Optimization of cGMP purification and expansion of umbilical cord blood-derived T-regulatory cells in support of first-in-human clinical trials.

BACKGROUND AIMS: Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft-versus-host disease (GVHD). We previously completed a "first-in-human" clinical trial using in vitro expanded umbilical cord blood (UCB)-derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation. METHODS: To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture. RESULTS: aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced. DISCUSSION: Expansion culture with cGMP aAPCs and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.

Successful "in-flight" activation of natural killer cells during long-distance shipping.

BACKGROUND: Natural killer (NK) cells have shown promise in the treatment of malignancy. However, the widespread use of these cells may be limited by both the lack of resources and the expertise needed to manufacture them and the apparent need to use only fresh cells. The NHLBI-sponsored Production Assistance for Cellular Therapies group was established to provide the resources and expertise to carry out cell therapy research, including support of clinical trials. Here we describe the qualification of in transit activation of an NK-cell therapy product in preparation for a Phase I clinical trial at a distant medical center. STUDY DESIGN AND METHODS: Nonmobilized apheresis mononuclear cell collections were CD3+ cell depleted, placed into culture bags with interleukin (IL)-2, and shipped from Minneapolis/Saint Paul, Minnesota, to Columbus, Ohio, and back to Minneapolis/Saint Paul, under warm, monitored temperatures. Products underwent quality control (QC) testing including cell count, immunophenotyping, viability, endotoxin, sterility culture, and cytotoxicity assays. One product tested the relative importance of IL-2 and controlled incubation. RESULTS: The length of shipment ranged from 14 to 16 hours, and temperatures were well controlled. QC testing was acceptable based upon previous in-house experience. Controlled incubation was not necessary for successful activation of NK cells, but IL-2 appeared essential. CONCLUSION: The need for novel cell therapies to be infused as fresh products may be a limitation for various cell types. However, we have shown that NK cells can be successfully shipped in the fresh state (allowing 48 hr from apheresis to product infusion) for use at clinical centers. Although IL-2 is critical for NK-cell activation, a 37 °C, 5% CO2 incubator is not.

The Minnesota Molecular and Cellular Therapeutics Facility: a state-of-the-art biotherapeutics engineering laboratory.

Molecular-, gene-, cellular-, and tissue-based therapies have become increasingly acceptable modes of clinical therapy. Regulatory requirements and oversight have increased, and the need for facilities suited for production of such therapies has become more apparent. The Minnesota Molecular and Cellular Therapeutics Facility is a state-of-the-art laboratory at the University of Minnesota, Saint Paul, Minn, that was designed to support production of biologic products for use in clinical trials. A talented staff experienced in the medical, scientific, technical, and regulatory aspects of the development, production, and administration of such products complements the special design and construction of the facility. Hematopoietic stem cells (HSCs) are manipulated for transplant, and current clinical trials involving novel therapies include the use of allogeneic natural killer (NK) cells and tumor vaccines for the treatment of various malignancies and suicide gene-transduced T cells for the prevention of graft-vs-host disease (GVHD) after bone marrow transplantation. Other therapies, including marrow-derived multipotent adult progenitor cells (MAPCs), umbilical cord blood (UCB) stem cells, regulatory T cells, skeletal myoblasts, and monoclonal antibodies, will be used to treat a spectrum of disease and are in various phases of development. Here we provide an overview of the Minnesota Molecular and Cellular Therapeutics (MMCT) Facility, detailing our approach to the manufacture of novel therapeutics and highlighting current and future activities.

Clinical production and therapeutic applications of alloreactive natural killer cells.

Recent advances have improved our understanding of natural killer (NK) cell-mediated alloreactivity after hematopoietic cell transplantation (HCT) or with adoptive transfer. NK cells contribute to a graft-versus-leukemia effect and may play a role in preventing graft-versus-host disease or controlling infectious diseases after allogeneic HCT. New discoveries in NK cell biology, including characterization of NK cell receptors and their interactions with self-HLA molecules and a better understanding of the mechanism of NK cell education have led to the development of novel strategies to exploit NK cell alloreactivity against tumors. While early studies using autologous NK cells lacked efficacy, the use of adoptively transferred NK cells to treat hematopoietic malignancies has been expanding. The production of allogeneic donor NK cells requires efficient removal of T- and B cells from clinical-scale leukapheresis collections. The goal of this chapter is to review NK cell biology, NK cell receptors, the use of NK cells as therapy and then to discuss the clinical decisions resulting in our current good manufacturing practices processing and activation of human NK cells for therapeutic use.

Massive ex vivo expansion of human natural regulatory T cells (T(regs)) with minimal loss of in vivo functional activity.

Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation. Natural CD4(+)CD25(+) regulatory T cells (nT(regs)) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nT(regs) has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nT(regs) could be purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial number of nT(regs) in UCB units is limited, and average yield after expansion was only 1 × 10(9) nT(regs). Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nT(regs). PB nT(regs) were purified under GMP conditions and expanded 80-fold to yield 19 × 10(9) cells using anti-CD3 antibody-loaded, cell-based artificial antigen-presenting cells (aAPCs) that expressed the high-affinity Fc receptor and CD86. A single restimulation increased expansion to ~3000-fold and yield to >600 × 10(9) cells while maintaining Foxp3 expression and suppressor function. nT(reg) expansion was ~50 million-fold when flow sort-purified nT(regs) were restimulated four times with aAPCs. Indeed, cryopreserved donor nT(regs) restimulated four times significantly reduced GVHD lethality induced by the infusion of human T cells into immune-deficient mice. The capability to efficiently produce donor cell banks of functional nT(regs) could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.

Good manufacturing practices production of natural killer cells for immunotherapy: a six-year single-institution experience.

BACKGROUND: Natural killer (NK) cells, a subset of lymphocytes and part of the innate immune system, play a crucial role in defense against cancer and viral infection. Herein is a report on the experience of clinical-scale, good manufacturing practices (GMPs) production of NK cells to treat advanced cancer. STUDY DESIGN AND METHODS: Two types of NK cell enrichments were performed on nonmobilized peripheral blood mononuclear cell apheresis collections with a cell selection system (CliniMACS, Miltenyi): CD3 cell depletion to enrich for NK cells and CD3 cell depletion followed by CD56 cell selection to obtain a more pure NK cell product. After overnight incubation with interleukin-2 (IL-2), cells were washed, resuspended in 5 percent human serum albumin, and then released for infusion. RESULTS: A total of 70 NK cell therapy products have been manufactured for patient infusion since 2000. For the CD3 cell-depleted NK cell products, the mean purity, recovery, and viability were 38, 79, and 86 percent, respectively. For the CD3 cell-depleted/CD56 cell-enriched NK cell products, the mean purity, recovery, and viability were 90, 19, and 85 percent, respectively. Gram stain, sterility, and endotoxin testing were all within acceptable limits for established lot release. Compared to the resting processed cells, IL-2 activation significantly increased the function of cells in cytotoxicity assays. CONCLUSION: Clinical-scale production of NK cells is efficient and can be performed under GMPs. The purified NK cell product results in high NK cell purity with minimal contamination by T cells, monocytes, and B cells, but it requires more time for processing and results in a lower NK cell recovery when compared to NK cell enrichment with CD3 cell depletion alone. Additional laboratory studies and results from clinical trials will identify the best source and type of NK cell product.

Cellular engineering of HSV-tk transduced, expanded T lymphocytes for graft-versus-host disease management.

Engineering donor T lymphocytes with inducible 'suicide genes', such as herpes simplex virus thymidine kinase, has potential to improve safety and efficacy in allogeneic transplantation by facilitating management of graft-versus-host disease. Elective administration of a relatively nontoxic pro-drug would induce in vivo negative selection of engineered lymphocytes specifically, sparing other donor hematopoietic cells. The engineered cells must retain immunologic function, and undergo negative selection in response to clinically attainable plasma concentrations of pro-drug. The cell engineering process itself, typically involving activation, transduction, ex vivo expansion, and selection, must produce clinically useful numbers of genetically modified cells at high purity. We discuss development of a cellular engineering manufacturing process that yields transduced, expanded T lymphocytes meeting these requirements.