines. CPEC displayed anti-leukemic activity with IC50 (after 3 days of incubation) ranging from 6 to 15 nM. Subsequently the in vivo activity of CPEC against primary human ALL was evaluated in a xenogeneic model of human ALL using NOD/scid mice inoculated with primary human ALL cells. In the model, only a marginal anti-leukemic activity was observed at 1.5 mg kg(-1) (5 days per week) and 5 mg kg(-1) (2 days per week), however, this activity was associated with severe systemic toxicity. The observed toxicity was not specific for the NOD/scid model, as toxicity at comparable treatment intensity was also observed in Balb/c mice. In conclusion, although CPEC showed antitumor activity against human ALL cells in vitro, its activity in the in vivo human leukemia model was only marginal and accompanied by severe toxicity.
Assuntos
Antineoplásicos/toxicidade , Citidina/análogos & derivados , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Animais , Citidina/toxicidade , Humanos , Cariotipagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
To optimally utilize therapeutic monoclonal antibodies in the treatment of B-cell acute lymphoblastic leukemia (B-ALL) understanding their mechanisms of action and the factors influencing these mechanisms is required. We show strong correlations between target antigen expression levels and sensitivity to complement-dependent cytotoxicity (CDC) induced by rituximab, ofatumumab, or alemtuzumab in a panel of cell lines derived from primary B-ALL cells and in primary B-ALL samples. Simultaneous loss of expression of membrane-bound complement regulatory proteins (mCRP) CD55 and CD59 due to glycophosphatidylinositol-anchor deficiency, significantly increased sensitivity to CDC. Accordingly, induced increase in CD55 or CD59 expression protected cells against CDC. The extent of protection co-depended on antigen expression and antibody concentration. In contrast, natural variation in mCRP expression could not be used as a single factor to predict sensitivity to CDC. In conclusion, sensitivity of B-ALL cells to CDC was predominantly determined by antibody concentration and target antigen expression.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígenos de Neoplasias/genética , Biomarcadores , Antígenos CD55/genética , Antígenos CD55/metabolismo , Antígenos CD59/genética , Antígenos CD59/metabolismo , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismoRESUMO
OBJECTIVE: Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation is largely unsuccessful in the treatment of acute lymphoblastic leukemia (ALL). To allow identification of the causes of this failure, we established an animal model of DLI in human ALL. METHODS: NOD/scid mice were inoculated with primary human ALL cells. Cells from five different patients were studied. After engraftment, DLI was performed by infusion of human leukocyte antigen (HLA)-identical donor T cells or HLA-disparate donor T cells. RESULTS: DLI resulted in expansion of activated, leukemia-reactive T cells in all donor-patient combinations. After 40 days of expansion, T cells abruptly declined in numbers and displayed loss of cytotoxicity. At this moment, remissions were observed in three of five donor-patient combinations. In animals engrafted with the two unresponsive ALL, remissions could be achieved when HLA-disparate DLI was given. CONCLUSION: Our results indicate that the inefficacy of DLI in ALL may be due to the limitation of the proliferative capacity of ALL-reactive T cells and that the antileukemic efficacy during the limited time span of proliferation depends on the antigenic disparity between the donor and the patient. The model can be used to study whether alternative strategies may result in more sustained antileukemic responses after DLI in ALL.
Assuntos
Efeito Enxerto vs Leucemia/imunologia , Leucemia Experimental/imunologia , Doadores Vivos , Transfusão de Linfócitos , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Linfócitos T/imunologia , Adulto , Animais , Proliferação de Células , Citotoxicidade Imunológica/imunologia , Feminino , Humanos , Imunidade Celular/imunologia , Transfusão de Linfócitos/métodos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Transplante de Células-Tronco , Linfócitos T/transplante , Transplante HomólogoRESUMO
PURPOSE: Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established. EXPERIMENTAL DESIGN: We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative. RESULTS: Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2. CONCLUSIONS: Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Inibidores de Fosfoinositídeo-3 Quinase , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Aminopiridinas/farmacologia , Sinergismo Farmacológico , Everolimo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Imidazóis/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Morfolinas/farmacologia , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Naftiridinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Imatinib should be avoided in women planning to become pregnant or during pregnancy, due to a higher risk of congenital malformations. However, it is not known whether imatinib affects future potential for fertility. Here we analysed ovaries and testes from adult mice receiving imatinib, focusing on testicular and ovarian functions. Seven male and 7 female mice were orally treated with 150 mg/kg body weight/day imatinib for two months. No effects on folliculogenesis or spermatogenesis could be observed postmortem by histological examinations, suggesting that, at least in two mouse models of imatinib treatment this tyrosine kinase inhibitor does not reduce fertility.
Assuntos
Modelos Animais de Doenças , Leucemia Experimental/tratamento farmacológico , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Espermatogênese/efeitos dos fármacos , Animais , Benzamidas , Relação Dose-Resposta a Droga , Feminino , Mesilato de Imatinib , Leucemia Experimental/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Proteínas Tirosina Quinases/antagonistas & inibidoresRESUMO
OBJECTIVE: B-lineage acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphatic blastic phase in adults have poor prognoses despite intensive chemotherapy. Novel targeted treatment modalities emerge, but their evaluation requires relevant in vitro models of lymphoblastic leukemia. Presently available cell lines do not fully represent this heterogeneous disease. Available in vitro culturing protocols do not support long-term proliferation of primary cells. We therefore aimed to develop a culture system that allows long-term proliferation of primary human B-lineage lymphoblastic leukemia. MATERIALS AND METHODS: Primary lymphoblastic leukemia cells were cultured in a defined serum-free medium, in the absence or presence of human hematopoietic growth factors or serum. RESULTS: In the defined serum-free medium, cells from 12 of 34 cases immediately proliferated in vitro. In the absence of hematopoietic growth factors and serum these cases proliferated for more than 1 year without signs of exhaustion. The culturing system supported different subtypes of lymphoblastic leukemia. Two chronic myeloid leukemia in lymphatic blastic phase, four bcr/abl-positive ALL, one etv6/abl-positive ALL, 2 e2a-pbx1-positive ALL, and one t(9;11)-positive ALL could be long-term expanded, as well as two ALL that displayed nontypical cytogenetics. Not all bcr/abl- or e2a-pbx1-positive ALL proliferated in vitro, demonstrating heterogeneity within these subtypes. The proliferating bcr/abl- and etv6/abl-positive cells displayed sensitivity to imatinib, demonstrating that their proliferation depended on the activity of these oncoproteins. CONCLUSION: The serum-free culturing system may be a valuable instrument in the study of ALL cell biology, as well as in the evaluation of novel targeted therapeutics.
Assuntos
Técnicas de Cultura de Células/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proliferação de Células , Meios de Cultura Livres de Soro , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Leucemia de Células B/patologia , Fatores de TempoRESUMO
Fusion kinases (FK) like BCR/ABL1 mediate leukemic transformation and represent therapeutic targets. Fusion of ETV6 (ETS translocation variant 6, previously known as TEL) to ABL1 due to t(9;12) has been observed in various hematological malignancies. ETV6/ABL1 and BCR/ABL1 FK display similar activity but they may not be identical in function. Here we present the generation of an ETV6/ABL1 positive human acute lymphoblastic leukemia (ALL) cell line, ALL-VG. The cell line expressed ETV6/ABL1 fusion transcripts and displayed sensitivity to imatinib with an IC(50) of 0.1 microM. Karyotyping did not reveal overt t(9;12), suggesting a cryptic translocation. Fluorescent in situ hybridization and array-based comparative genomic hybridization were performed to characterize the rearrangement. ETV6/ABL1 fusion was demonstrated to result from insertion of a duplicated 300 to 1300 kb region of 9q34 that contained the distal portion of the ABL1 gene, into the ETV6 locus on 12p13. With this insertion, an 1150 to 1750 kb region of 12p13 that contained the distal portion of the ETV6 gene as well the cyclin dependent kinase inhibitor (CDKN) 1B gene was lost. Furthermore, the cells displayed a del(9)(p21.1 approximately p23), typically associated with loss of CDKN2A and CDKN2B. The ALL-VG cell line may serve as a tool for the study of ETV6/ABL1.