RESUMEN
The recombinant CD3 immunotoxin, A-dmDT(390)-bisFv(UCHT1), composed of the catalytic and translocation domains of diphtheria toxin fused to two single chain Fv fragments of an anti-CD3epsilon monoclonal antibody was administered to five patients with cutaneous T cell lymphoma (CTCL) by eight 15 min intravenous infusions over four days. Side effects were fever, chills, nausea, hypoalbuminemia, transaminasemia and reactivation of EBV and CMV. Half-life of drug was 40 min. Anti-immunotoxin antibodies developed in all patients after two weeks. Two patients had partial remissions lasting 1 and 6+ months. The agent is undergoing further dose escalation and shows promising results in this disease.
Asunto(s)
Toxina Diftérica/uso terapéutico , Inmunotoxinas/uso terapéutico , Linfoma de Células T/terapia , Neoplasias Cutáneas/terapia , Anciano , Complejo CD3/inmunología , Toxina Diftérica/efectos adversos , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Inmunotoxinas/efectos adversos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
Macroautophagy (hereafter referred to as autophagy) can increase or decrease the amount of cell death in response to various stimuli. To test whether autophagy also controls the characteristics associated with dying cells, we studied tumor cell killing by epidermal growth factor receptor-targeted diphtheria toxin (DT-EGF). DT-EGF kills epithelial and glioblastoma tumor cells with similar efficiency but by different mechanisms that depend on whether the cells activate autophagy when treated with the drug. Dying cells in which autophagy is induced selectively release the immune modulator high-mobility group B1 (HMGB1) without causing lysis of the cell membrane and classical necrosis. Conversely, cells that are killed by DT-EGF where autophagy is blocked, activate caspases but retain HMGB1. These data suggest that it may be feasible to manipulate the immunogenicity of dying cells by increasing or decreasing autophagy.
Asunto(s)
Autofagia/inmunología , Glioblastoma/inmunología , Proteína HMGB1/inmunología , Factores Inmunológicos/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias Glandulares y Epiteliales/inmunología , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Toxina Diftérica/farmacología , Factor de Crecimiento Epidérmico/farmacología , Glioblastoma/metabolismo , Proteína HMGB1/metabolismo , Humanos , Factores Inmunológicos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaAsunto(s)
Antineoplásicos/farmacología , Toxina Diftérica/farmacología , Interleucina-3/farmacología , Leucemia Mieloide/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Enfermedad Aguda , Clorometilcetonas de Aminoácidos/farmacología , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Benzamidas/antagonistas & inhibidores , Caspasas/fisiología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Toxina Diftérica/toxicidad , Sistemas de Liberación de Medicamentos , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Humanos , Interleucina-3/toxicidad , Leucemia Mieloide/patología , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/fisiología , Piridinas/antagonistas & inhibidores , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/efectos de los fármacos , Receptores de Interleucina-3/efectos de los fármacos , Proteínas Recombinantes de Fusión/toxicidad , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Células U937/efectos de los fármacosRESUMEN
Targeted toxins are fusion proteins that combine a targeting molecule that selectively binds to and enters tumor cells with a protein toxin that kills the target cells. These molecules represent an exciting approach to develop effective cancer-specific therapeutics that have few side effects on normal tissues and numerous such toxins are in various stages of pre-clinical and clinical development to treat a wide variety of tumors. In this review, we discuss this strategy, describe ways that the toxins activate the apoptosis machinery and discuss future developments in this field.
Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Toxinas Biológicas/uso terapéutico , Toxina Diftérica/farmacología , Células HL-60 , Humanos , Modelos Biológicos , Transducción de SeñalRESUMEN
The novel fusion protein DT(388)IL3, composed of the catalytic and translocation domains of diphtheria toxin (DT(388)) fused with a Met-His linker to human interleukin 3 (IL-3), was tested for anti-leukemia efficacy in an in vivo model of differentiated human acute myeloid leukemia (AML). Six-week-old female SCID mice were irradiated with 350 cGy, inoculated 24 h later with 20 million (i.v., i.p., or s.c.) TF1 cells transfected with the v-SRC oncogene, and treated i.p., starting 24 h later, with up to five daily injections of saline, DT(388)IL3 (2 microg), DT(388)GMCSF (2 microg), DAB(389)IL2 (2 microg), or cytarabine (80 microg) or two weekly injections of anti-CD33-calicheamicin conjugate (5 microg). Animals were monitored twice daily, and moribund animals killed and necropsied. Control animals had a median disease-free survival (DFS) of 37 days (i.v., n = 45), 35 days (i.p., n = 20), and 21 days (s.c., n = 20), respectively. Only 5/49 (10%) of the DT(388)IL3 treated i.v. inoculated animals died with leukemia. Median DFS with i.v., i.p. and s.c. tumor inoculated animals was prolonged by fusion protein treatment to >120 days, 66 days and 31 days (P < 0.001, = 0.0003, and = 0.0006), respectively. Median DFS with s.c. tumor inoculated animals was also prolonged by other active anti-leukemia agents (DT(388)GMCSF, cytarabine and anti-CD33-calicheamicin) relative to controls by 67%, 172% and 47% (P < 0.0001, <0.0001, and =0.0004), respectively. In contrast, median DFS with s.c. tumor inoculated animals treated with DAB(389)IL2 non-significantly reduced by 13% relative to controls (P = 0.21). Thus, DT(388)IL3 fusion protein demonstrates in vivo anti-leukemia efficacy and warrants further preclinical development for treatment of chemo-resistant, IL-3 receptor positive AML patients.
Asunto(s)
Antineoplásicos/uso terapéutico , Toxina Diftérica/uso terapéutico , Interleucina-3/uso terapéutico , Leucemia Mieloide/tratamiento farmacológico , Proteínas Recombinantes de Fusión/uso terapéutico , Enfermedad Aguda , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Citarabina/administración & dosificación , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Humanos , Huésped Inmunocomprometido , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Inyecciones Subcutáneas , Interleucina-2/administración & dosificación , Leucemia Mieloide/mortalidad , Leucemia Mieloide/patología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacosRESUMEN
The objective of this work was to determine the safety and efficacy of gemtuzumab ozogamicin in patients with poor prognosis acute myeloid leukemia (AML). Patients with the following diagnoses/characteristics were treated with 1-3 infusions of gemtuzumab ozogamicin at a dose of 9 mg/m2: (1) relapse of AML < or = 6 months of first complete remission (CR); (2) AML refractory to chemotherapy at initial induction or at first relapse; (3) AML in second or greater relapse; (4) myeloid blast crisis of chronic myeloid leukemia (CML); (5) untreated patients > or = 70 years or > or = 55 years with abnormal cytogenetics (excluding inv 16, t(15;17) and t(8;21)) and/or an antecedent hematologic disorder; (6) refractory anemia with excess blasts in transformation (RAEBT). Forty-three patients, ages 19-84 (mean 62), were treated, including 7 patients with untreated AML age > 70 years, 2 with untreated RAEBT, 14 with AML first salvage (first remission 0-6 months), 15 with AML > or = second salvage and 14 with myeloid blast phase of CML. The overall response rate was 14%, with 4/43 (9%) patients achieving CR and 2/43 (5%) achieving CR without platelet recovery. The most significant toxicity was neutropenic fever, which occurred in 84% of patients. In conclusion, in patients with relapsed/refractory AML, gemtuzumab ozogamicin has a comparable response rate to single-agent chemotherapy and may offer a more favorable toxicity profile.
Asunto(s)
Aminoglicósidos , Antibacterianos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Leucemia Mieloide/tratamiento farmacológico , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/toxicidad , Anticuerpos Monoclonales/toxicidad , Anticuerpos Monoclonales Humanizados , Femenino , Gemtuzumab , Humanos , Leucemia Mieloide/complicaciones , Leucemia Mieloide/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Inducción de Remisión , Medición de Riesgo , Terapia Recuperativa , Resultado del TratamientoRESUMEN
Most cancer patients receive chemotherapy drugs that target DNA or the cell division apparatus. Many of these patients develop multidrug-resistant tumor cells, thus, novel methods to overcome drug resistance are needed. One approach is to target tumor cell protein synthesis. Peptide toxins, which catalytically inactivate protein synthesis, have been re-engineered to selectively bind and intoxicate tumor cells. Diphtheria toxin (DT), a member of the class of peptide toxins, has been subjected to structural and genetic analysis and protein engineering for several decades. In this review, we will examine the structure, function, synthesis and pharmacology of anticancer DT conjugates.
Asunto(s)
Antineoplásicos/uso terapéutico , Toxina Diftérica/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Antineoplásicos/farmacología , Toxina Diftérica/farmacología , Humanos , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Diphtheria fusion proteins are a novel class of agents for the treatment of chemotherapy resistant AML. We prepared DT(388)IL3 composed of human interleukin-3 (IL3) fused to the catalytic and translocation domain of diphtheria toxin (DT(388)) and assessed its activity on patient AML blasts. The number and affinity of IL3 receptors in circulating blasts was measured using a radiolabeled IL3 agonist (SC-65461). Ninety-two percent of patients' blasts had both high and low affinity IL3 receptors. DT(388)IL3 cytotoxicity (>1 log cell kill) was seen in nine of 25 samples (36%). There was a significant correlation between DT(388)IL3 log cell kill and blast high affinity IL3 receptor density (P=0.0044). These results show that specific high affinity IL3 binding is one factor important in the sensitivity of patients' leukemic blasts to DT(388)IL3.
Asunto(s)
Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Toxina Diftérica/farmacología , Interleucina-3/farmacología , Leucemia Mieloide Aguda/metabolismo , Receptores de Interleucina-3/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Células Madre/efectos de los fármacos , Enfermedad Aguda , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Leucemia Mieloide Aguda/patología , Unión Proteica , Células Tumorales CultivadasRESUMEN
DT(388)-GM-CSF, a targeted fusion toxin constructed by conjugation of human granulocyte-macrophage colony-stimulating factor (GM-CSF) with the catalytic and translocation domains of diphtheria toxin, is presently in phase I trials for patients with resistant acute myeloid leukemia. HL-60/VCR, a multidrug-resistant human myeloid leukemia cell line, and wild-type HL-60 cells were used to study the impact of DT(388)-GM-CSF on metabolism of ceramide, a modulator of apoptosis. After 48 hours with DT(388)-GM-CSF (10 nM), ceramide levels in HL-60/VCR cells rose 6-fold and viability fell to 10%, whereas GM-CSF alone was without influence. Similar results were obtained in HL-60 cells. Examination of the time course revealed that protein synthesis decreased by about 50% and cellular ceramide levels increased by about 80% between 4 and 6 hours after addition of DT(388)-GM-CSF. By 6 hours this was accompanied by activation of caspase-9, followed by activation of caspase-3, cleavage of caspase substrates, and chromatin fragmentation. Hygromycin B and emetine failed to elevate ceramide levels or induce apoptosis at concentrations that inhibited protein synthesis by 50%. Exposure to C(6)-ceramide inhibited protein synthesis (EC(50) approximately 5 microM) and decreased viability (EC(50) approximately 6 microM). Sphingomyelinase treatment depleted sphingomyelin by about 10%, while increasing ceramide levels and inhibiting protein synthesis. Diphtheria toxin increased ceramide and decreased sphingomyelin in U-937 cells, a cell line extremely sensitive to diphtheria toxin; exposure to DT(388)-GM-CSF showed sensitivity at less than 1.0 pM. Diphtheria toxin and conjugate trigger ceramide formation that contributes to apoptosis in human leukemia cells through caspase activation and inhibition of protein synthesis.
Asunto(s)
Apoptosis , Ceramidas/biosíntesis , Toxina Diftérica/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Inhibidores de la Síntesis de la Proteína/farmacología , Enfermedad Aguda , Caspasa 3 , Caspasa 9 , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Células HL-60 , Humanos , Cinética , Leucemia Mieloide/tratamiento farmacológico , Esfingomielina Fosfodiesterasa/farmacología , Esfingomielinas/metabolismo , Células U937RESUMEN
Most patients with acute myeloid leukemia (AML) respond initially to combination chemotherapy but later relapse. These patients often die from progressive disease or toxicities of further chemotherapy. At relapse, the patients' blasts are usually resistant to the drugs to which the patient has been exposed and frequently to other cytotoxic agents as well. Nevertheless, a number of these patients may be salvaged and achieve remissions with allogeneic stem cell transplants. In such cases, the pre-transplant conditioning regimens do not appear to account for the entire anti-leukemic efficacy. Immunological mechanisms for blast killing appear critical. There is tissue culture, animal and clinical evidence that stimulated donor T cells can recognize and kill leukemic blasts through recognition of alloantigens, differentiation antigens or leukemia-specific antigens as targets. We will review the molecular mechanisms for the generation of anti-leukemic T cells and discuss methods to improve the specificity and intensity of anti-leukemic T cell responses in the setting of allogeneic stem cell transplants, donor lymphocyte infusions, autologous anti-leukemic T cell infusions, and vaccine use in AML patients.
Asunto(s)
Inmunoterapia , Leucemia Mieloide/terapia , Linfocitos T/inmunología , Enfermedad Aguda , Animales , Ensayos Clínicos como Asunto , Trasplante de Células Madre Hematopoyéticas , Humanos , Leucemia Mieloide/inmunologíaRESUMEN
Patients with chemotherapy-resistant acute myeloid leukaemia are rarely cured by non-allogeneic transplant therapies. Multiple new investigational agents have become available for treatment of these patients and there are few tools to permit rational drug and clinical trial selection. In this review, we describe the chemical and biological properties of some of these agents and some of their initial clinical activity to date. The selected agents react with either cell surface molecules or signal pathway intermediates and include antibody and antibody conjugates to CD33 and CD45, a fusion protein directed to the granulocyte-macrophage colony-stimulating factor receptor, an anti-sense oligonucleotide to Bcl2, a farnesyl transferase inhibitor, and a protein kinase C agonist/inhibitor. The challenge for the next decade will be how to select patients for particular molecularly targeted therapeutics and how to combine these agents.
Asunto(s)
Antineoplásicos/uso terapéutico , Inmunoterapia , Leucemia Mieloide Aguda/terapia , Transducción de Señal/efectos de los fármacos , Animales , Antígenos de Superficie/inmunología , Antineoplásicos/inmunología , Ensayos Clínicos como Asunto , Resistencia a Antineoplásicos , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/fisiopatologíaRESUMEN
We are conducting a Phase I trial of a fusion toxin (DT-GM) for the treatment of relapsed or refractory acute myeloid leukemia (AML). The fusion toxin consists of a truncated diphtheria toxin (DT) linked to human granulocyte-macrophage colony stimulating factor (GM). Prior to beginning the Phase I trial, our first goal was to determine whether healthy controls and adult AML patients had preexisting antibodies able to inhibit DT-GM. Sera from 5 of the 9 controls completely neutralized DT-GM by an in vitro bioassay to assess the inhibition of DT-GM. Sera from 43 patients with AML were tested by bioassay and a specific enzymoimmunoassay (EIA) for anti-DT-GM antibodies. Forty-two of 43 samples were positive by EIA, and 5 patients (11.6%) showed complete neutralization of DT-GM in the bioassay. Anti-DT-GM concentrations were significantly higher in samples demonstrating neutralization than in samples demonstrating no neutralization (P = 0.003). In the Phase I trial of DT-GM prior to therapy, none of 28 patients exhibited neutralization by bioassay, but 89% were positive by EIA. After the first course of DT-GM, 23% developed neutralizing antibodies by the bioassay, and 64% of patients exhibited an increase in their anti-DT-GM antibody concentrations by EIA. Further studies are needed to determine the clinical impact of the anti-DT-GM antibodies and whether the neutralization bioassay can be replaced by our EIA.
Asunto(s)
Toxina Diftérica/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Leucemia Mieloide/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos/sangre , Anticuerpos/inmunología , Especificidad de Anticuerpos , Toxina Diftérica/genética , Toxina Diftérica/inmunología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Leucemia Mieloide/sangre , Leucemia Mieloide/inmunología , Masculino , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
We have developed an in vivo model of differentiated human acute myeloid leukemia (AML) by retroviral infection of the cytokine-dependent AML cell line TF-1 with the v-Src oncogene. When injected either intravenously or intraperitoneally into 300 cGy irradiated SCID mice, animals formed multiple granulocytic sarcomas involving the adrenals, kidneys, lymph nodes and other organs. The mean survival time was 34+/-10 days (n = 40) after intravenous injection and 24+/-3 days (n = 5) after intraperitoneal injection of 20 million cells. The cells recovered from leukemic animals continued to express interleukin-3 receptors and remained sensitive to the diphtheria fusion protein DT388IL3. Further, these granulocytic sarcoma-derived cells grew again in irradiated SCID mice (n = 10). The cytogenetic abnormalities observed prior to inoculation in mice were stably present after in vivo passage. Similar to the results with v-Src transfected TF-1 cells, in vivo leukemic growth was observed with TF-1 cells transfected with the human granulocyte-macrophage colony-stimulating factor gene (n = 5) and with TF-1 cells recovered from subcutaneous tumors in nude mice (n = 5). In contrast, TF-1 cells expressing v-Ha-Ras (n = 5), BCR-ABL (n = 5), or activated Raf-1 (n = 44) did not grow in irradiated SCID mice. This is a unique, reproducible model for in vivo growth of a differentiated human acute myeloid leukemia and may be useful in the assessment of anti-leukemic therapeutics which have human-specific molecular targets such as the interleukin-3 receptor.
Asunto(s)
Genes src/fisiología , Leucemia Mieloide Aguda/patología , Animales , Aberraciones Cromosómicas , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Receptores de Interleucina-3/análisis , Células Tumorales CultivadasRESUMEN
The alpha subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor has several isoforms that result from alternative splicing events. Two forms, alpha-1 and alpha-2, have intracytoplasmic sequences that are identical within a membrane-proximal domain but differ completely distally. Variant and mutated GM-CSF receptor alpha subunits, along with the beta subunit (beta(c) protein) were expressed in M1 murine leukemia cells. and the ability of the receptors to signal for differentiation events and to activate Jak/Stat signaling pathways was examined. All cell lines expressing both alpha and beta(c) proteins exhibited high-affinity binding of radiolabeled human GM-CSF. Receptor alpha subunits with intact membrane-proximal intracellular domains could induce expression of the macrophage antigen F4/80 and down-regulate the expression of CD11b. Addition of recombinant human GM-CSF to cells expressing alpha-1 subunits induced the expression of CD86 and tyrosine phosphorylation of Jak-2 and its putative substrates SHPTP-2, Stat-5, and the GM-CSF receptor beta(c) subunit. Cells containing alpha subunits that lacked a distal domain (term-3) or had the alternatively spliced alpha-2 distal domain showed markedly decreased ability to support tyrosine phosphorylation of Jak-2 and its substrates or to up-regulate CD86. Ligand binding induced stable association of the alpha-1 subunit and beta(c) protein. In contrast, the alpha-2 subunit did not stably associate with the beta(c) subunit. These data identify potential molecular mechanisms for differential signaling of the alpha-1 and alpha-2 proteins. The association of unique signaling events with the 2 active GM-CSF alpha subunit isoforms offers a model for variable response phenotypes to the same ligand.
Asunto(s)
Antígenos de Diferenciación/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteínas de la Leche , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Transactivadores/metabolismo , Animales , Antígenos CD/efectos de los fármacos , Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , Antígeno B7-2 , Proteínas de Unión al ADN/fisiología , Humanos , Janus Quinasa 2 , Antígeno de Macrófago-1/efectos de los fármacos , Antígeno de Macrófago-1/metabolismo , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Ratones , Mutación , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína , Subunidades de Proteína , Proteínas Tirosina Quinasas/fisiología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor de Transcripción STAT5 , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Transactivadores/fisiología , Transducción Genética , Células Tumorales CultivadasRESUMEN
Diphtheria fusion proteins are chimeric proteins consisting of the catalytic and translocation domains of diphtheria toxin (DT(388)) linked through an amide bond to one of a variety of peptide ligands. The ligand targets the molecule to cells and the toxin enters the cell, inactivates protein synthesis and induces cell death. Diphtheria fusion proteins directed to human myeloid leukemic blasts are a novel class of therapeutics for patients with chemotherapy refractory myeloid leukemia. Because of the presence of interleukin-3 (IL3) receptors on myeloid leukemic progenitors and its absence from mature myeloid cells, we synthesized four bacterial expression vectors encoding DT(388) fused to human IL3. Different molecules were engineered to assess the effects of modifications on yield, purity and potency of product. The constructs differed in the size of the linker peptide between the DT(388) and IL3 domains and in the presence or absence of an oligohistidine tag on the N- or C-terminus. Escherichia coli were transformed and recombinant protein induced and purified from inclusion bodies. Similar final yields of 3-6 mg of purified protein per liter of bacterial culture were obtained with each of the four molecules. Purity ranged from 70 to 90% after partial purification by anion-exchange, size-exclusion chromatography and/or nickel affinity chromatography. Proteins were soluble and stable at 4 degrees C and -80 degrees C in phosphate-buffered saline at 0.03-0.5 mg/ml. The fusion proteins showed predicted molecular weights by SDS-PAGE, HPLC and tandem mass spectrometry and had full ADP-ribosylating activities. Each was immunoreactive with antibodies to DT(388) and IL3. Each of the fusion proteins with the exception of the one with an N-terminal oligohistidine tag showed full IL3 receptor binding affinity (K:(d) = 3 nM) and potent and selective cytotoxicity to IL3 receptor positive human myeloid leukemia cell lines (IC(50) = 5-10 pM). In contrast, the N-terminal histidine-tagged fusion protein bound IL3 receptor with a 10-fold lower affinity and was 10-fold less cytotoxic to IL3 receptor positive blasts. Thus, we report a series of novel, biologically active DT(388)IL3 fusion proteins for potential therapy of patients with receptor positive myeloid leukemias.
Asunto(s)
Toxina Diftérica/química , Receptores de Interleucina-3/química , Proteínas Recombinantes de Fusión/química , Secuencia de Bases , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Cartilla de ADN , Electroforesis en Gel Bidimensional , Humanos , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Patients with acute myeloid leukemia frequently develop chemotherapy resistant blasts. To overcome multidrug resistance, a diphtheria toxin fusion protein (DTIL3) was engineered by fusing the catalytic and translocation domains of diphtheria toxin (DT) to human interleukin-3 (IL-3). However, when blasts were isolated from patients and tested for colony growth inhibition by DTIL3, only a third of the samples showed sensitivity to the fusion protein. Prior to clinical development, we need to be able to identify which patients are likely to respond to therapy with DTIL3. In this report, we compared the inhibition of thymidine incorporation in human leukemia cell lines by DTIL3 to the IL-3 receptor number and affinity. We found DTIL3 was cytotoxic to four of the eight cell lines tested with half-maximal inhibition of thymidine incorporation (IC(50)) from 1 to 50 pM. The IL-3 receptor density for these cell lines ranged from 0 to 2635 receptors per cell. The dissociation constant for an IL-3 high-affinity receptor agonist was 0.5 nM for cell lines with receptors. We found a correlation for the cell lines between the presence of high-affinity IL-3 receptors and sensitivity to DTIL3 (p = 0.03). These results suggest the variability in sensitivity of patient leukemic progenitors to DTIL3 may be due in part to the presence or absence of high-affinity IL-3 receptors.
Asunto(s)
Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Toxina Diftérica/farmacología , Interleucina-3/metabolismo , Interleucina-3/farmacología , Leucemia/metabolismo , Humanos , Leucemia/patología , Unión Proteica , Receptores de Interleucina-3/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Células Tumorales CultivadasRESUMEN
Leukemic blasts from patients with acute phase chronic myeloid leukemic and refractory acute myeloid leukemia are highly resistant to a number of cytotoxic drugs. To overcome multi-drug resistance, we engineered a diphtheria fusion protein by fusing human interleukin-3 (IL3) to a truncated form of diphtheria toxin (DT) with a (G4S)2 linker (L), expressed and purified the recombinant protein, and tested the cytotoxicity of the DTLIL3 molecule on human leukemias and normal progenitors. The DTLIL3 construct was more cytotoxic to interleukin-3 receptor (IL3R) bearing human myeloid leukemia cell lines than receptor-negative cell lines based on assays of cytotoxicity using thymidine incorporation, growth in semi-solid medium and induction of apoptosis. Exposure of mononuclear cells to 680 pM DTLIL3 for 48 h in culture reduced the number of cells capable of forming colonies in semi-solid medium (colony-forming units leukemia) > or =10-fold in 4/11 (36%) patients with myeloid acute phase chronic myeloid leukemia (CML) and 3/9 (33%) patients with acute myeloid leukemia (AML). Normal myeloid progenitors (colony-forming unit granulocyte-macrophage) from five different donors treated and assayed under identical conditions showed intermediate sensitivity with three- to five-fold reductions in colonies. The sensitivity to DTLIL3 of leukemic progenitors from a number of acute phase CML patients suggests that this agent could have therapeutic potential for some patients with this disease.
Asunto(s)
Toxina Diftérica/farmacología , Interleucina-3/farmacología , Leucemia Mieloide/patología , Células Madre Neoplásicas/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Toxina Diftérica/genética , Toxina Diftérica/aislamiento & purificación , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Interleucina-3/genética , Interleucina-3/aislamiento & purificación , Proteínas de Neoplasias/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Receptores de Interleucina-3/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Ensayo de Tumor de Célula MadreRESUMEN
Targeted toxins, consisting of tumor-selective ligands coupled to polypeptide toxins, represent a new class of cancer therapeutics that kills malignant cells by inactivating cytosolic protein synthesis and inducing apoptosis. A number of these molecules have been produced under good manufacturing practice conditions and given systemically to patients with a variety of neoplasms. The promising results to date and the remaining pharmacological hurdles are discussed.
Asunto(s)
Antineoplásicos/uso terapéutico , Citotoxinas/uso terapéutico , Inmunotoxinas/uso terapéutico , Neoplasias/tratamiento farmacológico , Antineoplásicos/efectos adversos , Citotoxinas/efectos adversos , Diseño de Fármacos , Humanos , Inmunotoxinas/efectos adversosRESUMEN
Combination chemotherapy produces remissions in patients with acute myeloid leukemia (AML). However, the majority of patients ultimately relapse and die with cytotoxic drug resistant blasts. Novel agents which circumvent resistance are needed. One such class are AML-cell surface targeted proteins. These genetically engineered polypeptides are hybrid molecules composed of two moieties--a haptophore which triggers AML cell binding and a toxophore which kills the cell. The haptophore or ligand portion consists of a monoclonal antibody or antibody fragment or a cytokine. These peptides react with cell surface receptors or antigens on AML cells. The haptophore is genetically or chemically linked to the toxophore. The toxophore may consist of an antibody Fc domain which triggers antibody-dependent cell cytotoxicity, a DNA-damaging cytotoxic drug, a radionuclide or a protein synthesis-inactivating peptide toxin. The toxophore may provide a cell death signal that overcomes standard resistance phenotypes. Further, the targeting provided by the haptophore may reduce normal tissue toxicities. This review describes some of the properties of the cell surface molecular targets, the reactive haptophores and toxophores and how these functional peptides have been optimally combined to kill leukemic blasts in patients with AML.
Asunto(s)
Aminoglicósidos , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Antígenos de Superficie/inmunología , Antineoplásicos/uso terapéutico , Inmunotoxinas/uso terapéutico , Leucemia Mieloide/terapia , Enfermedad Aguda , Animales , Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Superficie/metabolismo , Muerte Celular/inmunología , Ensayos Clínicos como Asunto , Gemtuzumab , Humanos , Hibridomas/inmunología , Hibridomas/metabolismo , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Radioisótopos de Yodo/uso terapéutico , Leucemia Mieloide/inmunología , Leucemia Mieloide/patología , Antígenos Comunes de Leucocito/inmunología , Ligandos , Ratones , Células Madre Neoplásicas/patología , Radioinmunoterapia/métodos , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
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.