Obtaining DCs
DCs can be obtained in large numbers from in vitro cell culture of either peripheral blood monocytes or bone marrow progenitors. The most common method for obtaining clinical-grade
DCs in vitro from leukapheresis product uses CD14+ monocytes (or plastic adherent monocytes) and growth factors that include granulocyte
macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4 or GM-CSF plus IL-13.7,9 The other approach includes the use of CD34+ cells with combinations of stem-cell factor, GM-CSF, Flt3-ligand, plus tumor
necrosis factor-alpha (TNF)-α.
Production Improvements
Improvements in the design and manufacture of DC products for therapy are being actively addressed. Efforts to optimize DC-based
therapy for clinical use include the establishment of quality control measures for consistent generation of high-quality therapeutic
vaccines as well as for reliable monitoring of responses to DC-based vaccines in patients receiving this therapy. The major
objective of this review is to discuss strategies for the evaluation of therapeutic DC products at the time they are ready
for adoptive transfer and for the development of methods that would reliably measure in vivo effectiveness of DC in inducing desirable immune responses in subjects receiving DC-based therapy.
DC GENERATION
Figure 1. A schema for in vitro generation of human dendritic cells (DCs) from peripheral blood monocytes
|
Figure 1 presents a schema for generating human DC from peripheral blood monocytes. Apheresis products are processed by elutriation
using an Elutra cell separation system (Gambro BCT, Lakewood, CO) to recover a monocyte fraction. The fraction purity is ascertained
using flow cytometry by determining the frequency of CD14+ cells. Culture of the recovered monocytes in the presence of IL-4
(1,000 IU/mL) and GM-CSF (1,000 IU/mL) for six days yields immature DCs (iDCs). These cells excel in antigen uptake or endocytosis.9 Complex antigens taken up by iDCs are processed by APM in the cytosol. Next, iDCs are matured in the mix of cytokines (IL-1b,
IL-6, TNF-α, and interferons) to yield mature DCs (mDCs). The latter are excellent peptide-presenting cells and represent
a final product that can be released for therapy, provided it meets established release criteria.
The method of DC generation described above may vary, depending on how the monocytes are isolated from peripheral blood mononuclear
cells and on conditions adopted for their culture and maturation. Monocyte purity, culture medium, cytokine content, and culture
vessels exert considerable influence on the quality of the final DC product.21 Production methods vary from manual approaches using plastic flasks to semi-automated functionally closed systems for monocyte
culture. The latter offer a simple and economical method for meeting good manufacturing practice (GMP) requirements and facilitate
process standardization and the reproducible production of high-quality DCs.
LOADING ANTIGENS ONTO DCs
Table 1. Tumor-derived antigens commonly used for DC loading in preparing vaccines
|
A DC product designed for therapy must be loaded with tumor antigens to complete the manufacturing process and to generate
APCs that elicit the desirable anti-tumor responses in vivo after their adoptive transfer. A variety of defined and undefined antigens have been used for DC loading (Table 1). Defined
antigens, such as peptides, proteins, cDNA, or mRNA, may elicit tumor-specific T-cell responses targeting a well-defined epitope
overexpressed in vivo in tumor cells. Defined antigens also allow for targeted immune monitoring following vaccination.
Undefined antigens, including apoptotic tumor cells, tumor cell lysates or extracts, tumor cell–DC fusions, and total genomic
DNA or total mRNA derived from tumor cells, have the potential to generate anti-tumor responses targeting a broad spectrum
of epitopes in the tumor.
Both defined and undefined antigens have disadvantages (Table 1). Therefore, selecting a DC payload is not simple, and requires
an understanding of the antigenic characteristics of the tumor as well as the immunogenic potential of the tumor antigens
under consideration. It is essential to remember that complex antigens have to be processed by DCs before presentation, and
should be delivered to iDCs, wherease mDCs are preferred for pulsing with peptides (Table 1). Technical issues relevant to
the most efficient delivery of proteins, DNA, or mRNA to DC also must be considered.
|
