Allogeneic ABCB5+ Mesenchymal Stem Cells for Treatment-Refractory Chronic Venous Ulcers: A Phase I/IIa Clinical Trial

Open AccessPublished:October 25, 2021DOI:https://doi.org/10.1016/j.xjidi.2021.100067
      A significant number of chronic venous ulcers (CVUs) fail to heal despite guideline-conform standards of care. Skin-derived ABCB5+ mesenchymal stem cells can dampen the sustained IL-1β‒driven inflammation present in chronic wounds. On the basis of their wound healing‒facilitating effects in a mouse CVU model and an autologous first-in-human study, ABCB5+ mesenchymal stem cells have emerged as a potential candidate for cell-based advanced therapy of nonhealing CVUs. In this interventional, multicenter, single-arm, phase I/IIa clinical trial, subjects whose CVUs had emerged as standard therapy resistant received one or two topical applications of 1 × 106 allogeneic ABCB5+ mesenchymal stem cells per cm2 wound area, in addition to standard treatment. Of 83 treatment-emergent adverse events, only three were judged related to the cell product; they were mild or moderate and recovered without sequelae. Wound size markedly decreased from baseline to week 12, resulting in a median wound size reduction of 76% (full analysis set, n = 31), 78% (per-protocol set, n = 27), and 87% (subset of responders, n = 21). In conclusion, the study treatment was well-tolerated and safe. The treatment elicited a profound wound size reduction within 12 weeks, identifying ABCB5+ mesenchymal stem cells as a potential candidate for adjunctive therapy of otherwise incurable CVUs. These results justify the conduct of a larger, randomized, controlled trial to confirm clinical efficacy.

      Abbreviations:

      CVU (chronic venous ulcer), FAS (full analysis set), IQR (interquartile range), MSC (mesenchymal stem cell), PP (per-protocol set), TEAE (treatment-emergent adverse event)

      Introduction

      Although venous leg ulcers can often be successfully treated, a significant number of ulcers become chronic (
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      Prognostic factors associated with healing of venous leg ulcers: a multicentre, prospective, cohort study.
      ;
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      • Edwards H.E.
      Risk factors for delayed healing in venous leg ulcers: a review of the literature.
      ) (Table 1). Even after 5 years of repeated conservative therapy, 8% of chronic venous ulcers (CVUs) were still remaining unresolved (
      • Callam M.J.
      • Harper D.R.
      • Dale J.J.
      • Ruckley C.V.
      Chronic ulcer of the leg: clinical history.
      ). From a pathophysiologic perspective, CVUs are unable to progress through the normal wound repair pattern (
      • Gurtner G.C.
      • Werner S.
      • Barrandon Y.
      • Longaker M.T.
      Wound repair and regeneration.
      ;
      • Raffetto J.D.
      Pathophysiology of wound healing and alterations in venous leg ulcers-review.
      ;
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      • Liang H.
      • Clarke E.
      • Jackson C.
      • Xue M.
      Inflammation in chronic wounds.
      ). Instead, they remain stuck in an inflammatory state characterized by defective transition of proinflammatory M1 macrophages to granulation-promoting M2 macrophages, which is accompanied by an excess release of ROS and proinflammatory cytokines, including IL-1β and TNF-α. The sustained oxidative attack induces a senescence program in wound fibroblasts associated with the release of proinflammatory cytokines, chemokines, and matrix-degrading metalloproteinases, whereas IL-1β and TNF-α trigger a self-perpetuating cycle of autocrine recruitment and activation of M1 macrophages. Ultimately, the wound is arrested in a nonhealing state (
      • Jiang D.
      • Scharffetter-Kochanek K.
      Mesenchymal stem cells adaptively respond to environmental cues thereby improving granulation tissue formation and wound healing.
      ;
      • Krzyszczyk P.
      • Schloss R.
      • Palmer A.
      • Berthiaume F.
      The role of macrophages in acute and chronic wound healing and interventions to promote pro-wound healing phenotypes.
      ;
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      • Peters T.
      • Wieschalka S.
      • Baican C.
      • Baican A.
      • Peter H.
      • et al.
      An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice.
      ).
      Table 1Reported Healing Failure Rates of Venous Leg Ulcers Under Standard Therapy
      Treatment DurationFailure RateSourcen
      4 weeks89%Control group from an RCT (
      • Cullen B.M.
      • Serena T.E.
      • Gibson M.C.
      • Snyder R.J.
      • Hanft J.R.
      • Yaakov R.A.
      Randomized controlled trial comparing collagen/oxidized regenerated cellulose/silver to standard of care in the management of venous leg ulcers.
      )
      27 patients
      12 weeks38%Retrospective cohort study (
      • Margolis D.J.
      • Berlin J.A.
      • Strom B.L.
      Risk factors associated with the failure of a venous leg ulcer to heal.
      )
      260 patients
      41%Control group from an RCT (
      • Cullen B.M.
      • Serena T.E.
      • Gibson M.C.
      • Snyder R.J.
      • Hanft J.R.
      • Yaakov R.A.
      Randomized controlled trial comparing collagen/oxidized regenerated cellulose/silver to standard of care in the management of venous leg ulcers.
      )
      27 patients
      55%US Wound Registry data (
      • Fife C.E.
      • Eckert K.A.
      • Carter M.J.
      Publicly reported wound healing rates: the fantasy and the reality.
      )
      97,420 ulcers
      55%Retrospective cohort study (
      • Gelfand J.M.
      • Hoffstad O.
      • Margolis D.J.
      Surrogate endpoints for the treatment of venous leg ulcers.
      )
      29,189 patients (56,488 ulcers)
      57%Control groups from 20 RCTs (
      • Fife C.E.
      • Eckert K.A.
      • Carter M.J.
      Publicly reported wound healing rates: the fantasy and the reality.
      )
      1,372 patients
      65%Control group from an RCT (
      • Bianchi C.
      • Cazzell S.
      • Vayser D.
      • Reyzelman A.M.
      • Dosluoglu H.
      • Tovmassian G.
      • et al.
      A multicentre randomised controlled trial evaluating the efficacy of dehydrated human amnion/chorion membrane (EpiFix®) allograft for the treatment of venous leg ulcers.
      )
      57 patients
      85%Control group from an RCT (
      • Hayes P.D.
      • Harding K.G.
      • Johnson S.M.
      • McCollum C.
      • Téot L.
      • Mercer K.
      • et al.
      A pilot multi-centre prospective randomised controlled trial of RECELL for the treatment of venous leg ulcers.
      )

      Failure rate dependent on basal wound size:

      ≤10 cm: 75%

      >10 cm: 93%
      26 patients
      16 weeks56%Control group from an RCT (
      • Bianchi C.
      • Cazzell S.
      • Vayser D.
      • Reyzelman A.M.
      • Dosluoglu H.
      • Tovmassian G.
      • et al.
      A multicentre randomised controlled trial evaluating the efficacy of dehydrated human amnion/chorion membrane (EpiFix®) allograft for the treatment of venous leg ulcers.
      )
      57 patients
      20 weeks55%Control group of an RCT (
      • Kelechi T.J.
      • Mueller M.
      • Hankin C.S.
      • Bronstone A.
      • Samies J.
      • Bonham P.A.
      A randomized, investigator-blinded, controlled pilot study to evaluate the safety and efficacy of a poly-N-acetyl glucosamine-derived membrane material in patients with venous leg ulcers.
      )
      20 patients
      24 weeks14‒23%RCT on compression therapy (
      • Finlayson K.J.
      • Courtney M.D.
      • Gibb M.A.
      • O'Brien J.A.
      • Parker C.N.
      • Edwards H.E.
      The effectiveness of a four-layer compression bandage system in comparison with Class 3 compression hosiery on healing and quality of life in patients with venous leg ulcers: a randomised controlled trial.
      )
      103 patients
      24%Prospective study (
      • Gohel M.S.
      • Taylor M.
      • Earnshaw J.J.
      • Heather B.P.
      • Poskitt K.R.
      • Whyman M.R.
      Risk factors for delayed healing and recurrence of chronic venous leg ulcers--an analysis of 1324 legs.
      )
      1,186 patients (1,324 legs)
      25%Prospective study (
      • Guest M.
      • Smith J.J.
      • Sira M.S.
      • Madden P.
      • Greenhalgh R.M.
      • Davies A.H.
      Venous ulcer healing by four-layer compression bandaging is not influenced by the pattern of venous incompetence.
      )
      198 legs
      26‒34%RCT on compression therapy (
      • Polignano R.
      • Bonadeo P.
      • Gasbarro S.
      • Allegra C.
      A randomised controlled study of four-layer compression versus Unna's Boot for venous ulcers.
      )
      68 patients
      35%RCT comparing surgery and compression with compression alone (
      • Barwell J.R.
      • Davies C.E.
      • Deacon J.
      • Harvey K.
      • Minor J.
      • Sassano A.
      • et al.
      Comparison of surgery and compression with compression alone in chronic venous ulceration (ESCHAR study): randomised controlled trial.
      )
      500 patients
      35–44%Retrospective cohort study (
      • Margolis D.J.
      • Berlin J.A.
      • Strom B.L.
      Risk factors associated with the failure of a venous leg ulcer to heal.
      ,
      • Margolis D.J.
      • Berlin J.A.
      • Strom B.L.
      Which venous leg ulcers will heal with limb compression bandages?.
      )

      Failure rate dependent on basal wound size and duration before treatment:

      ≤5 cm and ≤6 months: 5–7%

      >5 cm and >6 months: 63–87%
      260 patients
      38%Retrospective cohort study (
      • Margolis D.J.
      • Allen-Taylor L.
      • Hoffstad O.
      • Berlin J.A.
      The accuracy of venous leg ulcer prognostic models in a wound care system.
      )

      Failure rate dependent on basal wound size and duration before treatment:

      ≤10 cm and ≤12 months: 29%

      >10 cm and >12 months: 78%
      20,793 patients
      45%Retrospective cohort study (
      • Gelfand J.M.
      • Hoffstad O.
      • Margolis D.J.
      Surrogate endpoints for the treatment of venous leg ulcers.
      )
      29,189 patients (56,488 ulcers)
      51%Control group of an RCT (
      • Falanga V.
      • Margolis D.
      • Alvarez O.
      • Auletta M.
      • Maggiacomo F.
      • Altman M.
      • et al.
      Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Human Skin Equivalent Investigators Group.
      )
      129 patients
      56%Control group of an RCT (
      • Jull A.
      • Wadham A.
      • Bullen C.
      • Parag V.
      • Weller C.
      • Waters J.
      Wool-derived keratin dressings versus usual care dressings for treatment of slow healing venous leg ulceration: a randomised controlled trial (Keratin4VLU).
      )
      72 patients
      30 weeks29%Retrospective cohort study (
      • Margolis D.J.
      • Berlin J.A.
      • Strom B.L.
      Risk factors associated with the failure of a venous leg ulcer to heal.
      )
      260 patients
      1 year13%Prospective study on compression therapy (
      • Milic D.J.
      • Zivic S.S.
      • Bogdanovic D.C.
      • Karanovic N.D.
      • Golubovic Z.V.
      Risk factors related to the failure of venous leg ulcers to heal with compression treatment.
      )
      189 patients
      30‒31%Prospective study on compression therapy (
      • Ashby R.L.
      • Gabe R.
      • Ali S.
      • Adderley U.
      • Bland J.M.
      • Cullum N.A.
      • et al.
      Clinical and cost-effectiveness of compression hosiery versus compression bandages in treatment of venous leg ulcers (Venous leg Ulcer Study IV, VenUS IV): a randomised controlled trial.
      )
      453 patients
      40%Control group of an RCT (
      • Klonizakis M.
      • Tew G.A.
      • Gumber A.
      • Crank H.
      • King B.
      • Middleton G.
      • et al.
      Supervised exercise training as an adjunct therapy for venous leg ulcers: a randomized controlled feasibility trial.
      )
      21 patients
      2 years20%Survey (
      • Callam M.J.
      • Harper D.R.
      • Dale J.J.
      • Ruckley C.V.
      Chronic ulcer of the leg: clinical history.
      )
      600 patients
      5 years8%Survey (
      • Callam M.J.
      • Harper D.R.
      • Dale J.J.
      • Ruckley C.V.
      Chronic ulcer of the leg: clinical history.
      )
      600 patients
      Abbreviations: RCT, Randomized controlled trial; US, United States.
      Among proinflammatory mediators, IL-1β plays a predominant role in chronic wound development (
      • Harrell C.R.
      • Markovic B.S.
      • Fellabaum C.
      • Arsenijevic N.
      • Djonov V.
      • Volarevic V.
      The role of interleukin 1 receptor antagonist in mesenchymal stem cell-based tissue repair and regeneration.
      ). Its counterpart, the naturally occurring receptor antagonist IL-1RA, is crucial for the alleviation of IL-1‒driven inflammation (
      • Gabay C.
      • Lamacchia C.
      • Palmer G.
      IL-1 pathways in inflammation and human diseases.
      ). In a mouse chronic wound model mimicking the healing-impairing iron overload occurring in CVU tissue (
      • Sindrilaru A.
      • Peters T.
      • Wieschalka S.
      • Baican C.
      • Baican A.
      • Peter H.
      • et al.
      An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice.
      ), adaptive IL-1RA release by locally injected mesenchymal stem cells (MSCs) expressing ABCB5 (
      • Frank N.Y.
      • Pendse S.S.
      • Lapchak P.H.
      • Margaryan A.
      • Shlain D.
      • Doeing C.
      • et al.
      Regulation of progenitor cell fusion by ABCB5 P-glycoprotein, a novel human ATP-binding cassette transporter.
      ) dampened the IL-1β‒driven unrestrained M1 activation and promoted a shift toward M2 macrophages (
      • Vander Beken S.
      • de Vries J.C.
      • Meier-Schiesser B.
      • Meyer P.
      • Jiang D.
      • Sindrilaru A.
      • et al.
      Newly defined ATP-binding cassette subfamily B Member 5 positive dermal mesenchymal stem cells promote healing of chronic iron-overload wounds via secretion of interleukin-1 receptor antagonist.
      ).
      ABCB5+ MSCs can be retrieved from skin tissue, expanded to a clinical scale, isolated as a highly pure, vital and viable cell population, and manufactured as a readily available advanced-therapy medicinal product (
      • Ballikaya S.
      • Sadeghi S.
      • Niebergall-Roth E.
      • Nimtz L.
      • Frindert J.
      • Norrick A.
      • et al.
      Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: off-the-shelf GMP-manufactured donor-independent ATMP.
      ) with a favorable safety and tolerability profile (
      • Tappenbeck N.
      • Schröder H.M.
      • Niebergall-Roth E.
      • Hassinger F.
      • Dehio U.
      • Dieter K.
      • et al.
      In vivo safety profile and biodistribution of GMP-manufactured human skin-derived ABCB5-positive mesenchymal stromal cells for use in clinical trials.
      ). In mice, systemically applied ABCB5+ MSCs survived in the skin across a fully allogeneic barrier for at least 17 days (
      • Schatton T.
      • Yang J.
      • Kleffel S.
      • Uehara M.
      • Barthel S.R.
      • Schlapbach C.
      • et al.
      ABCB5 identifies immunoregulatory dermal cells.
      ). In a first-in-human trial, topically applied patient-derived (autologous) ABCB5+ MSCs facilitated the healing of standard therapy‒resistant CVUs (
      • Kerstan A.
      • Niebergall-Roth E.
      • Esterlechner J.
      • Schröder H.M.
      • Gasser M.
      • Waaga-Gasser A.M.
      • et al.
      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.
      ). In this study, we report on a phase I/IIa clinical trial (Figure 1a) evaluating the safety and efficacy of donor-derived, allogeneic ABCB5+ MSCs for topical treatment of CVUs in a high-medical-need population.
      Figure thumbnail gr1
      Figure 1Trial design, flow diagram, and patient characteristics. (a) Scheme of the trial design. 1Only subjects who did not reach month-12 visit before June 30, 2020 and were not scheduled for a planned safety follow-up visit in June 2020 were subjected to an end-of-study visit. (b) Study participants flow chart. For EoS visit, see a. 2Most frequent reasons for ineligibility were ulcer <1.5 cm2 or >100 cm2 (n = 268), ulcer not matching the CVU definition specified in the protocol (n = 207), patient aged >85 years (n = 110), BMI <20 or >45 (n = 101), and infected ulcer (n = 94). 3Owing to cardiac failure. 4Owing to pulmonary embolism. (c) Tukey’s boxplots of patient characteristics at screening and baseline wound size of all treated subjects (n = 31). BMI, body mass index; CVU, chronic venous ulcer; EoS, end of study; MSC, mesenchymal stem cell.

      Results

       Progress of the study

      Patients were enrolled between November 2017 and January 2020. Forced by the COVID-19 pandemic, which was associated with critical issues, including staffing shortages, impairments of supply chains, and increased infection risk for the elderly and/or comorbid study participants, recruitment and treatment were discontinued as of April 2020. After consultation with the ethics committee and the regulatory authority, the trial was prematurely completed as of the end of June 2020. At that time, all treated subjects had completed the efficacy follow-up. Participants who had already entered the safety follow-up period but were not scheduled for a safety visit in June 2020 were subjected to a supplementary end-of-study visit (Figure 1a and b).

       Participants

      In total, 1,013 patients with CVU were assessed for eligibility, 58 of whom could be enrolled. Of these, 27 failed the screening period, so 31 subjects (16 male, 15 female) were treated (Figure 1b). In these subjects, only slight intraindividual wound sizes changes had occurred during the screening period (median duration of 34 days, interquartile range [IQR] = 28–41 days), which resulted in a median wound size reduction of ‒1% (IQR = ‒10 to 17%) (Figure 2a, run in). At baseline, the median age of the subjects was 75 (IQR = 66‒80) years, median bodyweight was 95 (IQR = 78‒112) kg, median body mass index was 31 (IQR = 27.4‒35.8) kg/m2, and median wound size was 6.79 (IQR = 3.21‒17.05) cm2 (Figure 1c).
      Figure thumbnail gr2
      Figure 2Wound healing progress during the run-in and the treatment and efficacy follow-up period. (a) Percent wound size reduction during ≥4-week screening (run-in) period and during 12-week treatment/efficacy follow-up (presented as a reduction from baseline) in the FAS (n = 31) (upper panel) and PP (n = 27) (lower panel). (b) Percent wound size reduction from baseline at week 12 (last observation carried forward) in the FAS (upper panel) and PP (lower panel). Subjects whose wounds diminished or enlarged by >25% (dashed red lines) during the screening period did not qualify for study treatment. Subjects who had wound size reductions of at least 30% from baseline (indicated by light green dashed lines) at week 12 were considered responders. Error bars indicate median and interquartile range; ∗∗∗P < 0.001 versus baseline, two-sided Wilcoxon signed-rank test. FAS, full analysis set; PP, per-protocol set.
      Of the 31 treated participants, 22 subjects received both, and nine subjects received only one topical cell application. Of the nine subjects who received only one, three subjects did because they had been enrolled under previous protocol versions before the second application was amended to the protocol, one subject did because of death (see details in the following paragraph), two subjects did because their wounds were already closed at the week-6 visit, and three subjects did because of the COVID-19 pandemic‒related treatment discontinuation. Two subjects discontinued study participation because of death (cardiac failure, pulmonary embolism; not related to study treatment): one was during efficacy, and the other was during safety follow-up (Figure 1b).
      Four subjects had major protocol deviations: only week-2 efficacy assessment available owing to premature discontinuation, per-protocol wound size assessment at baseline missing, use of a wound irrigation solution, and exclusion criterion (potentially wound healing‒affecting condition) not met. These subjects were analyzed in the full analysis set (FAS) but were excluded from the per-protocol set (PP) (for a definition of the analysis sets, see Materials and Methods and statistical analysis.)

       Safety outcomes

      During the whole-study period, 83 treatment-emergent adverse events (TEAEs) were reported by 27 of 31 subjects (Table 2). Most TEAEs were mild or moderate, singular events; nine TEAEs reported by four subjects were severe. Nine TEAEs reported by seven subjects were serious (Table 3); two of them (cardiac failure and pulmonary embolism) had a fatal outcome. Only three TEAEs (increased wound exudation, mild; erythema, moderate; venous ulcer pain, moderate) were judged related to the cell product. These events recovered without sequelae.
      Table 2Adverse Events
      EventNumber of EventsNumber (%) of Subjects
      Any adverse event
      Includes pretreatment-emergent (occurring between giving written consent and first cell application) and treatment-emergent (occurring between first cell application and the end of safety follow-up) adverse events.
      9628 (90)
      Any TEAE8327 (87)
      Any serious TEAE
      None of the serious TEAEs was related to study treatment.
      97 (23)
      Any TEAE33 (10)
      Frequent TEAEs by MedDRA system organ class
      Only TEAEs that were reported by at least two subjects.
       General disorders and administration site conditions6 (19)
      Condition aggravated3 (10)
      Oedema peripheral2 (6)
       Infections and infestations9 (29)
      Nasopharyngitis3 (10)
      Wound infection2 (6)
       Skin and subcutaneous tissue disorders22 (71)
      Allergic contact dermatitis2 (6)
      Irritant contact dermatitis4 (13)
      Eczema, other2 (6)
      Pruritus2 (6)
      Skin ulcer12 (39)
      Stasis dermatitis2 (6)
      Venous ulcer pain5 (16)
      Abbreviations: MedDRA, Medical Dictionary for Regulatory Activities; TEAE, treatment-emergent adverse event.
      Adverse events are reported for the safety analysis set (n = 31).
      1 Includes pretreatment-emergent (occurring between giving written consent and first cell application) and treatment-emergent (occurring between first cell application and the end of safety follow-up) adverse events.
      2 None of the serious TEAEs was related to study treatment.
      3 Only TEAEs that were reported by at least two subjects.
      Table 3Serious Treatment-Emergent Adverse Events
      MedDRA System Organ Class

      Preferred Term
      Number (%) of Subjects
      Cardiac disorders1 (3)
       Cardiac failure
      Event had a fatal outcome.
      1 (3)
      General disorders and administration site conditions1 (3)
       Malaise1 (3)
      Infections and infestations2 (6)
       Cellulitis
      Moderate cellulitis originating from a superinfected nontarget (not cell-treated) ulcer, starting 9 months after the first and only cell application.
      1 (3)
       Wound infection
      Severe postoperative wound infection after toe amputation due to foot deformation caused by rheumatoid arthritis, starting 11 months after the first and only cell application.
      1 (3)
      Musculoskeletal and connective tissue disorders1 (3)
       Foot deformity1 (3)
      Renal and urinary disorders1 (3)
       Renal amyloidosis1 (3)
      Respiratory, thoracic, and mediastinal disorders1 (3)
       Pulmonary embolism
      Event had a fatal outcome.
      1 (3)
      Skin and subcutaneous tissue disorders2 (6)
       Skin ulcer
      Worsening of the CVU, starting 3 months after the second cell application, required hospitalization owing to poor social situation of the subject.
      ,
      Worsening of a skin ulcer at the contralateral foot, starting 4 weeks after the first cell application.
      2 (6)
      Any event7 (23)
      Abbreviation: MedDRA, Medical Dictionary for Regulatory Activities.
      Serious treatment-emergent adverse events are reported for the safety analysis set (n = 31). None of these events was related to the study treatment.
      1 Event had a fatal outcome.
      2 Moderate cellulitis originating from a superinfected nontarget (not cell-treated) ulcer, starting 9 months after the first and only cell application.
      3 Severe postoperative wound infection after toe amputation due to foot deformation caused by rheumatoid arthritis, starting 11 months after the first and only cell application.
      4 Worsening of the CVU, starting 3 months after the second cell application, required hospitalization owing to poor social situation of the subject.
      5 Worsening of a skin ulcer at the contralateral foot, starting 4 weeks after the first cell application.
      During efficacy follow-up, no clinically relevant changes in vital signs occurred (Table 4). A total of 14 new (not present at screening) physical examination findings were documented in 11 subjects (Table 5). Five findings (36%) represented improvements.
      Table 4Vital Signs
      ParameterBaseline (Day 0) (n = 31)Change at Week 12 (n = 28)
      Body temperature (°C)36.7 (0.5)‒0.1 (0.5)
      Blood pressure (mm Hg)
       Systolic135 (21)‒4 (16)
       Diastolic76 (11)1 (10)
      Heart rate (bpm)73 (9)1 (13)
      Abbreviation: bpm, beats per minute.
      Vital signs are reported for the safety analysis set (n = 31). Data are presented as mean (SD).
      Table 5Changes in Physical Examination Findings from Screening Visit
      Subject
      Subjects presenting with changes in physical examinations; they are numbered consecutively.
      Organ SystemVisit atSpecification
      1ExtremitiesWeek 12Oedema lower legs significantly reduced
      SkinWeek 12Target ulcer almost closed, newly occurred nontarget ulcer
      2ExtremitiesWeek 12Ulcer left leg closed
      HeadWeek 12Scar occipital
      3SkinWeek 6.1
      Week 6.1: this visit was intended for the second cell application, scheduled 1–5 days after the week-6 efficacy follow-up visit.
      Skin irritation left lower leg
      4SkinWeek 6.1
      Week 6.1: this visit was intended for the second cell application, scheduled 1–5 days after the week-6 efficacy follow-up visit.
      Plaster allergy left lower leg
      Documented as TEAE (allergic contact dermatitis) not related to study treatment.
      5SkinWeek 6.1
      Week 6.1: this visit was intended for the second cell application, scheduled 1–5 days after the week-6 efficacy follow-up visit.
      Scar from carpal tunnel surgery
      SkinWeek 12Pruritus
      Documented as TEAE (pruritus) not related to study treatment.
      6SkinWeek 12Target ulcer and nontarget ulcers smaller
      7SkinWeek 12Clavus left hallux
      Documented as TEAE (hyperkeratosis) not related to study treatment.
      8SkinWeek 12Target ulcer closed
      9SkinWeek 6.1
      Week 6.1: this visit was intended for the second cell application, scheduled 1–5 days after the week-6 efficacy follow-up visit.
      Intertrigo at the right mamma
      Documented as TEAE (allergic contact dermatitis) not related to study treatment.
      10SkinWeek 12Stasis dermatitis at the right foot/ankle
      Documented as TEAE (stasis dermatitis) not related to study treatment.
      11EarsWeek 6.1
      Week 6.1: this visit was intended for the second cell application, scheduled 1–5 days after the week-6 efficacy follow-up visit.
      Disorder of the tuba auditiva
      Abbreviation: TEAE, treatment-emergent adverse event.
      These changes are reported for the safety analysis set (n = 31).
      1 Subjects presenting with changes in physical examinations; they are numbered consecutively.
      2 Week 6.1: this visit was intended for the second cell application, scheduled 1–5 days after the week-6 efficacy follow-up visit.
      3 Documented as TEAE (allergic contact dermatitis) not related to study treatment.
      4 Documented as TEAE (pruritus) not related to study treatment.
      5 Documented as TEAE (hyperkeratosis) not related to study treatment.
      6 Documented as TEAE (allergic contact dermatitis) not related to study treatment.
      7 Documented as TEAE (stasis dermatitis) not related to study treatment.

       Efficacy outcomes

      Efficacy assessments were performed on the FAS (n = 31) and the PP (n = 27) as specified in the Materials and Methods (see Statistical analysis). The primary efficacy outcome, median wound size reduction from baseline to week 12, was 76% (FAS) and 78% (PP) (Figure 2b). A summary of the secondary efficacy outcomes (see Material and Methods) is given in Table 6.
      Table 6Summary of the Secondary Efficacy Outcomes
      ParameterFAS (n = 31)PP (n = 27)Details
      Absolute wound size reduction, (cm2)
      Mean (SD).
       Change from baseline at week 122.4 (14.0)
      n = 30.
      5.2 (6.6)Table 7
      Complete wound closure
       Subjects with complete closure at week 12, n (%)6 (20)
      n = 30.
      6 (22)Table 9
       Subjects with complete closure at any time up to week 12, n (%)7 (23)7 (26)Table 9
       Time to complete closure, days
      Median (95% CI).
      Not reachedNot reachedFigure 3a
      ≥30% wound size reduction
       Subjects with ≥30% reduction at week 12 (responders), n (%)21 (70)
      n = 30.
      21 (78)Table 9
       Subjects with ≥30% reduction at any time up to week 12, n (%)26 (84)24 (89)Table 9
       Time to ≥30% reduction, days
      Median (95% CI).
      21 (12; 27)15 (9; 27)Figure 3b
      Reopening after complete wound closure
       Subjects with wounds reopened at week 12, n (%)1 (3.2)1 (3.7)n.a.
      Granulation, % of wound area
       Day 0not evaluablenot evaluablen.a.
       Week 12not evaluablenot evaluablen.a.
      Epithelialization, % of wound area
       Day 0not evaluablenot evaluablen.a.
       Week 12not evaluablenot evaluablen.a.
      Exudation
       Wounds with low exudation, n (%)
      Day 014 (45)11 (41)Table 10
      Week 1218 (62)
      n = 29.
      16 (62)
      n = 26.
      Table 10
       Wounds with moderate exudation, n (%)
      Day 015 (48)14 (52)Table 10
      Week 1210 (34)
      n = 29.
      10 (38)
      n = 26.
      Table 10
      Pain score
      Mean (SD).
       Day 03.6 (3.2)n.a.Table 11
       Week 122.5 (2.2)
      n = 30.
      n.a.Table 11
      QOL
      Owing to space limitations, the Short Form (36) Health Survey subscale scores (which remained virtually unchanged during the efficacy follow-up) are not shown in this table but are given in Table 12.
       Dermatology Life Quality Index
      Median (IQR).
      Day 09.5 (4–16)
      n = 30.
      n.a.Table 12
      Week 126.0 (3–12)
      n = 30.
      n.a.Table 12
      Abbreviations: CI, confidence interval; FAS, full analysis set; IQR, interquartile range; n.a., not applicable; PP, per-protocol set.
      Detailed results are given in Tables 7 and Table 9, Table 10, Table 11, Table 12.
      1 Mean (SD).
      2 n = 30.
      3 Median (95% CI).
      4 n = 29.
      5 n = 26.
      6 Owing to space limitations, the Short Form (36) Health Survey subscale scores (which remained virtually unchanged during the efficacy follow-up) are not shown in this table but are given in Table 12.
      7 Median (IQR).
      In more detail, wound size reduction was most pronounced during the first 2 weeks after the first MSC application. Thereafter, the median wound size continued to decrease until the end of efficacy follow-up at week 12 (Figure 2a and Table 7; for absolute wound size measurements by subject and visit, see Table 8).
      Table 7Absolute Wound Size Reduction from Baseline by Visit
      Visit atFAS (n = 31)PP (n = 27)
      nDifference from Baseline (cm2)
      Mean (SD).
      nDifference from Baseline (cm2)
      Mean (SD).
      Week 2312.6 (3.6)272.8 (3.8)
      Week 3293.3 (4.2)263.2 (4.1)
      Week 4293.6 (4.4)273.6 (4.2)
      Week 6284.1 (5.8)254.4 (5.4)
      Week 8294.1 (6.6)264.2 (6.1)
      Week 10284.6 (6.2)255.0 (6.1)
      Week 12302.4 (14.0)275.2 (6.6)
      Abbreviations: FAS, full analysis set; PP, per-protocol set.
      1 Mean (SD).
      Table 8Absolute Wound Size Measurements by Visit
      Subject No.Wound Size (cm2)
      Day 0Week 2Week 3Week 4Week 6Week 8Week 10Week 12
      124.620.419.218.112.910.010.811.1
      227.724.825.826.725.727.623.124.4
      32.41.40.80.60.40.60.10.3
      43.22.11.71.00.80.30.20.0
      59.68.58.18.59.611.210.610.9
      62.31.71.61.31.31.10.70.5
      715.610.29.910.89.112.66.64.7
      89.85.65.33.84.02.82.61.3
      95.55.66.08.913.613.010.29.4
      104.64.5
      113.22.01.91.61.51.51.01.3
      123.21.21.30.51.30.30.3
      133.92.72.11.34.42.94.0
      1417.65.64.54.21.20.30.20.3
      1522.520.910.912.611.96.513.385.3
      1618.910.19.88.87.67.95.83.3
      1725.910.89.98.95.96.36.04.1
      188.27.46.06.66.23.86.96.3
      1914.711.611.611.49.910.313.412.4
      2017.012.214.811.712.18.28.96.2
      2110.08.56.37.29.26.95.42.4
      2239.342.643.543.644.348.948.550.0
      238.96.35.13.23.01.20.7
      241.71.11.00.90.70.30.10.1
      253.82.62.52.03.73.65.8
      264.84.52.71.61.73.70.30.1
      274.02.82.62.11.81.20.60.4
      281.91.61.00.30.10.00.00.0
      294.82.62.92.40.90.90.71.0
      306.85.95.85.04.4
      311.40.30.00.00.00.00.00.0
      Abbreviation: No., number.
      In six subjects (20% for FAS and 22% for PP), the wound was completely closed at week 12 (Table 9). A further subject presented with complete wound closure at the visit intended for the second cell application; however, the wound had enlarged at the subsequent visits (89% wound size reduction at week 12 from baseline). The median time to complete wound closure was not reached (Figure 3a).
      Table 9Subjects with Complete Wound Closure and with ≥30% Wound Size Reduction by Visit
      Visit atComplete Wound Closure≥30% Wound Size Reduction
      FAS (n = 31)PP (n = 27)FAS (n = 31)PP (n = 27)
      nSubjects with Complete Wound Closure, n (%)nSubjects with Complete Wound Closure, n (%)nSubjects with 30% Wound Size Reduction, n (%)nSubjects with 30% Wound Size Reduction, n (%)
      Week 2310 (0)270 (0)3114 (45)2713 (48)
      Week 3291 (3)261 (4)2920 (69)2618 (69)
      Week 4291 (3)271 (4)2920 (69)2719 (70)
      Week 6282 (7)252 (8)2821 (75)2519 (76)
      Week 8293 (10)263 (12)2921 (72)2620 (77)
      Week 10283 (11)253 (12)2820 (71)2519 (76)
      Week 12306 (20)276 (22)3021 (70)2721 (78)
      Any time317 (23)277 (26)3126 (84)2724 (89)
      Abbreviations: FAS, full analysis set; PP, per-protocol set.
      Figure thumbnail gr3
      Figure 3Time-to-event and subgroup analyses. (a, b) Kaplan–Meier plots for the time to (a) full wound closure and (b) first 30% wound size reduction, expressed as the probability of the first occurrence of the event at a respective day during the efficacy follow-up period in the FAS (n = 31), PP (n = 27), and the subgroup of responders (n = 21). Subjects without event were censored at the date of the last available wound size assessment (indicated by small vertical ticks). Vertical dashed lines indicate the median time to event (not reached for full wound closure). Note that nearly all (except two) responders had reached 30% wound closure already by week 4 (day 28). (c) Tukey’s boxplots of the primary efficacy outcome parameter (percent wound size reduction from baseline at week 12) in the FAS (last observation carried forward), PP, and the subgroup of responders. FAS, full analysis set; PP, per-protocol set.
      Wound size reduction by at least 30% at week 12 was observed in 70% (21 of 30; FAS) and 78% (21 of 27; PP) of subjects (Table 9). These subjects were considered responders. The median time to first 30% wound size reduction was 21 days (95% confidence interval = 12‒27; FAS) and 15 days (95% confidence interval = 9‒27; PP) (Figure 3b). The wound healing progress of three representative responders is illustrated in Figure 4.
      Figure thumbnail gr4
      Figure 4Wound healing progress during the treatment and efficacy follow-up period in three representative subjects in the subgroup of responders. All subjects had consented to publication. MSC, mesenchymal stem cell.
      Most subjects showed low or moderate wound exudation. The group of subjects with low wound exudation increased from 45 to 62% (FAS) and from 41 to 62% (PP), whereas the percentage of subjects with moderate wound exudation decreased from 48 to 34% (FAS) and from 52 to 38% (PP) (Table 10). Owing to data inconsistencies resulting from measurement difficulties, formation of granulation and epithelial tissue was not evaluable.
      Table 10Wound Exudation by Visit
      Visit atFAS (n = 31)PP (n = 27)
      No. (%) of SubjectsNo. (%) of Subjects
      nLowModerateHighnLowModerateHigh
      Day 03114 (45)15 (48)2 (7)2711 (41)14 (52)2 (7)
      Day 1–3319 (29)18 (58)4 (13)278 (30)15 (56)4 (15)
      Week 13112 (39)16 (52)3 (10)2711 (41)14 (52)2 (7)
      Week 23113 (42)18 (58)0 (0)2713 (48)14 (52)0 (0)
      Week 32914 (48)14 (48)1 (3)2611 (42)14 (54)1 (4)
      Week 42813 (46)14 (50)1 (4)2612 (46)14 (54)0 (0)
      Week 62818 (64)10 (36)0 (0)2516 (64)9 (36)0 (0)
      Week 82916 (55)12 (41)1 (3)2615 (58)10 (39)1 (4)
      Week 102816 (57)12 (43)0 (0)2514 (56)11 (44)0 (0)
      Week 122918 (62)10 (34)1 (3)2616 (62)10 (38)0 (0)
      Abbreviations: FAS, full analysis set; No., number; PP, per-protocol set.
      Wound exudation was rated as low, moderate, or high according to
      • Romanelli M.
      • Vowden K.
      • Weir D.
      Exudate management made easy. Wounds International.
      .
      The mean pain score decreased slightly from 3.6 ± 3.2 at baseline to 2.5 ± 2.2 at week 12 (Table 11). The Short Form (36) Health Survey subscale scores remained virtually unchanged, whereas the median Dermatology Life Quality Index dropped from 9.5 (IQR = 4–16) at baseline to 6.0 (IQR = 3–12) at week 12 (Table 12).
      Table 11Pain Score by Visit
      Visit atnScore
      Pain was rated using a 0–10 point numerical rating scale with 0 = no pain and 10 = worst pain imaginable.
      ,
      Mean (SD).
      Day 0313.6 (3.2)
      Day 1–3312.8 (2.7)
      Week 1313.3 (2.9)
      Week 2312.9 (2.7)
      Week 3292.4 (2.4)
      Week 4292.8 (2.4)
      Week 6282.6 (2.2)
      Week 8292.6 (2.0)
      Week 10282.5 (2.0)
      Week 12302.5 (2.2)
      The scores are reported for the full analysis set (n = 31).
      1 Pain was rated using a 0–10 point numerical rating scale with 0 = no pain and 10 = worst pain imaginable.
      2 Mean (SD).
      Table 12QOL Scores by Visit
      ScaleDay 0 (n = 31)Week 4 (n = 29)Week 8 (n = 29)Week 12 (n = 30)
      Short Form (36) Health Survey subscale scores
       Subscales
      Physical functioning
      Transformed scale (0–100).
      45 (30–65)45 (25–75)40 (25–70)45 (25–70)
      Role functioning (physical)
      Transformed scale (0–100).
      25 (0–100)75 (0–100)50 (0–75)38 (0–75)
      Role functioning (emotional)
      Transformed scale (0–100).
      100 (0–100)100 (33–100)67 (0–100)67 (0–100)
      Social functioning
      Transformed scale (0–100).
      75 (50–100)88 (63–100)88 (63–100)88 (63–100)
      Mental health
      Transformed scale (0–100).
      64 (52–80)68 (60–80)68 (56–76)60 (48–80)
      Bodily pain
      Transformed scale (0–100).
      51 (22–74)62 (41–74)52 (41–74)53 (41–74)
      Vitality
      Transformed scale (0–100).
      45 (35–60)50 (45–65)50 (35–65)45 (35–65)
      General health perceptions
      Transformed scale (0–100).
      52 (37–65)52 (40–65)50 (40–70)52 (35–62)
       Health transition
      Raw scale.
      3 (3–3)3 (2–3)3 (2–3)3 (2–3)
      Dermatology Life Quality Index
       Summary score9.5 (4–16)
      n = 30.
      6.5 (2–11)
      n = 28.
      6.0 (2–8)6.0 (3–12)
      Abbreviation: IQR, interquartile range.
      Scores are reported for the full analysis set (n = 31). Data are presented as median (IQR).
      1 Transformed scale (0–100).
      2 Raw scale.
      3 n = 30.
      4 n = 28.

       Posthoc analyses

      Posthoc analyses were performed on the subgroup of responders. In this group, median wound size reduction from baseline at week 12 was 87% (IQR = 73–97%) (Figure 3c), and 29% (6 of 21) of responders had complete wound closure at week 12. All except two responders had achieved the first 30% wound size reduction by week 4; the median time to first 30% wound size reduction was 14 days (95% confidence interval = 8‒22) (Figure 3b).
      Another posthoc analysis was conducted to ascertain a possible relationship between the wound size change during the screening period and the wound healing progress after MSC treatment. A Spearman’s rank correlation test revealed a weak association (r = 0.39, P = 0.03) between percent wound size reduction during screening and percent wound size reduction from baseline at week 12 (Figure 5a). Whereas most (8 of 10) of the nonresponders had ≤5% wound size reduction during screening, almost half (10 of 21) of the responders fell also in this category (Figure 5a). A separate analysis of the subjects with low (≤5%) wound size reduction or wound enlargement during screening showed median postbaseline reductions that were still highly significant at each postbaseline visit, although they were numerically smaller than in the FAS (Figure 5b). On the other end, in the subjects with high (>15%) wound size reduction during screening, the wounds also improved further during the treatment/efficacy period because it became obvious, for example, from wound size reductions of 31 to 85% (median = 65%) 4 weeks after the first cell application compared with 17‒23% (median = 20%) during the ≥4-week screening period (Figure 5c).
      Figure thumbnail gr5
      Figure 5Assessment of association between wound size reduction during run-in and following MSC treatment. (a) Spearman’s rank correlation analysis between percent wound size reduction during screening and percent wound size reduction from baseline at week 12 in the full analysis set. (b, c) Percent wound size reduction from baseline during the treatment and efficacy period in subjects with (b) low (‒25 to 5%) and (c) high (15–25%) wound size reduction during screening. Error bars indicate median and interquartile range; ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.001 versus baseline, two-sided Wilcoxon signed-rank test. MSC, mesenchymal stem cell.
      A further posthoc analysis was conducted to compare the wound healing progress between the subjects who had received both versus those who had received only one cell application. In both groups, the median percent wound size reduction from baseline was significant at all postbaseline visits. There was also no obvious difference in the median percent wound size reduction from baseline at week 12 between the two-dose and the one-dose groups (77% vs. 72% for the FAS and 78% vs. 72% for the PP). The percentage of responders did also not differ between the two-dose and the one-dose groups (15 of 22 [68%] vs. 6 of 9 [67%] for the FAS and 15 of 19 [79%] vs. 6 of 8 [75%] for the PP]) (Figure 6).
      Figure thumbnail gr6
      Figure 6Comparison of the wound healing progress during the treatment and efficacy follow-up period in the subjects receiving both versus those receiving only one-cell dose. (a, b) Percent wound size reduction from baseline in subjects receiving (a) subjects receiving both versus (b) those receiving one-cell dose in the FAS. (c, d) Percent wound size reduction from baseline in (c) subjects receiving both versus (d) those receiving one-cell dose in the PP. Subjects who had wound size reductions of at least 30% from baseline (indicated by light green dashed lines) at week 12 were considered responders. Error bars indicate median and interquartile range; ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.001 versus baseline, two-sided Wilcoxon signed-rank test. FAS, full analysis set; PP, per-protocol set; MSC, mesenchymal stem cell.

      Discussion

      On the basis of evidence that locally applied ABCB5+ MSCs can dampen IL-1β‒driven M1 macrophage overactivation present in nonhealing wounds (
      • Vander Beken S.
      • de Vries J.C.
      • Meier-Schiesser B.
      • Meyer P.
      • Jiang D.
      • Sindrilaru A.
      • et al.
      Newly defined ATP-binding cassette subfamily B Member 5 positive dermal mesenchymal stem cells promote healing of chronic iron-overload wounds via secretion of interleukin-1 receptor antagonist.
      ), ABCB5+ MSCs have been considered a potential treatment option for patients suffering from noncurable CVUs (
      • Kerstan A.
      • Niebergall-Roth E.
      • Esterlechner J.
      • Schröder H.M.
      • Gasser M.
      • Waaga-Gasser A.M.
      • et al.
      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.
      ). To identify patients who were indeed in high need of an advanced wound closure strategy, all subjects underwent a two-step selection procedure. Basically, only such patients whose target CVUs qualified as therapy resistant, that is, having failed to improve within 3 months or to heal within 12 months of optimal phlebological treatment before enrolment (
      European Dermatology Forum
      Evidence-based (S3) guidelines for diagnostics and treatment of venous leg ulcers.
      ), were enrolled. Furthermore, according to the anticipated mode of action of MSC therapy, that is, to advance wounds that are stalled in the inflammation phase of wound healing into the next stage of wound healing, we needed to verify that at the time of the first MSC application, the wound was indeed stalled. To this end, all enrolled subjects underwent at least 4-week screening period with standard-of-care treatment, during which the ulcer size was required to be static, defined as not changing by ≥25%. This rigorous selection process markedly outreached the approach advocated by the United States Food and Drug Administration, that is, to exclude subjects whose ulcer decreases by ≥30–50% during an initial 1–2 week standard-of-care period (
      United States Department of Health and Human Services, Food and Drug Administration
      Guidance for industry: chronic cutaneous ulcer and burn wounds – developing products for treatment.
      ). As a result, 21% of the enrolled therapy-resistant subjects were subsequently excluded again because their wounds appeared not clearly static. Finally, only 3.1% of the overall 1,013 prescreened patients with CVU could participate in this study (Figure 1b). In this way, we enrolled a study population for which a particularly high likelihood of healing failure at standard-of-care conditions can be presumed.
      In this highly therapy-refractory population, topically applied ABCB5+ MSCs elicited a median wound size reduction of 76% (FAS) and 78% (PP) after 12 weeks (Figure 2b). Similar results (63% reduction) were observed in a previous first-in-human pilot trial with patient-derived (autologous) ABCB5+ MSCs (
      • Kerstan A.
      • Niebergall-Roth E.
      • Esterlechner J.
      • Schröder H.M.
      • Gasser M.
      • Waaga-Gasser A.M.
      • et al.
      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.
      ). In this study, using donor-derived cells, we show that ABCB5+ MSCs display their wound healing-promoting capacity also in an allogeneic therapy setting. Because donor-derived ABCB5+ MSCs can be expanded to a clinical scale and manufactured as an off-the-shelf available, standardized advanced-therapy medicinal product of proven biological activity (
      • Ballikaya S.
      • Sadeghi S.
      • Niebergall-Roth E.
      • Nimtz L.
      • Frindert J.
      • Norrick A.
      • et al.
      Process data of allogeneic ex vivo-expanded ABCB5+ mesenchymal stromal cells for human use: off-the-shelf GMP-manufactured donor-independent ATMP.
      ), the hurdles associated with autologous therapy strategies, mainly potential interdonor variations and a long waiting time until treatment initiation owing to the time-consuming cell expansion process, can be overcome. Whether the greater effect on wound size reduction observed in this study than the autologous pilot trial may be attributed to the allogeneic approach and/or to the higher cells dose (1 × 106 vs. 5 × 105 cells/cm2 wound area) remains to be elucidated.
      When viewed over time (Figure 2a), the effect of the applied cells appears most pronounced during the first weeks of treatment. This was expected considering that the action of ABCB5+ MSCs on wound healing relies on rather transient immunomodulatory activity that re-establishes a regenerative local environment, which enables the wound to resume physiologic healing. In line with this concept, subjects whose ulcers had not achieved a roughly 30% decrease at 4 weeks of study treatment had a high likelihood of emerging as nonresponders, staying below 30% wound size reduction until week 12 (Figures 2 and 3b). Similar observations were reported from other studies on CVU healing (
      • Cardinal M.
      • Eisenbud D.E.
      • Phillips T.
      • Harding K.
      Early healing rates and wound area measurements are reliable predictors of later complete wound closure.
      ;
      • Chaby G.
      • Senet P.
      • Ganry O.
      • Caudron A.
      • Thuillier D.
      • Debure C.
      • et al.
      Prognostic factors associated with healing of venous leg ulcers: a multicentre, prospective, cohort study.
      ), supporting a 30% wound size decrease as a suitable discriminator between potential responders and nonresponders. Moreover, in this study, the second cell dose at 6 weeks did neither enhance the percent wound size reduction achieved at week 12 nor increase the percentage of responders over the subjects who received only the first cell dose at day 0 (Figure 6). It may therefore be speculated that patients who will not respond to ABCB5+ MSC therapy can be detected already at around 4 weeks, which would help to adjust therapy decisions early as advocated by current treatment guidelines (
      • Marston W.
      • Tang J.
      • Kirsner R.S.
      • Ennis W.
      Wound Healing Society 2015 update on guidelines for venous ulcers.
      ;
      • O’Donnell Jr., T.F.
      • Passman M.A.
      • Marston W.A.
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      • Dalsing M.
      • Kistner R.L.
      • et al.
      Management of venous leg ulcers: clinical practice guidelines of the Society for Vascular Surgery® and the American Venous Forum.
      ).
      The phenomenon that some patients (30% of FAS and 22% of PP in this study) emerge as nonresponders is widely known in MSC therapy approaches (
      • Caplan A.I.
      Cell-based therapies: the nonresponder.
      ;
      • Levy O.
      • Kuai R.
      • Siren E.M.J.
      • Bhere D.
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      • et al.
      Shattering barriers toward clinically meaningful MSC therapies.
      ). In this trial, the strongly standardized quality of the cell product rules out potential differences in product quality as a cause of variation in the treatment responses (Supplementary Table S1). In addition, as shown in a posthoc subgroup analysis, achievement of responder status did not appear to be impacted by the wound size changes that had occurred during the run-in period (Figure 5). When comparing the characteristics that have been considered as unfavorable predictors for CVU healing, including higher patient age, higher body mass index, larger baseline wound size, and lower ankle‒brachial index (
      • Gohel M.S.
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      ;
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      ;
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      ,
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      ;
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      • Kirsner R.S.
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      ;
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      ;
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      ;
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      ;
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      ;
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      Associations between patient, treatment, or wound-level factors and venous leg ulcer healing: wound characteristics are the key factors in determining healing outcomes.
      ), there were no obvious differences between the responders and the nonresponders that could be called responsible for failure of treatment response (Figure 7). The absence of a clear association between treatment success and independent risk factors is not surprising, given the multifactorial etiology of impaired CVU healing, which involves more numerous factors such as nutritional status, mobility, and comorbidities (
      • Gohel M.S.
      • Taylor M.
      • Earnshaw J.J.
      • Heather B.P.
      • Poskitt K.R.
      • Whyman M.R.
      Risk factors for delayed healing and recurrence of chronic venous leg ulcers--an analysis of 1324 legs.
      ) or, on a cellular level, the differential expression or polymorphisms of genes associated with tissue inflammation, regeneration, or iron homeostasis (
      • Charles C.A.
      • Tomic-Canic M.
      • Vincek V.
      • Nassiri M.
      • Stojadinovic O.
      • Eaglstein W.H.
      • et al.
      A gene signature of nonhealing venous ulcers: potential diagnostic markers.
      ;
      • Gemmati D.
      • Federici F.
      • Catozzi L.
      • Gianesini S.
      • Tacconi G.
      • Scapoli G.L.
      • et al.
      DNA-array of gene variants in venous leg ulcers: detection of prognostic indicators.
      ;
      • Tognazzo S.
      • Gemmati D.
      • Palazzo A.
      • Catozzi L.
      • Carandina S.
      • Legnaro A.
      • et al.
      Prognostic role of factor XIII gene variants in nonhealing venous leg ulcers.
      ;
      • Zamboni P.
      • Gemmati D.
      Clinical implications of gene polymorphisms in venous leg ulcer: a model in tissue injury and reparative process.
      ). To further investigate and understand what segregates responders from nonresponders could help detect the predictors of response to ABCB5+ MSC therapy, which might enable to identify patients with a high likelihood of nonresponsiveness already before treatment initiation.
      Figure thumbnail gr7
      Figure 7Comparison of patient characteristics at screening and baseline wound size between all treated subjects (n = 31) and subgroups of responders (n = 21) and nonresponders (n = 9). Depicted are Tukey’s boxplots. ABI, ankle‒brachial index; BMI, body mass index.
      Nevertheless, the majority of subjects (70 and 78% for FAS and PP, respectively) responded to the study treatment, and these responders achieved a substantial median wound size reduction from baseline at week 12 of 87% (Figure 3c). Even though partial wound closure is a clinically less meaningful outcome than full wound closure, it is considered valid to “indicate relevant biological activity and help guide subsequent trials design” (
      United States Department of Health and Human Services, Food and Drug Administration
      Guidance for industry: chronic cutaneous ulcer and burn wounds – developing products for treatment.
      ). After all, in the present poor-prognosis population, 20% (FAS), 22% (PP), and 29% (responders) of subjects achieved full-wound closure. Moreover, it seems reasonable to expect that this rate would increase if the follow-up period was extended, given that at 12 weeks, the median time to full wound closure was not reached (Figure 3a), whereas the median wound size reduction was still increasing (Figure 2a). Another intriguing question to be studied in a subsequent trial is whether a higher cell dose would increase the healing effect, considering that in a preclinical dose selection study, a dose-dependent effect of ABCB5+ MSCs on wound size reduction was seen (
      • Kerstan A.
      • Niebergall-Roth E.
      • Esterlechner J.
      • Schröder H.M.
      • Gasser M.
      • Waaga-Gasser A.M.
      • et al.
      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.
      ).
      Naturally, the present conclusions are limited by factors typically associated with early-phase trials, mainly a relatively small sample size and an open, noncomparative design. Even though all wounds had been judged refractory to standard treatment, we cannot rule out that part of the observed improvements can be attributed to additional attention and care—for example, an optimized wound dressing strategy—the subjects may have received during the trial. Furthermore, wound healing can be affected by various patient-individual factors that were not controlled for. In addition, not all patients received a second cell dose. Nevertheless, we conclude that donor-derived ABCB5+ MSCs emerge as a promising candidate for adjunctive therapy of otherwise incurable CVUs. The very low rate of treatment-related adverse events verified good tolerability and overall safety of the cell product. Together, the results justify the conduct of a subsequent larger study with a randomized controlled, dose-ranging design, an extended efficacy follow-up period, and enhanced outcome parameters to validate the potential benefit and optimize the treatment strategy.

      Materials and Methods

       Participants

      Adults (aged 35–85 years) were eligible if they had a lower leg CVU (sized 1.5‒100 cm2) confirmed by Doppler ultrasonography and unremarkable ankle‒brachial index (0.9–1.3) and were judged as therapy resistant according to the European Dermatology Forum S3 guideline (
      European Dermatology Forum
      Evidence-based (S3) guidelines for diagnostics and treatment of venous leg ulcers.
      ), that is, having shown no healing tendency within 3 months or having not healed within 1 year of optimum phlebological therapy.
      The main exclusion criteria were involvement of underlying muscle, tendon, or bone; diabetes; treatment-requiring peripheral artery disease; acute or untreated deep vein thrombosis; ulcer infection; adjacent skin disorders; other potentially wound healing‒affecting conditions; surgical procedures within 2 months before treatment; use of active wound care agents within 2 weeks before treatment; and current use of systemic glucocorticoids, immunosuppressants, or cytotoxic drugs.

       Trial design

      The study was a national, multicentric (nine sites in Germany), single-arm, phase I/IIa trial comprising screening (≥4 weeks), treatment and efficacy follow-up (weeks 1–12), and safety follow-up (until the end of month 12) periods (Figure 1a). During screening, the wound was required to be static, that is, subjects whose ulcers enlarged or diminished by >25% under standard of care were excluded.
      The trial complied with the principles of the Helsinki Declaration and Good Clinical Practice. The study protocol and all other relevant documents had been approved by the local independent ethics committees (lead: Ethics Committee at the University of Würzburg, Germany; reference number 63/17_ff-sc) and the competent regulatory authority (Paul Ehrlich Institute, Langen, Germany). Before any trial-related activities/procedures, all participants gave written informed consent. The trial was registered with EudraCT (2017-000233-31) and ClinicalTrials.gov (NCT03257098).

       Interventions

      Treatment consisted of up to two topical applications of 1 × 106 allogeneic ABCB5+ MSCs (suspended in Ringer’s lactate solution containing 2.5% human serum albumin and 0.4% glucose at a target concentration of 1 × 107 cells/ml) per cm2 wound area at 6 weeks apart. The cells were delivered as Good Manufacturing Practice‒conforming standardized advanced-therapy medicinal products of proven vitality, viability, and biological activity (potency) (for product release data, see Supplementary Table S1). Originally, only one cell application was planned. The second application was amended to the protocol only after first-in-human data (
      • Kerstan A.
      • Niebergall-Roth E.
      • Esterlechner J.
      • Schröder H.M.
      • Gasser M.
      • Waaga-Gasser A.M.
      • et al.
      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.
      ) suggested that a second application at 6 weeks after the first cell dose might provide additional benefit. For cell application, the wound was debrided, and after the bleeding had entirely stopped, the cell suspension was carefully dropped and evenly distributed on the wound surface. Thereafter, cells were allowed to settle for 15–30 minutes, and then the wound was covered with a waterproof film dressing (Tegaderm; 3M, Neuss, Germany) to hold the cell suspension in place. After 1–3 days, the film dressing was replaced by a foam (Mepilex; Mölnlycke, Düsseldorf, Germany or Biatain; Coloplast, Hamburg, Germany) or microbe-binding (Cutimed Sorbact; BSN Medical GmbH, Hamburg, Germany) dressing. In addition, participants received standard compression dressings. Dressings were changed during follow-up at the discretion of the investigator, depending on the subject’s individual wound situation.

       Outcome measures

      Safety outcome measures included adverse events (during efficacy and safety follow-up) and vital signs and changes in physical examination findings (during efficacy follow-up). The primary efficacy endpoint was percent wound size reduction at week 12 or the last available post-baseline measurement. Secondary efficacy endpoints were percent and absolute wound size reduction at predefined visits, the proportion of subjects achieving complete and 30% wound closure, time to complete and time to 30% wound closure, reopening after complete wound closure, granulation, epithelialization, wound exudation, pain, and QOL.

       Outcome determination

      Wound healing was documented by standardized digital photographs, and wound sizes were measured by the investigator using PictZar (BioVisual, Elmwood Park, NJ) planimetry software (98% accuracy, 94% inter-rater reliability, 98% intra-rater reliability according to
      • Wendelken M.E.
      • Berg W.T.
      • Lichtenstein P.
      • Markowitz L.
      • Comfort C.
      • Alvarez O.M.
      Wounds measured from digital photographs using photodigital planimetry software: validation and rater reliability.
      ). Formation of granulation and epithelial tissue was estimated by the investigator in the percentage of wound area from standardized wound photographs. Wound exudation was rated by the investigator as low (dry), moderate (moist), and high (wet), according to the criteria defined by the World Union of Wound Healing Societies (
      • Romanelli M.
      • Vowden K.
      • Weir D.
      Exudate management made easy. Wounds International.
      ). Pain was rated by the participant using a 0‒10‒point numerical rating scale with 0 meaning no pain and 10 meaning worst imaginable pain. QOL was assessed using the participant-reported Short Form (36) Health Survey and Dermatology Life Quality Index questionnaires.

       Sample size

      Enrolment followed a Simon optimal two-stage design, with responders defined as subjects showing at least 30% wound size reduction at week 12. The sample size required to achieve 80% power at a 5% significance level was calculated to be 37 subjects using PASS 13 software (NCSS, East Kaysville, UT). This enabled the option to terminate the trial if ≤6 or ≥14 of the first 18 treated subjects were responders. Because 13 of 18 subjects emerged as responders in an interim analysis, recruitment was continued. However, forced by the emerging COVID-19 pandemic in early 2020, the trial was prematurely completed. At that time, 31 subjects had been treated.

       Statistical analysis

      Safety assessments were performed on the safety analysis set (n = 31), which included all subjects who received at least one cell dose. All efficacy assessments were performed on the FAS, which included all subjects of the safety analysis set who underwent wound size assessments at baseline and at least one post-baseline visit (n = 31). In addition, the wound assessment parameters were also analyzed on the PP, which is a subset of the FAS, including all subjects of the FAS who had no major protocol deviations (n = 27).
      Normally (D'Agostino–Pearson normality test) distributed parameters are presented as mean ± SD, and non-normally distributed parameters are presented as median and IQR. Time to complete wound closure and to attain 30% wound size reduction was analyzed using the Kaplan–Meier method. Statistical significance of percent wound size changes from baseline was tested against the null hypothesis (median change = 0) using a two-sided Wilcoxon signed-rank test. Spearman’s rank correlation analyses were performed to test for associations between variables.

       Data availability statement

      The individual participant data related to this article cannot be made publicly available for ethical/privacy reasons. The datasets are available from the corresponding author on reasonable request.

      Conflict of Interest

      NYF and MHF are inventors or coinventors of United States and international patents assigned to Brigham and Women’s Hospital (Boston, MA) and/or Boston Children’s Hospital (Boston, MA), licensed to TICEBA (Heidelberg, Germany) and RHEACELL (Heidelberg, Germany). MHF and KSK serve as scientific advisors to TICEBA and RHEACELL and participate in corporate-sponsored research collaborations with RHEACELL. KD, AKD, KK, and HS are employees of RHEACELL. ENR, JE, SB, and SS are employees of TICEBA. CG and MAK are Chief Executive Officer and Chief Scientific Officer, respectively, of RHEACELL and TICEBA. The remaining authors state no conflict of interest.

      Acknowledgments

      The authors thankfully acknowledge FGK Clinical Research GmbH (Munich, Germany) for expert support in project management, monitoring, and data analysis and the Coordination Centre for Clinical Trials (KKS) at the University of Heidelberg (Heidelberg, Germany) for advice on statistical analysis. We also gratefully thank the tissue donors and the tissue retrieval facilities: Aesthetic Quartier Heidelberg GmbH (Germany) (Director: Joachim Beck) and Cologne Dermatology (Germany) (Director: Wolfgang G. Philipp-Dormston). The trial was sponsored by RHEACELL (Heidelberg, Germany). The contributions by NYF and MHF to this work were supported by the National Institutes of Health /National Eye Institute grants RO1EY025794 and R24EY028767. DPO declared a grant from TICEBA to Brigham and Women's Hospital (Boston, MA).

      ORCIDs

      Charlotte von Engelhardt: http://orcid.org/0000-0003-1888-9015
      Hannes Matthias Schröder: http://orcid.org/0000-0002-0427-5867
      Karin Scharffetter-Kochanek: http://orcid.org/0000-0002-9655-685X

      Author Contributions

      Conceptualization: AKe, KD, MAK, GFM, DPO, NYF, KSK, MHF, MGa; Data Curation: KD, ENR, AKD, KK, MAK, HMS; Formal Analysis: ENR, AKe, KD, MAK; Investigation: AKe, MS, GD, MJ, TG, UMP, CEB, CVE, AKl, CP, MGa, AMWG, MGo; Methodology: AKe, KD, MAK, GFM, DPO, NYF, KSK, MHF, MGa; Project Administration: KD ENR AKD KK MAK; Resources: JE, HMS, SB, SS, CG; Validation: JE, HMS, SB, SS, CG, AKe; Visualization: ENR, KD, AKD, KK, MAK, HMS; Writing - Original Draft Preparation: ENR; Writing - Review and Editing: AKe, MS, GD, MJ, TG, UMP, CEB, CVE, AKl, CP, MGa, AMWG, MGo, KD, ENR, AKD, KK, MAK, JE, HMS, SB, SS, CG, GFM, DPO, NYF, KSK, MHF

      Supplementary Material

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