ESTABLISHING THE QUALITY OF THERAPEUTIC DC PRODUCTS
The quality of therapeutic DC products has to be assessed before they can be released for clinical use. This is usually accomplished
by: first determining the phenotypic characteristics of DCs using multicolor flow cytometry that confirms cell maturity and
expression of co-stimulatory and migration-related molecules on DC; and then measuring the DCs' ability to induce antigen-specific
CTL from naive peripheral blood mononuclear cells (PBMC) precursors. The latter is difficult to execute because of the requirement
for functional T-cell assays (e.g., cytotoxicity or ELISPOT), and for prior IVS with DCs as well as the availability of defined
antigens or targets. For this reason, various surrogate methods for measuring DC functions have been introduced, including
intercellular adhesion molecule (ICAM) expression, cytokine production, endocytosis of latex beads or mannose receptor expression
on the cell surface.25,26 These methods are useful in defining the functional status of DC, but do not provide information relevant to their ability
to mediate antigen-specific T-cell responses.
Most current anti-tumor vaccines depend on the use of ex vivo manipulated autologous products, thus representing "personalized" therapy. A DC product generated from cells of one individual
is referred to as a DC batch. Patient-to-patient variability in starting material makes it difficult to ensure DC batch uniformity. Inter-batch uniformity
would be easier to achieve with normal allogeneic DCs than with autologous DC products. Although "off-the-shelf" allogeneic
DC products might be more practical and uniform, the possibility of viral transmission and rapid in vivo clearance of allogeneic cells introduces another set of concerns and regulatory issues. Once a batch of DCs is made and its
quality tested, it can be aliquoted into sterile vials, and the cells cryopreserved for multiple vaccinations. However, any
further manipulation of the vaccine, such as washing or adjusting cell numbers, will necessitate testing for sterility and
endotoxin levels before vaccine delivery. From a practical viewpoint, the same batch of DCs can deliver multiple vaccines
provided no additional manipulations of DCs are performed.
Current Standards for Release of Therapeutic Dendritic Cell Products
Although there are no widely accepted release criteria for therapeutic DCs, the cells should meet predefined sterility, viability,
purity, and stability standards (according to 21 CFR 211) as well as defined phenotypic and functional criteria. Assays used for DC characterization and product release are summarized
in Table 2. Current FDA recommendations for release of therapeutic DC products specify sterility, viability, purity, phenotypic
characterization, and stability measurements, but no functional assays for Phase 1 and 2 studies (FDA current GTP final rule;
21 CFR 211). A definition of release criteria based on expected results of these assays is made before the implementation of DC
production and is adhered to for all DC batches produced by a facility. For example, if DC viability of 80% is defined as
a release standard, any DC batch with viability <80% would be rejected. The development of potency assays for DC is encouraged
throughout the early-phase clinical trials, but is not a requirement for DC release.
Table 2. Procedures and assays required to evaluate dendtric cell products generated for therapy
Sterility testing. Sterility testing of DC products requires 14-day incubation and ensures the absence of aerobic or anaerobic microorganisms.
Results are not available until after DC administration. Therefore, a Gram stain is performed immediately before DC release
for therapy. Negative results from the Gram stain and the availability of the established plan of action necessary in case
a contaminated DC product is administered are sufficient criteria for release of DC products. The products also must be mycoplasma
free. In this instance, DNA hybridization, or RT-PCR–based assays, which take several hours to complete, are performed instead
of 28-day mycoplasma culture assays. Endotoxin levels are measured in the final DC product, which must be endotoxin free.
For the mycoplasma and endotoxin assays, medium and cells must be submitted for testing.
Viability, purity, and stability. Viability, measured microscopically by a trypan blue-dye test or flow cytometry, using PI or 7 amino-actinomycin D (7-AAD),
is necessary to show that most DCs in culture are alive. Viability measurements are especially useful after freezing and thawing
DCs aliquoted for multiple vaccinations. Purity determinations use flow cytometry to evaluate the presence of lymphocytes
(CD3+, CD19+ or CD56/16+) and/or monocytes (CD14+) in a DC product. In general, purity of 80% or higher is desirable, although
the overall requirement for the purity level still remains to be established and will be dictated by the overall immunopotency
of the final product. Stability assays measure DCs' ability to retain viability and activity over time at room temperature
or in the cold, as well as possible detrimental effects of freezing and thawing. Stability assays are especially critical
when a DC-based product is divided into smaller batches that are cryopreserved to be thawed for administration to patients
for multiple dosing over long treatment intervals.