November 15, 2005 (Vol. 25, No. 20)

Applications Go Beyond Vaccines and Gene Therapy to include Drug Discovery Research

Advancing Therapeutic Vaccines

Introgen Therapeutic’s (Austin, TX) p53-based INGN 225 cancer vaccine yielded promising safety and efficacy results in a Phase II trial in advanced small cell lung cancer. The personalized therapeutic vaccine employs a patient’s dendritic cells transfected with an adenoviral vector carrying the human p53 gene. It stimulates the immune system into destroying cancer cells and sensitizes solid tumors to the effects of chemotherapy.

Therion Biologics’ (Cambridge, MA) pipeline includes its lead therapeutic cancer vaccine products, PANVAC-VF, in Phase III clinical trials to treat metastatic pancreatic cancer and earlier stage trials in breast, lung, and colorectal cancer, as well as PROSTVAC-VF, which has entered Phase II testing in patients with hormone refractory metastatic prostate cancer.

BioVex (Cambridge, MA) will initiate a Phase II trial of its OncoVEXGM-CSF oncolytic vaccine in malignant melanoma during the fourth quarter. The company is also planning Phase II trials of OncoVEXGM-CSF in pancreatic, head and neck, and lung cancer.

BioVex is evaluating its ImmunoVEX vaccines, which stimulate dendritic cells, as treatments for melanoma and a range of chronic infectious diseases. Both the OncoVEX and ImmunoVEX vaccine platforms utilize a herpes simplex-based viral vector.

BioVex’s functional genomics platform, NeuroVEX, relies on herpes simplex virus to deliver genes representing potential drug targets for diseases affecting the nervous system.

Cell Genesys (S. San Francisco, CA) is focusing its efforts on two main product lines, its GVAX vaccines in Phase III development for prostate cancer and in Phase II testing for the treatment of leukemia and pancreatic cancer, as well as CG0070, an oncolytic virus therapy that delivers the GM-CSF gene, and is currently being tested in patients with recurrent bladder cancer.

Mounting interest in the use of viral vector technology for vaccine and gene therapy applications is being driven by recent developments in these industry sectors.

Widespread media attention on the potential for a global influenza pandemic linked to the spread of the H5N1 avian flu strain, approval of the first gene therapy product in China, and the expanded use of viral vector technology in vaccine production have all contributed to the enthusiasm in these fields.

Technological advances such as the development of novel viral-based gene delivery methods, improvements in the design and production of viral vectors, and advances in large-scale manufacturing and purification of vaccine and gene therapy products, have all helped broaden the use of viral vector technology.

Fears of a human influenza pandemic linked to the H5N1 avian flu strain among birds in Asia and Europe have led to intense scrutiny of global preparedness to prevent and treat a large-scale flu outbreak.

In an effort to respond to this concern, sanofi pasteur (Lyon, France and Swiftwater, PA) announced a $100 million contract with the U.S. Department of Health and Human Services (HHS) in September. The agreement focuses on production and storage, in bulk concentrate form, of a vaccine that would help protect against a mutated version of H5N1 influenza that could spread among humans. The U.S. government is currently testing an investigational vaccine developed using conventional egg-based techniques and delivered by Sanofi in May 2004.

Earlier this year, HHS awarded a contract to Sanofi Pasteur to accelerate the development of a cell culture-based production process for manufacturing influenza vaccine.

MedImmune (Gaithersburg, MD) as well has entered into a collaborative agreement with the National Institute of Allergy and Infectious Diseases (NIAID) to develop investigational pandemic influenza vaccines. The company will apply the same technology it used to develop its FluMist product, an FDA-approved intranasal, live, attenuated influenza vaccine.

The firm will produce vaccine seed lots “for a number of different virus strains that we would not normally see in an annual cycle,” according to George Kemble, Ph.D., vp of vaccines research and development and general manager of MedImmune’s California facilities. One of these vaccine seeds will be based on the H5N1 strain of avian influenza.

“We will be using a cDNA-based approach called plasmid rescue, in which we individually clone six of the gene segments of the vaccine strain and two of the wild-type strain and combine them; we then rescue [the virus] following introduction of the cDNAs into mammalian cells,” explains Dr. Kemble. The company then produces the vaccine using conventional egg-based production techniques.

At the 2005 Williamsburg Bioprocessing “Viral Vectors and Vaccines” conference, held recently, Ajit Subramanian of MedImmune presented the company’s early R&D work using alternative, cell-based methods to produce influenza virus.

In September, MedImmune filed with the FDA for approval of a refrigerator-stable form of FluMist for use in healthy individuals between 5 to 49 years. The company is hoping to gain approval of this CAIV-T (cold adapted influenza vaccine, trivalent) for use in younger children as well.

Earlier this year, MedImmune completed construction of a new bulk vaccine manufacturing facility in Speke, England. The facility has the capacity to produce up to 15 million bulk doses per month, or about 90 million doses per influenza manufacturing season.

In October, Crucell (Leiden, The Netherlands) announced that Merck & Co. would use Crucell’s PER.C6 production technology to develop an adenovirus-based vaccine against hepatitis C.

Merck already uses the PER.C6 human cell line technology to produce its adenovirus-based HIV vaccine that is in Phase II proof-of-concept trials. Crucell is applying the technology to develop an inactivated split virus influenza vaccine and an inactivated whole virus West Nile vaccine.

Crucell is also leveraging its adenovirus-based AdVac vector technology to create recombinant vaccines against the Ebola virus and malaria. It has signed an agreement with the U.S. Navy to develop AdVac-based vaccines against anthrax and plague as well as to test them in nonhuman primates.

“We’re seeing positive trends on the adenovirus side in terms of both gene therapy and vaccines,” says Anthony Green, Ph.D., a scientist at Puresyn (Malvern, PA). Dr. Green points to the positive results of animal studies from both a safety and efficacy perspective and the growing number of companies using adenovirus as part of a vaccine or gene therapy strategy.

“The issues that were problematic in the gene therapy field forced researchers to go back and address safety as part of their development process,” Dr. Green says. “How you make your material and how clean it is”the process, independent of the vector”can affect both biological activity and safety,” of the product, he adds.

Specializing in technology for large-scale production and purification of recombinant adenoviral vectors for use in gene therapy and vaccine applications, Puresyn developed the Adenopure adenovirus purification kits. These can replace cesium chloride gradients and chromatographic methods used to separate and purify recombinant adenovirus.

Puresyn plans to expand its contract manufacturing services and to add GMP services within the next 12 to 15 months, according to Mickey Flynn, president of Puresyn.

Optimism that the gene therapy industry in the U.S. will see its first product come to market and realize commercial success, overcoming safety issues that plagued early product development efforts, stems from the growing number of gene therapy products advancing through the pipeline as well as the successful launch of a recombinant adenovirus-based gene therapy product in China in April 2004. Gendicine contains the human p53 gene and is approved for the treatment of head and neck squamous cell carcinoma.

Lentiviral Vectors

Virxsys (Gaithersburg, MD) initiated a Phase II dosing and efficacy trial earlier this year of its HIV gene therapy product, VRX496. The therapeutic agent comprises a lentiviral vector carrying an antisense molecule that targets the HIV envelope gene and blocks HIV replication. Delivery of the antisense gene into the CD4 T cells of an HIV-infected patient would create a population of immune cells capable of resisting HIV infection.

Virxsys is initially developing gene therapy products against HIV and cancer for use with ex vivo cell processing. It is also pursuing the formulation of in vivo injectable genetic medicines and the application of its lentiviral gene delivery technology for vaccine production.

Lentigen (Baltimore, MD) is leveraging its lentiviral vector gene delivery and expression technology to target several markets. This includes the research market, where it provides a web-based custom vector design service. Researchers can use its LentiDesign tool to cut and paste gene and promoter sequences in order to create a tailored, research-grade vector that Lentigen can then produce.

The key advantages of lentiviral vectors as compared to electroporation, chemical transfection methods, and other virus-based gene delivery strategies are its high efficiency and stable transfection characteristics, points out Boro Dropulic, Ph.D., founder and CEO of Lentigen.

“We are getting in excess of 90% transduction efficiency on average,” in many different cell lines tested, says Dr. Dropulic. He believes lentiviral vectors are particularly useful for gene over-expression or knock-down experiments as part of target validation studies and drug specificity testing in drug discovery.

Lentigen is also focusing its core competency on developing a contract services business for GMP lentiviral vector manufacturing to provide clinical-grade material for companies developing lentiviral-based gene therapy products. In the future, according to Dr. Dropulic, the company will develop its own proprietary lentiviral gene therapy products. Lentigen is optimizing a scaleable manufacturing process based on its LentiMax production system.

Another facet of Lentigen’s evolving business strategy will involve offering lentiviral vector kits to the research community. This will afford scientists the capability to create vectors incorporating their own gene sequences.

As Research Tools

Viral vectors are becoming increasingly attractive as enabling tools in drug discovery research. For example, GE Healthcare (Piscataway, NJ) is launching an Adenoviral Vector Gene Delivery system for use in cell-based assays for lead compound profiling and drug target validation, in conjunction with the company’s IN Cell Analyzer automated microscopy platform.

The system comprises a range of recombinant adenoviral preparations called Ad-A-Gene vectors that contain a gene of interest fused to either green fluorescent protein or a response element that controls the expression of nitroreductase.

GE Healthcare “chose adenovirus because of its broad host range effects and efficiency of transfection,” says John Anson, head of lead discovery at the company. The first set of about 50 different vectors to be introduced will incorporate genes associated with protein translocation and gene activation.

The ability to study multiple genes in a single experiment and in particular more than one gene in a given pathway, allows researchers to “interrogate a pathway at multiple points and look for off-target effects,” Anson says. We are especially “interested in genes associated with branch points in pathways,” he adds.

Purification Remains a Key Issue

One of the key challenges in viral vector purification today, according to Sibylle Herzer, Ph.D., staff scientist in the protein separations division of GE Healthcare, is the persistent academic mindset, with a focus on small-scale molecular biology and an emphasis on designing the best possible vector, without sufficient consideration given to producting the vector and developing a scaleable, cost-efficient process that could be used to manufacture an economical, safe, and commecially successful product.

In general, the products on the market for plasmid or viral vector purification were developed for the protein separations market. These biomolecules however, are so much larger than typical proteins that the same dynamics do not apply. The use of larger chromatography beads with greater surface area can overcome some of the problems associated with virus purification on media composed of smaller diameter beads.

High volume throughput, economical virus capture can be achieved at process scale using ion exchange media that restricts virus or plasmid particles from diffusing into the support matrix and essentially soaks up the contaminants, filtering them out of the process flow in a gentle manner that minimizes shear stress.

Dr. Herzer points to three main GE Healthcare product lines that offer advantages for the capture and polish steps in purifying large biomolecules: the ktaCrossflow chromatography and filtration system with a variety of hollow fiber and cassette options. This can be used to optimize process parameters for scale-up; the Capto line of BioProcess media, including Capto Q and Capto MMC, with additional product launches planned over the next 12 to 18 months; and Q and SP Sepharose Big Beads.

Responding to an emerging trend toward the development of more purified, clinical-grade material early in the research and product development cycle for gene therapy and vaccine products, Pall (East Hills, NY) has introduced new membrane chromatography systems. These are aimed at improving product purity during the critical capture and polishing steps in downstream processing.

Earlier Implementation

In particular the May 2004 European regulatory directive called for earlier implementation of GMP standards in the production of investigational new products intended for use in humans, points out Helene Pora, Ph.D., director of vaccine applications at Pall. The directive raised the bar for product purity before a biopharmaceutical enters Phase I clinical trials, requiring that a well-defined, validated manufacturing process be in place.

These regulatory constraints, combined with existing time and cost efficiency pressures, are driving demand for higher throughput purification methods. Pall developed its Mustang XT5000 membrane chromatography media to overcome problems of low throughput in virus capture and polishing applications caused by the large size of virus particles and the relatively small size of standard chromatography media.

The Mustang membrane has an anion exchange support with pendant quaternary amine functional groups and a pore size of 0.8 m. The functional groups are readily accessible, and binding is not dependent on diffusion, explains Dr. Pora. Membrane chromatography is capable of processing a batch of hundreds of liters of process fluid in about an hour.

Dr. Pora also points to a trend toward the use of disposable, single-use systems and aseptic processing technology in viral vector production. This minimizes the risk of human contact with viral products and of product contamination. It also eliminates the need to implement and validate complex clean-in-place and sterilize-in-place procedures, notes Dr. Pora. She points to Pall’s Kleenpak line of disposable filter systems and aseptic connectors as products developed in recognition of this emerging transition to disposable processing components.

More Efficient Virus Production

The use of microcarriers in vaccine manufacturing is gaining popularity, according to Cory Card, senior scientist and development team leader at Hyclone Laboratories (Logan, UT). Microcarriers provide increased surface area for the growth of attachment-dependent cells compared to cell culture in roller bottles. High cell densities of 20 g/L and greater, translate into high virus titers.

Hyclone has introduced six versions of its new HyQ Sphere microcarrier product line, which includes collagen-coated beads suitable for serum-containing cultures and animal-derived component-free (ADCF) beads. Some beads carry a charge, whereas others are coated with factors selected to enhance cell attachment.

Card describes a trend toward increased use of animal-free systems for virus production. Vaccine manufacturing has generally lagged behind in this area, largely because the cell lines used to grow the viruses tend to thrive in a serum-based culture environment. Also, existing vaccine products are exempt from regulatory pressures to develop animal-free processes.

Rising concerns over transmission of prion diseases, such as bovine spongiform encephalopathy, are leading new vaccine manufacturers to opt for cell culture media, free of animal-derived components.

Using a hybrid business model to augment the capital resources required for its in-house cell therapy development efforts, MaxCyte (Gaithersburg, MD) licenses the company’s flow electroporation system for bulk production of viral vectors and also, as an enabling technology to produce cells for use as therapeutic agents. Leading MaxCyte’s product pipeline is a cell therapy currently in Phase I/II clinical studies for the treatment of chronic lymphocytic leukemia, with Phase II trials planned for 2006.

The need to develop or license a packaging cell line is a key drawback in the conventional process used to produce viral vectors, according to Madhusudan Peshwa, Ph.D., vp of R&D at MaxCyte. In contrast, MaxCyte’s technology works with any defined cell line that is qualified for production purposes.

“We circumvent the need to have viral banks by using the key structural component plasmids that make up a virus,” and loading these components as either DNA plasmids or as RNA directly into any production cell line, concurrently using an electroporation-based system.

“Because of the increased efficiency of loading and expression in cells, we can engineer the system to generate higher yields of functionally active viral particles,” Dr. Peshwa says. Although much of the development work is done at small scale, scale-up is essentially linear from laboratory to clinical scale.

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