Graft Engineering and Cell Processing

Routine Minimal Manipulation for Volume Reduction or ABO Incompatibility

The most widely used form of manipulation in the hematopoietic progenitor cell (HPC) processing facility probably is the removal of erythrocytes and plasma to overcome incompatibility between donor and recipient ( Table 103.2 ). This process is performed using techniques that were developed by the blood banking industry.

Plasma depletion to remove donor antibodies that may react with recipient cells is achieved by centrifugation of the graft, usually in a transfer pack, at approximately 2000 g for 10 minutes at ambient temperature. The pack is then placed in a plasma expresser, which compresses the product bag so that plasma can be forced out and into a separate collection bag. Plasma depletion can also be used to reduce the volume of ABO-compatible grafts when the donor is larger than the recipient.

Erythrocyte depletion removes incompatible donor erythrocytes that would stimulate a reaction by the recipient upon administration. Most facilities establish a maximum volume of incompatible RBCs that can be infused with HPC; exceeding this limit can result in hemolysis and a transfusion reaction. Depletion of erythrocytes can be achieved most simply by centrifugation at approximately 3000 g for approximately 10 minutes at ambient temperature. The leukocyte-rich buffy coat is then collected from the interface between the plasma layer and the RBCs.

Erythrocyte depletion can also be achieved by sedimenting erythrocytes using hydroxyethyl starch (Hetastarch). This promotes RBC sedimentation by the formation of erythrocyte rouleaux. The hematocrit of the product is first adjusted to ∼25% by the addition of normal saline, and 6% Hetastarch (Hespan) is added at a volume:volume ratio of 1:6 to 1:7. Sedimentation can be performed under gravity or accelerated by centrifugation.

A more rigorous erythrocyte depletion is achieved by centrifugation of the collection on a Ficoll-Hypaque density gradient. The cells are layered onto a density gradient cushion of Ficoll-Hypaque and centrifuged at 400 g for approximately 30 minutes. The mononuclear cells collect at the interface and are removed and washed before use. This process depletes erythrocytes, platelets, and granulocytes. The overall nucleated cell recovery is lower than with other techniques in which granulocytes are retained.

Automated devices are available for preparing buffy coats and density gradient-enriched cells. For larger volumes, the COBE 2991 Cell Processor from Terumo can be used. This requires a minimum volume of 150 mL RBCs for operation and may therefore not be suitable for pediatric processing. It is capable of preparing buffy coats and density-separated cell preparations using a functionally closed disposable set.

The COBE Spectra from Terumo is commonly used to collect PBPC by apheresis. The device is also less widely used in the processing facility to enrich mononuclear cells from BM.

For smaller starting volumes, the Sepax device from Cytiva (formerly GE Cell Therapy) ( Fig. 103.1A ) can be used. It has found widespread application in cord blood banks for buffy coat preparation (with or without the addition of hydroxyethyl starch) and has also been used for volume reduction of PBPC collections, density gradient separation of BM, and cell washing. The device has a small footprint, uses functionally closed disposables, and provides a print-out of operations.

The AXP II System for cord blood processing is available from Thermogenesis. The device is designed for enriching mononuclear cells from cord blood that are transferred to the processing set, which is then placed into the AXP device. This fits into a centrifuge bucket, and during spinning, the red and mononuclear cells are collected into separate bags, and the plasma is retained in the processing set. The PXP System performs a similar procedure on marrow harvests. Both devices provide closed sterile systems, and each system comes with software that enables data tracking to assist with regulatory compliance.

Purging of Autologous Grafts

Autologous HPC can be used for recipients lacking a human leukocyte antigen (HLA)-matched related or unrelated donor. It has been proposed that occult viable tumor cells collected with the graft and returned to the patient could act as a source for disease relapse. Gene marking studies have supported this hypothesis. As a result, much effort has been exerted to develop methods for the ex vivo detection and removal of tumor cells from autologous grafts ( Table 103.3 ). Techniques have included incubation with chemotherapeutic drugs, such as 4-hydroperoxy cyclophosphamide (4-HC), photosensitizing agents, and antisense oligonucleotides. Alternatively, tumor-directed monoclonal antibodies (MAb) can be used to identify the cells and effect their removal. The MAb-coated tumor cells can be eliminated by the addition of serum complement or by capturing them on a solid phase, such as a column matrix, a plastic sheet, or magnetic particles. These particles may be large (5 μm diameter—Dynabeads, Invitrogen), so they can be collected with the attached tumor cells in a standard magnetic field. The matrix material may be much smaller, such as iron-dextran nanoparticles (Miltenyi Biotec Bergisch Gladbach, Germany) or ferrofluids (Ferrotec Santa Clara, CA), which coat the cells. These are then collected on a metal matrix placed in a field generated by permanent magnets. Such systems are capable of depleting 4 to 6 logs of tumor cells from a graft. However, even at such high efficiencies, and given the limits of our ability to detect residual tumor cells, the clinical value of purging autologous grafts is debatable. There has also been a decline in interest in purging techniques because of the potential benefits of a graft-versus-tumor (GVT) effect detected in recipients of allogeneic grafts.

T-Cell Depletion of Allogeneic Products

T cells in HPC grafts have the potential to cause severe or lethal GVHD or exert a beneficial GVT effect. Considerable work has been done to determine whether or not these opposing effects are produced by distinct subpopulations 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 subpopulations of T cells have been identified as candidate effector subpopulations; however, there is still no widespread consensus as to which subsets should be targeted.

Methods are available for Pan-T cell elimination from grafts using approaches similar to those used for purging tumor cells (see Table 103.3 ). Early techniques included the use of soybean agglutinin to aggregate the majority of non-progenitor cells and the 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 possible by using MAb directed toward the antigens that currently are 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 MAb for these procedures. Several potential target antigens have been identified and separation techniques implemented. They range from pan–T-cell depletions using MAb to CD3 and CD2, depletions of helper and cytotoxic T cells using MAb against CD4 and CD8, to stimulation and removal of alloreactive populations by targeting activation antigens. In addition, methods have been developed for the selective manipulation of natural killer (NK) cells, natural killer T (NKT) cells, myeloid suppressor populations, and MSCs for administration to stem cell transplant recipients to prevent or treat GVHD.

Efforts have recently focused on the depletion of αβ-positive T cells from the graft. This spares the donor-derived alloreactive NK and γδ T cells, which may provide a tumor-directed response. Promising early clinical results have been obtained using this approach. Twelve patients received marrow or PBPC grafts depleted of ∼4 logs of αβ T cells, and all were alive at 3 months, and after 1 year, two had died. There was no severe GVHD (grade III to IV) post-infusion. A systematic review of the impact of γδ cells on the outcome of transplants concluded from 11 studies (919 patients), with a median follow-up of 30 months, that high γδ numbers were associated with lower relapse, fewer viral infections, and higher overall and disease-free survivals; however, no association was found between high γδ numbers and GVHD incidence.

Regulatory T Cells

A different approach for GVHD is to administer T _reg cells post-transplant to prevent and treat GVHD. A first-in-man clinical study was performed using in vitro expanded T _reg from partially HLA-matched third-party umbilical cord blood units that were administered to 23 patients receiving double cord blood transplants. Results were compared to 108 historical controls. No T _reg acute toxicities were seen, and there was a reduced incidence of grades II-IV acute GVHD but since this was a Phase 1 study, it was not designed to demonstrate efficacy. Later studies support the finding that donor T _reg infusions early after transplant can prevent GVHD after allogeneic transplantation. There are several questions still to be addressed. These include the source of T _reg cells (cord versus peripheral blood—cord blood generally yields cells of a higher purity but lower numbers); whether the cells should be expanded in vitro before use; the best method for their purification; and the best conditions for expansion.

T _reg represents a rare cell population whose phenotype cannot be categorically defined. They are generally enriched using their expression of CD25, sometimes incorporating prior depletion of C8 and CD19-positive cells. This leads to an enrichment of CD4 ⁺ CD25 ⁺ FOXP3 ⁺ cells which can be further increased to 87% purity by further in vitro expansion. The optimal method for enrichment appears to be the separation of CD4 ⁺ CD25 ^hi CD127 ^lo cells. Peripheral blood T _reg cells can be effectively expanded in vitro by stimulation with CD3 and CD28 MAb and high concentrations of IL-2. Alternatively, artificial antigen-presenting cells (APCs) primed with CD3 (and CD28) MAb have been shown to preserve FOXP3 expression while achieving cell expansion. Expansion of cord blood T _re can be further improved by using APC additionally primed with OX40L or 4-IBBL. Rapamycin can also be used to enhance TGF-β-dependent FOXP3 expression and to limit the activation and expression of effector T cells. It should be noted that cryopreservation of T _reg may impair their function.

Positive Selection of Progenitor Cells

Methods that eliminate or physically remove either T cells or tumor cells from grafts are referred to as negative selection techniques . They are affected by variables such as target antigen expression, the sensitivity of detection technologies for quantitating separation efficiency, and other technical hurdles. These may be difficult to control in order to achieve the ideal composition of the graft, and the target level of residual T cells, or T cell subsets, remains to be established. A dose of 10 ⁵ T cells/kg is generally regarded as the goal to minimize the risk of GVHD while facilitating engraftment. There are also no approved devices for negative selection, so these types of procedures must be performed under an IND.

For many years, the goal was to replace the negative selection with a procedure in which HPC populations could be enriched by positive selection. This would effectively deplete T cells, or tumor cells, from allogeneic and autologous grafts, respectively. The problem was the lack of a method for identifying the target HSC until the CD34 antigen was identified on a small population of progenitor cells, including the pluripotent cells required for hematopoietic transplantation. The subsequent availability of MAb directed against this antigen made possible the development of techniques for the enrichment of these cells. Immobilization of the antibodies on a matrix (e.g., plastic sheets),cellulose, and magnetic particles were used as the primary approach, and several devices were commercially developed (see Table 103.3 ). The first to achieve FDA approval for use with apheresis products were the Baxter Isolex 300i, which used Dynal 5 μm magnetic beads as the separation modality and released CD34 ⁺ cells from the beads using a competitive binding peptide. This device was taken over by Nexell, which ultimately went out of business in 2002.

The alternative is the CliniMACS system (Miltenyi Biotec, Fig. 103.1C ), which currently has regulatory approval in Europe, but still requires an IND for use in the United States for other than one specific indication (the depletion of T cells by CD34 ⁺ cells selection for the treatment of acute myeloid leukemia). This uses anti-CD34 or anti-CD133 nanoparticles to effect separation. The labeling with and removal of unbound MAb reagent is performed manually. The CD34 reagent-treated cells are then processed on the device, where they are retained on a column located in a high-gradient magnetic field. Non-labeled cells flow through the column and are collected in the negative fraction. The labeled cells are recovered from the column after several automated separations and washing cycles by removing the magnetic field. The nanoparticles remain on the CD34/133-positive cells; however, they are biocompatible and may be infused into the recipient. Recently the company has come up with REAlease nanoparticles that can be removed from the targeted cells, however, these are not yet clinically approved. The CliniMACS device normally achieves purities over 90% with yields of approximately 60%. This results in the passive depletion of 4 to 6 logs of allogeneic T cells or autologous tumor cells. The device may be used with a variety of MAb directed against antigens expressed by various types (T, B, and NK cells), potentially allowing it to be used as a platform for multiple types of graft manipulation.

Miltenyi has also introduced the Prodigy device ( Fig. 103.1B ), which can automatically perform many of the steps requiring manual intervention on the CliniMACS. Additional features provide the potential ability to fully automate the preparation of a variety of cellular therapy products (e.g., CAR-T cells) in a functionally closed system. The regulatory status of these devices should be discussed with the appropriate regulatory agencies before clinical use.

Positive selection techniques may, however, passively deplete from the graft certain cells that could be of potential benefit to the recipient. These include some stromal elements, GVT-mediating T cells, and other populations that may facilitate engraftment. As our understanding of the identity of these populations improves, it may be possible to recover them from the normally discarded negative fraction and add them back to the CD34 ⁺ cells or to administer them in the post-transplant period as donor leukocyte infusions).

Table 103.3 lists various methods for manipulation of HSC grafts to achieve tumor removal from autologous products and T cells depletion by HPC enrichment.