First‐in‐human high‐cumulative‐dose stem cell therapy in idiopathic pulmonary fibrosis with rapid lung function decline

Abstract Previous phase I studies demonstrated safety and some beneficial effects of mesenchymal stem cells (MSCs) in patients with mild to moderate idiopathic pulmonary fibrosis (IPF). The aim of our study was to evaluate the safety, tolerability, and efficacy of a high cumulative dose of bone marrow MSCs in patients with rapid progressive course of severe to moderate IPF. Twenty patients with forced ventilation capacity (FVC) ≥40% and diffusing capacity of the lung for carbon monoxide (DLCO) ≥20% with a decline of both >10% over the previous 12 months were randomized into two groups: one group received two intravenous doses of allogeneic MSCs (2 × 108 cells) every 3 months, and the second group received a placebo. A total amount of 1.6 × 109 MSCs had been administered to each patient after the study completion. There were no significant adverse effects after administration of MSCs in any patients. In the group of MSC therapy, we observed significantly better improvement for the 6‐minute walk distance in 13 weeks, for DLCO in 26 weeks, and for FVC in 39 weeks compared with placebo. FVC for 12 months in the MSCs therapy group increased by 7.8% from baseline, whereas it declined by 5.9% in the placebo group. We did not find differences between the groups in mortality (two patients died in each group) or any changes in the high‐resolution computed tomography fibrosis score. In patients with IPF and a rapid pulmonary function decline, therapy with high doses of allogeneic MSCs is a safe and promising method to reduce disease progression.


| INTRODUCTION
Idiopathic pulmonary fibrosis (IPF) is the most common of the interstitial lung diseases, a progressing chronic disease with unknown origin characterized by the development of fibrotic transformation of the lung parenchyma, predominantly in the older population. 1 The prevalence of IPF appears to be increasing, potentially explained by population aging. 2 The survival median of patients with IPF is 3 to 5 years; at the same time, the individual survival of patients may be extremely variable. [3][4][5] Conventionally, the IPF clinical course can be categorized into three types based on the rate of the forced ventilation capacity (FVC) decline: slow deterioration (less than 10% decline in FVC over 6 months or no changes in the disease indicators for many months), intermittent deterioration with episodes of exacerbations and rapid deterioration (more than 10% decline in FVC for 6 months). 3,6,7 For the last type, before the era of antifibrotic medications, the life expectancy of patients did not usually exceed 2 years from the time of diagnosis. [6][7][8] Besides acute exacerbations, a poor prognosis might be suspected in patients with IPF with such predictors as low values of FVC and diffusing capacity of the lung for carbon monoxide (DLCO), old age, and a high rate of pulmonary function decline. 3,9 The majority of clinical studies on the drug therapy of IPF did not evaluate the pretreatment functional reduction rate 10,11 ; therefore, it is difficult to judge its efficiency in patients with a fast lung function decline.
After a large number of successful preclinical studies, a treatment with mesenchymal stem cells (MSCs) is considered a potential new direction for lung fibrosis therapy. 12,13 The potential effects of MSCs in pulmonary fibrosis are related to their ability to produce a large number of biologically active substances with anti-inflammatory, immunosuppressive, and angiogenic properties. 12 The results of the published clinical trials on the cell therapy for IPF confirmed the safety of MSCs; however, all of these studies included patients with mild to moderate impairment of lung function, without taking into account the rate of their preceding decline. Also, the total dose of administered cells did not exceed 2 × 10 8 . 14- 16 We performed the first clinical trial in this field in patients who had a fast lung function decline, using a significantly more intensive intervention with respect to both the individual MSCs dose and the frequency of dose administration. Twenty patients were included into the study (age 33-74 years, 11 males, 9 females), all of whom had a histologically or radiologically confirmed pattern of usual interstitial pneumonia (UIP), with a history of a lung function decline (FVC or DLCO) ≥10% over the last 12 months and a current FVC ≥40% of predicted and DLCO ≥20% of predicted. The complete list of inclusion/exclusion criteria is shown in Supporting Information Appendix S1. According to the study protocol, those patients that met the eligibility criteria were randomized into two groups, with 10 patients per group: group 1 received four series of MSCs intravenous infusions, repeated after 12 weeks. Each of the series included two intravenous infusions performed in a 7-day interval. Each infusion contained a suspension of 200 million cells in 400 mL of normal saline. The last MSCs administration was performed at 39 weeks after the study beginning. The total number of MSCs administered in group 1 was 1.6 × 10 9 for the patients who completed the protocol. Group 2 received a placebo (400 mL of normal saline) with the same schedule. Sixteen patients completed the study ( Figure 2). The total characteristics of enrolled patients in each group are presented in Table 1. None of the patients took antifibrotic drugs because of their inaccessibility in the country during the study. Most of patients took prednisone as a single drug approved by the current

Lessons learned
• The primary objective was the evaluation of the safety and tolerability of repeated infusions of high doses of bone marrow-derived MSCs up to the total cumulative dose of 2 billion cells in subjects with rapidly progressing idiopathic pulmonary fibrosis.
• The evaluation was based on the number and severity of AEs related to the infusion during 52 weeks of follow-up.
• The secondary objective was evaluation of the main lung function parameters, such as forced ventilation capacity and diffusing capacity of the lung for carbon monoxide.
• The stem cell treatment has been found safe and well tolerable.
• Patients in the main group had their lung function increased, as compared to the placebo group, in which the continued decline of the lung function was observed.
• These findings allow us to conclude that such stem cell therapy is effective for the treatment of rapidly progressing idiopathic pulmonary fibrosis.

Significance statement
The results of this first-in-human clinical trial revealed that a high cumulative dose of mesenchymal stem cells (MSCs) is safe and well tolerated by patients with idiopathic pulmonary fibrosis with a rapid lung function decline. During the treatment period, the patients in the main group experienced increased lung function; however, the patients in the placebo group experienced a continued decline in lung function. Thus, this study shows the safety, tolerability, and potential benefits of greater doses of MSCs than those used earlier in patients with idiopathic pulmonary fibrosis, and these findings might move future trials toward a new step in stem cells transplantation. national guidelines. The diagnosis of IPF was based on the results of a multidisciplinary discussion (MDD) according to American Thoracic Society/European Respiratory Society criteria. 1 The primary endpoint was the evaluation of treatment-related adverse events (AEs), related to timing of each dosing regimen of the MSCs suspension. Vital signs (including body temperature, pulse rate, respiration rate, and blood pressure), oxygen saturation, 12-lead electrocardiogram, and clinical laboratory tests (including hematology, chemistry, and urinalysis) were  17,18 The 6MWTD was performed according to the АТS protocol. 19 A semiquantitative assessment of the HRCT pulmonary fibrosis score was performed by two independent radiologists using the method of Oda et al. 20 2.2 | MSCs preparation

| Isolation of human MSCs
For half of the patients (n = 5), the bone marrow for MSCs separation was obtained from young (20-35 years of age), healthy blood relatives. In cases of the absence of young, healthy relatives or their refusal (n = 5), we used the bone marrow from healthy donors with the same sex as the patient. In these cases, donors for each MSC injection were randomized. Each donor signed the informed T A B L E 1 Descriptive statistics for the groups at the baseline point*

| Osteogenic, adipogenic, and chondrogenic differentiation
The cultured MSCs (passage 5) were differentiated into the osteo- A Nikon Eclipse Ci microscope (Nikon Instruments Inc., New York) was used for the image capture.

| Karyotyping
Karyotypes were analyzed in MSCs from the first and the last (4 or 5) passages that were isolated and cultured in vitro using the chromosome multicolor fluorescence in situ hybridization (mFISH) technique. 22 The  ThermoFisher). ote: There were no significant differences between the study groups compared using the general linear model. Abbreviations: AEs, adverse events; IPF, idiopathic pulmonary fibrosis; LRTI, lower respiratory tract infection; URTI, upper respiratory tract infection.

| Statistical analysis
HRCT-based fibrosis score was calculated in accordance with the recommendations by Kundel and Polansky, using Cohen's kappa coefficient with 95% confidence limits. 23 A safety analysis was performed in the safety population. All AEs were compared in two groups by Fisher's exact criteria. A two-sided probability threshold of .05 was considered statistically significant. The descriptive data are presented as mean ± SD and the other data as median and interquartile range. Note: Data are presented as median (interquartile range). Abbreviations: 6MWTD, 6-minute walk test difference; DLCO, diffusing capacity of the lung for carbon monoxide; FVC, forced ventilation capacity; HRCT, high-resolution computed tomography; ND, no data. *Statistically significant differences (P < .05) between the study groups. **Statistically significant differences (P < .01) between the study groups.

| Safety
Of the 20 initially recruited patients, 16 completed the study. Adverse effects were more frequently observed in the group receiving MSCs; among them were fever and chills, predominantly in the first day after infusion (Table 2). However, these reactions were mild and did not require study discontinuation. Light fever after an The statistical analysis did not reveal any therapy-associated significant AEs (Table 2).
F I G U R E 3 Secondary endpoints dynamics. A-C, Differences (delta) of real values (y-axis) of forced ventilation capacity (FVC; A), diffusing capacity of the lung for carbon monoxide, B, and 6MWD, C, from the baseline level, during the treatment period (median, minimum and maximum, 25% and 75% quartiles are shown). D, Change from the baseline in FVC median (% of predicted) during 12 months before the treatment and during the treatment (by week 52) in the main group and the control. In the legend, 3, 6, 9, and 12 mean the values after 3, 6, 9, and 12 months, respectively

| Overall effects of MSCs versus placebo
After the first 3 months of treatment (administration of 400 million MSCs), we did not observe a statistically significant difference between the groups with regard to any of the studied indices, except for the 6MWTD, which increased by 15.3% in the MSCs group and decreased by 2.7% in the placebo group (Table 3) factors, and so forth. [30][31][32][33] Interestingly, in the therapy group, no episode of acute exacerbation was registered in any of the patients for the year-long follow-up period ( Figure 3D). This fact allows an assumption that the MSC therapy with In our study, we did not measure the levels of fibrocytes in the blood and MSC in the BAL liquid; however, we did not find radiological signs of fibrosis in the active therapy group.
The HRCT-based fibrosis score did not show statistically significant differences between the study group and the placebo group. This might be due to either the small number of study participants or the fact that The proangiogenic mechanism of the MSC effect is verified by the data obtained in the animal models of diabetes, physical overexertion, and diaphragm dysfunction, demonstrating that administration of different MSC types resulted in augmented angiogenesis in the muscle tissue, increase in the blood flow, and enhancement of the mitochondrial reserve and muscle function. [36][37][38][39] The proangiogenic potential is also confirmed by the recent proteomic studies showing overexpression of the proteins associated with angiogenesis in the MSCs' secretome, the angiogenic effect being proved with the tube formation assay. 40 It is of note that many proangiogenic factors are simultaneously anti-inflammatory; therefore, the classic point of view on the MSCs as inducers of only anti-inflammatory cytokines probably requires reconsideration. 40  The limitations of the current study include a small number of study participants as well as performance of treatment at a single center, which may explain the observed oscillations in the dynamics of certain indices (eg, substitution of an increase in DLCO by a decrease between 26 and 39 weeks) as well as the absence of significant differences in the fibrosis score assessment based on the HRCT data. Furthermore, the procedure was not blind for the researchers. We also could not investigate the distribution and survival of the transplanted cells in the recipients' bodies, their influence on the cytological analysis of the BAL fluid, or biomarkers of the fibrotic activity.

| CONCLUSION
We have shown in this study that high cumulative dose of transplanted bone marrow-derived MSCs is safe and well tolerated.
The safety of the MSCs therapy has been proved by the fact that even application of an extremely high dose of stem cells (the highest one on the record) has not been associated with any significant adverse effects, except for a transitory fever during the first day after the infusion noted in 4 out of 10 patients. During the treatment period, patients in the main group had their lung function increased, as compared with the baseline and the same parameters in the placebo group, in which the continued decline of lung function was observed. Thus, we state the safety, tolerability, and potential benefits of greater doses of MSCs than those used earlier in patients with IPF and other pulmonary diseases, and these findings can move future trials toward a new step in stem cells transplantation.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author.