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Chapter 97 Graft Engineering and Cell Processing 1545

Genes can be introduced into DLI or alloreactive-depleted cells to antigen receptor often incorporates other moieties that are designed
express the herpes simplex virus-1 thymidine kinase (HSV-tk). If to dampen inhibitory signals from the cancer or to amplify a benefi-
GVHD develops, Ganciclovir is administered to the recipient, result- cial immune response. Manufacture of these cells requires preparation
ing in suicide of the transduced donor leukocytes. of an appropriate construct and delivery system (viral or nonviral
Alternative suicide genes based on the dimerization of Fas or vector) that is used to modify the expanded T-cell population. Release
Caspase-9 in the apoptotic pathway have been developed to circum- testing is similar to that required for other gene-modified cellular
vent the problem of immunogenicity of HSV-tk. The inducible therapeutics (see Fig. 97.4).
Caspase construct also encodes a selection marker, such as CD19,
that can be used to enrich the transduced population. In the event
that GVHD develops after administration of the Caspase-transduced Natural Killer Cells
cells, the suicide activation drug can be administered to the recipient
to eradicate the transduced T cells by dimerization of Caspase 9 and NK cells are effectors from the innate immune system that also
activation of the apoptosis pathway. mediate antiviral and antitumor immunity. Studies have shown that
haploidentical NK cells infused after lymphodepleting chemotherapy
can have antitumor effects. Most facilities use a simple positive selec-
Antigen-Specific Cytotoxic T Lymphocytes tion with clinical scale immunomagnetic methods involving CD56
selection or CD3 depletion for such products. These procedures
Two major prerequisites for generating antigen-specific cytotoxic T must be done under an IND because of lack of commercially avail-
lymphocytes (CTLs) are the identification of appropriate viral (in the able devices and reagents for this indication. Recent evidence has
case of EBV-associated lymphomas) or tumor target antigens and the suggested that cryopreserved NK cells have impaired functionality
availability of suitable antigen-presenting cells (APCs). After being on recipients, in contrast to cells that are infused fresh. This poses
identified, CTL lines can be generated by coculturing T cells with an additional challenge to the manufacturer, who must coordi-
APCs that express the target antigen. The lines are then expanded by nate testing and possible shipment of the fresh product. It is not
restimulation with the antigen of choice and the addition of cytokines clear whether the same finding may apply to other cell therapy
such as IL-2. products.
To generate antigen-specific CTLs, it is necessary to have a good When preparing an IND application for NK cell enrichment,
source of APCs and a source of antigen to present to T cells. A variety care should be taken to ensure that the MAbs used for the selec-
of APCs have been evaluated, including fibroblasts, monocytes, and tion or depletion are of the highest quality available. A certificate
DCs, using different sources of antigen, including virus lysate or of analysis for these reagents should be submitted with the IND
lysate of antigen-positive cells, peptides, or transduction of the APC, application, and the agency will probably require detailed informa-
such as lymphoblastoid cells (LCLs) and DCs with an immunodomi- tion on their manufacturing (i.e., to ensure that the process includes
nant antigen. Use of lysates as the antigen source can be problematic robust procedures for virus inactivation or removal). The same type of
because they are likely to be variable and difficult to standardize. information may be required for any ancillary reagents (e.g., buffers
Alternatively, many processing laboratories may not be experienced and protein sources used to supplement buffers). Preclinical data
in handling virus and virus-infected cells. demonstrating that the enrichment technique is effective should be
LCLs prepared using laboratory strains of EBV make excellent provided and accompanied by data from clinical scale validation
APCs for use in manufacturing EBV-specific T cells. They present enrichments, providing evidence that the NK cells can be separated
EBV antigens efficiently, and they express high levels of costimulatory with acceptable viability, purity, and yield for the proposed study. The
molecules. The normal procedure for generating the LCL is coincu- CMC section of the IND application should include information on
bation with EBV derived from the tamarin B95-8 cell line. This line the methods used to store, label, and administer the cells. Engineered
has undergone extensive testing and has been approved for use as a APCs, such as the K562-mb15-41BBL cell line, if used to stimulate
source of “clinical” EBV. The resulting LCLs are cultured in media NK cells, should initially have been grown and tested as a Master Cell
containing acyclovir to eliminate any residual EBV. The LCLs are Bank, from which a Working Cell Bank is generated and tested for
used to repeatedly stimulate T cells (as a mononuclear cell fraction routine use.
of peripheral blood leukocytes), resulting in a population that is
directed toward the immunodominant EBV antigens. These CTLs
can be frozen while release testing is performed. They are delivered Dendritic Cells
frozen to the bedside, where they are thawed and administered
intravenously. Release testing consists of the routine tests described DCs are powerful APCs that can be used in vitro or in vivo to elicit
earlier for Type 351 products. For allogeneic CTLs, functionality is immune response to the antigen they are presenting. A number of
usually assessed by cytotoxicity assays, which must demonstrate less studies have evaluated tumor antigen-primed DCs for treatment of
than 10% killing of autologous blasts. hematologic malignancies and solid tumors. Antigen-primed DCs are
One limitation of this approach is that expansion of virus-specific used to target T cells in vitro toward specific antigens. These cells
T cells to achieve adequate dose levels is extremely time consuming. then can be used therapeutically to eliminate viruses or tumor cells
All of these procedures require several weeks to generate APCs. More bearing that antigen. DCs can be derived from BM or cord blood
recently accelerated methods of production have been developed. and mobilized, and resting peripheral blood obtained from normal
These include the use of direct T-cell stimulation using mixtures of donors and patients. The normal procedure consists of enriching
peptides for overlapping regions of the target antigen(s) (PepMix, monocytes from the source material, which has been accomplished
JPT) and the use of disposable bioreactors that provide improved gas by plastic adherence or CD14-based immunomagnetic selection (or
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exchange, thereby markedly improving rates of T-cell expansion. by depletion of CD19 B cells and CD2 T cells). An alternative
An alternative strategy is to manufacture and bank third-party approach is use of elutriation to collect an enriched monocyte frac-
allogeneic EBV-specific CTLs with a range of HLA types so that an tion from the donor. A purpose-built elutriation system for collection
“off-the-shelf” product is available. Clinical responses have been of monocytes is available (the Elutra, Terumo BCT). This method
described for patients who received partially matched allogeneic has also been used successfully to enrich monocytes from cryopre-
CTLs. The generation of banks of CTLs requires careful donor served mobilized apheresis collections.
screening and HLA typing to allow at least partial matching with the The monocyte fraction can be cultured in polystyrene tissue
intended recipient. culture flasks or gas-permeable bags in culture medium containing
The use of T cells transduced to express chimeric antigen receptors IL-4 and granulocyte-macrophage colony-stimulating factor to
directed against tumor antigens has produced very promising results induce DC differentiation. This is followed by culture in medium
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in early-phase clinical trials in CD19 leukemias. The chimeric containing proinflammatory mediators to accelerate maturation. A