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1.
Mol Ther Methods Clin Dev ; 19: 330-340, 2020 Dec 11.
Article in English | MEDLINE | ID: mdl-33145369

ABSTRACT

The recombinant adeno-associated virus (AAV) vector is one of the most utilized viral vectors in gene therapy due to its robust, long-term in vivo transgene expression and low toxicity. One major hurdle for clinical AAV applications is large-scale manufacturing. In this regard, the baculovirus-based AAV production system is highly attractive due to its scalability and predictable biosafety. Here, we describe a simple method to improve the baculovirus-based AAV production using the ExpiSf Baculovirus Expression System with a chemically defined medium for suspension culture of high-density ExpiSf9 cells. Baculovirus-infected ExpiSf9 cells produced up to 5 × 1011 genome copies of highly purified AAV vectors per 1 mL of suspension culture, which is up to a 19-fold higher yield than the titers we obtained from the conventional Sf9 cell-based system. When mice were administered the same dose of AAV vectors, we saw comparable transduction efficiency and biodistributions between the vectors made in ExpiSf9 and Sf9 cells. Thus, the ExpiSf Baculovirus Expression System would support facile and scalable AAV manufacturing amenable for preclinical and clinical applications.

2.
Stem Cells Transl Med ; 2(1): 43-52, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23283488

ABSTRACT

Although cancer is largely seen as a disease stemming from genetic mutations, evidence has implicated epigenetic regulation of gene expression as a driving force for tumorigenesis. Epigenetic regulation by histone modification, specifically through polycomb group (PcG) proteins such as EZH2 and BMI-1, is a major driver in stem cell biology and is found to be correlated with poor prognosis in many tumor types. This suggests a role for PcG proteins in cancer stem cells (CSCs). We hypothesized that epigenetic modification by EZH2, specifically, helps maintain the CSC phenotype and that in turn this epigenetic modifier can be used as a reporter for CSC activity in an in vitro high-throughput screening assay. CSCs isolated from pancreatic and breast cancer lines had elevated EZH2 levels over non-CSCs. Moreover, EZH2 knockdown by RNA interference significantly reduced the frequency of CSCs in all models tested, confirming the role of EZH2 in maintenance of the CSC population. Interestingly, genes affected by EZH2 loss, and therefore CSC loss, were inversely correlated with genes identified by CSC enrichment, further supporting the function of EZH2 CSC regulation. We translated these results into a novel assay whereby elevated EZH2 staining was used as a reporter for CSCs. Data confirmed that this assay could effectively measure changes, both inhibition and enrichment, in the CSC population, providing a novel approach to look at CSC activity. This assay provides a unique, rapid way to facilitate CSC screening across several tumor types to aid in further CSC-related research.


Subject(s)
Biomarkers, Tumor/metabolism , Neoplastic Stem Cells/metabolism , Polycomb Repressive Complex 2/metabolism , Biomarkers, Tumor/genetics , Breast Neoplasms , Cell Line, Tumor , Chromatin Immunoprecipitation , Enhancer of Zeste Homolog 2 Protein , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Histones/metabolism , Humans , Neoplastic Stem Cells/physiology , Oligonucleotide Array Sequence Analysis , Pancreatic Neoplasms , Polycomb Repressive Complex 2/genetics , RNA Interference , Transcriptome
3.
Drug Metab Dispos ; 41(4): 774-84, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23318685

ABSTRACT

Peginesatide, a polyethylene glycol (PEG)ylated peptide-based erythropoiesis-stimulating agent, stimulates the erythropoietin receptor dimer that governs erythropoiesis. Studies were designed to determine the erythropoietic response, pharmacokinetics (PK), tissue distribution, metabolism, and excretion of peginesatide in nonhuman primates following a single i.v. dose. The PK profile of peginesatide (0.1-5 mg/kg) is characterized by low, dose-dependent plasma clearance; small volume of distribution; and long half-life. The peginesatide PK profile following a single i.v. dose is consistent with the sustained erythropoiesis. Biodistribution quantitative whole-body autoradiography demonstrated high peginesatide levels in bone marrow (i.e., primary hematopoietic site) as well as other known hematopoietic sites persisting through at least 3 weeks at 2.1 mg/kg. Microautoradiography analysis at 48 hours postdose revealed uniform and high distribution of radioactivity in the bone marrow and splenic red pulp with less extensive distribution in the renal cortex (glomeruli, associated ducts, interstitial cells). Radioactivity in the kidney was most prominent in the outer medullary and papillary interstitium. At 2 weeks after dosing, cumulative radioactivity recovery in the urine and feces was 60 and 7% of the administered dose, respectively, with most of the radioactivity associated with the parent molecule. In conclusion, the PK characteristics are consistent with a PEGylated peptide of a 45-kDa molecular mass, specifically low volume of distribution and long half-life. Drug was localized principally to hematopoietic sites, and nonspecific tissue retention was not observed. The nonhuman primate data indicate that peginesatide is metabolically stable and primarily excreted in the urine.


Subject(s)
Hematinics/administration & dosage , Hematinics/pharmacokinetics , Peptides/administration & dosage , Peptides/pharmacokinetics , Administration, Intravenous , Animals , Biological Availability , Bone Marrow/diagnostic imaging , Bone Marrow/metabolism , Dose-Response Relationship, Drug , Erythropoiesis/drug effects , Hematinics/metabolism , Hematinics/pharmacology , Kidney/diagnostic imaging , Kidney/metabolism , Macaca fascicularis , Male , Peptides/metabolism , Peptides/pharmacology , Radionuclide Imaging , Spleen/diagnostic imaging , Spleen/metabolism
4.
Xenobiotica ; 42(7): 660-70, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22188389

ABSTRACT

The pharmacokinetics (PK) (absorption, distribution, metabolism, excretion) of peginesatide, a synthetic, PEGylated, investigational, peptide-based erythropoiesis-stimulating agent (ESA), was evaluated in rats. The PK profile was evaluated at 0.1-5 mg·kg(-1) IV using unlabeled or [(14)C]-labeled peginesatide. Mass balance, tissue distribution and metabolism were evaluated following IV administration of 5 mg·kg(-1) [(14)C]-peginesatide, with tissue distribution also evaluated by quantitative whole-body autoradiography (QWBA) following an IV dose of 17 mg·kg(-1) [(14)C]-peginesatide. Plasma clearance was slow and elimination was biphasic with unchanged peginesatide representing >90% of the total radioactivity of the total radioactive exposure. Slow uptake of the radiolabeled compound from the vascular compartment into the tissues was observed. Biodistribution to bone marrow and extramedullary hematopoietic sites, and to highly vascularized lymphatic and excretory tissues occurred. A predominant degradation event to occur in vivo was the loss of one PEG chain from the branched PEG moiety to generate mono-PEG. Renal excretion was the primary mechanism (41%) of elimination, with parent molecule (67%) the major moiety excreted. In conclusion, elimination of [(14)C]-peginesatide-derived radioactivity was extended, retention preferentially occurred at sites of erythropoiesis (bone marrow), and urinary excretion was the primary elimination route.


Subject(s)
Hematinics/pharmacokinetics , Peptides/pharmacokinetics , Absorption/physiology , Animals , Autoradiography , Hematinics/urine , Male , Metabolic Clearance Rate , Peptides/urine , Rats , Rats, Sprague-Dawley , Tissue Distribution
5.
Article in English | MEDLINE | ID: mdl-20516612

ABSTRACT

The recombinant N-terminal domain of human ephrin type-A receptor 2 (rEphA2) has been crystallized in complex with the recombinantly produced Fab fragment of a fully human antibody (1C1; IgG1/kappa). These are the first reported crystals of an ephrin receptor bound to an antibody. The orthorhombic crystals belonged to space group C222(1) (the 00l reflections obey the l = 2n rule), with unit-cell parameters a = 78.93, b = 120.79, c = 286.20 A. The diffraction of the crystals extended to 2.0 A resolution. However, only data to 2.55 A resolution were considered to be useful owing to spot overlap caused by the long unit-cell parameter. The asymmetric unit is most likely to contain two 1C1 Fab-rEphA2 complexes. This corresponds to a crystal volume per protein weight (V(M)) of 2.4 A(3) Da(-1) and a solvent content of 49.5%. The three-dimensional structure of this complex will shed light on the molecular basis of 1C1 specificity. This will also contribute to a better understanding of the mechanism of action of this antibody, the current evaluation of which as an antibody-drug conjugate in cancer therapy makes it a particularly interesting case study.


Subject(s)
Antigen-Antibody Complex/chemistry , Immunoglobulin Fab Fragments/chemistry , Peptide Fragments/chemistry , Receptor, EphA2/chemistry , Antigen-Antibody Complex/immunology , Crystallization , Crystallography, X-Ray , Humans , Immunoglobulin Fab Fragments/immunology , Peptide Fragments/immunology , Receptor, EphA2/immunology
6.
Birth Defects Res B Dev Reprod Toxicol ; 89(2): 155-63, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20437475

ABSTRACT

BACKGROUND: Aperi- and postnatal reproduction toxicity study was conducted in rats treated with Hematide, a synthetic PEGylated peptidic erythropoiesis stimulating agent (ESA). METHODS: Hematide, at IV doses of 0, 0.5, 3, and 15 mg/kg, was administered from implantation through lactation on gestation days (GDs) 5 and 18 and lactation day (LD) 13. RESULTS: Hematide induced pronounced polycythemia in all Hematide-treated dams. On LDs 2 and 21, hemoglobin (Hgb) increases above control levels were 3.1, 5.2, and 5.0 g/dL and 4.1, 5.1, and 5.5 g/dL at the 0.5, 3, and 15 mg/kg/dose, respectively. There were no effects on parturition, lactation, or maternal behavior in the F0 generation female rats. A slight decrease in pup viability on postpartum days 2-4 and lower body weights and/or body weight gain for the F1 generation were associated with pronounced polycythemia and decreases in maternal body weight gain and/or food consumption at > or =3 mg/kg/dose. Hematide fetal exposure was negligible. No Hematide effect, other than on growth and survival, was noted on developmental, functional, mating, and fertility end points in the F1 generation rats, and no effect on litter or fetal parameters was observed in the F2 generation. The maternal no-observed-adverse-effect level (NOAEL) for Hematide was 0.5 mg/kg, and the NOAEL for parturition and maternal behavior was 15 mg/kg. The NOAEL for F1 pup viability and growth was 0.5 mg/kg/dose. CONCLUSIONS: In conclusion, the Hematide-associated adverse findings were attributed to exaggerated erythropoiesis (pronounced and prolonged polycythemia) resulting from administration of an ESA to pregnant animals.


Subject(s)
Hematinics/toxicity , Maternal Exposure , Peptides/toxicity , Polyethylene Glycols/toxicity , Reproduction/drug effects , Teratogens/toxicity , Animals , Animals, Newborn/growth & development , Behavior, Animal/drug effects , Body Weight/drug effects , Embryo, Mammalian/drug effects , Embryo, Mammalian/embryology , Embryonic Development/drug effects , Female , Fetal Development/drug effects , Hematinics/classification , Injections, Intravenous , Lactation/drug effects , Longevity/drug effects , Male , Maternal Behavior/drug effects , Peptides/classification , Polycythemia/chemically induced , Polycythemia/physiopathology , Polyethylene Glycols/classification , Pregnancy , Rats , Reproduction/physiology , Teratogens/classification
7.
Drug Chem Toxicol ; 33(1): 28-37, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19995307

ABSTRACT

The subchronic toxicity of Hematide™, a synthetic PEGylated peptidic erythropoiesis-stimulating agent (ESA), was evaluated in CD-1 mice at intravenous doses of 0, 1, 5, 25, and 125 mg/kg administered once every 3 weeks for 3 months. Hematide displayed sustained plasma levels with reduced clearance and prolonged half-lives up to 59.4 hours that translated into sustained, pronounced polycythemia, bone marrow hyperplasia, and splenic and liver extramedullary hematopoiesis. Toxicological findings were considered to be secondary to exaggerated pharmacology, rather than a direct drug effect, and included mortality at ≥25 mg/kg/dose. The no-observed-adverse-effect-level was determined to be 5 mg/kg.


Subject(s)
Peptides/toxicity , Polyethylene Glycols/toxicity , Animals , Bone Marrow/drug effects , Bone Marrow/pathology , Dose-Response Relationship, Drug , Female , Half-Life , Hematopoiesis, Extramedullary/drug effects , Hyperplasia/chemically induced , Male , Mice , No-Observed-Adverse-Effect Level , Peptides/administration & dosage , Peptides/pharmacokinetics , Polycythemia/chemically induced , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/pharmacokinetics
8.
Basic Clin Pharmacol Toxicol ; 104(2): 155-63, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19143750

ABSTRACT

Hematide is a synthetic peptide-based, PEGylated erythropoiesis-stimulating agent, which is being developed for the chronic treatment of anaemia associated with chronic renal failure. To support the safety of long-term dosing of chronic renal failure patients, a comprehensive toxicology programme was implemented including rat subchronic and chronic studies. Rats were administered 0, 0.1, 1 and 10 mg/kg of Hematide every 3 weeks for 3 months via subcutaneous injection or for 6 months via intravenous injection. The dosing period was followed by a 6-week follow-up period. The primary pharmacology of Hematide resulted in erythroid polycythemia as measured by elevated haemoglobin levels that were time- and dose-dependent. The pharmacology profiles were similar regardless of administration route. For example, for male rats at Day 90, subcutaneous dosing resulted in haemoglobin increases of 2.7, 4.5 and 6.9 g/dl for 0.1, 1 and 10 mg Hematide/kg respectively, compared to 2.8, 5.7 and 7.4 g/dl increases for intravenous dosing. Histopathological changes were related to the prolonged severe polycythemia induced in normocythemic animals administered an erythropoiesis-stimulating agent. The findings included extramedullary haematopoiesis in the spleen and liver, bone marrow hypercellularity and organ congestion. Microscopic findings were reversible, demonstrating a return towards control findings within 6 weeks following cessation of dosing. Systemic exposures, based on both area under the curve (AUC) and maximum concentration (C(max)), were substantially greater for intravenous than subcutaneous administration. No Hematide-specific antibodies were detected. In conclusion, Hematide is a potent erythropoiesis-stimulating agent, and the studies provide support for the safety of clinical development, including chronic dosing, for the treatment of anaemia associated with chronic renal failure.


Subject(s)
Hematinics/adverse effects , Hematinics/pharmacology , Peptides/adverse effects , Peptides/pharmacology , Polyethylene Glycols/adverse effects , Polyethylene Glycols/pharmacology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Hematinics/administration & dosage , Hematinics/pharmacokinetics , Hematopoiesis, Extramedullary/drug effects , Hemoglobins/analysis , Injections, Intravenous , Injections, Subcutaneous , Male , Organ Size/drug effects , Peptides/administration & dosage , Peptides/pharmacokinetics , Polycythemia/chemically induced , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/pharmacokinetics , Rats , Rats, Sprague-Dawley
9.
Haematologica ; 93(9): 1376-9, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18519511

ABSTRACT

Hematide is a synthetic peptide-based, pegylated erythropoiesis stimulating agent in clinical development for treatment of anemia. To support chronic clinical dosing requirements, a 9-month repeat dose IV monkey safety study was undertaken. Animals received 0, 0.2, 2 or 20 mg/kg hematide IV every three weeks for nine months followed by a 14-week recovery. Hematide administration was associated with time and dose-dependent polycythemia. Histological findings were related to exaggerated pharmacology that was secondary to the administration of an erythropoiesis stimulating agent to a normocythemic animal. In conclusion, these results support the use of repeated administration of hematide for the correction of anemia.


Subject(s)
Drug-Related Side Effects and Adverse Reactions , Hematinics/pharmacology , Macaca fascicularis , Peptides/pharmacology , Polyethylene Glycols/pharmacology , Animals , Disease , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Erythropoiesis/drug effects , Peptides/adverse effects , Polyethylene Glycols/adverse effects
10.
Drug Chem Toxicol ; 31(2): 229-44, 2008.
Article in English | MEDLINE | ID: mdl-18330784

ABSTRACT

The pharmacology, toxicokinetics, and safety of Hematide, a synthetic peptidic erythropoiesis-stimulating agent (ESA), were characterized. Hematide was given intravenously (0, 0.5, 5, and 50 mg/kg) weekly for five weeks with a 6- (rat) and 12-week (monkey) recovery period. The pharmacological action of Hematide resulted in polycythemia. Histopathology consistent with drug-induced exaggerated pharmacology was observed primarily in rats. Secondary sequelae resulting from pronounced polycythemia was considered the cause of deaths in rats and a single high-dose monkey. Toxicokinetic analysis indicated prolonged exposure. In conclusion, Hematide is a potent ESA and the safety and efficacy profile support clinical development.


Subject(s)
Erythropoiesis/drug effects , Peptides/administration & dosage , Peptides/toxicity , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/toxicity , Animals , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , Injections, Intravenous , Macaca fascicularis , Male , Peptides/pharmacokinetics , Polycythemia/chemically induced , Polyethylene Glycols/pharmacokinetics , Rats , Rats, Sprague-Dawley , Species Specificity
11.
Mol Immunol ; 41(10): 985-1000, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15302161

ABSTRACT

A panel of anti-human CD2 monoclonal antibodies (mAb) and soluble human CD58 (LFA-3) were tested for binding to human peripheral blood mononuclear cells (PBMCs), recombinant human CD2 and mononuclear cells from Cynomolgus, Rhesus and African green monkey, Stump-tail, Pig-tail and Assamese macaque, Chimpanzee and Baboon. This analysis revealed that whilst some antibodies recognized all species, there were differential binding profiles with others. Three antibodies, MEDI-507, 6F10.3 and 4B2, recognized CD2 from human and Chimpanzee but not that from the other primates. We have cloned eight of the previously unknown primate CD2 molecules and report here their sequences for the first time. This analysis revealed that 12 amino acids formed a common set of residues in the extra cellular domain of human and Chimpanzee CD2. Using a "knock-in" mutagenesis approach starting with Baboon CD2, which does not bind MEDI-507, 6F10.3 and 4B2, we have identified three residues in the adhesion domain of human CD2 which are critical for its binding to these mAbs. These residues, N18, K55 and T59 define a region located outside of the previously described binding regions on CD2. Affinity measurements of the mutants revealed a variety of degrees of binding restoration for MEDI-507, 6F10.3 and 4B2, indicating that there are fine differences within a given epitope. Furthermore, the analysis of the competition of several of the anti-human CD2 antibodies with each other and CD58 demonstrated the existence of a continuum of overlapping epitopes on human CD2, which is in contrast to the commonly held belief that epitopes on human CD2 are clearly segregated.


Subject(s)
Antibodies/immunology , CD2 Antigens/immunology , Animals , CD2 Antigens/chemistry , CD2 Antigens/genetics , CD58 Antigens/immunology , Epitope Mapping , Haplorhini/genetics , Haplorhini/immunology , Humans , Pan troglodytes/genetics , Pan troglodytes/immunology , Sequence Analysis, Protein , T-Lymphocytes/immunology
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