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

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Methods for the Automated Manufacturing of an Autologous Dendritic-Cell Immunotherapy
The authors developed automated equipment that uses functionally closed disposables to perform cellular and ribonucleic processing.


Pharmaceutical Technology
Volume 35, pp. s12-s17

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.

Generating a dendritic-cell immunotherapy


Figure 1: Overview of an autologous dendritic cell-manufacturing process for oncology and infectious-disease indications. (ALL IMAGES ARE COURTESY OF THE AUTHORS)
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.

In addition to the amplified RNA from the patient's disease, an RNA-encoding cluster of differentiation 40 ligand (CD40L) is added to the RNA payload. The purpose of adding this CD40L RNA is to provide the CD40–CD40L ligation signal required by the DC to induce IL-12 secretion (3–5). IL-12 is linked to the functionality of the DC because IL-12 secretion is one of the three signals required for a typical adaptive immune response (6). A technique for quantifying the release of IL-12 from the DC immunotherapy is in development as the drug product's potency assay (7).

This CD40L RNA is generated in bulk from a plasmid with one batch of CD40L RNA used for several batches of the DC immunotherapeutic drug product (8). For each batch of CD40L RNA, the plasmid is linearized, and uncapped CD40L RNA is generated using IVT methods. The uncapped CD40L RNA is capped and polyadenylated to generate the final CD40L RNA that is added to the RNA payload during electroporation. This process of maturing the DCs before electroporating them and adding CD40L RNA to the RNA payload has been called the postmaturation electroporation CD40L or the PME CD40L process (9). Dendritric cells resulting from this maturation process expand the central and effector memory T cells (CD8+CD28+) associated with favorable clinical outcomes (10).


Table I: Results of the cellular process based on elutriation, culture bags, and PME-CD40L maturation methods for manufacturing clinical-scale batches of RCC and HIV.
Following electroporation with the amplified RNA from the tumor or viral sample and the CD40L RNA, the DCs are cultured for 4 h with GM-CSF and IL-4 to recover, translate the RNAs, and process and present the resulting tumor or viral peptides. After culture, the DCs are harvested, formulated in autologous plasma collected during leukapheresis and cryoprotectants (i.e., dimethyl sulfoxide and dextrose), and frozen in multiple vials. Each vial is a single dose of drug product. These vials are stored cryogenically and shipped individually to the clinical site for administration to each subject. Implementing this cellular process based on elutriation, culture bags, and the PME-CD40L maturation method, yields a mean number of doses produced per batch greater than 20 for the RCC and HIV indications (see Table I). This method provides multiple years of dosing for a patient from a single leukapheresis.


Figure 2: Post-thaw immunophenotyping results confirmed the identity and quality of the dendritic-cell drug products generated for renal-cell carcinoma (RCC) and human immunodeficiency virus (HIV) after implementing the cellular process based on elutriation, culture bags, and PME-CD40L maturation methods. CD is cluster of differentiation, and HLA is human leukocyte antigen.
The drug-product release testing includes post-thaw total viable cell count and viability to verify the dose strength and immunophenotyping for identity (see Table I and Figure 2). Cell-surface markers CD80, CD86, CD83, and CD209 identify the cells as mature DCs with the appropriate co-stimulatory molecules to generate an immunostimulatory T-cell response. CD14 is a monocyte marker; therefore, the low percentage confirms that the monocytes were converted to DCs. Human leukocyte antigen-DR indicates the presence of major histocompatibility complex Class II receptor for peptide antigen presentation. These data demonstrate the consistency in the formulation and quality of the DCs despite the significant biological variability in the starting materials. The consistency in results between batches for each disease indication and for the two different disease indications establishes that the clinical manufacturing methods are robust.


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