Long‐term efficacy and safety of intravenous injection of clonal mesenchymal stem cells derived from bone marrow in five adults with moderate to severe atopic dermatitis

Atopic dermatitis is a chronic and relapsing inflammatory skin disease that is treated with immunosuppressants. However, long‐term use of immunosuppressants may cause toxicity and severe side‐effects. To confirm the long‐term efficacy and safety of clonal mesenchymal stem cell therapy, we performed investigator‐initiated clinical trials and long‐term observation in five adult patients with moderate to severe atopic dermatitis that was refractory to conventional treatments. The clinical response assessment values such as Eczema Area and Severity Index (EASI) improved significantly at 16 weeks, and 80% (4/5) of the patients achieved EASI‐50 after one or two treatment cycles. Patients were observed for long‐term efficacy and safety for an average of 38 weeks (range, 16–86) and showed no serious side‐effects. Among the cytokines tested, CCL‐17, interleukin (IL)‐13, and IL‐22 significantly decreased at the end‐point of the five participants, two patients who maintained good clinical response over 84 weeks showed increased IL‐17 cytokine levels in the blood.

prior approval from the Korean Ministry of Food and Drug Safety.
The isolation protocol, called the sub-fractionation culturing method (SCM), was applied to generate single bone marrow cell-derived clonal MSC for this trial. 16,17 The multipotency including the capacity to differentiate into the three major mesenchymal lineages (osteoblasts, adipocytes, and chondrocytes) and the expression pattern of specific cell surface markers of general MSC were confirmed in all clonal MSC lines utilized in this trial. The trial was conducted over a period of 16 weeks (treatment for 4 weeks and follow-up for 12 weeks) and was followed by an observation period. In this clinical trial, 1.0 × 10 6 cells/ kg dose of SCM-CGH (fresh stem cell product) or SCM-AGH (frozen stem cell product) (SCM Lifescience) was administrated intravenously three times every 2 weeks at 2-4.5 mL/min infusion rate to enrolled patients. Participants were enrolled with an individual protocol for fresh or frozen form and one patient (S004) was injected with the frozen form of clonal MSC for two cycles while the other patients were injected with the fresh form. Concomitant medications such as corticosteroid-free emollients, low-potency (groups 6 and 7) topical corticosteroids, antihistamines, or anti-inflammatory drugs were permitted during study participation. Other detailed of the study design are in the Supplemental Study Protocol.
After 16 weeks of the trial, we followed up to confirm the period during which the patient's improved symptoms are maintained by oral antihistamines and/or topical medications including lowpotency (groups 6 and 7) topical corticosteroids without additional use of systemic steroids and systemic immunomodulators. Longterm safety and effectiveness were evaluated every 2 months. If the symptoms worsened during follow-up in patients who had improved clinical symptoms during the trial cycle, an additional cycle of treatments was conducted. Evaluation and follow-up of the additional cycle of treatments was conducted in the same way as the first trial.

| Participants
This clinical trial included healthy adults with moderate to severe AD diagnosed according to the criteria of Hanifin and Rajka. 18 The inclusion criteria included moderate to severe AD (Scoring Atopic Dermatitis [SCORAD] > 20), age from 20 to 60 years, and persistent symptoms (≥6 months). The exclusion criteria are in the Supplemental Study Protocol.

| Measurement of cytokine biomarkers
During the treatment cycle and follow-up period, blood was collected, allowed to clot at room temperature for 30 min, and then centrifuged for 10 min at 400 g. Serum samples were aliquoted, frozen at −80°C, and thawed immediately prior to analysis. Quantification of CCL-17

| Statistical analyses
Statistical analyses were performed using R software version 3.6.0 (R Foundation). Wilcoxon signed-rank test was performed to evaluate the statistical significance of the change in clinical and experimental assessment values between the scores documented at the baseline visit and the end-point. For statistical tests, the level of significance was set at p < 0.05.

| Characteristics of patients
This clinical trial included five patients who did not respond to conventional treatments (Table S2)

| Improvement of AD using clonal MSC
All primary efficacy end-points including a mean reduction of EASI, SCORAD, body surface area (BSA), and Investigator Global Assessment (IGA) in both cycles were statistically significant (Figure 1a and Table   S3). Both cycles were treated using the same protocol, and only S005 in the second cycle received two additional injections of MSC due to exacerbation of AD symptoms during the injection period. EASI reduction at the end-point for each patient were as follows: S001, 87% in the first cycle; S002, 82% in the first cycle and 56% in the second cycle; S003, 33% in the first cycle and 50% in the second cycle; S004, 5% in the first cycle and 51% in the second cycle; and S005, 46% in the first cycle and 37% in the second cycle. S001 achieved more than 50% reduction in EASI (EASI-50 response) in the first cycle ( Figure S2), and S002 achieved EASI-50 response in both cycles ( Figures S3 and S4). S003 and S004 did not achieve EASI-50 response in the first cycle, but achieved it in the second cycle. S005 did not achieve EASI-50 response in both cycles. For secondary efficacy end-points, a reduction in eosinophil count and eosinophil cationic protein was observed, but it was not statistically significant, and total IgE in the undiluted serum was similar at the end of both cycles ( Figure S1 and Table S3). Most primary and secondary end-points were evaluated at 12 weeks after clonal MSC therapy, and only those of the additional cycle for S003 were evaluated at 17 weeks.

| Long-term efficacy and safety of MSC therapy
Participants were followed up for an average of 38 weeks (range, 16-86) after screening to check how long the improved symptoms were

| Cytokine biomarkers in response to treatment
We measured several biomarkers (CCL-17, CCL-22, IL-13, IL-18, IL-22, and IgE) using ELISA with the appropriate dilution factor (Table S1). CCL-17, IL-13, and IL-22 significantly decreased at the end-point, and other biomarkers showed decreasing trends during the experiments (Figure 3 and Table S4). As a result of measuring IgE, difference was not observed in the pre-and post-treatment groups in total IgE in the undiluted serum ( Figure S1), but as a result of measuring IgE by diluting the serum, a decrease in IgE was clearly observed in patients 1 and 2, whose symptoms were clearly improved ( Figure 3 and