Off-the-shelf human umbilical cord mesenchymal stromal cell product in acute-on-chronic liver failure: A multicenter phase I/II clinical trial

Acute-on-chronic liver failure (ACLF) is a complex clinical syndrome characterized by high 28-day and 90-day mortality rates, ranging from 13% to 93%, resulting from the acute deterioration of liver function in patients with chronic liver disease. Transplantation of umbilical cord-derived mesenchymal stem cells (UC-MSCs) at different cell dosages has been shown to improve hepatic function in patients with decompensated cirrhosis or ACLF.^[1,2] Notably, most clinical studies to date have used freshly prepared MSCs. This trial aimed to evaluate the tolerability, safety, and efficiency of off-the-shelf UC-MSC products in patients with ACLF.

Study design: This study aimed to investigate the safety and efficacy of an off-the-shelf UC-MSC preparation (Tianjin AmCellGene Engineering Co., Ltd., China) as a therapy for patients with ACLF. This phase Ib and IIa clinical trial was conducted at three centers (Xijing Hospital, Beijing Youan Hospital, and The Fifth Medical Center of Chinese PLA General Hospital) between February 2022 and April 2024 in China. The study was approved by the Ethics Committee of Xijing Hospital (YS20211029-1; Chinese Clinical Trial Registry, ChiCTR2100050852). All patients signed the informed consent. Phase Ib trial was a single-arm, open, multicenter, multi-dose clinical trial designed to evaluate human safety, tolerability, and dose range (dose-escalation phase). Phase IIa trial was also a single-arm, open, multicentre trial with an expanded sample size to evaluate the clinical efficacy of multiple administrations (dose-expansion phase). Based on preclinical findings, the dose-escalation strategy followed the traditional “3 + 3” sequential enrollment approach. If no dose-limiting toxicity (DLT) was observed according to the Common Terminology Criteria for Adverse Events (CTCAE) v5.0, and no patient developed grade 3 or higher drug-related toxicity, including any drug-related toxicity that exceeded baseline and was considered clinically significant and/or unacceptable, within 7 days after the final dose, the next patient was enrolled. As no DLTs were observed in the three individuals in the one-dose group within 21 days of the final dose, the next dose group was initiated. According to the geometric progression increment method, three dose levels, 1.0 × 10⁶ cells/kg, 2.0 × 10⁶ cells/kg, and 4.0 × 10⁶ cells/kg, were established. Each dose was administered once weekly for 3 consecutive weeks via intravenous injection.

Participants and procedures: ACLF was defined according to the Guidelines for Diagnosis and Treatment of Liver Failure (2018) issued by the Chinese Medical Association.^[3] The inclusion criteria were as follows: (1) Patients with ACLF in the early or middle stages. Early stage: total bilirubin (TBIL) ≥171 μmol/L or daily increase ≥17.1 μmol/L; 30 % <prothrombin activity (PTA) ≤40% (or 1.5 ≤international normalized ratio [INR] <1.9); no complications or extra-hepatic organ failure. Middle stage: 20% <PTA ≤30% (or 1.9 ≤INR <2.6), with one complication and/or one extrahepatic organ failure. (2) Age between 18 and 70 years. The main exclusion criteria were as follows: (1) Active gastrointestinal bleeding; (2) severe infections; (3) malignancies; (4) alcoholic liver disease with abstinence for <6 months; (5) pregnancy or lactation; (6) previous or planned liver transplantation; (7) refusal by the patient or their family.

Endpoints: Safety endpoints included: (1) Any adverse events (AEs) related to UC-MSC treatment occurring during the clinical study; (2) determination of DLT and maximal tolerable dose (MTD). Primary efficacy endpoints included: Patient survival time without liver transplantation and clinical improvement rate assessed at 1, 2, 3, 4, 5, 8, 14, and 26 weeks after treatment. Clinical improvement was defined as meeting the following criteria: (1) Significant improvement in clinical symptoms and signs; (2) TBIL <5 × upper limit of normal, PTA >40%, or INR <1.5.

Preparation of UC-MSCs: Third-party UC-MSCs were provided by Tianjin AmCellGene Co. Ltd. (Tianjin, China) and approved by the China National Medical Products Administration. The UC-MSCs were derived from human umbilical cord tissue obtained from healthy donors, cryopreserved with suitable cryoprotectants, and stored in liquid nitrogen until passages P3. The final product, based on P6 hUC-MSCs, was also stored in liquid nitrogen. UC-MSCs were packaged in 50 mL cryopreservation-protected bags, each containing 2.0 × 10⁷ cells.

Statistical analysis: All statistical analyses were primarily descriptive and conducted using SPSS version 26.0 (IBM Corp, Armonk, NY, USA) or GraphPad Prism 9.0 (GraphPad; La Jolla, CA, USA). Quantitative data are presented as medians (minimum and maximum), and qualitative data as n (%). Statistical differences between the different cell dose groups were calculated using the independent sample t-test or Wilcoxon signed-rank test, with P values <0.05 considered statistically significant. Summary tables for AEs included all AEs that occurred during the study period. Survival curves were plotted using the Kaplan-Meier method and analysed using the log-rank test. The full analysis set (FAS), which included participants who received at least one treatment after enrolment, was used as the primary dataset for the security analysis. The per-protocol set (PPS), comprising participants who fully complied with the protocol, was used for the efficacy analysis.

Patient characteristics: A total of 34 patients with ACLF were successfully enrolled, including 9 patients in the dose-escalation phase and 25 in the dose-expansion phase [Supplementary Figure 1, http://links.lww.com/CM9/C573]. Three patients received 1.0 × 10⁶ cells/kg, 15 received 2.0 × 10⁶ cells/kg, and 16 received 4.0 × 10⁶ cells/kg. All 34 patients were included in the FAS, and 31 were included in the PPS sets. Baseline patient characteristics are presented in Supplementary Table 1, http://links.lww.com/CM9/C573. Most patients were male (27/34, 79%) and had hepatitis B virus (HBV)-related ACLF (19/34, 56%). Of all patients, 71% (24/34 ) had early-stage ACLF and 29% (10/34) had middle-stage ACLF. No significant differences were observed among the three dose groups in terms of age, sex, etiology, stage, alanine transaminase, aspartate aminotransferase, TBIL, direct bilirubin (DBIL), albumin (ALB), INR, PTA, model for end-stage liver disease, white blood cell count, hemoglobin, platelet count, glucose, and serum creatinine.

Dose-escalation results: According to the dose-escalation principle, nine patients participated in the dose-escalation phase: three received 1.0 × 10⁶ cells/kg, three received 2.0 × 10⁶ cells/kg, and three received 4.0 × 10⁶ cells/kg. None of the patients developed DLT during the evaluation period. Therefore, 4.0 × 10⁶ cells/kg was determined to be the MTD. None of the patients died during the observation period; therefore, clinical improvement rates were further evaluated. As shown in Table 1, one-third of patients with ACLF treated with 1.0 × 10⁶ cells/kg achieved clinical improvement at 4 weeks, and 50% achieved clinical improvement at 26 weeks. In contrast, more than two-thirds of patients treated with 2.0 × 10⁶ cells/kg or 4.0 × 10⁶ cells/kg showed clinical improvement at 4 weeks, and all patients in these groups achieved clinical improvement at 26 weeks. The cumulative clinical improvement rates across the three dose groups at weeks 1, 2, 3, 4, 5, 8, 14, and 26 were 23% (5/22), 77% (17/22), and 79% (19/24), respectively (P <0.05).

Table 1.

Analysis of clinical improvement rates in ACLF patients after UC-MSCs therapy during the dose-escalation phase and the dose-expansion phase.

Weeks after UC-MSC treatment)	Dose-escalation phase				Dose-expansion phase
	1.0 × 106 cells/kg (N = 3)	2.0 × 106 cells/kg (N = 3)	4.0 × 106 cells/kg (N = 3)	P values	2.0 × 106 cells/kg (N = 13)	4.0 × 106 cells/kg (N = 15)	P values
Week 1	0/3	1/3	1/3	1.000	3/13	3/15	1.000
Week 2	0/3	2/3	1/3	0.679	4/13	2/15	0.372
Week 3	0/3	2/3	2/3	0.357	5/13	4/15	0.689
Week 4	1/3	2/3	3/3	0.679	5/10	7/14	1.000
Week 5	1/3	3/3	3/3	0.250	6/10	8/14	1.000
Week 8	1/3	3/3	3/3	0.250	6/11	9/13	0.675
Week 14	1/2	2/2	3/3	0.571	8/10	9/13	0.660
Week 26	1/2	2/2	3/3	0.571	8/8	10/12	0.495
Total	5/22	17/22	19/24	<0.05	45/88	52/111	0.548

Data were shown as n/N. ACLF: Acute-on-chronic liver failure; UC-MSCs: Umbilical cord-derived mesenchymal stem cells.

Clinical efficacy after the dose-expansion phase: During the dose-escalation phase, no DLTs occurred, and clinical improvement in patients treated with 2.0 × 10⁶ cells/kg or 4.0 × 10⁶ cells/kg was superior to that treated with 1.0 × 10⁶ cells/kg. Therefore, 2.0 × 10⁶ cells/kg and 4.0 × 10⁶ cells/kg were selected for the dose-expansion cohort. As shown in Supplementary Table 2, http://links.lww.com/CM9/C573, four patients in the moderate-dose group died between 3 and 23 weeks after cell therapy, and three patients in the high-dose group died between 6 and 11 weeks after cell therapy. The primary causes of death were gastrointestinal bleeding, liver failure, or infection. The survival rates of patients with ACLF at weeks 4 and 12 following completion of the entire UC-MSCs infusion protocol were 88% and 79%, respectively. The survival curves for patients treated with the three cell doses showed no statistically significant difference (P = 0.792) [Supplementary Figure 2, http://links.lww.com/CM9/C573].

The clinical improvement rates between the moderate-dose and high-dose groups during the dose-expansion phase were similar at weeks 4, 8, and 26, as well as across all cumulative time points (45/88 [51%] vs. 52/111 [47%], P = 0.548) [Table 1]. Supplementary Figure 3 [http://links.lww.com/CM9/C573] illustrates the trends in TBIL, DBIL, PTA, INR, ALB, Child-Turcotte-Pugh (CTP), and other related parameters over time across different dose groups. After treatment, liver function, coagulation parameters, and CTP scores improved significantly in all three groups. From 4 weeks onwards, the moderate- and high-dose groups exhibited a progressively more significant improvement trend.

Safety and AEs: A total of 550 AEs were recorded in 34 patients [Supplementary Table 3, http://links.lww.com/CM9/C573]. Of these, 304 AEs were grade 1, 166 were grade 2, 57 were grade 3, 13 were grade 4, and 10 were grade 5. A total of 81 AEs were considered possibly related to UC-MSC therapy, including 26 AEs in eight patients (53%) in the moderate-dose group and 55 AEs in 15 patients (94%) in the high-dose group. Ten AEs were worse than grade 3 according to the CTCAE associated with UC-MSC therapy. These included two AEs in two patients (13%) in the moderate-dose group and eight AEs in five patients (31%) in the high-dose group.

As shown in Supplementary Table 4, http://links.lww.com/CM9/C573, the most common AEs following UC-MSC therapy were fever, decreased platelet count, leukopenia, neutropenia, anemia, decreased lymphocyte count, hyponatremia, hypokalemia, insomnia, gastrointestinal hemorrhage, leukocytosis, hyperkalemia, hyperglycemia, and COVID-19 infection. Among these, the grade ≥3 AEs potentially related to MSC therapy included decreased platelet count, leukopenia, neutropenia, and anemia. No serious treatment-related AEs leading to treatment discontinuation were observed.

To better evaluate the efficacy and safety of UC-MSCs in the treatment of ACLF, this study focused on patients in the early and middle stages of the disease. As expected, a total of seven out of the 34 patients with ACLF died from liver failure between 3 and 23 weeks after UC-MSC therapy. This strategy helped rule out deaths caused by UC-MSC treatment and, simultaneously, allowed for a comprehensive evaluation of UC-MSC-related AEs.

The MSC dosages used for ACLF treatment have varied widely, ranging from (0.75 ± 0.50) × 10⁶/kg to 5 × 10⁷/kg or (3.4 ± 3.8) × 10⁸/kg.^[4,5] In the present study, 4.0 × 10⁶ cells/kg was determined to be the MTD. Meanwhile, cumulative clinical improvement rates in patients treated with 2.0 × 10⁶ cells/kg or 4.0 × 10⁶ cells/kg were superior to those treated with 1.0 × 10⁶ cells/kg. However, neither short-term mortality nor clinical improvement rates differed significantly between the moderate- and high-dose groups following the dose-expansion phase. This may be attributed to the following factors: (1) The sample size of the study was relatively small; (2) Most enrolled patients with ACLF were in the early or middle stages of disease; (3) The efficacy of the two cell doses may have been equivalent. Nevertheless, these findings support the need for further investigation in future phase III and IV clinical trials.

To date, systematic observations and detailed documentation of AEs associated with UC-MSC therapy remain limited. In the present study, all patients were monitored for AEs related to UC-MSC treatment throughout the clinical trial. These findings highlight the need for greater attention to safety monitoring in future clinical treatment processes.

Funding

This study was supported by grants from the National Key Research and Development Program of China (No. 2020YFA0710804), Key Research and Development Program of Shaanxi (No. 2023ZDLSF-34), and Clinical Research Project of Air Force Military Medical University (Nos. 2021LC2105 and 2023LC2311).

Conflicts of interest

None.

Supplementary Material

cm9-138-2347-s001.docx ^{(267.5KB, docx)}