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

ADVERTISEMENT

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.


BioPharm International
Volume 24, Issue 12, pp. 50-54

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.



blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

AbbVie/Shire Deal Officially Off
October 20, 2014
Amgen Sues Sanofi and Regeneron over Patent for mAb Targeting PCSK9
October 20, 2014
EMA Works to Speed Up Ebola Treatment
October 20, 2014
Lilly to Close Manufacturing Facility in Puerto Rico
October 17, 2014
BioReliance Introduces New Predictive Assays
October 17, 2014
Author Guidelines
Source: BioPharm International,
Click here