Methods for the Automated Manufacturing of an Autologous Dendritic-Cell Immunotherapy

The author describes the development of automated equipment that uses functionally closed disposables to perform cellular and ribonucleic acid processing.
Dec 01, 2011
Volume 24, Issue 12

The dendritic cell (DC), the most powerful antigen-presenting cell in the immune system, has been a popular choice as a basis for personalized or autologous cellular immunotherapies. For these autologous DC immunotherapies, a batch of drug product is generated for each individual patient using his or her own cells. Scientists use three main approaches to obtain or generate DCs for a patient.

One approach is to isolate circulating DCs directly from the blood or from white blood cells collected through leukapheresis. The number of circulating DCs in the peripheral blood is extremely low, which limits the potential yields and doses that can be obtained using this approach. The advantage of this approach is that the DCs require minimal manipulation once isolated. Scientists essentially load the DCs with the antigen or antigens of interest against which an immune response is desired.

The two other approaches involve isolating precursor cells and culturing them to generate DCs. These precursor cells are either hematopoietic stem cells (HSCs) or monocytes. HSCs require longer culturing times to generate DCs compared with monocytes and require the patient to be mobilized (e.g., pretreatment with granulocyte colony-stimulating factor) before the leukapheresis. The advantage of the HSC approach is the ability to proliferate the cells before differentiation. Monocytes reduce culture time and manipulations compared with HSCs with the limitation that the cells have with no proliferation capacity. Therefore, the key to autologous DC processing from monocytes is managing losses and optimizing recoveries at each step to ensure an efficient process.


Figure 1: Overview of an autologous dendritic cell-manufacturing process for oncology and infectious-disease indications. (ALL FIGURES ARE COURTESY OF THE AUTHOR)
Using monocytes, Argos Therapeutics developed a robust method for generating its autologous DC therapy from leukapheresis for clinical trials in renal-cell carcinoma (RCC) and human immunodeficiency virus (HIV) indications (see Figure 1). The monocytes are isolated from the leukapheresis using the Elutra Cell Separation System (Caridian BCT). This instrument uses elutriation, also known as counterflow centrifugation, to fractionate the cells in the leukapheresis based primarily on cell volume, thus providing a final fraction of enriched monocytes. These monocytes can be cultured immediately to generate DCs, or can be frozen to be thawed and cultured when the antigen is available. Monocytes are cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) for 5 days to generate immature DCs in culture bags. Maturation media containing tumor necrosis factor α, interferon γ, and prostaglandin E2 is added, and the DCs are cultured overnight to mature before the antigen is introduced.

Antigen is added to the DCs in the form of ribonucleic acid (RNA) using electroporation. The antigen RNAs are amplified for each patient from samples of his or her tumor or virus (1, 2). This method provides antigen that is unique to that individual and tailors the DC immunotherapy for that individual. This technique, however, makes the manufacturing more complex than alternative approaches in which the antigen is universal for all patients and can be manufactured in bulk quantities. The RNA is manufactured for each patient by isolating the total tumor or viral RNA from the patient's sample. That isolated RNA is converted to first-strand cDNA through reverse transcription and amplified by polymerase chain reaction (PCR) using nonsequence-specific primers and methods, thus making them universal for all samples. RNA is generated using the resulting amplified cDNA through an in vitro transcription (IVT) reaction and is post-transcriptionally capped to ensure a high capping efficiency. Using this process, milligrams of amplified RCC messenger RNA (mRNA) are generated consistently from micrograms of total RNA. For HIV, RNAs for the quasispecies of gag, vpr, rev, and nef proteins in the viral sample are amplified (2). From isolated viral RNA, the concentration of which cannot be measured by standard spectrophotometric methods, milligrams of RNA for each antigen are amplified for each patient.

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