Evaluation of Dendritic Cell Products Generated for Human Therapy and Post-Treatment Immune Monitoring - - BioPharm International

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Evaluation of Dendritic Cell Products Generated for Human Therapy and Post-Treatment Immune Monitoring


BioPharm International
Volume 21, Issue 3

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.


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