Page 1739 - Hematology_ Basic Principles and Practice ( PDFDrive )

1544 Part IX Cell-Based Therapies

manufacturing the cells and the criteria that will be used to determine subset markers. When other constituents of the product may adversely
whether they can be released for clinical use. Many manufacturing affect the activity of effector cells, it may be necessary to set an upper
procedures use reagents and materials that are not approved for limit for contamination by these cells in the clinical product. Most
human use, and the application should include information about facilities perform some type of functional assay as a part of the release
these materials and what testing will be performed before they can process, which in the case of TILs may be cytokine release or cytotoxic
be used for manufacturing the clinical product. Justification should activity toward tumor cells of the appropriate histologic type.
be provided for the use of nonapproved media and supplements and
any potentially undesirable ancillary products, such as antibiotics.
Evidence should be provided on the efficacy of their removal before Allodepleted Cells
administration of the product.
The release criteria for autologous T-cell products are similar to The major drawback of DLI is the significant risk of GVHD, which
those described for generic Type 351 products. They include tests for is the most important source of treatment-related mortality. Given
sterility, endotoxin, mycoplasma, and identity and purity (i.e., by that the frequency of tumor or viral antigen-specific T cells in most
flow cytometry); some test of potency is recommended, but is not cases is considerably lower than that of alloreactive T cells, it is neces-
usually required for phase I studies. A draft of the certificate of analy- sary to expand antigen-specific T cells ex vivo not only to enhance
sis, which will be used for release, should be included in the IND antitumor activity, but also to try to separate GVHD from GVT
submission. effect. An alternative method for separating GVHD from GVT effect
is to selectively deplete alloreactive donor T cells ex vivo. Selective
allodepletion is performed by removing donor T cells that express
Tumor-Infiltrating Lymphocytes activation markers after coculture with nonleukemic recipient cells.
Activation markers investigated include CD25 and CD69 and the
TILs are cells harvested from tumor sites and are expanded ex vivo increased sensitivity of activated T cells to photosensitizing dyes. A
with IL-2. As the cells are being expanded, increased tumor specificity number of phase I/II studies using immunotoxin directed against
is achieved by pulsing the cells with tumor-specific peptides or CD25 have demonstrated the feasibility of this approach, with
transducing them with a retrovirus encoding a tumor-specific T-cell accelerated reconstitution of virus-specific and total T-cell numbers
receptor. A major limitation is that patients must have preexisting and low rates of severe GVHD. Relapse rates remained high in this
lymphocytes that can both respond to tumor and be expanded ex study, which probably reflects the high-risk nature of the patient
vivo. Transfer of these cells has led to tumor regression in 50% of population.
lymphodepleted patients with metastatic melanoma. Potential recipients of allodepleted cells often receive a CD34-
A number of methods have been developed for isolation of TILs selected HPC graft. As a part of the CD34 selection process, a nega-
from tumors. They include mechanical disruption, enzymatic treat- tively selected population of cells that includes T cells becomes
ment, differential centrifugation approaches, and positive immuno- available. However, this fraction usually is not used as the source of
magnetic selection. The method of choice depends on the type of cells for allodepletion. These cells usually have been obtained from
tissue from which the cells are to be extracted and the availability of donors who received granulocyte-macrophage colony-stimulating
reagents, such as MAbs, to enrich the T-cell population of interest. factor for mobilization of peripheral blood progenitor cells, and
As with T-cell depletion of allogeneic grafts, the predominant effector during processing for CD34 selection they were exposed to anti-
TIL cell population is not fully characterized, and the availability of CD34 MAbs. Therefore, it is preferable to obtain cells for allodeple-
this information should facilitate the design of more effective separa- tion from a peripheral blood draw of the donor before administration
tion techniques. A number of approved enzymes and centrifugation of growth factor for mobilization. The T-cell–enriched fraction is
media are available, and several companies have suitable MAbs that usually obtained by centrifugation on a Ficoll-Hypaque density
have been prepared under GMP conditions but have not been sub- cushion. The cells are washed and coincubated with irradiated recipi-
mitted for FDA approval. ent mononuclear cells, which act as stimulators. A convenient source
The extracted cells are expanded ex vivo, usually starting in semio- for stimulator cells is obtained by generation of a cell line from the
pen systems such as cluster plates. The initial populations may be donor by infection of his or her peripheral blood mononuclear cells
tested in a cytotoxicity assay in an attempt to identify the effector with a laboratory strain of EBV. The coincubation step produces
population, which can then be selected for expansion. The expansion stimulation of alloreactive donor T cells with resulting expression of
phase usually progresses through a number of types of cultures as cell activation markers, which can then be targeted to remove the alloreac-
numbers increase. These may progress from plates to T flasks and tive population. Methods for elimination include cytotoxic drugs
gas-permeable bags to hollow fiber and culture bag bioreactors. The (e.g., methotrexate), anti-CD25 MAbs conjugated to the toxin ricin,
culture medium contains IL-2 as the primary cytokine, although and immunomagnetic selection. All of the regulatory issues associated
other agents alone and in combination may be used to promote with the manufacturing and release of Type 351 products apply to
outgrowth of specific cell subpopulations. allodepleted T-cell products. Release criteria include routine assays
Some investigators have used irradiated tumor cells to restimulate for sterility and purity. HLA typing may be included as a confirma-
TILs during culture; others have performed selective separations to tion that the cells in the product are of donor origin. Functional
enrich the effector cells during expansion. Highly characterized tissue assays may include demonstration that the allodepleted cells fail to
culture media, such as the serum-free lymphocyte medium AIM V proliferate when cultured in a primary mixed lymphocyte reaction
(Thermo Fisher Scientific), have been used for expansion. The goal (MLR). If a suicide gene has been introduced (see next section),
is to use the simplest medium with the fewest additives that will testing will include demonstration that it can be efficiently
support growth of functional cells. When possible, the media should activated.
be free of animal serum and proteins to simplify the regulatory issues.
However, the FDA is aware that complex media and serum combina-
tions may be required to support growth of some cell types and is Suicide Gene-Transduced Lymphocytes
willing to consider them, particularly if the cells can be washed into
an approved excipient for administration. Although treatment with DLI has led to remission in patients with
Initiation of a phase I study using TILs or TIL subpopulations disease after HSC transplantation, unmanipulated cells also contain
will not require complete characterization of the effector cell popula- alloreactive T cells and can induce GVHD. The incidence of GVHD
tion, but some preliminary information should be available that ranges from 55% to 90% and is associated with a treatment-related
allows quantification of the putative effectors for dosing purposes. In mortality rate of about 20%. Approaches that maintain the GVT
most cases, flow cytometric analysis will be used, and the target effect while decreasing the incidence of GVHD have been evaluated
antigens may be pan–T-cell markers or specific combinations of T and include transduction of donor T cells with a “suicide gene.”