David H. McKenna

Massive ex Vivo Expansion of Human Natural Regulatory T Cells (T regs ) with Minimal Loss of in Vivo Functional Activity

A good manufacturing grade–compatible approach generates massive numbers of natural regulatory T cells that retain suppressive function in vivo. Cross-Checking Graft-Versus-Host Disease Fighting in hockey is a long-standing tradition: Stitches and gap-toothed smiles are badges of honor among these aggressive athletes. Yet, a balance must be maintained between the occasional high stick and an all-out melee. Black-and-white striped referees serve to uphold this balance, breaking up fights and preventing the bench-clearing brawl. Regulatory T cells (Tregs) are the referees of the adaptive immune system. They prevent the enforcers, cytotoxic T cells, from an overly exuberant response and, in the case of a bone marrow transplant, from attacking the patient’s own tissues. This process, called graft-versus-host disease (GVHD), is one of the risks of transplantation and differs from organ rejection. However, using Tregs to prevent GVHD has been limited by low Treg numbers and altered function after expansion in vitro. Hippen et al. now report a new way to expand Tregs to numbers much larger than those previously achieved while maintaining their ability to selectively suppress self-attacking cytotoxic T cells in vivo. Umbilical cord blood can be used to expand functional natural Tregs (nTregs); however, the initial number of nTregs in cord blood is limited. Therefore, the authors used peripheral blood as a source of nTregs for expansion. Using good manufacturing practice conditions and artificial antigen-presenting cells designed to stimulate T cell expansion, Hippen et al. expanded nTregs 80-fold after only one stimulation; they then showed that these multiplied cells maintained suppressor function. Stimulation of the nTreg population up to four times expanded the numbers of functional cells ~50 million–fold. When injected into mice at the same time as human T cells, these expanded Tregs significantly reduced mortality resulting from GVHD. Such large numbers of functional nTregs could be used to establish donor banks that would keep human GVHD and autoimmunity in check. Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation. Natural CD4+CD25+ regulatory T cells (nTregs) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nTregs has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nTregs could be purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial number of nTregs in UCB units is limited, and average yield after expansion was only 1 × 109 nTregs. Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nTregs. PB nTregs were purified under GMP conditions and expanded 80-fold to yield 19 × 109 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 × 109 cells while maintaining Foxp3 expression and suppressor function. nTreg expansion was ~50 million–fold when flow sort–purified nTregs were restimulated four times with aAPCs. Indeed, cryopreserved donor nTregs 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 nTregs could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.

A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BACKGROUND Natural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancer. METHODS Patients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m(2) × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometry. RESULTS Twenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30-65) years. Mean NK cell dose was 2.16 × 10(7)cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = <0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansion. CONCLUSIONS Adoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.

Mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices

Mesenchymal stem cells (MSCs) have recently generated great interest in the fields of regenerative medicine and immunotherapy due to their unique biologic properties. In this review we attempt to provide an overview of the current clinical status of MSC therapy, primarily focusing on immunomodulatory and regenerative or tissue repair applications of MSCs. In addition, current manufacturing is reviewed with attention to variation in practices (e.g., starting material, approach to culture and product testing). There is considerable variation among the 218 clinical trials assessed here; variations include proposed mechanisms of action, optimal dosing strategy, and route of administration. To ensure the greatest likelihood of success in clinical trials as the field progresses, attention must be given to the optimization of MSC culture.

Mesenchymal stem (stromal) cells for treatment of ARDS: a phase 1 clinical trial

BACKGROUND No effective pharmacotherapy for acute respiratory distress syndrome (ARDS) exists, and mortality remains high. Preclinical studies support the efficacy of mesenchymal stem (stromal) cells (MSCs) in the treatment of lung injury. We aimed to test the safety of a single dose of allogeneic bone marrow-derived MSCs in patients with moderate-to-severe ARDS. METHODS The STem cells for ARDS Treatment (START) trial was a multicentre, open-label, dose-escalation, phase 1 clinical trial. Patients were enrolled in the intensive care units at University of California, San Francisco, CA, USA, Stanford University, Stanford, CA, USA, and Massachusetts General Hospital, Boston, MA, USA, between July 8, 2013, and Jan 13, 2014. Patients were included if they had moderate-to-severe ARDS as defined by the acute onset of the need for positive pressure ventilation by an endotracheal or tracheal tube, a PaO2:FiO2 less than 200 mm Hg with at least 8 cm H2O positive end-expiratory airway pressure (PEEP), and bilateral infiltrates consistent with pulmonary oedema on frontal chest radiograph. The first three patients were treated with low dose MSCs (1 million cells/kg predicted bodyweight [PBW]), the next three patients received intermediate dose MSCs (5 million cells/kg PBW), and the final three patients received high dose MSCs (10 million cells/kg PBW). Primary outcomes included the incidence of prespecified infusion-associated events and serious adverse events. The trial is registered with ClinicalTrials.gov, number NCT01775774. FINDINGS No prespecified infusion-associated events or treatment-related adverse events were reported in any of the nine patients. Serious adverse events were subsequently noted in three patients during the weeks after the infusion: one patient died on study day 9, one patient died on study day 31, and one patient was discovered to have multiple embolic infarcts of the spleen, kidneys, and brain that were age-indeterminate, but thought to have occurred before the MSC infusion based on MRI results. None of these severe adverse events were thought to be MSC-related. INTERPRETATION A single intravenous infusion of allogeneic, bone marrow-derived human MSCs was well tolerated in nine patients with moderate to severe ARDS. Based on this phase 1 experience, we have proceeded to phase 2 testing of MSCs for moderate to severe ARDS with a primary focus on safety and secondary outcomes including respiratory, systemic, and biological endpoints. FUNDING The National Heart, Lung, and Blood Institute.

Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein

Haploidentical natural killer (NK) cell infusions can induce remissions in some patients with acute myeloid leukemia (AML) but regulatory T-cell (Treg) suppression may reduce efficacy. We treated 57 refractory AML patients with lymphodepleting cyclophosphamide and fludarabine followed by NK cell infusion and interleukin (IL)-2 administration. In 42 patients, donor NK cell expansion was detected in 10%, whereas in 15 patients receiving host Treg depletion with the IL-2-diphtheria fusion protein (IL2DT), the rate was 27%, with a median absolute count of 1000 NK cells/μL blood. IL2DT was associated with improved complete remission rates at day 28 (53% vs 21%; P = .02) and disease-free survival at 6 months (33% vs 5%; P < .01). In the IL2DT cohort, NK cell expansion correlated with higher postchemotherapy serum IL-15 levels (P = .002), effective peripheral blood Treg depletion (<5%) at day 7 (P < .01), and decreased IL-35 levels at day 14 (P = .02). In vitro assays demonstrated that Tregs cocultured with NK cells inhibit their proliferation by competition for IL-2 but not for IL-15. Together with our clinical observations, this supports the need to optimize the in vivo cytokine milieu where adoptively transferred NK cells compete with other lymphocytes to improve clinical efficacy in patients with refractory AML. This study is registered at clinicaltrials.gov, identifiers: NCT00274846 and NCT01106950.

Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs

Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs.

Therapeutic Potential of Mesenchymal Stem Cells for Severe Acute Lung Injury

Preclinical studies indicate that allogeneic human mesenchymal stem cells (MSC) may be useful for the treatment of several clinical disorders, including sepsis, acute renal failure, acute myocardial infarction, and more recently, acute lung injury (ALI). This article provides a brief review of the biologic qualities of MSC that make them suitable for the treatment of human diseases, as well as the experimental data that provide support for their potential efficacy for critically ill patients with acute respiratory failure from ALI. The article then discusses which patients with ALI might be the best candidates for cell-based therapy and provides a template for the regulatory and practical steps that will be required to test allogeneic human MSC in patients with severe ALI. There is a dual focus on how to design trials for testing both safety and efficacy.

Cell loss and recovery in umbilical cord blood processing: a comparison of postthaw and postwash samples

BACKGROUND: Engraftment after umbilical cord blood (UCB) transplantation is highly dependent on nucleated cell (NC) and CD34+ cell content. Current standard postthaw (PT) processing includes a wash step to remove dimethyl sulfoxide (DMSO), lysed red cells, and stroma. The contribution of the wash step to cell loss and ultimately the dose of cells available for transplant have yet to be systematically reported. This study examines the effect of the wash step as well as that of PT storage on various quality control variables of UCB units.

Umbilical cord blood-derived T regulatory cells to prevent GVHD: kinetics, toxicity profile and clinical effect

We studied the safety and clinical outcomes of patients treated with umbilical cord blood (UCB)-derived regulatory T cells (Tregs) that expanded in cultures stimulated with K562 cells modified to express the high-affinity Fc receptor (CD64) and CD86, the natural ligand of CD28 (KT64/86). Eleven patients were treated with Treg doses from 3-100 × 10(6) Treg/kg. The median proportion of CD4(+)FoxP3(+)CD127(-) in the infused product was 87% (range, 78%-95%), and we observed no dose-limiting infusional adverse events. Clinical outcomes were compared with contemporary controls (n = 22) who received the same conditioning regimen with sirolimus and mycophenolate mofetil immune suppression. The incidence of grade II-IV acute graft-versus-host disease (GVHD) at 100 days was 9% (95% confidence interval [CI], 0-25) vs 45% (95% CI, 24-67) in controls (P = .05). Chronic GVHD at 1 year was zero in Tregs and 14% in controls. Hematopoietic recovery and chimerism, cumulative density of infections, nonrelapse mortality, relapse, and disease-free survival were similar in the Treg recipients and controls. KT64/86-expanded UCB Tregs were safe and resulted in low risk of acute GVHD.

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.