Darin Sumstad

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.

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.

Phase I/II Trial of StemRegenin-1 Expanded Umbilical Cord Blood Hematopoietic Stem Cells Supports Testing as a Stand-Alone Graft

Clinical application of umbilical cord blood (UCB) as a source of hematopoietic stem cells for transplantation is limited by low CD34+ cell dose, increased risk of graft failure, and slow hematopoietic recovery. While the cell dose limitation is partially mitigated by using two UCB units, larger-dosed single units would be preferable. We have evaluated the feasibility and safety of StemRegenin-1 (SR-1), an aryl hydrocarbon receptor antagonist that expands CD34+ cells, by placing one of the two units in expansion culture. SR-1 produced a 330-fold increase in CD34+ cells and led to engraftment in 17/17 patients at a median of 15 days for neutrophils and 49 days for platelets, significantly faster than in patients treated with unmanipulated UCB. Taken together, the marked expansion, absence of graft failure, and enhanced hematopoietic recovery support testing of SR-1 expansion as a stand-alone graft and suggest it may ameliorate a limitation of UCB transplant.

A multicenter comparison study between the Endosafe® PTS™ rapid-release testing system and traditional methods for detecting endotoxin in cell-therapy products

BACKGROUND Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Healths Department of Transfusion Medicine, USA. METHODS Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing. RESULTS Valid paired results, based on each centers LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases. DISCUSSION The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.

Complement Fragment 3a Priming of Umbilical Cord Blood Progenitors: Safety Profile

Preclinical data showed that priming CD34(+) hematopoietic progenitor cells with complement fragment 3a (C3a) improved homing and engraftment. Thus, we hypothesized that priming of umbilical cord blood (UCB) hematopoietic progenitors with C3a would facilitate homing and could potentially be used to address the need for improved engraftment after UCB transplantation. We primed 1 of 2 UCB units for double UCB transplantation after nonmyeloablative conditioning. This design provided adequate safety and the potential to observe skewed long-term chimerism in favor of the C3a-primed unit as a surrogate measure of efficacy. C3a priming of 1 UCB unit did not result in infusional toxicity. Increased grades 1 to 3 hypertension were the only infusional adverse events observed in 9 (30%) patients. We observed no activation of inflammatory or coagulation pathways downstream of C3a. As tested, C3a priming did not impair engraftment, but did not skew chimerism toward the treated unit. As compared with historical controls, mortality and survival were not adversely affected. Thus, before any additional clinical studies, C3a priming to promote engraftment will require further preclinical optimization.

Shipping of therapeutic somatic cell products

BACKGROUND AIMS Shipment of therapeutic somatic cells between a current good manufacturing practice (cGMP) facility and a clinic or between different cGMP facilities requires validated standard operating procedures (SOP). Under National Heart Lung & Blood Institute (NHLBI) sponsorship, the Production Assistance for Cellular Therapies (PACT) group conducted a validation study for the shipping SOP it has created, including shipments of cryopreserved somatic cells, fresh peripheral blood specimens and apheresis products. METHODS Comparisons of pre- and post-shipped cells and cell products at the three participating facilities included measurements of viability, phenotypic profiles and cellular functions. The data were analyzed at the University of Pittsburgh Biostatistics Facility. RESULTS No consistent shipping effects on cell viability, phenotype or functions were detected for cryopreserved and shipped peripheral blood mononuclear cells (PBMC), monocytes, immature dendritic cells (iDC), NK-92 or cytotoxic T cells (CTL). Cryopreserved mesenchymal stromal cells (MSC) had a significantly decreased viability after shipment, but this effect was in part because of inter-laboratory variability in the viable cell counts. Shipments of fresh peripheral blood and apheresis products for the generation of CTL and dendritic cells (DC), respectively, had no significant effects on cell product quality. MSC were successfully generated from fresh bone marrow samples shipped overnight. CONCLUSIONS This validation study provides a useful set of data for guiding shipments of therapeutic somatic cells in multi-institutional clinical trials.

Loss of integrity of umbilical cord blood unit freezing bags: description and consequences

BACKGROUND: Umbilical cord blood (UCB) is now a commonly used resource for hematopoietic stem cell (HSC) transplantation; great effort has been put forth in standardizing protocols for processing, storage, and testing of UCB units. Because UCB units are selected on an individual basis to maximize the chance of engraftment, loss of container integrity may have adverse effects on patient outcome.

Interlaboratory assessment of a novel colony-forming unit assay: A multicenter study by the cellular team of Biomedical Excellence for Safer Transfusion (BEST) collaborative

BACKGROUND: Interlaboratory scoring performances were determined using a traditional 14‐day colony‐forming unit (CFU) assay and a new 7‐day CFU assay.

Transfusion-associated graft-versus-host disease: a perspective from a cell therapy laboratory

Transfusion-associated graft-versus-host disease (TAGVHD) is a rare complication of blood transfusion. TA-GVHD occurs from recipient exposure to foreign T-lymphocytes. While immunocompetent patients can contract TA-GVHD, immunocompromised patients are at the greatest risk of contracting the disease. TA-GVHD is nearly always fatal, and there is currently no treatment; thus, prevention of the disease is of the greatest importance. Irradiation and leukoreduction are the most common strategies for prevention of TA-GVHD. Pathogen inactivation also prevents the transfusion of functional T-lymphocytes, but the technology is currently unavailable in the United States. Irradiation remains the most accepted approach for patients known to be at risk for TA-GVHD. However, errors in identifying such patients and failures to irradiate products before issuing have occurred. TA-GVHD has occurred after infusion of leukoreduced products; however, leukoreduction has decreased the incidence of TA-GVHD. Certainly, while leukoreduction is more universally performed, there are limitations to this practice. According to the most recent FDA Guidance, up to 1 ¥ 10 white blood cells (WBCs) may remain per product (red blood cells [RBCs], apheresis platelets [PLTs]) with no recommendation for limits of specific WBC subsets. AABB Standards require that no more than 5 ¥ 10 WBCs be present after leukoreduction. Although all WBC populations are significantly reduced after filtration, depending on the method and filter used, the percent reduction differs among WBC subsets. Using a mean lymphocyte content of 40% for peripheral blood, adherence to either FDA Guidance or AABB Standards would result in approximately 0.4 ¥ 10 to 2 ¥ 10 total T-lymphocytes remaining in the product. Natural killer (NK) cells are a subset of human lymphocytes that are critical to the function of the innate immune system. Nonirradiated NK cell therapy products have been infused into immunocompromised cancer patients at the University of Minnesota for several years. Briefly, peripheral blood mononuclear cells are collected by apheresis from HLA-haploidentical, related donors. Reduction of T-lymphocytes is performed by CD3 selection using the CliniMACS cell separation system (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany). The CD3depleted product is incubated overnight with interleukin-2, washed, and then resuspended in 5% human serum albumin before infusion. Products are infused less than 24 hours after completion of the apheresis procedure. In this analysis CD3-depleted NK cell products were administered to 49 patients (40 acute myeloid leukemia/ myelodysplastic syndrome; 4 breast cancer; 4 ovarian cancer; 1 non-Hodgkin lymphoma) after medical therapy (60 mg/kg cyclophosphamide ¥two doses and 25 mg/m fludarabine ¥five doses). The mean nucleated cell dose was 3.34 ¥ 10 cells/kg (range, 1.58 ¥ 10-8.02 ¥ 10 cells/ kg) with a mean NK cell purity of 38%. While providing NK cells to patients, these products also include residual T-lymphocytes (mean, 0.47% of infused product) with a mean dose of 1.34 ¥ 10 cells/kg (range, 9.79 ¥ 106.62 ¥ 10 cells/kg). The mean absolute number of T-lymphocytes infused was 1.01 ¥ 10 cells (range, 8.70 ¥ 10-5.26 ¥ 10 cells), roughly 25-fold greater than that expected with FDA Guidelines for RBCs and apheresis PLTs. Although substantial T-lymphocyte doses have been given to this immunocompromised population, TA-GVHD has not been observed. This observation is particularly remarkable given the fact that the products are fresh and from related, haploidentical donors, factors which increase the risk for development of TA-GVHD. Interestingly, short-term engraftment and in vivo expansion of donor NK cells were seen in several patients who received these products. It is unclear what prevented Tlymphocyte engraftment and consequent TA-GVHD to occur with these patients. It may be that this lymphoreducing chemotherapy is not as immunosuppressive as previously thought. Additionally, several studies including a recent report have suggested that NK cells have a protective role against GVHD. We conclude that under some settings, higher T-cell numbers from haploidentical donors can be infused without obvious ill effects. This suggests that biology of TA-GVHD is not completely explained by cell dose alone. Further studies on the biology of TA-GVHD are warranted and may change transfusion practices and have implications for hematopoietic cell transplantation.