A production method has been developed for the purification of pharmaceutical-grade plasmid DNA for in vivo gene therapy. This method has been applied to the purification of VCL-1005, which is a eukaryotic plasmid expression vector that codes for the production of the HLA-B7 protein. Purified VCL-1005 is formulated with a cationic lipid and injected directly into established tumors of HLA-B7-negative patients with advanced cancers to heighten the patient's immune response against the cancer. The purification of pharmaceutical-grade plasmid DNA requires the development of highly reproducible and scaleable processing methods that meet regulatory standards similar to those required for the manufacture of recombinant protein pharmaceuticals. Defined pharmaceutical standards of purity, potency, efficacy, and safety are routinely met by the process described in this study. The scaleable purification method described here is a combination of highly reproducible unit operations; alkaline lysis, precipitation, and size-exclusion chromatography. The advantages over existing DNA purification methods include improved plasmid purity and the elimination of undesirable process additives such as toxic organic extractants and animal-derived enzymes. The overall process yield of purified plasmid DNA from fermentation through final column purified product is greater than 50%. Contaminating Escherichia coli DNA levels are reproducibly below 1% as measured by Southern analysis. Endotoxin levels are less than 0.03 endotoxin units/micrograms plasmid DNA and residual protein is undetectable. This process was used to produce 100 mg of VCL-1005 for use in an active clinical protocol.

Production of large quantities of highly purified plasmid DNA is essential for gene therapy. A low-copy-number pBR322-derived plasmid (VCL1005) was converted to a high-copy-number plasmid (VCL1005G/A) by incorporating a G-->A mutation that affects initiation of DNA replication from the ColE1 origin of replication. Because the phenotypic effect of this mutation is enhanced at an elevated temperature, a further increase in yield was achieved by changing the growth temperature from 37 degrees C to 42 degrees C at mid-log phase during batch and fed-batch fermentation. The combined effect of the single base-pair change and the elevated growth temperature produced an overall yield of 2.2 grams of plasmid DNA available for recovery from a 10-liter fed-batch fermentation compared to 0.03 grams from a 10-liter batch fermentation, a 70-fold increase in yield. The plasmid DNA isolated from this process contained lower levels of RNA and chromosomal DNA contaminants, simplifying downstream processing.

The primary sequence of the N-terminal somatomedin B (SMB) domain of native vitronectin contains 44 amino acids, including a framework of four disulfide bonds formed by 8 closely spaced cysteines in sequence patterns similar to those found in the cystine knot family of proteins. The SMB domain of vitronectin was isolated by digesting the protein with endoproteinase Glu-C and purifying the N-terminal 1-55 peptide by reverse-phase high performance liquid chromatography. Through a combination of techniques, including stepwise reduction and alkylation at acidic pH, peptide mapping with matrix-assisted laser desorption ionization mass spectrometry and NMR, the disulfide bonds contained in the SMB domain have been determined to be Vitronectin is a large glycoprotein with wide ranging distribution and function. The hallmark feature of the vitronectin structure is a series of distinct functional domains that allow it to interact both with itself and with a number of other ligands in a variety of environments including the circulation, extracellular matrix, and platelets (1-4). Of particular interest is the N-terminal somatomedin B (SMB) 1 domain of vitronectin. This domain contains the high affinity binding site for the serpin PAI-1. The interaction between PAI-1 and vitronectin is important to the function of both proteins in thrombolysis, cell adhesion, and pericellular proteolysis (1, 5-9). 2 Equally important for the adhesive properties of vitronectin are binding sites for cell-surface receptors, including integrins and uPAR, that are housed within this small N-terminal domain (2, 10 -15).Because it is known that the SMB domain provides a high affinity binding site for PAI-1 and that this interaction stabilizes PAI-1 in its physiologically active form, the structure of the SMB domain of vitronectin has been hotly pursued. Computational predictions for the structure of this domain using a threading algorithm were challenging compared with other domains from vitronectin (16), as there were no reported structures at the time for homologues of this small domain containing four disulfides. Nevertheless, there are over 100 homologues in the sequence data base, suggesting that this folding motif has been conserved in evolution. Only recently have three-dimensional structures describing the SMB domain become available from two different approaches. First, an x-ray structure has been reported on a co-crystal of PAI-1 and a recombinant form of the SMB domain expressed in Escherichia coli (17). Subsequently, we completed the determination of a solution structure for the SMB domain purified from circulating vitronectin that was isolated from human plasma (18). Although the two structures differ in overall fold, they share a common feature, a single ␣-helix that contains key amino acids for PAI-1 binding. Not surprisingly, a recent report using NMR on a recombinant SMB arrived at a similar structure to that observed in the co-crystal with PAI-1 (19).Key to understanding the structure of the SMB domain is defining the correct dis...

Mouse Interleukin 4 is a 20-kDa glycoprotein, synthesized by activated T lymphocytes and mast cells, which regulates the growth and/or differentiation of a broad spectrum of target cells of the immune system, including B and T lymphocytes, macrophages, and hematopoietic progenitor cells. Using an inducible recA promoter and the g10-L ribosome-binding site, recombinant non-glycosylated interleukin 4 (IL-4) was expressed as 17% of total cellular protein in Escherichia coli inclusion bodies, as a reduced, inactive 14.5-kDa polypeptide. The protein was refolded and aggregates dissociated when three disulfide bonds were reformed by slowly decreasing the concentration of guanidine hydrochloride and cysteine. The oxidized monomer was purified to homogeneity by sequential ion-exchange and size exclusion chromatography. When compared with native IL-4, E. coli-derived IL-4 displayed an identical specific activity of 4-7 x 10(7) units/mg. This recombinant IL-4 contained a three-amino-acid NH2-terminal extension, which did not affect its biological activity. Purified biologically active protein consisted of three isoforms as shown by two-dimensional gel electrophoresis, with a pI greater than 9.0. These data suggest that neither glycosylation nor the NH2 terminus of mouse IL-4 play a critical role in contributing to its in vitro biological activity.

The potential applications of using plasmid DNA for immunization and other gene therapy approaches have been discussed in an increasing number of publications in the past few years. Injection of mouse muscle with naked DNA (plasmid DNA in saline) resulted in significant episomal expression from a number of encoded reporter genes such as firefly luciferase, chloramphenicol acetyltransferase, and β-galactosidase (1). DNA vaccination has been shown to induce neutralizing antibodies against the gene product, helper T-cell responses of the Th1 phenotype, and cytotoxic T lymphocyte responses (2). Vaccination with plasmid DNA stimulates immunogenicity and provides protection against various infectious diseases in pre-clinical animal models. Examples include hepatitis B in chimpanzees (3), bovine herpes virus in mice (4), influenza A virus in ferrets (5), human immunodeficiency virus in rhesus monkeys (6), Mycobacterium tuberculosis in mice (7,8), malaria in mice (9,10), and genital herpes simplex virus in guinea pigs (11). Recently, DNA vaccines for the protection against influenza (Merck Research Laboratories, Rahway, NJ), malaria (Vical Inc., San Diego, CA), and HIV (Apollon Inc., Philadelphia, PA), have entered phase I human clinical trials. Rapid progress has been made in the areas of adjuvants for DNA vaccines (12), route of immunization (13), industrial scale fermentation and pharmaceutical grade purification (14). One major interest in the commercial development of DNA vaccines, especially for developing countries, is to increase DNA vaccine stability at room temperature, to reduce the requirement for costly cold storage, and to extend product shelf-life.