RESUMO
E7777 is a recombinant cytotoxic fusion protein composed of the diphtheria toxin fragments A and B and human interleukin-2. It shares an amino acid sequence with denileukin diftitox, but has improved purity and an increased percentage of active monomer. We undertook a multicenter, single-arm phase II study of E7777 in patients with relapsed or refractory peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL) to evaluate its efficacy, safety, pharmacokinetics, and immunogenicity. A total of 37 patients were enrolled, of which 17 and 19 patients had PTCL and CTCL, respectively, and one patient with another type of lymphoma (extranodal natural killer/T-cell lymphoma, nasal type), diagnosed by the Central Pathological Diagnosis Committee. Among the 36 patients with PTCL and CTCL, objective response rate based on the independent review was 36% (41% and 31%, respectively). The median progression-free survival was 3.1 months (2.1 months in PTCL and 4.2 months in CTCL). The common adverse events (AEs) observed were increased aspartate aminotransferase (AST) / alanine aminotransferase (ALT), hypoalbuminemia, lymphopenia, and pyrexia. Our results indicated that a 9 µg/kg/d dose of E7777 shows efficacy and a manageable safety profile in Japanese patients with relapsed or refractory PTCL and CTCL, with clinical activity observed across the range of CD25 expression. The common AEs were manageable, but increase in ALT / AST, hypoalbuminemia, and capillary leak syndrome should be carefully managed during the treatment.
Assuntos
Interleucina-2/administração & dosagem , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma de Células T Periférico/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Proteínas Recombinantes de Fusão/administração & dosagem , Administração Intravenosa , Sítios de Ligação , Toxina Diftérica/administração & dosagem , Toxina Diftérica/efeitos adversos , Toxina Diftérica/química , Toxina Diftérica/genética , Toxina Diftérica/farmacocinética , Esquema de Medicação , Feminino , Humanos , Interleucina-2/efeitos adversos , Interleucina-2/química , Interleucina-2/genética , Interleucina-2/farmacocinética , Japão , Linfoma Cutâneo de Células T/sangue , Linfoma de Células T Periférico/sangue , Masculino , Recidiva Local de Neoplasia/sangue , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacocinética , Análise de Sobrevida , Resultado do TratamentoRESUMO
Tagraxofusp (tagraxofusp-erzs) [Elzonris™] is an intravenously administered CD123-directed cytotoxin (composed of human interleukin-3 and a truncated diphtheria toxin payload) that was developed by Stemline Therapeutics, Inc. for the treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN). In December 2018, tagraxofusp received its first global approval in the USA for the treatment of BPDCN in adults and in paediatric patients aged 2 years and older. A centralized registration application for the use of tagraxofusp in patients with BPDCN is under review in the EU. This article summarizes the milestones in the development of tagraxofusp leading to its first global approval for the treatment of BPDCN.
Assuntos
Antineoplásicos/farmacocinética , Toxina Diftérica/farmacocinética , Interleucina-3/farmacocinética , Neoplasias Cutâneas/tratamento farmacológico , Administração Intravenosa , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Criança , Pré-Escolar , Toxina Diftérica/administração & dosagem , Toxina Diftérica/efeitos adversos , Toxina Diftérica/uso terapêutico , Aprovação de Drogas , Feminino , Humanos , Interleucina-3/administração & dosagem , Interleucina-3/efeitos adversos , Interleucina-3/uso terapêutico , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Resultado do Tratamento , Estados Unidos , United States Food and Drug Administration , Adulto JovemRESUMO
Research on diphtheria and anthrax toxins over the past three decades has culminated in a detailed understanding of their structure function relationships (e.g., catalytic (C), transmembrane (T), and receptor binding (R) domains), as well as the identification of their eukaryotic cell surface receptor, an understanding of the molecular events leading to the receptor-mediated internalization of the toxin into an endosomal compartment, and the pH triggered conformational changes required for pore formation in the vesicle membrane. Recently, a major research effort has been focused on the development of a detailed understanding of the molecular interactions between each of these toxins and eukaryotic cell factors that play an essential role in the efficient translocation of their respective catalytic domains through the trans-endosomal vesicle membrane pore and delivery into the cell cytosol. In this review, I shall focus on recent findings that have led to a more detailed understanding of the mechanism by which the diphtheria toxin catalytic domain is delivered to the eukaryotic cell cytosol. While much work remains, it is becoming increasingly clear that the entry process is facilitated by specific interactions with a number of cellular factors in an ordered sequential fashion. In addition, since diphtheria, anthrax lethal factor and anthrax edema factor all carry multiple coatomer I complex binding motifs and COPI complex has been shown to play an essential role in entry process, it is likely that the initial steps in catalytic domain entry of these divergent toxins follow a common mechanism.
Assuntos
Proteína Coatomer/metabolismo , Citosol/efeitos dos fármacos , Toxina Diftérica/farmacocinética , Células Eucarióticas/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Linhagem Celular , Citosol/metabolismo , Toxina Diftérica/química , Toxina Diftérica/genética , Células Eucarióticas/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Transporte ProteicoRESUMO
Diphtheria toxin has been well characterized in terms of its receptor binding and receptor mediated endocytosis. However, the precise mechanism of the cytosolic release of diphtheria toxin fragment A from early endosomes is still unclear. Various reports differ regarding the requirement for cytosolic factors in this process. Here, we present data indicating that the distribution of actin filaments due to cytochalasin D action enhances the retention of diphtheria toxin in early endosomes. Treating cells with cytochalasin D reduces the cytosolic fragment A activity and leads to changes in the intracellular distribution and size of early endosomes with toxin cargo. F-actin and eukaryotic elongation factor 2 can promote fragment A release from toxin-loaded early endosomes in an in vitro translocation system. Moreover, these proteins bind to toxin-loaded early endosomes in vitro and promote each other's binding. They are thus thought to be involved in the cytosolic release of fragment A. Finally, ADP-ribosylation of eukaryotic elongation factor 2 is shown to inhibit fragment A release and, via a feed-back mechanism, to account for the minute amounts of fragment A normally found in the cytosol.
Assuntos
Citoesqueleto de Actina/metabolismo , Citocalasina D/farmacologia , Toxina Diftérica/farmacologia , Fator de Iniciação 2 em Eucariotos/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fragmentos de Peptídeos/farmacologia , Venenos/farmacologia , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Toxina Diftérica/farmacocinética , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fator de Iniciação 2 em Eucariotos/fisiologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Venenos/farmacocinéticaRESUMO
Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor with median survival of only 12 to 15 months under the current standard of care. To both increase tumor specificity and decrease nonspecific side effects, recent experimental strategies in the treatment of GBM have focused on targeting cell surface receptors, including the transferrin (Tf) receptor, that are overexpressed in many cancers. A major limitation of Tf-based therapeutics is the short association of Tf within the cell to deliver its payload. We previously developed two mutant Tf molecules, K206E/R632A Tf and K206E/K534A Tf, in which iron is locked into each of the two homologous lobes. Relative to wild-type Tf, we showed enhanced delivery of diphtheria toxin (DT) from these mutants to a monolayer culture of HeLa cells. Here, we extend the application of our Tf mutants to the treatment of GBM. In vitro treatment of Tf mutants to a monolayer culture of glioma cells showed enhanced cellular association as well as enhanced delivery of conjugated DT. Treatment of GBM xenografts with mutant Tf-conjugated DT resulted in pronounced regression in vivo, indicating their potential use as drug carriers.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Toxina Diftérica/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Glioblastoma/tratamento farmacológico , Imunotoxinas/administração & dosagem , Transferrina/administração & dosagem , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Cricetinae , Toxina Diftérica/farmacocinética , Feminino , Engenharia Genética/métodos , Glioblastoma/metabolismo , Humanos , Imunotoxinas/genética , Imunotoxinas/farmacocinética , Camundongos , Camundongos Nus , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Transferrina/genética , Transferrina/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: To generate and evaluate a modular recombinant transporter (MRT) for targeting 211 At to cancer cells overexpressing the epidermal growth factor receptor (EGFR). METHODS AND MATERIALS: The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[211 At]astato-5-guanidinomethylbenzoate (SAGMB), its 125 I analogue SGMIB, or with 131 I using Iodogen. Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines. RESULTS: The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [211 At]astatide for all three cell lines. CONCLUSION: The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted alpha-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this 211 At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor.
Assuntos
Astato/uso terapêutico , Núcleo Celular/efeitos da radiação , Portadores de Fármacos/uso terapêutico , Receptores ErbB/metabolismo , Partículas alfa/uso terapêutico , Antígenos Transformantes de Poliomavirus/metabolismo , Astato/farmacocinética , Benzoatos/farmacocinética , Benzoatos/uso terapêutico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Intervalos de Confiança , Di-Hidropteridina Redutase/farmacocinética , Toxina Diftérica/farmacocinética , Portadores de Fármacos/farmacocinética , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Proteínas de Escherichia coli/farmacocinética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Guanidina/análogos & derivados , Guanidina/farmacocinética , Guanidina/uso terapêutico , Guanidinas/farmacocinética , Guanidinas/uso terapêutico , Hemeproteínas/farmacocinética , Humanos , NADH NADPH Oxirredutases/farmacocinética , Radioimunoterapia/métodosRESUMO
Denileukin diftitox (Ontak) represents an example of a fused molecule that targets cells bearing high affinity interleukin-2 receptors internalized via receptor-mediated endocytosis in an acidified vesicle. Denileukin diftitox is proteolytically cleaved within the endosome liberating the enzymatically active portion of the diphtheria toxin, the A fragment. Diphtheria toxin fragment A is released into the cytosol inhibiting the protein synthesis through the ADP-ribosylation of the elongation factor-2, and leading to cell death. This review focuses on the clinical trials that led to the FDA approval of the drug for cutaneous T cell lymphoma in the US, and investigational studies demonstrating drug-activity against B and T-cell non-Hodgkin's lymphoma, chronic lymphocytic lymphoma and acute graft versus disease within allogeneic hematopoietic stem cell transplant.
Assuntos
Antineoplásicos/uso terapêutico , Toxina Diftérica/uso terapêutico , Doença de Hodgkin/tratamento farmacológico , Interleucina-2/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Antineoplásicos/farmacocinética , Ensaios Clínicos como Assunto , Toxina Diftérica/farmacocinética , Transplante de Células-Tronco Hematopoéticas , Doença de Hodgkin/terapia , Humanos , Interleucina-2/farmacocinética , Leucemia Linfocítica Crônica de Células B/terapia , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêutico , Segurança , Estados Unidos , United States Food and Drug AdministrationRESUMO
Despite advances in our knowledge about the genesis, molecular biology, and natural history of malignant gliomas and the use of a multi-disciplinary approach to their treatment, patients harboring this diagnosis continue to face a grim prognosis. At the time of diagnosis, patients typically undergo surgery for the establishment of a histologic diagnosis, the reduction of tumor burden, and the relief of mass effect, with the maintenance of the patient's neurological function in mind. This is followed by the administration of adjuvant therapeutics, including radiation therapy and chemotherapy. Many investigational agents with laboratory evidence of efficacy against malignant gliomas have not met their promise in the clinical setting, largely due to the barriers that they must overcome to reach the tumor at a therapeutically meaningful concentration for a durable period of time. The relevant aspects of the blood-brain barrier, blood-tumor barrier, and blood-cerebrospinal fluid barrier, as they pertain to the delivery of agents to the tumor, will be discussed along with the strategies devised to circumvent them. This discussion will be followed by a description of agents currently in preclinical and clinical development, many of which are the result of intense ongoing research into the molecular biology of gliomas.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Convecção , Sistemas de Liberação de Medicamentos , Glioma/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/farmacocinética , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Toxina Diftérica/administração & dosagem , Toxina Diftérica/farmacocinética , Exotoxinas/administração & dosagem , Exotoxinas/farmacocinética , Glioma/metabolismo , Humanos , Interleucina-13/administração & dosagem , Interleucina-13/farmacocinética , Interleucina-4/administração & dosagem , Interleucina-4/farmacocinética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacocinética , Transferrina/administração & dosagem , Transferrina/análogos & derivados , Transferrina/farmacocinética , Fator de Crescimento Transformador alfa/administração & dosagem , Fator de Crescimento Transformador alfa/farmacocinéticaRESUMO
DTAT13, a novel recombinant bispecific immunotoxin (IT) consisting of truncated diphtheria toxin, an amino-terminal (AT) fragment of the urokinase-type plasminogen activator (uPa), and a fragment of human IL-13 was assembled in order to target receptors on glioblastoma multiforme (GBM) and its associated neovasculature. Previous in vitro studies confirmed the efficacy of DTAT13 against various GBM cell lines expressing both IL-13 receptor or uPA receptor, and previous in vivo testing demonstrated the efficacy of DTAT13 in significantly inhibiting a range of xenograft tumors and showed that DTAT13 was 160- and 8-fold less toxic to the parental fusion IT, DTAT and DTIL13, respectively. To further understand the properties of DTAT13, pharmacokinetic/biodistribution experiments were performed. Binding analysis revealed that the IL-13 domain functioned independently of the uPA domain and that the K (d) for each binding domain was essentially the same as that of DTIL13 and DTAT. Flow cytometry studies indicated that DTAT13 bound better than DTAT or DTIL13. Analysis of the rate of protein synthesis inhibition in U87 MG cells by DTAT13 compared to DTAT revealed a faster rate of inhibition with DTAT13 compared to DTAT. The rate of protein synthesis inhibition of DTAT13 was identical to that of DTIL13 in U373 MG cells. Intracranial biodistribution studies revealed that DTAT13 was able to cross to the contralateral hemisphere unlike DTIL13 but similar to DTAT. These studies show that DTAT13 has properties encompassing those of both DTIL13 and DTAT and warrants further consideration for clinical development.
Assuntos
Toxina Diftérica/farmacocinética , Glioblastoma/metabolismo , Imunotoxinas/farmacocinética , Interleucina-13/farmacocinética , Neovascularização Patológica/imunologia , Ativador de Plasminogênio Tipo Uroquinase/farmacocinética , Animais , Encéfalo/metabolismo , Toxina Diftérica/uso terapêutico , Feminino , Glioblastoma/irrigação sanguínea , Glioblastoma/tratamento farmacológico , Glioblastoma/imunologia , Humanos , Imunotoxinas/uso terapêutico , Interleucina-13/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêutico , Ativador de Plasminogênio Tipo Uroquinase/uso terapêuticoRESUMO
KS Biomedix (formerly Avicenna Medica; now a subsidiary of the Xenova group) and Nycomed, together with Japanese licensee Sosei and Chinese licensee PharmaEngine, are developing TransMID, a transferrin-mediated diphtheria toxin delivery system for the potential treatment of adult, recurrent, inoperable, high-grade glioma (as TransMID-107R). It is also under investigation for other forms of brain cancer, including early brain cancer (as TransMID-107N), metastatic brain cancer (as TransMID-107M) and pediatric brain cancer (as TransMID-107P). TransMID is currently undergoing phase III clinical trials.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Toxina Diftérica/uso terapêutico , Glioma/tratamento farmacológico , Adulto , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Criança , Ensaios Clínicos como Assunto , Toxina Diftérica/efeitos adversos , Toxina Diftérica/farmacocinética , HumanosRESUMO
Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to low pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.
Assuntos
Aciltransferases/deficiência , Membrana Celular/metabolismo , Toxina Diftérica/farmacocinética , Lipídeos de Membrana/metabolismo , Transporte Proteico/fisiologia , Esfingolipídeos/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Toxina Diftérica/farmacologia , Relação Dose-Resposta a Droga , Taxa de Depuração Metabólica , Transporte Proteico/efeitos dos fármacos , Serina C-PalmitoiltransferaseRESUMO
The fusion toxin DT388IL3 composed of the catalytic and translocation domains of diphtheria toxin (DT388) linked to interleukin-3 (IL3) was administered to 6 cynomolgus monkeys which possessed cross-reactive IL3 receptors. Groups of 2 animals (1 male and 1 female) received up to 6 every other day slow intravenous infusions of 40, 60, or 100 microg/kg DT388IL3. Monkeys given 40 or 60 microg/kg showed mild or moderate transient malaise and anorexia, respectively, without evidence of organ damage by blood tests or histopathology. Animals treated at 100 microg/kg showed severe malaise and anorexia. The female monkey had moderate to severe vasculitis in multiple tissues. Necropsies were performed on the 40 microg/kg monkeys on day 14 and the 100 microg/kg monkeys on days 6 and 7. DT388IL3 plasma half-life was approximately 30 min with a peak concentration of 0.45 microg/ml or 10,000 pM (IC50 for AML blasts treated in vitro was 6 pM). Immune responses were minimal in 4 animals tested at 12 days and 2 animals tested at 30 days post treatment with anti-DT388IL3 levels < 1 microg/ml. Bone marrow aspirates were obtained on all animals at day 19 or at necropsy and revealed myeloid suppression in the females and myeloid hyperplasia in the males irrespective of dose groups. The maximal tolerated dose of 60 microg/kg for 6 doses is markedly higher than other recombinant diphtheria toxins and provides a dose level sufficient for anti-leukemic activity in vitro and in rodent models. Thus, we propose this agent is a promising drug for AML patients.
Assuntos
Toxina Diftérica/farmacocinética , Toxina Diftérica/toxicidade , Interleucina-3/farmacocinética , Interleucina-3/toxicidade , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/toxicidade , Animais , Anorexia/induzido quimicamente , Células da Medula Óssea/efeitos dos fármacos , Feminino , Meia-Vida , Humanos , Infusões Intravenosas , Macaca fascicularis , Masculino , Dose Máxima Tolerável , Receptores de Interleucina-3/imunologia , Receptores de Interleucina-3/metabolismo , Vasculite/induzido quimicamenteRESUMO
In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.
Assuntos
Fusão de Membrana/fisiologia , Respirovirus/fisiologia , Animais , Antígenos Virais/análise , Células COS , Linhagem Celular , Vírus Defeituosos/genética , Vírus Defeituosos/fisiologia , Toxina Diftérica/análise , Toxina Diftérica/farmacocinética , Técnica Indireta de Fluorescência para Anticorpo , Genoma Viral , Células HeLa , Humanos , Cinética , Lipossomos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/farmacocinética , Respirovirus/genética , Transfecção , Replicação ViralRESUMO
Genetic engineering has led to the development of fusion protein toxins, which are targeted effector molecules that combine a targeting ligand such as a growth factor with a cytocidal moiety such as a plant or bacterial toxin. The first genetically constructed family of fusion proteins used diphtheria toxin as the toxophore for receptor-binding domain substitution. Diphtheria toxin consists of three domains: an enzymatically active adenosine diphosphate (ADP) ribosyltransferase domain, a hydrophobic transmembrane domain, and a C-terminal receptor-binding domain. Introduction of the enzymatically active domain into the cytosol via receptor-mediated endocytosis results in inhibition of protein synthesis by ADP ribosylation of elongation-factor 2. DAB(486)IL-2, in which the native receptor-binding domain of diphtheria toxin was replaced with the full-length interleukin-2 (IL-2) gene, was capable of intoxicating high-affinity IL-2 receptor-bearing cells in vitro with an IC(50) of 10(-10) M; whereas, cells lacking the full component of the high-affinity IL-2 receptor (p55, p75, p64) were relatively resistant (IC(50) of 10(-8) M). Because of a short in vivo half-life of DAB(486)IL-2, efforts to reengineer the molecule were undertaken, leading to the DAB(389)IL-2 construct, which had a twofold to threefold higher Kd than DAB(486)IL-2 and a longer half-life, resulting in a tenfold increase in potency. Thus far, the clinical activity of both IL-2 chimeric fusion toxins has been similar, with the DAB(389)IL-2 molecule displaying more favorable pharmacokinetics.
Assuntos
Toxina Diftérica , Imunossupressores , Imunotoxinas , Interleucina-2 , Proteínas Recombinantes de Fusão , Ensaios Clínicos como Assunto , Toxina Diftérica/química , Toxina Diftérica/farmacocinética , Toxina Diftérica/farmacologia , Humanos , Imunossupressores/química , Imunossupressores/farmacocinética , Imunossupressores/farmacologia , Imunotoxinas/química , Imunotoxinas/farmacocinética , Imunotoxinas/farmacologia , Interleucina-2/química , Interleucina-2/farmacocinética , Interleucina-2/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.
Assuntos
Toxina Diftérica/farmacocinética , Endocitose/fisiologia , Glicosilfosfatidilinositóis/metabolismo , Receptores de Superfície Celular/metabolismo , beta-Ciclodextrinas , Adolescente , Transporte Biológico/efeitos dos fármacos , Ciclodextrinas/farmacologia , Toxina Diftérica/metabolismo , Dinaminas , Endossomos/metabolismo , GTP Fosfo-Hidrolases/genética , Expressão Gênica , Glicosilfosfatidilinositóis/genética , Células HeLa , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Lisossomos/metabolismo , Mutação , Nistatina/farmacologia , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Diphtheria toxin enters the cytosol of mammalian cells where it inhibits cellular protein synthesis, leading to cell death. Recently we found that the addition of a signal for N-end-rule-mediated protein degradation to diphtheria toxin substantially reduced its intracellular stability and toxicity. These results prompted us to construct a toxin containing a degradation signal that is removable through the action of a viral protease. In principle, such a toxin would be preferentially stabilized, and thus activated, in cells expressing the viral protease in the cytosol, i.e. virus-infected cells, thereby providing a specific eradication of these cells. In the present work we describe the construction of toxins that contain a signal for N-end-rule-mediated degradation just upstream of a cleavage site for the protease from HIV type 1 (HIV-1 PR). We show that the toxins are cleaved by HIV-1 PR exclusively at the introduced sites, and thereby are converted from unstable to stable proteins. Furthermore, this cleavage substantially increased the ability of the toxins to inhibit cellular protein synthesis. However, the toxins were unable to selectively eradicate HIV-1-infected cells, apparently due to low cytosolic HIV-1 PR activity, since we could not detect cleavage of the toxins by HIV-1 PR in infected cells. Alternative strategies for the construction of toxins that can specifically be activated by viral proteases are discussed.
Assuntos
Antígenos de Bactérias , Toxinas Bacterianas/farmacocinética , Biotransformação , Toxina Diftérica/farmacocinética , Protease de HIV/metabolismo , Sequência de Aminoácidos , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Sequência de Bases , Primers do DNA , Toxina Diftérica/genética , Toxina Diftérica/farmacologia , Células HeLa , Humanos , Hidrólise , Dados de Sequência Molecular , Mutagênese , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
We developed a fusion toxin consisting of the catalytic and translocation domains of diphtheria toxin linked to human granulocyte-macrophage colony-stimulating factor (GM-CSF) (DTGM) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to determine the toxicity and pharmacokinetics of DTGM in cynomolgus monkeys (Macacca fascicularis), which possess cross-reactive GM-CSF receptors. Four groups of young adult monkeys (6 males and 12 females) were treated with five daily bolus iv infusions of 1, 5, 7.5, and 10 microgram/kg DTGM. Monkeys (2 males and 2 females) treated at 1 microgram/kg/day showed no significant side effects. Monkeys (2 males and 2 females) treated at 5 microgram/kg/day showed Grade 1-2 thrombopenia (NCI common toxicity criteria) on day 9. In contrast, monkeys (6 females) treated at 7.5 microgram/kg/day developed Grade 3 neutropenia, Grade 1-2 thrombopenia, Grade 1-3 anemia, and Grade 1-3 hypoalbuminemia. The neutropenia developed by day 4 in the 7.5 microgram/kg/day monkeys and by day 3 or 5 in the 10 microgram/kg/day monkeys and resolved in both groups by day 9, but the thrombopenia, anemia, and hypoalbuminemia persisted until day 16. Monkeys (2 male and 2 female) treated with 10 microgram/kg/day showed Grade 4 neutropenia that resolved by day 8 and Grade 2-3 anemia, hypoalbuminemia, and thrombopenia. Three of the animals developed sepsis. DTGM plasma half-life was 30 min with a peak concentration of 0.1 microgram/mL or 2 nM (1000-fold higher than the IC50 in vitro for AML blasts). Immune responses were minimal in all animals tested at 14 and 28 days with anti-DTGM levels <1 microgram/mL. All four animals at 10 microgram/kg died or were euthanized, and necropsies were performed. Animals necropsied on days 4 and 6 showed marked apoptosis and hypoplasia in the marrow, which was completely resolved for animals necropsied on day 9. No injury to other organs, including kidney, heart, liver, central nervous system, or lung, was seen. The drug was selectively toxic to malignant or differentiated myeloid cells with little toxicity to myeloid progenitors or other organs. Minimal effects in nontarget tissues make DTGM a promising candidate chemotherapeutic agent.
Assuntos
Contagem de Células Sanguíneas/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Fatores Estimuladores de Colônias/farmacologia , Toxina Diftérica/toxicidade , Imunoglobulina G/sangue , Animais , Fatores Estimuladores de Colônias/imunologia , Toxina Diftérica/imunologia , Toxina Diftérica/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Humanos , Macaca fascicularis , Masculino , Fatores de TempoRESUMO
The effect of vacuolating toxin (VacA) from Helicobacter pylori on endosomal and lysosomal functions was studied by following procathepsin D maturation and epidermal growth factor (EGF) degradation in HeLa cells exposed to the toxin. VacA inhibited the conversion of procathepsin D (53 kDa) into both the intermediate (47 kDa) and the mature (31 kDa) form. Nonprocessed cathepsin D was partly retained inside cells and partly secreted in the extracellular medium via the constitutive secretion pathway. Intracellular degradation of EGF was also inhibited by VacA with a similar dose-response curve. VacA did not alter endocytosis, cell surface recycling, and retrograde transport from plasma membrane to trans-Golgi network and endoplasmic reticulum, as estimated by using transferrin, diphtheria toxin, and ricin as tracers. Subcellular fractionation of intoxicated cells showed that procathepsin D and nondegraded EGF accumulate in lysosomes. Measurements of intracellular acidification with fluorescein isothiocyanate-dextran revealed a partial neutralization of the lumen of endosomes and lysosomes, sufficient to account for both mistargeting of procathepsin D outside the cell and the decreased activity of lysosomal proteases.
Assuntos
Proteínas de Bactérias/toxicidade , Catepsina D/metabolismo , Precursores Enzimáticos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Catepsina D/biossíntese , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Toxina Diftérica/farmacocinética , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HeLa , Helicobacter pylori , Humanos , Cinética , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Ricina/farmacocinética , Transferrina/metabolismoAssuntos
Difteria/complicações , Miocardite/etiologia , Acidose/induzido quimicamente , Acidose/metabolismo , Animais , Difteria/metabolismo , Toxina Diftérica/farmacocinética , Toxina Diftérica/farmacologia , Toxina Diftérica/toxicidade , Coração/efeitos dos fármacos , Humanos , Miocardite/metabolismo , Miocárdio/metabolismoRESUMO
Acute myeloid leukemia (AML) is the most common form of acute leukemia. Contemporary chemotherapy regimens fail to cure most patients with AML. We have genetically engineered a recombinant diphtheria toxin human granulocyte macrophage colony-stimulating factor (GMCSF) chimeric fusion protein (DTctGMCSF) that specifically targets the GMCSF receptor on fresh human AML cells and myeloid leukemia cell lines. At a nontoxic dose level, DTctGMCSF therapy was superior to the standard chemotherapeutic agents 1-beta-D-arabinofuranosylcytosine and Adriamycin, resulting in 60% long-term event-free survival of severe combined immunodeficient mice challenged with an otherwise invariably fatal cell dose of the human HL-60 myeloid leukemia. Notably, systemic exposure levels of DTctGMCSF, which were found to be therapeutic in the severe combined immunodeficient mouse xenograft model of human HL-60 myeloid leukemia, could be achieved in cynomolgus monkeys without any significant nonhematological toxicities. The recombinant DTctGMCSF fusion toxin might be useful in the treatment of AML patients whose leukemias have recurred and developed resistance to contemporary chemotherapy programs.