Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates.

Using lentiviral vector products in clinical applications requires an accurate method for measuring transduction titer. For vectors lacking a marker gene, quantitative polymerase chain reaction is used to evaluate the number of vector DNA copies in transduced target cells, from which a transduction titer is calculated. Immune Design previously described an integration-deficient lentiviral vector pseudotyped with a modified Sindbis virus envelope for use in cancer immunotherapy (VP02, of the ZVex platform).

Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors.

It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo.

Filamentous, mixed micelles of triblock copolymers enhance tumor localization of indocyanine green in a murine xenograft model.

Polymeric micelles formed by the self-assembly of amphiphilic block copolymers can be used to encapsulate hydrophobic drugs for tumor-delivery applications. Filamentous carriers with high aspect ratios offer potential advantages over spherical carriers, including prolonged circulation times. In this work, mixed micelles composed of poly(ethylene oxide)-poly[(R)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO) and Pluronic F-127 (PF-127) were used to encapsulate a near-infrared fluorophore.

The delivery of doxorubicin to 3-D multicellular spheroids and tumors in a murine xenograft model using tumor-penetrating triblock polymeric micelles.

Doxorubicin (DOX) is an effective chemotherapeutic against a wide range of solid tumors. However, its clinical use is limited by severe side effects such as cardiotoxicity as well as inherent and acquired drug resistance of tumors. DOX encapsulation within self-assembled polymeric micelles has the potential to decrease the systemic distribution of free drug and enhance the drug accumulation in the tumor via the enhanced permeability and retention (EPR).

Evaluation of temperature-sensitive, indocyanine green-encapsulating micelles for noninvasive near-infrared tumor imaging.

Purpose: Indocyanine green (ICG), an FDA-approved near infrared (NIR) dye, has potential application as a contrast agent for tumor detection. Because ICG binds strongly to plasma proteins and exhibits aqueous, photo, and thermal instability, its current applications are largely limited to monitoring blood flow. To address these issues, ICG was encapsulated and stabilized within polymeric micelles formed from the thermo-sensitive block copolymer Pluronic F-127, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), to increase the stability and circulation time of ICG.

Biophysical characterization of a soluble CD40 ligand (CD154) coiled-coil trimer: evidence of a reversible acid-denatured molten globule.

The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multimeric state and its tendency to form molten globules has not been elucidated. Corroborating evidence of a trimerized molecule in aqueous solution was obtained from size-exclusion chromatography, laser light scattering, and analytical ultracentrifugation.

Release of PEGylated granulocyte-macrophage colony-stimulating factor from chitosan/glycerol films.

We have prepared a new formulation for mucosal delivery of GM-CSF or PEGylated GM-CSF based on a chitosan carrier plus added glycerol to control the rate of release of the protein. Thin dry films comprised of various weight ratios of chitosan to glycerol and containing either granulocyte-macrophage colony-stimulating factor (GM-CSF) or PEGylated GM-CSF, PEG-(GM-CSF), were prepared. The amount of GM-CSF or PEG-(GM-CSF) released from the chitosan/glycerol films was determined using size exclusion high performance liquid chromatography (HPLC-SEC).

Protein release from alginate matrices.

There are a variety of both natural and synthetic polymeric systems that have been investigated for the controlled release of proteins. Many of the procedures employed to incorporate proteins into a polymeric matrix can be harsh and often cause denaturation of the active agent. Alginate, a naturally occurring biopolymer extracted from brown algae (kelp), has several unique properties that have enabled it to be used as a matrix for the entrapment and/or delivery of a variety of biological agents.

Characterization of the isoforms of PIXY321, a granulocyte-macrophage-colony stimulating factor-interleukin-3 fusion protein, separated by preparative isoelectric focusing on immobilized pH gradients.

We present here the purification and the characterization of the isoforms of PIXY321, a genetically engineered fusion of granulocyte-macrophage-colony stimulating factor and interleukin-3 expressed in yeast. The isoforms of PIXY321 were isolated using preparative isoelectric focusing (IEF) on immobilized pH gradients. Analysis of the collected fractions on analytical IEF gels showed that PIXY321 was resolved into four discrete isoforms of isoelectric point (pI) 5.

Minimization of recombinant human Flt3 ligand aggregation at the Tm plateau: a matter of thermal reversibility.

This study elucidates the importance of thermal reversibility as it pertains to the minimization of recombinant human Flt3 ligand aggregation and its potential role for determining solution conditions that can achieve the greatest long-term storage stability. Both thermal reversibility and Tm were evaluated as microcalorimetric parameters of stability within the range extending from pH 6 to 9, where the Tm was shown to plateau near 80 degrees C. Within this region, the reversibility was shown to decrease from 96.

The development of site-specific drug-delivery systems for protein and peptide biopharmaceuticals.

The desire to deliver protein and peptide biopharmaceuticals conveniently and effectively has led to intense investigation of site-specific drug-delivery systems. Despite challenges, progress towards the convenient noninvasive delivery of proteins and peptides has been achieved through specific routes of administration. In addition, the delivery of proteins and peptides to specific sites of action has been utilized to lower the total delivered dose, to gain access to specific organs or body compartments and to concentrate a therapeutic dose at a specific site of pharmacological action.

Interleukin-1 receptor (IL-1R) liquid formulation development using differential scanning calorimetry.

Purpose: To elucidate the solution conditions that confer stability of aqueous IL-1R using differential scanning calorimetry (DSC).

Methods: Optimal pH conditions were determined by monitoring degradation products encountered during accelerated studies (at elevated temperatures) using SDS-PAGE. At the pH optimum, DSC screened for excipients that enhanced thermal stability by shifting the Tm to higher values.

Stability of thiotepa (lyophilized) in 0.9% sodium chloride injection.

The stability of thiotepa in a new formulation of the drug was studied. Vials of Thioplex (Immunex), a relatively new lyophilized formulation of thiotepa, were reconstituted with sterile water and diluted with 0.9% sodium chloride injection in polyvinyl chloride infusion bags to thiotepa concentrations of 0.

Characterization of poly(glycolide-co-D,L-lactide)/poly(D,L-lactide) microspheres for controlled release of GM-CSF.

Purpose: This study describes the preparation and characterization of a controlled release formulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) encapsulated in poly(glycolide-co-D,L-lactide) (PLGA) and poly(D,L-lactide) (PLA) microspheres.

Methods: GM-CSF was encapsulated in PLGA/PLA microspheres by a novel silicone oil based phase separation process. Several different blends of PLGA and low molecular weight PLA were used to prepare the microspheres.

Structure-function studies of interleukin 15 using site-specific mutagenesis, polyethylene glycol conjugation, and homology modeling.

Interleukin (IL)-15 is a multifunctional cytokine that shares many biological activities with IL-2. This functional overlap, as well as receptor binding subunits shared by IL-15 and IL-2, suggests tertiary structural similarities between these two cytokines. In this study, recombinant human IL-15 was PEGylated via lysine-specific conjugation chemistry in order to extend the circulation half-life of this cytokine.

Enhancement of bone ingrowth by transforming growth factor-beta.

Enhancement of bone ingrowth with transforming growth factor-beta was evaluated in a canine model. Ten dogs had bilateral implantation of a titanium-fiber-metal-coated rod in the proximal part of the humerus. A three-millimeter gap between the outer surface of the porous coating and the surrounding cancellous bone was created to impair bone ingrowth.

Acceleration of wound healing in aged rats by topical application of transforming growth factor-beta(1).

The impaired wound healing associated with aging may reflect inadequate secretion or delivery of cytokines. Transforming growth factor-beta(1) is a mitogenic polypeptide with beneficial effects on wound healing. In the present study we questioned whether topical administration of transforming growth factor-beta(1) could improve the wound healing process in aged rats in vivo.

Biodegradable polymers for protein and peptide drug delivery.

We have reviewed a large cross-section of degradable polymeric delivery systems for protein and peptide pharmaceuticals. These systems include monolithic type devices in which the drug is dispersed throughout the polymer and protein-polymer conjugates where the drug is covalently bound to the polymer. These delivery systems have unique challenges associated with their development that are related to both protein stability and protein release kinetics.

The enhancement in wound healing by transforming growth factor-beta 1 (TGF-beta 1) depends on the topical delivery system.

Transforming growth factor-beta 1 (TGF-beta 1) has beneficial effects on wound healing. However, the ideal method for its administration to the wound site remains unknown. Our aim was to analyze the release of TGF-beta 1 from different formulations and to study whether the changes in wound healing by TGF-beta 1 depend on its topical delivery system.

Novel delivery system for inducing quiescence in intestinal stem cells in rats by transforming growth factor beta 1.

Background/aims: Intestinal mucosa, a tissue in a dynamic state of rapid cellular proliferation, is often adversely affected by cytotoxic drugs. The purpose of this study was to develop an oral delivery system targeting transforming growth factor (TGF) beta 1 locally and analyze its effects on the epithelial stem cells of gastrointestinal mucosa.

Methods: Rats were treated with recombinant TGF-beta 1 in alginate beads perorally or with recombinant TGF-beta 1 in phosphate-buffered saline perorally or intraperitoneally.

Stimulation of bone healing by transforming growth factor-beta 1 released from polymeric or ceramic implants.

The ability to transforming growth factor-beta 1 (TGF-beta 1), to stimulate bone healing was evaluated in a rat critical calvarial defect model. Both a low dose and a high dose of TGF-beta 1 were incorporated into two different types of implants: one made from a composite of poly(lactic-co-glycolic acid) (PLPG) (50:50) and demineralized bone matrix (DBM), and the other from calcium sulfate (CaSO 4). Scanning electron microscopy showed that the CaSO 4 implants were more porous than the PLPG/DBM samples.

The stabilization of a human IgM monoclonal antibody with poly(vinylpyrrolidone).

An IgM anti-group B Streptococcus monoclonal antibody (4B9) was found to undergo irreversible heat-induced aggregation at 50 degrees C. A variety of excipients was tested for their ability to inhibit antibody aggregation. The amount of 4B9 aggregation, which was determined by analysis on a size-exclusion HPLC, was significantly reduced in the presence of low concentrations [between 0.

Attempts to stabilize a monoclonal antibody with water soluble synthetic polymers of varying hydrophobicity.

Proteins are subject to a variety of physical and chemical reactions that lead to a loss of activity. These reactions are a particular problem in controlled-release devices, where temperatures and protein concentrations are high. Current approaches to increasing protein stability include the addition of saccharides, amino acids, or polymers.