Ex vivo-expanded highly pure ABCB5+ mesenchymal stromal cells as Good Manufacturing Practice-compliant autologous advanced therapy medicinal product for clinical use: process validation and first in-human data

Abstract

Background aim: Mesenchymal stromal cells (MSCs) hold promise for the treatment of tissue damage and injury. However, MSCs comprise multiple subpopulations with diverse properties, which could explain inconsistent therapeutic outcomes seen among therapeutic attempts. Recently, the adenosine triphosphate-binding cassette transporter ABCB5 has been shown to identify a novel dermal immunomodulatory MSC subpopulation.

Methods: The authors have established a validated Good Manufacturing Practice (GMP)-compliant expansion and manufacturing process by which ABCB5+ MSCs can be isolated from skin tissue and processed to generate a highly functional homogeneous cell population manufactured as an advanced therapy medicinal product (ATMP). This product has been approved by the German competent regulatory authority to be tested in a clinical trial to treat therapy-resistant chronic venous ulcers.

Results: As of now, 12 wounds in nine patients have been treated with 5 × 105 autologous ABCB5+ MSCs per cm2 wound area, eliciting a median wound size reduction of 63% (range, 32-100%) at 12 weeks and early relief of pain.

Conclusions: The authors describe here their GMP- and European Pharmacopoeia-compliant production and quality control process, report on a pre-clinical dose selection study and present the first in-human results. Together, these data substantiate the idea that ABCB5+ MSCs manufactured as ATMPs could deliver a clinically relevant wound closure strategy for patients with chronic therapy-resistant wounds.

Trial registration: ClinicalTrials.gov NCT03257098 NCT03267784 NCT03529877 NCT03860155.

Keywords: ABCB5; GMP manufacturing; advanced therapy medicinal product; chronic wound; mesenchymal stromal cells; venous ulcer.

Copyright © 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.

Morphology and growth behavior of cultured ABCB5+ MSCs. (A) Cultured MSCs at about 80% confluency, displaying a spindled-shaped appearance. Magnification ×200. (B) Live cell count measured over time (upper panel) in parallel with cell confluency as determined visually (lower panel) from representative donors. (C) Cdkn1α gene expression (means of relative quantification + SD) was determined at 30%, 70% and 99% confluency. (D) Cell cycle phase distribution expressed as mean (± SD) percentage of cells in G1, S and G2/M phases; n = 503 batches from 260 donors. SD, standard deviation. (Color version of figure is available online).

Figure 2.

Validation of the expansion process of ABCB5+ MSCs. (A,B) Validation of passage number. Individual CPDs (n = 15 donors, represented by different colors) (A) and mean (± SD) division rates (n = 15 donors) (B), plotted against the respective passage number, demonstrating continuous cell expansion during up to 16 passages. (C) Comparability and homogeneity of batches between different passages of a given donor and between different donors as assessed by microarray-based gene expression analysis performed on samples from four various passages per donor for eight donors. Within each donor, the quantile-normalized data sets from the three higher passages were compared with the lowest passage. From each of these between-passage comparisons, the median gene ratio (of all genes that had shown an average signal >20 and at least 2-fold differential regulation) was determined. These values were used to calculate the median gene ratio (mean ± SD) for each delta passage value over all eight donors; n = number of between-passage comparisons with the delta passage value specified. Original microarray data were uploaded in Gene Expression Omnibus (accession no. GSE145589) and are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE145589. (D) Relative amount (mean percentage ± SD) of ABCB5+ cells in culture, plotted against the respective passage number (n = 17 donors) (left panel), and absolute yield of ABCB5+ MSCs shown as single values from 500 batches derived from 260 donors, giving a mean count of 26.27 × 106 ABCB5+ MSCs per batch (represented by red line, SD = 19.76 × 106) (right panel). SD, standard deviation. (Color version of figure is available online).

Figure 3.

Effect of topical application of ABCB5+ MSCs on healing of experimental full-thickness skin wounds in NSG mice. (A–C) Wound sizes (means of both wounds of each animal) at 9 days (A), 11 days (B) and 13 days (C) after vehicle (control) or MSC application. (D) Total number of fully closed wounds at 13 days after vehicle (control) or MSC application, shown as percentage value for each group. (E–G) Immunohistochemical evaluation of the wounds for human CD31 (E), unspecific CD31 (antibody-detected mouse and human CD31) (F) and cytokeratin 14 (G). Expression was semi-quantitatively quantified as no (−), slight (1+), moderate (2+), strong (3+) and intense (4+) positivity. Shown are group means ± SD. Control: vehicle only (n = 12 wounds, six animals). Low dose: 1.875 × 105 ABCB5+ MSCs per wound (n = 18 wounds, nine animals; one animal was prematurely euthanized). Low to mid dose: 3.75 × 105 ABCB5+ MSCs per wound (n = 19 wounds, 10 animals; one wound was pathologically not evaluable). Mid to high: 7.5 × 105 ABCB5+ MSCs per wound (n = 20 wounds, 10 animals). High dose: 1.5 × 106 ABCB5+ MSCs per wound (n = 20 wounds, 10 animals). The high-dose group was excluded from wound size analysis (A–C) because of significantly smaller baseline wound size values (not shown). Non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.01 versus control. d., dose; IHC, immunohistochemistry; SD, standard deviation. (Color version of figure is available online).

Figure 4.

Effect of topical application of 5 × 105 autologous skin-derived ABCB5+ MSCs per cm2 wound area on healing of human chronic venous ulcers. (A) Photographs of the target wounds of three representative patients before (day 0, upper panel) and 12 weeks after (lower panel) cell application. For photographs of all patients who were eligible for efficacy evaluation, see supplementary Figure 4. (B) Wound closure kinetics before (during cell expansion) and throughout the 12-week period following cell application (efficacy follow-up). Shown are all patients who were eligible for efficacy evaluation. For patient 8, wound size data at week 4 are missing. Increase in the wound after week 6 coincided with two changes in routine wound care; namely, less frequent (once instead of twice weekly) dressing changes and a switch to less absorbent dressing (Mepilex instead of Biatain). For patient 9, wound size data at week 6 are missing; instead, week 7 measurement is included in the graph. Red shaded areas highlight the first 6 weeks of efficacy follow-up, during which wound healing was most pronounced. w6, week 6; w7, week 7; w12, week 12. (Color version of figure is available online).