No abstract

Allogeneic umbilical cord blood (UCB) cells have recently been used for transplantation following high-dose chemotherapy. However, the numbers of total cells, including progenitor cells, harvested are low compared with bone marrow or peripheral blood progenitor cell harvests. Therefore, we evaluated the potential of UCB cells for their ability to expand granulocyte-macrophage colony-forming cells (GM-CFC) and burst-forming unit-erythroid (BFU-E) cells over 10 days. We used an ammonium chloride lysing buffer to eliminate the majority of contaminating red blood cells. An average recovery of 61% of the starting number of white blood cells was obtained, while retaining 100% of the CD34+ cells. Ex vivo expansion cultures were established in Teflon cell culture bags (American Fluoroseal Corp, Columbia, MD) in 25 ml defined medium (Amgen Inc, Thousand Oaks, CA) containing 100 ng/ml each of stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), and megakaryocyte growth and development factor. Either unselected UCB cells or CD34+ UCB cells, selected with Magnetic Activation Cell Sorting technology (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany), were incubated for 10 days at 37 degrees C without refeeding. Unselected UCB cells seeded at 1 X 10(6)/ml produced an average expansion of 1.4-fold in total cells, 0.8-fold in GM-CFC, and 0.3-fold in BFU-E cells. By contrast, CD34+ selected UCB cells seeded at 1.0 X 10(4)/ml produced an average expansion of 113-fold in total cells, 72.6-fold in GM-CFC, and 49-fold in BFU-E cells. These data demonstrate that CD34+ cell selection is necessary for optimal expansion of both GM-CFC and BFU-E cells. The cell numbers thus obtained postexpansion may be sufficient for transplantation in adults.

The Amgen Cell Selection Device (ACSD) is a fully automated system based on the research scale magnetic-activated cell separation (MACS) system (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) for the selection of CD34+ cells. Leukapheresis products (LP) (n = 30) from normal donors mobilized with recombinant human granulocyte colony-stimulating factor (rhG-CSF) were selected with the ACSD to evaluate the performance of this system. The starting LP contained a median of 0.51% CD34+ cells (range 0.21%-1.54%) and a median WBC count of 3.0 x 10(10) (range 1-4.7 x 10(10) cells). After selection on the ACSD a mean purity of 91.5% +/- 0.6% CD34+ cells was obtained, with a median purity of 95.5% CD34+ cells. A median of 98 x 10(6) total CD34+ cells were recovered postselection, with a range of 31-323 x 10(6) cells collected from the LP. This represented a mean recovery of 81.7% +/- 6% of CD34+ cells and a median of 78% compared with starting CD34+ cell numbers in the LP. FACS analysis of the selected products demonstrated a 4-5 log depletion of T cell subsets, including CD3, CD4, CD8, and CD56 subsets. These data demonstrate the high performance obtained with the ACSD resulting in a final product of greater than 90% purity of CD34+ cells. CD34+ cells selected with the ACSD represent an ideal product for clinical applications, such as tumor cell purging, T cell depletion for allogeneic transplant, ex vivo expansion, and gene therapy.

Ex vivo expansion of cord blood (CB) cells requires CD34+ cell selection before expansion to obtain optimal numbers of progenitor cells. As a preliminary step to preclinical development of CB expansion, we have evaluated two clinical scale selection devices, the Isolex 300i (Baxter Healthcare, Immunotherapy Division) and the CliniMACS (Miltenyi Biotech Inc.), for CD34+ cell selection from frozen CB products. As expansion of CB results in differentiation of cells, there may be a depletion of stem cells. Therefore, only a fraction of the CB should be expanded while a portion of the CB is maintained unmanipulated for infusion. After thawing of 40% fractions of each CB product, we observed >95% viable cells, with a median total WBC count of 1.8 x 10(8) cells. Use of the Isolex 300i resulted in a median purity of 51% CD34+ cells (n=8) and a median recovery of 34% CD34+ cells. Use of the CliniMACS resulted in a median purity of 54% CD34+ cells (n=10) and a median recovery of 80% CD34+ cells. The absolute number of CD34+ cells recovered after selection varied with samples from 6.7 x 10(4) to 3.2 x 10(6) CD34+ cells. Expansion of CD34+ cells from both systems resulted in >20-fold expansion of CFU-GM, with a median of 44-fold expansion. These data demonstrate the feasibility of selecting small fractions of frozen CB products using clinical scale CD34+ cell selection devices.

Background: Interleukin-7 receptor a (IL-7Ra) is associated with autoimmune disease. Blocking its activation by interleukin-7 (IL-7) with a therapeutic monoclonal antibody may reduce pathogenic T cells and effectively control the autoimmune response in these disorders.Methods: Two flow cytometry-based assays were developed and implemented to evaluate the interaction between cell surface IL-7Ra and an anti-IL-7Ra monoclonal antibody (Ab1). The receptor occupancy assay utilized competing and noncompeting commercial detection antibodies for "free" and "total" IL7Ra, respectively. STAT5 phosphorylation (pSTAT5) was measured as a proximal biomarker of IL-7Ra inhibition by Ab1.Results: Monkeys administered Ab1 had no free IL-7Ra detectable on the CD31 T cell surface at 0.25 hours postdose through day 4, in all treatment groups. Ab1 treatment resulted in a significant reduction in total IL-7Ra, dropping to 53%, 44%, and 55% on day 4 at 0.3, 3, and 30 mg/kg, respectively, compared to predose levels. There were treatment-related decreases in the ability of IL-7 to induce STAT5 phosphorylation in both CD41 and CD81 T cells in monkey blood samples from all treated animals from 0.25 hours through Day 4 postdose.Conclusions: The nonclinical receptor occupancy assay was developed and applied to detect free and total IL-7Ra on the surface of CD31 T cells in cynomolgus monkeys treated with Ab1. The results showed good correlation with the phosphorylation of STAT5 and serum concentration of Ab1. The approach for IL-7Ra occupancy and pSTAT5 measurements established in monkeys can be utilized in clinical trials for pharmacokinetic/pharmacodynamic evaluation of Ab1 effect in humans. V C 2015 Interna-