Research article A phase II study of human allogeneic liver-derived progenitor cell therapy for acute-on-chronic liver failure and acute decompensation Authors Frederik Nevens, Thierry Gustot, Pierre-François Laterre, Luc L. Lasser, Lyudmil E. Haralampiev, Victor Vargas, Desislava Lyubomirova, Agustin Albillos, Mustapha Najimi, Sébastien Michel, Ivaylo Stoykov, Noelia Gordillo, Yelena Vainilovich, Virginie Barthel, Nathalie Clerget-Chossat, Etienne M. Sokal Correspondence etienne.sokal@promethera.com (E.M. Sokal). Graphical abstract MELD-Na Child-Pugh 40 15 Completed 3-month follow-up Died during 3-month follow-up 30 Mean, SD, score Mean, SD, score 20 10 10 5 0 Pre1 1 2 3 Pre1 1 2 3 Months after the first dose Bilirubin CRP Leukocyte 50 100 30 Mean, SD, 1010 cells/L Mean, SD, mg/L 40 80 Mean, SD, mg/dl 20 30 60 20 40 10 10 20 0 0 0 Pre1 1 2 3 Pre1 1 2 3 Pre1 1 2 3 Months after the first dose Highlights Lay summary  In cirrhosis, ACLF and AD are acute phases associ- Patients with liver cirrhosis may suffer from the rapid ated with high risk of death. onset of organ failure or multiple organ failure asso-  HALPC therapy is classified as an ATMP that can ciated with a high risk of death in the short term. This potentially treat ACLF/AD. clinical study of 24 patients suggests that an advanced  Up to 2 i.v. infusions of 0.6–1.2×106 HALPC/kg body therapy based on the intravenous infusion of low weight appeared safe.  Post HALPC infusion, markers of systemic inflam- doses of human allogeneic liver-derived progenitor mation and altered liver function decreased grad- cells is safe and supports the next phase of clinical ually for the surviving patients. development of this type of therapy.  Day-28 and Month-3 survival rates were 83% and 71%; and no patient had ACLF at Month 3. https://doi.org/10.1016/j.jhepr.2021.100291 Research article A phase II study of human allogeneic liver-derived progenitor cell therapy for acute-on-chronic liver failure and acute decompensation Frederik Nevens,1 Thierry Gustot,2 Pierre-François Laterre,3 Luc L. Lasser,4,5 Lyudmil E. Haralampiev,6 Victor Vargas,7 Desislava Lyubomirova,8 Agustin Albillos,9 Mustapha Najimi,10 Sébastien Michel,11 Ivaylo Stoykov,11 Noelia Gordillo,11 Yelena Vainilovich,11 Virginie Barthel,11 Nathalie Clerget-Chossat,11 Etienne M. Sokal10,11,12,* 1 Department of Gastroenterology and Hepatology, University Hospitals, KU Leuven, Belgium; 2Department of Gastroenterology and Hepato- Pancreatology, C.U.B. Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium; 3Intensive Care Unit, Cliniques Universitaires Saint-Luc, UCLouvain, Brussels, Belgium; 4Gastroenterology Clinic, CHU Brugmann, Brussels, Belgium; 5Department of Hepatogastroenterology, CHU Brugmann, Brussels, Belgium; 6Department of Internal Diseases, Multiprofile Hospital for Active Treatment (MEDICA), Ruse, Bulgaria; 7Liver Unit, Hospital Vall d’Hebron, Universitat Autònoma Barcelona, CIBERehd, Barcelona, Spain; 8Department of Clinical Gastroenterology with Hepatology, Gastroenterology Clinic, University Multiprofile Hospital for Active Treatment “Georgi Stranski”, Pleven, Bulgaria; 9Gastroenterology and Hepatology, University Hospital Ramón y Cajal, Madrid, Spain; 10UCLouvain, Institute of Experimental and Clinical Research (IREC), Laboratory of Pediatric Hepatology and Cell Therapy (PEDI), Brussels, Belgium; 11Promethera Biosciences, Mont-Saint-Guibert, Belgium; 12Cliniques Universitaires Saint-Luc, UCLouvain, Pediatric Hepatology & Gastroenterology Unit, Brussels, Belgium JHEP Reports 2021. https://doi.org/10.1016/j.jhepr.2021.100291 Background & Aims: Human allogeneic liver-derived progenitor cells (HALPC, HepaStem®; Promethera Biosciences, Mont- Saint-Guibert, Belgium) are an advanced therapy medicinal product that could potentially alleviate systemic inflammation and ameliorate liver function in patients with acute-on-chronic liver failure (ACLF) or acute decompensation of cirrhosis (AD). Methods: This open-label phase II study was conducted in 9 centres in Belgium, Spain, and Bulgaria between 2016 and 2019. The primary objective was to assess the safety of HALPC therapy up to Day 28 and the secondary objectives were to assess its safety and preliminary efficacy up to Month 3. Results: The 24 treated patients (mean age: 51 years) were mostly male with an alcoholic cirrhosis. On pre-infusion Day 1, 15 patients had ACLF and 9 patients had AD. Two of the 3 initial patients treated with high HALPC doses (5×106 cells/kg body weight [BW]) had severe adverse bleeding events attributed to treatment. In 21 patients subsequently treated with lower HALPC doses (0.6 or 1.2×106 cells/kg BW, 1 or 2 times 7 days apart), no serious adverse events were related to treatment, and the other adverse events were in line with those expected in patients with ACLF and AD. Overall, markers of systemic inflammation and altered liver function decreased gradually for the surviving patients. The Day-28 and Month-3 survival rates were 83% (20/24) and 71% (17/24), and at Month 3, no patient had ACLF. Conclusions: The treatment of patients with ACLF or AD with up to 2 doses of 1.2×106 HALPC/kg BW appeared safe. The results of this study support the initiation of a proof-of-concept study in a larger cohort of patients with ACLF to further confirm the safety and evaluate the efficacy of HALPC therapy. Clinical Trials Registration: EudraCT 2016-001177-32. Lay summary: Patients with liver cirrhosis may suffer from the rapid onset of organ failure or multiple organ failure asso- ciated with a high risk of death in the short term. This clinical study of 24 patients suggests that an advanced therapy based on the intravenous infusion of low doses of human allogeneic liver-derived progenitor cells is safe and supports the next phase of clinical development of this type of therapy. © 2021 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction cirrhosis (AD) or acute-on-chronic liver failure (ACLF). Although The acute development of 1 or more major complications of the definitions of AD and ACLF can differ geographically, overall, cirrhosis can be classified as acute decompensation of liver these syndromes are characterised by single or multiple organ failure and a high risk of mortality.1–3 Notably, ACLF is also a condition linked with systemic inflammation and immune Keywords: Alcoholic liver disease; Stem cell; Liver regenerative medicine. dysfunction.4–7 The Day-28 and Month-3 transplant-free mor- Received 22 September 2020; received in revised form 16 March 2021; accepted 7 April 2021; available online 18 April 2021 tality rates have been estimated at, respectively, 32.8% and 51.2% * Corresponding author. Address: Promethera Biosciences, Watson & Crick Hill, Rue in ACLF patients, and 1.9% and 9.8% in patients with AD but Granbonpré 11, 1435 Mont-Saint-Guibert, Belgium. Tel.: +32-10-39-43-00; +32-2- without ACLF.1 When stratified by total bilirubin, the Day-28 and 764-13-86. Month-3 overall mortality rates have been estimated at, E-mail address: etienne.sokal@promethera.com (E.M. Sokal). Research article respectively, 26.4% and 39.4% in patients with ACLF with total assess the safety of different HALPC regimens up to Day 28; and bilirubin <5 mg/dl, and 63.7% and 76.3% in those with total the secondary objectives were to evaluate the clinical and bio- 8 bilirubin > −5 mg/dl. logical preliminary efficacy of the therapy, and to further assess ACLF can occur in 3–6% of patients with cirrhosis,2 and the its safety up to Month 3. prevalence of ACLF in hospitalised patients with AD ranges from 24% to 34%.2,9,10 ACLF can be classified into 3 grades, primarily based on the number of organ failures and with higher grading Patients and methods associated with higher mortality.1–3 Study design and participants At present, besides supportive care,3,11 there is no specific This open-label study was carried out in 9 centres in Belgium, treatment for ACLF that improves survival. Supportive care is Spain, and Bulgaria between December 2016 and October 2019. symptom-dependent and includes antibiotics for infections, The protocol, its amendments, and other relevant study docu- corticosteroids for severe alcoholic hepatitis, lactulose and mentation were approved by an independent ethics committee rifaximin for encephalopathy, vasopressors and albumin for in each participating country, and the study was conducted in hepatorenal syndrome. The Molecular Adsorbent Recirculating accordance with the Guideline for Good Clinical Practice of the System (MARS) and other artificial liver support devices can International Council for Harmonisation of Technical Re- improve cerebral and renal function but not ACLF prognosis.12,13 quirements for Pharmaceuticals for Human Use,29 the Declara- Circulatory support with vasopressors and intravenous (i.v.) al- tion of Helsinki (2013)30 and all applicable regulatory bumin infusion, which improves renal function, also does not requirements. HALPC (HepaStem®) were obtained from the appear to improve survival.14 Liver transplantation represents sponsor, Promethera Biosciences (Mont-Saint-Guibert, Belgium), the only definitive therapeutic option for patients with ACLF. and produced with an agreement of the Belgian Ministry of However, in contrast to patients with acute liver failure, most Health. Human tissue sourcing was collected and provided as patients with ACLF cannot be listed on the high-urgency trans- sourcing material by the hepatic tissue bank of the Cliniques plantation list, owing to advanced age, active alcoholism, un- Universitaires Saint-Luc, Brussels, Belgium, and by Promethera controlled infections, and multiple organ failure. Only 10–25% of LLC, Durham NC, USA, collecting organs from US organ pro- patients with ACLF receive transplants and 50–70% of the pa- curement organisations. All donors had not opted out from organ tients down for transplant surgery die on the waiting list.15 and tissue donation. Liver tissue was only obtained from organs Moreover, ACLF represents a large healthcare and economic or organ segments that could not be used for liver trans- burden as a result of prolonged hospitalisation and resource- plantation. All patients screened for the study provided written intensive treatment. Although defined differently from the informed consent. European Association for the Study of Chronic Liver Failure (EASL-CLIF) criteria, the number of ACLF hospitalisations in the Procedures USA rapidly increased by 6-fold during 10 years (2001–2011), Initially, eligible patients had Grade 1 or Grade 2 ACLF (based on leading to a 5-fold higher cost.16 the EASL-CLIF criteria) and the planned dose regimen was four The limited access to transplantation, as well as the absence infusions of 250×106 cells/dose. However, the protocol was of approved products, result in a persisting high unmet medical amended after a temporary halt of the study before which 3 need for ACLF. Accumulating preclinical and clinical evidence patients had been treated. In the amended protocol, eligible supports the therapeutic potential of bone-marrow-derived patients had (i) ACLF, or (ii) AD with the risk of developing ACLF mesenchymal stem cells (MSC) in various immune-mediated and an international normalised ratio (INR) > −1.2 and <2 (this diseases.17 These multipotent cells can differentiate into a vari- range was later changed to INR > −2). The dose and frequency of ety of cells of the mesodermal lineage, but their most relevant infusions were changed and adapted to the patient’s body medicinal properties are related to their potent immunomodu- weight (BW) to 0.6 or 1.2×106 cells/kg BW, administered either as latory properties.18 Umbilical or bone-marrow-derived MSC have 1 (initially) or 2 infusions, 7 days apart. been used to treat patients with ACLF, and were associated in one The main exclusion criteria included: (i) thrombosis of the study with a decrease in the model for end-stage liver disease portal vein; (ii) ongoing uncontrolled bleeding; (iii) septic shock (MELD) score, total bilirubin, and alanine aminotransferase;19 or non-controlled bacterial infection; (iv) circulatory failure; (v) and in another study with a significant increase in the 24- mechanical ventilation as a result of respiratory failure; (vi) week survival rate, an improvement of liver function and a treatment with corticosteroids for acute liver disease less than 1 lower incidence of severe infections.20 day before screening; (vii) previous organ transplantation and/or Human allogeneic liver-derived progenitor cells (HALPC) ongoing immunosuppressive treatments; (viii) postoperative therapy is classified as an advanced therapy medicinal product decompensation after hepatectomy; (ix) major surgeries/inva- (ATMP) by the European Medicines Agency and represents an sive procedures within 4 weeks before HALPC infusion; and (x) alternative potential stem cell treatment for ACLF. HALPC are MELD-Na score >35. derived from the parenchymal fraction of healthy human liver The screening period lasted up to 7 days (Fig. 1A). Patients tissue.21 HALPC have a liver-specific homing capacity after pe- were hospitalised in intensive care, intermediate, or standard ripheral i.v. infusion and have immunomodulatory and anti- units, depending on the severity of the patient’s disease. During fibrotic properties.22–26 The safety profile of HALPC therapy has the screening period, the patient’s comprehensive medical his- been extensively investigated in preclinical studies and HALPC tory was recorded, such as background condition leading to therapy has been safely administered in children with inborn cirrhosis, possible previous episode(s) of AD/ACLF, and factor(s) errors of metabolism.27,28 triggering AD/ACLF. The active study period lasted 28 days (±2 Here, we report the results of a phase II clinical study of days) and was divided into 2 periods; the treatment period HALPC therapy in cirrhotic patients with ACLF or with AD at risk during which HALPC therapy was administered, and the sur- of developing ACLF. The primary objective of the study was to veillance period. Patients were hospitalised during the screening JHEP Reports 2021 vol. 3 j 100291 2 A Dose 1 Day 1 Dose 2 Day 8 Screen Treatment period Surveillance period Safety and efficacy follow-up Interim: End: Max 7 days Day 1 to Day 14 Day 15 to Day 28 Month 1 Month 3 Month 12 B Screened, n = 35 10 Cohort 1: Cohort 2: Cohort 3: Cohort 4: Cohort 5: 1 or 2 doses 1 dose 2 doses 1 dose 2 doses 4-5x106 0.6-0.8x106 0.6x106 1.2x106 1.2x106 n=3 n=6 n=3 n=4 n=9 Full-analysis set ACLF1 = 2 ACLF1 = 2 ACLF1 = 2 ACLF1 = 4 ACLF1 = 2 ACLF2 = 1 ACLF2 = 4* AD = 4 AD = 1 AD = 3 • Not dosed 1* Safety-analysis set n=3 n=6 n=3 n=4 n=8 • Scheme of administration 1 1 2 • Stop rules not respected 1 1 Per-Protocol set n=2 n=5 n=3 n=3 n=5 • Liver transplant 1 3 Efficacy-analysis set n=3 n=3 n=4 n=8 (post amendment) • Death 1 2 1 3 Month 3 completion n=2 n=4 n=3 n=3 n=5 Fig. 1. Study design and patient disposition. (A) Schematic representation of the study design in accordance with the amended protocol. This article reports the study results of the interim analysis at Month 3. (B) Schematic description of patient disposition and allocation to cohorts and analysis sets. *Note that the AD/ ACLF grades are for diagnoses based on parameters assessed at screening; for the patient in Cohort 5 who was not dosed, the diagnosis was Grade 2 ACLF at screening. ACLF, acute-on-chronic liver failure; AD, acute decompensation of liver cirrhosis. and the treatment periods. Study visits were performed on Days HALPC therapy or its administration, and included clinically 1, 4, 8, 12, and 14 during the treatment period, and on Days 21 significant changes in clinical examinations, vital signs, labora- and 28 during the surveillance period. Subsequently, patients tory tests, cardiac Doppler ultrasound, and abdominal echog- entered the long-term follow-up period, including study raphy with Doppler. The relationship to HALPC therapy or study visits at Months 2 and 3 for the analysis of the study. All pa- procedures was based on the investigator’s assessment and tients received the same standard of care for liver disease confirmed by the safety monitoring committee and the sponsor’s throughout the study. In addition to receiving 1 bolus of glu- pharmacovigilance unit, and in line with the guideline for cocorticoids before HALPC infusion (see below), 8 patients ATMPs.31 received glucocorticoids before screening for severe alcoholic The secondary outcomes for preliminary efficacy assessments hepatitis, and 9 other patients received glucocorticoids after were at Day 28 and Month 3: (i) features of systemic inflam- the screening assessments or later for liver transplantation or mation such as C-reactive protein (CRP) level and leucocyte complications of ACLF. count, (ii) other serum biochemistry and haematological pa- rameters, and (iii) disease scores (CLIF-OF, CLIF-C ACLF, CLIF ACLF Outcomes grade, CLIF-C AD, MELD-Na, and Child-Pugh); and up to Month 3: The primary outcome was the occurrence of adverse events (AEs) (i) the occurrence of a fatal event, and (ii) the occurrence of a up to Day 28, graded by seriousness, severity, relationship to liver transplantation. JHEP Reports 2021 vol. 3 j 100291 3 Research article Table 1. Demographic characteristics at baseline (Day 1). activated partial thromboplastin time, thromboelastography Parameter N Value (TEG), and thrombin generation test (TGT) were performed before and at certain intervals up to 72 h after infusion. During Age* ± SD, years 24 50.5 ± 9.2 Female/male, n 24 7/17 the infusion, the patient was continuously monitored for po- Aetiology* 24 tential AEs. On the other days during the hospital stay, patients Alcoholic cirrhosis, n 23 were followed according to usual practice. Ulcero-haemorrhagic rectocolitis 1 complication, n ACLF Grade 1/Grade 2, n 24 10/5 Statistical analysis and sample size AD, n 24 9 The sample size was typical of a safety and preliminary efficacy Triggering factors* of AD/ACLF study evaluating cell-based ATMPs. The analyses were descrip- Active alcoholism/infection/hepatitis/other, n 24 20/3/1/3 tive and no formal hypotheses were assessed. Descriptive sta- Biochemistry mean ± SD (minimum–maximum) Creatinine (mg/dl) 24 1.0 ± 0.47 (0.33–2.4) tistics for quantitative variables consisted of number of observed INR 23 2.0 ± 0.5 (1.2–3.0) values, number of missing observations, mean, median, standard MELD-Na† 24 27 ± 4.2 (17–37) deviation (SD), minimum, and maximum. Categorical parame- Bilirubin (mg/dl) 24 20 ± 10 (7.2–35) ters were summarised using absolute (number of participants) Platelets (109/L) 23 132 ± 82 (40–292) and relative (percentages calculated based on non-missing data) Fibrinogen (g/L) 21 2.4 ± 1.1 (0.88–5.0) frequencies. Leucocytes (109/L) 24 12 ± 6.9 (2.0–29) Neutrophils (109/L) 17 10 ± 7.0 (0.88–26) CRP (mg/L) 23 29 ± 21 (0.23–83) AD, acute decompensation of liver cirrhosis; CRP, C-reactive protein; INR, interna- Results tional normalised ratio; MELD-Na, MELD, model for end-stage liver disease-sodium; Demography, disposition, and protocol amendment SD: standard deviation. * Parameters reported at screening; other parameters reported at baseline (Day 1). A total of 25 patients were enrolled and 24 patients received the † Note that no enrolled patient had a MELD-Na score >35 at screening. study treatment (Fig. 1B). Of the 24 treated patients (mean age: 51 years, mostly male; Table 1), 23 patients had alcoholic cirrhosis and 1 patient had biliary cirrhosis as a result of scle- The secondary outcome for safety was the occurrence of AEs rosing cholangitis associated with ulcerative colitis. At screening, of special interest (AESIs), including serious AEs (SAEs) with fatal 12 patients had Grade 1 ACLF and 4 patients had Grade 2 ACLF outcome, malignancies, liver transplantations, hospitalisations (Fig. 1B). At baseline (Day 1), 10 patients had Grade 1 ACLF, 5 for new episodes of ACLF, and AEs assessed by the investigator as patients had Grade 2 ACLF, and 9 patients had AD (Table 1). All possibly related to HALPC therapy. patients had bilirubin levels > −7.2 mg/dl (mean 20 mg/dl; maximum 35 mg/dl; Table 1). The triggering factors for AD or HALPC reconstitution and infusion ACLF were reported as alcohol intake in 20 cases, infection in 3 HALPC product (HepaStem®) was shipped in dry shippers filled cases, alcoholic hepatitis in 1 case, and listed as ‘other’ in 3 cases. with liquid nitrogen. Within 2 h before administration, HALPC A sentinel of 3 patients were treated first. The actual quantity were reconstituted in a diluent supplied by Promethera Bio- of cells injected in the first patient (with Grade 2 ACLF) was sciences to 5×106 cells/ml. Between 15 and 30 min before each negligible owing to the product sedimenting in the syringe. For infusion, a single bolus of 100 mg of hydrocortisone or equivalent subsequent patients, gentle agitation of the syringe during was administered to the patient. HALPC were administered to infusion secured proper infusion. the patient by i.v. infusion with gentle agitation of the syringe Each of the other 2 patients in the sentinel (Grade 2 ACLF and containing the cells during the infusion. AD at infusion) received approximately 5×106 cells/kg BW and On the same day before each infusion, a physical examination, both reported suspected unexpected serious adverse reactions an evaluation of vital signs, and blood tests were performed. (SUSARs). One SUSAR was severe bleeding at a jugular puncture Coagulation parameters, including INR, fibrinogen, platelets, site after the second infusion, and the other SUSAR consisted of Table 2. Adverse events (AEs) occurring during the first 28 days of the study (Safety Analysis set). AEs occurring during the active study period (within 28 days after HALPC infusion) Cohort 1 Cohort 2 Cohort 3 Cohort 4 Cohort 5 Total (N = 3) (N = 6) (N = 3) (N = 4) (N = 8) (N = 24) n (%) NoE n (%) NoE n (%) NoE n (%) NoE n (%) NoE n (%) NoE Any AE 3 (100) 11 6 (100) 46 1 (33) 1 4 (100) 23 7 (88) 40 21 (88) 121 Related to HALPC therapy 2 (67) 4 2 (33) 2 0 (0) 0 0 (0) 0 0 (0) 0 4 (17) 6 Related to study procedure 2 (67) 4 1 (17) 1 0 (0) 0 0 (0) 0 0 (0) 0 3 (12) 5 Leading to study stop 2 (67) 4 0 (0) 0 0 (0) 0 0 (0) 0 0 (0) 0 2 (8) 4 Any severe AE 3 (100) 5 3 (50) 9 0 (0) 0 2 (50) 2 3 (38) 9 11 (46) 25 Any serious AE 3 (100) 5 3 (50) 7 0 (0) 0 1 (25) 1 3 (38) 5 10 (42) 18 Related to HALPC therapy 2 (67) 4 0 (0) 0 0 (0) 0 0 (0) 0 0 (0) 0 2 (8) 4 Related to study procedure 2 (67) 4 0 (0) 0 0 (0) 0 0 (0) 0 0 (0) 0 2 (8) 4 Leading to study stop 2 (67) 4 0 (0) 0 0 (0) 0 0 (0) 0 0 (0) 0 2 (8) 4 Leading to death 1 (33) 1 0 (0) 0 0 (0) 0 1 (25) 0 2 (25) 2 4 (17) 4 n (%), number (percentage) of patients. HALPC, human allogeneic liver-derived progenitor cells; NoE, number of events. JHEP Reports 2021 vol. 3 j 100291 4 A 1-dose cohorts Neutrophils Cohort 1 and 2-dose cohorts C Platelets (1-dose cohorts) 50 Cohort 1 50 600 Cohort 2: 1 x LD Cohort 2: 1 x LD Cohort 3: 2 x LD 40 Cohort 3: 2 x LD 40 500 Cohort 4: 1 x HD Cohort 4: 1 x HD Cohort 5: 2 x HD 109 cells/L 400 109 cells/L Cohort 5: 2 x HD 30 30 300 20 20 200 10 10 100 0 0 0 4 8 12 14 21 28 4 8 12 14 21 28 Scr Pre1 0.2 1 2 3 4 8 11 13 20 27 r e1 r e1 Sc Sc Pr Pr (Pre2) Days after dosing Study day Fibrinogen B Dose 1 (1- and 2-dose cohorts) Dose 2 (2-dose cohorts) Dose 1 Platelets (2-dose cohorts) Dose 2 6 6 Cohort 2: 1 x LD 600 600 Cohort 3: 2 x LD 5 5 Cohort 4: 1 x HD 500 500 Cohort 5: 2 x HD 4 4 400 400 109 cells/L g/L 3 3 300 300 2 2 200 200 1 1 100 100 0 0 0 0 4 8 12 14 48 72 4 8 12 14 48 72 0.2 1 2 3 4 0.2 1 2 3 4 6 13 20 r e1 e2 r e1 e2 Sc Sc Pr Pr Pr Pr Hours after dosing Days after dosing Fig. 2. Haematology measurements in the Safety Analysis set during the treatment and surveillance periods of the study. (A) Neutrophil counts in Cohorts 2 and 4 (left graph) and in Cohorts 1, 3 and 5 (right graph). (B) Fibrinogen values after dosing in Cohorts 2 and 4 (left graph) and in Cohorts 3 and 5 (right graph). (C) Platelet counts in Cohorts 2 and 4 (upper graph) and in Cohorts 3 and 5 within 4 days after the first dose (lower left graph), and within 20 days after the second dose (lower right graph). HD, higher dose of 1.2×106 cells/kg BW; LD. lower dose of 0.6–0.8×106 cells/kg BW; Pre1, pre-dose 1 (Day 1); Pre2, pre-dose 2 (Day 8); Scr, screening. coagulopathy (clotting disorder) and severe persistent epistaxis non-serious and mild in intensity and were reported for 2 pa- after the single infusion. Consequently, the doses were modified tients in Cohort 2. One AE (flushing) was also related to study downward (to 0.6 or 1.2×106 cells/kg BW) and normalised to BW procedures and resolved on the same day. The other AE (non- for the remaining 21 patients (Fig. 1B). The allocation to treat- haemorrhagic vomiting) was followed by the Mallory-Weiss ment cohorts was also modified in light of that and because of a syndrome which was not related to HALPC therapy; these change in the method to estimate the number of cells adminis- sequelae resolved within 3 days. This latter patient also had an tered, such that the first 3 patients were assigned to Cohort 1, episode of bilious vomiting 2 weeks before inclusion in the and the other 21 patients were assigned to Cohorts 2–5. Given study. that the Per-Protocol set was smaller (N = 18) than the Safety Twenty-one of the 25 severe AEs, reported for 9 patients, Analysis set (SAS; N = 24) and that protocol deviations were were unrelated to HALPC therapy or study procedures, but were generally considered as not clinically important, the SAS was also typical of the clinical course of ACLF and AD (Table 2). Twelve of considered for the analysis of survival and efficacy parameters. the 21 AEs were serious, including 3 liver transplants and 4 that were fatal. The 4 fatalities occurred on Day 11 (decompensated Safety of HALPC therapy liver cirrhosis; Cohort 1), Day 7 (multiple organ failure; Cohort In total, 121 AEs were reported by 21 patients of the 24 patients 4), and Days 10 and 18 (septic shock and decompensated liver in the SAS during the active study period (Day 1–28; Table 2). cirrhosis, respectively; Cohort 5). The AEs were generally typical of the clinical course of ACLF and The haematological and biochemical parameters for the ma- AD, with the most frequent AEs being classified as gastrointes- jority of the SAS patients were not clinically significantly tinal disorders (36 AEs reported by 13 patients) and infections abnormal at baseline (Day 1) or during the active study period. and infestations (14 AEs reported by 10 patients). There was no consistent temporal connection between clinically No serious or severe AE was related to HALPC therapy or significant abnormal values and HALPC therapy. For example, study procedures with the regimens that included up to 2 doses clinically significant abnormal neutrophil counts were reported of 1.2×106 cells/kg BW (Cohorts 2–5; Table 2). The 4 serious and for 2 patients in Cohort 4 before treatment (Fig. 2A). During the severe AEs related to HALPC therapy and study procedures were active study period, clinically significant abnormal counts were reported for the 2 SUSAR patients in Cohort 1. Three of these AEs reported again for 1 of those patients in Cohort 4 who later died constituted the 2 SUSARs. Liver transplantation was the fourth of multiple organ failure on Day 7, and for 1 patient in Cohort 5 AE and was reported as related to HALPC therapy for the patient who died of septic shock on Day 10. who had the severe-bleeding SUSAR. Two other AEs related to There was no clear evidence that HALPC infusion in the dose HALPC therapy, flushing and non-haemorrhagic vomiting, were range 0.6–1.2×106 cells/kg BW increased the risk of bleeding. For JHEP Reports 2021 vol. 3 j 100291 5 Research article certain parameters associated with clotting, such as fibrinogen patients who had Class I anti-HLA antibodies at screening. and platelets, more frequent measurements with the amended However, for one of these 2 patients, the Class II anti-HLA anti- protocol were performed over the 3-day period(s) after dosing bodies were only detected after liver transplantation. In 4 other (Fig. 2B and C). Nevertheless, the values for these parameters patients without Class I (or Class II) anti-HLA antibodies at remained relatively stable, even though most fibrinogen con- screening, Class I and/or Class II anti-HLA antibodies were centrations and platelet counts at baseline and over the 3-day detected after treatment. However, 2 of those patients had also period after dosing were lower than normal (<2 g/l and <150 received blood transfusions. In 3 of the 6 patients with anti-HLA ×109/ml, respectively) for a number of patients (10/21 and 14/21, antibodies post-treatment, no anti-HLA antibodies were detec- respectively). ted in subsequent samples. As expected in patients with cirrhosis,32 mean values for factor VIII appeared elevated, whereas those for factor II and Clinical and biochemical observations following HALPC factor VII were low compared with expected normal ranges. The therapy levels of protein C, protein S, and antithrombin III were lower During the first 3 months of the study, 4 patients in the SAS (N = than the normal ranges at screening (not shown), as expected in 24) underwent liver transplantation and 7 patients died, patients with cirrhosis. However, no clinically significant de- including 1 of the liver transplant patients (Fig. 1B). Hence, the creases in coagulation factors were reported within 24 h after overall survival rate was 83% (20/24) at Day 28 and 71% (17/24) at HALPC infusion. Month 3, and the transplant-free survival rate was 71% (17/24) at Before infusion, D-dimer concentrations were in line with the Day 28 and 58% (14/24) at Month 3. When considering only the high concentration generally observed in patients with cirrhosis 15 patients with ACLF at Day 1, the overall survival rate was 73% (>5 mg/l).33,34 A transient increase, reaching its maximum at 4–8 (11/15) at Day 28 and 53% (8/15) at Month 3, and the transplant- h after HALPC infusion was observed in most patients. In the 4 h free survival rate was 67% (10/15) at Day 28 and 53% (8/15) at after infusion, fibrin clot formation or fibrinolysis appeared un- Month 3. Overall, the averages of the prognosis scores for the affected based on TEG data. Thrombin generation also appeared surviving patients in the SAS were lower at Month 3 than at Day unaffected as illustrated by endogenous thrombin potential peak 1 (Table 4), and no patient had ACLF at Month 3. values. Of the 21 patients included in the amended protocol (i.e. No thrombotic event was detected after HALPC infusion and Cohorts 2–5), 18 were assigned for further efficacy analysis (Ef- no specific signal indicating a perturbation in the coagulation ficacy Analysis set; Fig. 1B); hence, 3 patients in Cohort 2 (re- balance was detected except for 2 patients with pre-existing cipients of a single infusion of 0.6×106 cells/kg BW) were not coagulopathy who had coagulation failure and high INR at considered because they underwent liver transplantation within screening, which further increased after infusion. One of these 29 days after HALPC infusion. patients also had a very low fibrinogen level. The averages of the prognosis scores for these 18 patients No AEs arising from vital signs or physical examinations (e.g. improved over the 3-month follow-up, illustrated by the MELD- hypotension, hypertension, or findings associated with intestinal, Na (transplant prioritisation) and Child-Pugh scores (mortality pulmonary, cardiac, neurological systems, and skin) were related risk). At Day 28, Months 2 and 3, the average MELD-Na scores of to HALPC therapy or cell infusion. Liver parenchyma examination 21 (SD = 7.8), 15 (SD = 5.2) and 15 (SD = 5.2), respectively, were showed abnormalities in the majority of patients at baseline (15/ lower than at baseline (27; SD = 4.7). At Month 3, the MELD-Na 24), and at the end of the active study period at Day 28 (12/16). score was lower than the respective baseline for 12/13 (92%) The portal vein was patent for all patients at baseline and surviving patients, with MELD-Na score < −15 for 8/13 (61%) pa- remained as such up to Day 28. Abnormal cardiac Doppler ul- tients. At Day 28, Month 2, and Month 3, the average Child-Pugh trasound values were reported for 10/24 patients at baseline and scores of 8.8 (SD = 1.7), 7.4 (SD = 2.0) and 6.8 (SD = 1.5), 5/24 patients on Day 1. No major changes were observed in the respectively, were lower than at baseline (11; SD = 1.5). At Month 24 h after HALPC infusion. 3, the Child-Pugh score was lower than the respective baseline From Day 28 to Month 3, 3 more patients died from disease for 12/13 (92%) surviving patients, with Child-Pugh score − <6 for complications, 1 patient was hospitalised for AD and 1 patient 7/13 (53%) patients. received a liver transplant (Table 3). No additional laboratory Overall, markers of systemic inflammation (i.e. CRP and leu- abnormalities were reported. cocyte count) and altered liver function (i.e. serum biochemistry There was evidence of some immune recognition of HALPC in values that contributed to the MELD-Na and Child-Pugh scores) some patients. Class II anti-HLA antibodies were detected decreased gradually for the surviving patients over the 3-month (>1,500 mean fluorescence intensity) after treatment in 2 of the 4 follow up, most notably at Months 2 and 3 (Fig. 3A). The average Table 3. Adverse events of special interest (AESIs) occurring between Day 29 and Month 3 (Safety Analysis set). AESIs occurring between Day 29 and Month 3 Cohort 1 (N = 3) Cohort 2 (N = 6) Cohort 3 (N = 3) Cohort 4 (N = 4) Cohort 5 (N = 8) Total (N = 24) n (%) NoE n (%) NoE n (%) NoE n (%) NoE n (%) NoE n (%) NoE Any AESI 0 (0) 0 3 (50) 3 0 (0) 0 0 (0) 0 2 (25) 2 5 (25) 5 Leading to death 0 (0) 0 2 (33) 2 0 (0) 0 0 (0) 0 1 (12) 1 3 (12) 3 Liver transplant 0 (0) 0 1 (17) 1 0 (0) 0 0 (0) 0 0 (0) 0 1 (8) 1 Hospitalisation 0 (0) 0 0 (0) 0 0 (0) 0 0 (0) 0 1 (12) 1 1 (8) 1 n (%), number (percentage) of patients. NoE, number of events. JHEP Reports 2021 vol. 3 j 100291 6 MELD-Na Child-Pugh A 40 15 6 INR 5 Albumin Mean, SD, no units 30 Mean, SD, score 4 Mean, SD, g/dl Mean, SD, score 4 20 10 3 2 10 2 5 0 0 1 Pre1 1 2 3 Pre1 1 2 3 Pre1 1 2 3 Pre1 1 2 3 Months after the first dose Months after the first dose Creatinine Sodium B 50 Bilirubin 100 CRP 3 150 Mean, SD, mmol/L Mean, SD, mg/dl Mean, SD, mg/L 40 80 Mean, SD, mg/dl 2 140 30 60 20 40 1 130 10 20 0 0 0 120 Pre1 1 2 3 Pre1 1 2 3 Pre1 1 2 3 Pre1 1 2 3 Months after the first dose Months after the first dose Leukocyte Completed 3-month follow-up Died during 3-month follow-up 30 Completed 3-month follow-up Mean, SD, 1010 cells/L Died during 3-month follow-up Fig. 4. Additional haematology and serum biochemistry parameters in the 20 Efficacy Analysis set at baseline, Day 28 (Month 1), Month 2, and Month 3. (A) Means, standard deviations (SDs) and individual patient values for inter- national normalised ratio (INR; left graph) and albumin (right graph). (B) 10 Means, SDs and individual patient values for creatinine (left graph) and sodium (right graph). Baseline was pre-dose 1 (Pre1) on Day 1. Values for patients who died during the follow-up are indicated by square orange symbols. 0 Pre1 1 2 3 Months after the first dose bilirubin, CRP, and leucocyte values and their variability (SD) at Months 2 and 3 were lower than at baseline (Fig. 3B). Hence, bilirubin values declined to below 10 mg/dl for all surviving Fig. 3. Prognosis scores and representative serum biochemistry and hae- matology parameters in the Efficacy Analysis set at baseline, Day 28 (Month patients at Month 3, including those with CRP values > −20 mg/l at 1), Month 2, and Month 3. (A) Means, standard deviations (SDs), and indi- baseline. Furthermore, the averages and SDs of the INRs followed vidual patient values for model for end-stage liver disease-sodium (MELD-Na; a similar trend to that of bilirubin values (Fig. 4). By contrast, the left graph) and Child-Pugh scores (right graph). (B) Means, SDs, and individual average albumin score of 3.6 g/dl (SD = 0.6) at Month 3 was patient values for bilirubin (left graph), C-reactive protein (CRP; middle graph) marginally higher than at baseline (3.0 g/dl; SD = 0.6) and was at and leucocytes (right graph). Baseline was pre-dose 1 (Pre1) on Day 1. Values the low end of the normal reference range (3.5–5.4 g/dl; Fig. 4). for patients who died during the follow-up are indicated by square orange Creatinine and sodium levels appeared to be generally stable symbols. over the active study period suggesting that any kidney condi- tions did not worsen as could have occurred (Fig. 4). Discussion Table 4. Prognosis scores in the Safety Analysis set at baseline, Day 28, and ACLF is associated with single or multiple organ failure and a Month 3. high risk of mortality, whereas patients with AD may present at Day 1 (Baseline) Day 28 Month 3 maximum 1 non-renal organ failure and have lower mortality.1–3 Although a liver transplant represents the only definitive ther- Score* n Mean (SD) n Mean (SD) n Mean (SD) apeutic option, few ACLF patients receive one owing to advanced CLIF-OF 24 9.2 (1.4) 17 7.5 (1.7) 13 6.2 (0.4) age, active alcoholism, uncontrolled infections, and multiple or- CLIF-C ACLF 15 49.0 (6.9) 4 49.6 (5.7) 0 – gan failure, leaving an unmet medical need. Spontaneous re- CLIF-C AD 9 51.8 (8.3) 15 49.1 (8.5) 17 44.9 (6.5) MELD-Na 24 27.2 (4.2) 19 20.9 (8.0) 17 13.1 (5.2) covery may occur in a subset of patients under supportive care. Child-Pugh 24 10.8 (1.5) 16 8.9 (2.1) 16 6.4 (1.6) HALPC therapy is a medicinal product with a potential to treat ACLF, acute-on-chronic liver failure; AD, acute decompensation of liver cirrhosis; ACLF and improve this rate of recovery. Hence, we conducted an CLIF, chronic liver failure; MELD-Na, model for end-stage liver disease-sodium; SD, open-label phase II clinical study evaluating the safety and pre- standard deviation. liminary efficacy of HALPC infusions in patients with ACLF or AD. * Scores were calculated when data were available and allowed the score calculation. Scores were similar when data from patients who underwent liver transplantation The study population of 24 patients was small but typical of the were excluded. pathology1; and the analysis was descriptive with no formal JHEP Reports 2021 vol. 3 j 100291 7 Research article hypothesis testing (i.e. it was a feasibility study without a generally preserved in patients with AD/ACLF,40,41 was unaf- control group). fected after HALPC infusion. There was no specific signal indi- Initially, the dose of HALPC (5×106 cells/kg BW) was similar cating a perturbation in the coagulation balance (detected with to that administered with other candidate MSC treatments in the conventional coagulation tests and global tests of clot for- other clinical studies, and also shown to be safe in a previous mation [TEG and TGT]) following i.v. infusions of HALPC at doses trial of HALPC therapy in children with inborn errors of meta- of 0.6 or 1.2×106 cells/kg BW. bolism.27,35 However, as a consequence of severe adverse During the first 3 months of the study, 4 patients received bleeding events attributed to treatment in 2 of the first 3 patients liver transplants and 7 patients died (including a transplant (as explained below), the study was amended to reduce HALPC recipient). The 3 transplant recipients in Cohort 2 had the doses to 0.6 or 1.2×106 cells/kg BW, administered either as 1 or 2 operation before Day 28. Hence for the 24 treated patients – all infusions 7 days apart. For the subsequent 21 patients, no SAEs of whom had bilirubin levels above 5 mg/dl at screening – the were related to treatment, and other AEs were in line with those Day-28 and Month-3 survival rates were 83% and 71%, respec- expected in patients with ACLF and AD, suggesting that the tively, and the transplant-free survival rates were 71% and 58%, safety profiles of the amended HALPC regimens were acceptable. respectively. In the 15 ACLF patients, the survival rates were 73% The 2 severe bleeding events observed with the high dose and 53%, respectively. regimens may have arisen because the cells may have triggered The averages of the prognosis scores for the surviving patients the consumption of procoagulant factors from an already were generally lower (improved) at Month 3 than at baseline, depleted reservoir of those factors that is a typical feature of and no patient at Month 3 had ACLF. In those 18 patients, who cirrhosis.32 HALPC, as with other MSC or pancreatic islets, and via received 0.6–1.2×106 cells/kg BW and were not recipients of liver their expression of tissue factor which can activate the extrinsic transplants and bilirubin, CRP, and leucocyte values (markers of coagulation cascade, have known procoagulant activity.34–36 potential systemic inflammation) were generally lower at Month Nevertheless, MSC have been safely administered by the i.v. 3 than at baseline. Creatinine and sodium levels appeared to be route without anticoagulation medication, even if triggering generally stable over the 3-month period suggesting that any activation of the clotting system.34,37,38 In this study, some kidney conditions did not worsen. The suggestion of a progres- coagulation activity may have been manifested by the transient sive increase in albumin over time may have also indicated elevation of serum D-dimer concentration, which is typical of i.v. functional recovery. MSC and other cell-based treatments.33,34 With respect to the In conclusion, this clinical study suggests that the treatment coagulation factor profile, it was consistent with the known of patients with ACLF or AD with up to 2 doses of 1.2×106 profile for patients with cirrhosis39: slightly elevated levels of HALPC/kg BW is safe. This supports the next phase of clinical factor VIII, and low levels of factor VII, factor II, protein C, protein development of HALPC therapy, which is a proof-of-concept S, and antithrombin III. No clinically relevant drop in coagulation study in a larger cohort of patients with Grade 1 or Grade 2 factors was recorded 24 h after HALPC infusion. No thrombotic ACLF to confirm safety and to demonstrate preliminary evi- events were observed and thrombin generation, which is dence of efficacy. Abbreviations Authors’ contributions ACLF, acute-on-chronic liver failure; AD, acute decompensation of liver Supervised the design and conduct of the trial: ES, NC-C, VB, FN, TG. cirrhosis; AE, adverse event; AESI, AE of special interest; ATMP, advanced Participated in the acquisition, analysis, and interpretation of the therapy medicinal product; BW, body weight; CRP, C-reactive protein; data: ES, NC-C, VB, FN. EASL-CLIF, European Association for the Study of Chronic Liver Failure; Drafting the manuscript or revising it critically for important intel- HALPC, human allogeneic liver-derived progenitor cells; INR, interna- lectual content: all authors. tional normalised ratio; i.v., intravenous; MELD, model for end-stage liver disease; MSC, mesenchymal stem cells; SAE, serious AE; SAS, safety Data availability statement analysis set; SUSAR, suspected unexpected serious adverse reaction; TEG, The datasets generated during and/or analysed during the current study thromboelastography; TGT, thrombin generation test. are available from the corresponding author on reasonable request. Financial support Acknowledgements This work was supported and sponsored by Promethera Biosciences The authors thank all study participants. Matthew Morgan (MG Science during all stages of the trial and its analysis. The costs associated with the Communications, Belgium) provided scientific writing services and development and publishing of the manuscript, including scientific Frédéric Lin (Promethera Biosciences) provided editorial support in the writing assistance and statistical analyses were provided by Promethera development of this manuscript. Promethera Biosciences thanks the Biosciences. Walloon Region of Belgium for supporting this study under the grant Nr 7341 ‘HepaStem Clinical development: phase IIa in ACLF’. Conflicts of interest NC-C, IS, NG, YV, SM, and VB are employees of Promethera Biosciences. Supplementary data MN is a founder, patent holder, and consultant of Promethera Bio- Supplementary data to this article can be found online at https://doi.org/1 sciences and employee of UCLouvain. ES is a founder, patent holder, 0.1016/j.jhepr.2021.100291. consultant and board member of Promethera Biosciences and employee of UCLouvain. 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