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Chapter 97 Graft Engineering and Cell Processing 1539

Chapter 108). Considerable work has been done to determine
whether these opposing effects are produced by distinct subpopula-
tions of T lymphocytes. This would allow ex vivo manipulation of
allogeneic grafts to remove differentially the GVHD-producing T
cells while sparing those that mediate GVT responses. Various sub-
populations of T cells have been identified as candidate effector
subpopulations; however, there is no widespread consensus as to
which subsets should be targeted.
Methods are available for eliminating T cells from grafts using
approaches similar to those used for purging tumor cells. Early
techniques included use of soybean agglutinin to aggregate the major-
ity of nonprogenitor cells and rosetting of sheep erythrocytes with T
cells to facilitate their removal. Although successful, these techniques
are not “FDA friendly” and do not offer the specificity that likely is
required to engineer T-cell subpopulations in allogeneic grafts. This
is made possible by the use of MAbs directed toward the antigens
that are currently used to identify T-lymphocyte subpopulations. The
target population then can be removed with high efficiency using
immunomagnetic separation, as described previously for purging
autologous grafts. The challenge remains to identify the appropriate
target T-cell populations and to source clinical grade MAbs for these
procedures. A number of potential target antigens have been identi-
fied and separation techniques implemented. They range from
pan–T-cell depletions using antibodies to CD3 and CD2, to deple-
tions of helper and cytotoxic T cells using MAbs against CD4 and
CD8, to stimulation and removal of alloreactive populations by
targeting activation antigens.
Efforts have recently focused on depletion of αβ-positive T cells
Fig. 97.1 THE BIOSAFE SEPAX DEVICE. Used for automated processing from the graft. This spares the donor-derived alloreactive natural
of hematopoietic cells. A newer version of this device provides additional good killer (NK) and γδ T cells, which may provide a tumor-directed
manufacturing practice features. response. Promising early clinical results have been obtained using
this approach. An alternative approach is to administer regulatory T
cells (Tregs) posttransplant to prevent and treat GVHD. A first-in-
XpressTRAK software enables data tracking to assist with regulatory man clinical study was performed using in vitro-expanded Tregs from
compliance. partially HLA-matched third party umbilical cord blood units that
were administered to 23 patients receiving double-cord blood trans-
plants. Results were compared with 108 historical controls. No Treg
Purging of Autologous Grafts acute toxicities were seen, and there was a reduced incidence of grades
II–IV acute GVHD, but since this was a phase I study it was not
Autologous HPCs can be used for recipients lacking a human leuko- designed to demonstrate efficacy. Later studies support the finding
cyte antigen (HLA)-matched related or unrelated donor. It has been that donor Treg infusions can prevent GVHD after allogeneic trans-
proposed that occult viable tumor cells collected with the graft and plantation. Evidence that Tregs can be used to treat GVHD is still
returned to the patient could act as a source for disease relapse. under evaluation
Gene-marking studies have supported this hypothesis. As a result, Methods that eliminate or physically remove either T cells or
much effort has been exerted to develop methods for the ex vivo tumor cells from grafts are referred to as negative selection techniques.
detection and removal of tumor cells from autologous grafts. Tech- They are affected by variables such as target antigen expression,
niques have included incubation with chemotherapeutic drugs, such sensitivity of detection technologies for quantitating separation
as 4-hydroperoxycyclophosphamide, photosensitizing agents, and efficiency, and other technical hurdles. These may be difficult
antisense oligonucleotides. Alternatively, tumor-directed monoclonal to control in order to achieve the ideal composition of the graft
antibodies (MAbs) can be used to identify the cells and effect their and the target level of residual T cells, or T-cell subsets, remains
5
removal. The MAb-coated tumor cells can be eliminated by addition to be established. A dose of 10 T cells/kg is generally regarded
of serum complement, or by capturing them on a solid phase, such as the goal to minimize the risk of GVHD while facilitating
as a column matrix, a plastic sheet, or magnetic particles. These engraftment. There are also no approved devices for negative
particles may be large (5 µm diameter) so they can be collected, with selection, so these types of procedures must be performed under
the attached tumor cells, in a standard magnetic field. The matrix an IND.
material may be much smaller, such as nanoparticles or ferrofluids, For many years, the goal was to replace negative selection
which coat the cells. These are then collected on a metal matrix placed with a procedure in which HPC populations could be specifically
in a field generated by permanent magnets. Such systems are capable enriched by positive selection. This would effectively deplete T cells
of depleting 4–6 logs of tumor cells from a graft. However, even at or tumor cells from allogeneic and autologous grafts, respectively.
such high efficiencies, and given the limits of our ability to detect The problem was the lack of a method for identifying the target
residual tumor cells, the clinical value of purging autologous grafts is HPCs until the CD34 antigen was identified on a small popula-
debatable. There has also been a decline in interest in purging tech- tion of progenitor cells, including the pluripotent cells required
niques because of the potential benefits of a graft-versus-tumor for hematopoietic transplantation. The subsequent availability of
(GVT) effect detected in recipients of allogeneic grafts. MAbs directed against this antigen made possible the development
of techniques for enrichment of these cells. Immobilization of the
antibodies on a matrix (e.g., plastic sheets) and cellulose and magnetic
T-Cell Depletion of Allogeneic Products particles was used as the primary approach, and a number of devices
were commercially developed. The first to achieve FDA approval for
T cells in HPC grafts have the potential to cause severe or lethal use with apheresis products was the Baxter Isolex 300i, which uses
GVHD or potentially to exert a beneficial GVT effect (discussed in Dynal 5-µm magnetic beads as the separation modality and releases