Randomized placebo-controlled trial of CL2020, an allogenic muse cell–based product, in subacute ischemic stroke

Abstract

Effective treatments for stroke after the acute phase remain elusive. Muse cells are endogenous, pluripotent, immune-privileged stem cells capable of selectively homing to damaged tissue after intravenous injection and replacing damaged/lost cells via differentiation. This randomized, double-blind, placebo-controlled trial enrolled ischemic stroke patients with modified Rankin Scale (mRS) ≥3. Randomized patients received a single intravenous injection of an allogenic Muse cell-based product, CL2020 (n = 25), or placebo (n = 10), without immunosuppressant, 14–28 days after stroke onset. Safety (primary endpoint: week 12) and efficacy (mRS, other stroke-specific measures) were assessed up to 52 weeks. Key efficacy endpoint was response rate (percentage of patients with mRS ≤2 at week 12). To week 12, 96% of patients in the CL2020 group experienced adverse events and 28% experienced adverse reactions (including one Grade 4 status epilepticus), compared with 100% and 10%, respectively, in the placebo group. Response rate was 40.0% (95% CI, 21.1–61.3) in the CL2020 group and 10.0% (0.3–44.5) in the placebo group; the lower CI in the CL2020 group exceeded the preset efficacy threshold (8.7% from registry data). This randomized placebo-controlled trial demonstrated CL2020 is a possible effective treatment for subacute ischemic stroke.

Registry information: JAPIC Clinical Trials Information site (JapicCTI-184103, URL: https://www.clinicaltrials.jp/cti-user/trial/ShowDirect.jsp?japicId=JapicCTI-184103).

Introduction

Stroke is the second most common cause of death and disability worldwide. ¹ Although advances in acute-phase treatment have reduced mortality, population growth and longer lives have led to more stroke survivors, estimated at 80 million in 2016. ¹ After the acute phase, treatment for subacute stroke is limited to rehabilitation, which reaches its maximal effect at 3–6 months, after which patients live with burdensome sequelae. ² Therefore, treatments that are effective in the subacute stage are needed.

Ideally, treatment for subacute stroke would replenish lost neural cells and reconstruct neural circuits; stem cells may fulfill this purpose. Various stem cell types, including mesenchymal stem cells (MSCs) from bone marrow (BM), mononuclear cells from BM (BM-MNCs), and umbilical cord blood, have been trialed in stroke and represent a promising strategy. ^{3 , 4} However, no randomized placebo-controlled trial has demonstrated therapeutic efficacy of stem cells in the subacute phase. ³ IV or intra-arterial administration is preferred in the subacute phase, ³ and the lack of efficacy in previous trials may be explained by the low homing rate of cells to the stroke-affected, post-infarct region and their rapid disappearance after being trapped in the lung. ^{3 , 4} Importantly, they mainly act through a bystander effect rather than replenishing lost neural cells. ³

Multilineage-differentiating stress-enduring (Muse) cells are endogenous pluripotent stem cells distributed in the BM, blood, and organ connective tissues as cells positive for the surface marker of pluripotency, stage-specific embryonic antigen-3. ^{5 , 6} Because Muse cells are endogenous, there are few safety concerns with their therapeutic use. Because Muse cells detect sphingosine-1-phosphate, an injury signal produced by phosphorylation of membrane-bound sphingosine by damaged cells, they can be delivered intravenously and will selectively migrate to the damage site instead of being trapped in the lung. ⁷ Within injured tissue, Muse cells replenish damaged/lost cells by spontaneously differentiating into the appropriate cell types and integrating into the correct position to maintain tissue function; thus, they do not require gene introduction or cytokine treatment to be rendered pluripotent and to induce differentiation.^{6 –10} Furthermore, allogenic Muse cells can be used without human leukocyte antigen (HLA) matching or immunosuppressants, partly due to HLA-G expression, implicated in immunotolerance of immune-privileged tissues like placenta. ⁷ Finally, donor-derived Muse cells remain incorporated as functional cells in host tissue for an extended time after IV injection without immunorejection. ⁷

In rodent ischemic stroke models, human Muse cells differentiated spontaneously into neuronal and glial cells after engraftment into the peri-infarct area. ^{9 , 10} Muse cell–derived neurites were incorporated into the pyramidal tract, including the pyramidal decussation, and into the sensory tract, as demonstrated by somatosensory-evoked potentials and formation of synapses with host neurons. These actions led to statistically significant, meaningful functional recovery with no adverse effects for 2–3 months. ^{9 , 10}

The proliferation speed of Muse cells is comparable to fibroblasts, and their production can be expanded to clinical scale. ⁵ Recently, a clinical-grade allogenic Muse cell–based product, CL2020, was developed for IV administration. In an immunodeficient mouse stroke model, CL2020 administered intravenously during the subacute phase migrated to the infarct within 1 day, spontaneously differentiated into neural and glial cells, and improved forelimb motor function compared with vehicle for up to 22 weeks. ¹¹ When engrafted CL2020 cells were ablated by human-selective diphtheria toxin, functional recovery was abrogated, suggesting that the behavioral outcome was due to reconstruction of neuronal circuits by integrated CL2020 cells. ¹¹ Thus, specific homing to the post-infarct region after systemic administration and long-lasting structural regeneration by replenishment of lost cells and long-lasting reconstruction of neural circuits, distinct from a bystander effect, are keys to Muse cell therapeutic outcomes.

In Japan, there are standard treatments for the acute phase of ischemic stroke up to about 2 weeks after onset; however, treatments after the acute phase are limited to rehabilitation and management of potential risk factors for recurrence. ¹² Given the efficacy of CL2020 in the regeneration of damaged neuronal networks in a mouse model of stroke, ¹¹ we hypothesized that CL2020 may also be effective in patients in the subacute phase after an ischemic stroke. In agreement with the Japanese Pharmaceuticals and Medical Devices Agency (PMDA), this randomized, double-blind, placebo-controlled trial was an exploratory study of the safety and efficacy of a single IV injection of allogenic CL2020 without HLA matching or immunosuppressants in patients with subacute ischemic stroke who have moderate to severe neurological symptoms and physical dysfunction.

Material and methods

Data availability

To ensure patient privacy is maintained, patient-level data will not be available due to small, single-center study.

Results

Of 43 patients enrolled, 37 were randomized to CL2020 (n = 27) or placebo (n = 10) (Figure 1). Two patients allocated to CL2020 did not receive study treatment because of complications that were protocol deviations. One patient received CL2020 on day 29 after stroke (1 day outside the protocol-specified time frame) but was considered eligible for assessment. Consequently, 25 patients in the CL2020 group and 10 patients in the placebo group received study treatment. All patients completed to week 12; 33 patients completed to week 52. Four additional patients in the CL2020 group and 1 additional patient in the placebo group were excluded from week 28 and week 52 efficacy evaluations because of serious AEs or COVID-19–related difficulties attending the clinic. Except for mRS, which included the patient who died, there were 21 patients in the CL2020 group and 8 patients in the placebo group for week 52 efficacy measures (Supplementary Table 1). Patients who could not attend the clinic were assessed via telephone.

Demographics and baseline characteristics were similar in the 2 groups, with some minor imbalances likely due to chance (Table 1; Supplementary Table 2). One patient in the CL2020 group had baseline mRS 3; all other patients had mRS 4 or 5. Baseline NIHSS scores were numerically higher in the placebo group than in the CL2020 group. FMMS total score (P = 0.041) and EQ-5D-5L score (P = 0.026) was significantly higher in the CL2020 group than in the placebo group.

During the first 12 weeks after administration, AEs were experienced by 24 (96%) and 10 (100%) patients in the CL2020 and placebo groups, respectively, most commonly gastrointestinal disorders, including diarrhea (CL2020, 28%; placebo, 30%) and constipation (CL2020, 24%; placebo, 60%) (Table 2). The occurrence of psychiatric disorders (CL2020, 16%; placebo, 70%; P = 0.004) and insomnia (CL2020, 16%; placebo, 60%; P = 0.016) were significantly low in the CL2020 group compared with the placebo group. Up to week 12, ARs occurred in 7 (28%) patients in the CL2020 group and 1 (10%) patient in the placebo group. In the CL2020 group, 3 (12%) patients had hair discoloration from gray/white to black by week 12, and 3 additional patients had hair discoloration between week 12 and week 52, for a total of 6 patients (24%); no patients in the placebo group had this AR. A 56-year-old man had a serious AR of Grade 4 status epilepticus 86 days after CL2020 administration, which was treated with antiepileptic drugs. The patient recovered at day 93 but experienced 2 additional serious AEs that were considered sequelae of status epilepticus (epileptic encephalopathy on day 94; esophageal ulcer due to antiplatelet drugs on day 129). At study end, his symptoms were stable.

During the full 52 weeks, all patients had at least 1 AE (Table 2, Supplementary Table 3). Seven serious AEs occurred in 5 patients in the CL2020 group (status epilepticus [described above], epileptic encephalopathy, femur fracture, pneumonia aspiration, coronary artery stenosis, sepsis, esophageal ulcer) and 1 patient in the placebo group (epilepsy). Non-serious ARs occurred in 3 additional patients in the CL2020 group between week 12 and week 52 (total of 10 [40%]); no ARs occurred in the placebo group after week 12.

In the responder analysis, 10 (40%) patients in the CL2020 group had mRS 0–2 at week 12 compared with 1 (10%) patient in the placebo group (Figure 2). The lower limit of the 95% CI for the CL2020 response rate was 21.1%, far exceeding the assumed efficacy threshold of 8.7%. The estimated difference between groups in response rate at week 12 was 30.0% (95% CI, –7.8 to 65.2, mid-P = 0.080) and remained at ∼30% at week 52 (Supplementary Table 4). In subgroup analyses, there was no remarkable effect of sex, baseline mRS, baseline NIHSS, days from stroke onset to study treatment, presence/absence of reperfusion therapy, or stroke type on the response rate (Supplementary Figure 1).

The adjusted mean change from baseline in mRS score was numerically greater in the CL2020 group than in the placebo group after week 4 (P = 0.079 at week 12) (Figure 3(a); Supplementary Table 5). The difference between groups was largest between weeks 12 and 28 (Figure 3(b)). Across 52 weeks, the distribution of mRS scores in the CL2020 group shifted to mRS 1 or 2 (15/22 patients; 68% at week 52); notably, 2 (8%) patients achieved mRS 1 at week 12 and 7 (32%) patients at week 52 (Figure 3(c); Supplementary Table 6). No patients in the placebo group achieved mRS 1. The distribution of mRS scores was significantly different between groups at weeks 8 (P = 0.029) and 12 (P = 0.034) (Supplementary Table 6). The percentage of patients with mRS ≤1 was greater in the CL2020 group compared with the placebo group and stayed at ∼30% between weeks 28 and 52, although differences were not statistically significant (Supplementary Table 7). The percentage of patients whose mRS scores improved by ≥2 points was greater in the CL2020 group than in the placebo group, with the greatest difference at week 12 (Supplementary Table 8); between-group differences were not statistically significant. Shift analysis demonstrated that 12-week mRS tended towards lower in the CL2020 group, but the difference was not significant (common odds ratio of a shift in score on the mRS in the CL2020 group, 3.69; 95%CI, 0.83 – 16.47; P = 0.087).

Improvements in FMMS upper limb and total scores, but not lower limb scores, were greater in the CL2020 group compared with the placebo group and were statistically significant after week 4 (Figure 4; Supplementary Table 5 and Supplementary Figure 2). At week 52, 5/21 patients (23.8%) in the CL2020 group had NIHSS score ≤1, compared with 0/8 patients in the placebo group (Supplementary Table 9). Improvements in SIAS scores were numerically greater in the CL2020 group than in the placebo group. There were no apparent differences between groups in the change from baseline for NIHSS or BI. Changes from baseline in EQ-5D-5L scores were generally greater in the CL2020 than in the placebo group (significant at week 12; Supplementary Table 5 and Supplementary Figure 2).

Classification of evidence

This study provides Class I evidence that CL2020 improved functional outcomes in patients with subacute ischemic stroke with no major safety issues.

Discussion

This randomized, double-blind, placebo-controlled study evaluated the safety and efficacy of allogenic cells for treating patients with ischemic stroke–induced neurological deficits and physical dysfunction during the subacute phase. There were no major safety issues associated with a single IV injection of CL2020, although 1 patient experienced status epilepticus. In the key responder analysis, the lower 95% CI in the CL2020 group exceeded the threshold response rate of 8.7%, which was based on Japanese patient registry data ¹⁶ and supported by international data. ¹⁷ Further, the percentage of patients with mRS ≤2 at week 12 was higher in the CL2020 group than in the placebo group (40% vs 10%). Both the change from baseline and the distribution of mRS scores supported the efficacy of CL2020 for improving functional recovery for up to 52 weeks. In addition, the CL2020 group exhibited statistically significant improvements in FMMS upper limb and total scores. Symptoms continued to improve during the first 6 months, and numerical differences between CL2020 and placebo were maintained between 6 and 12 months. Taken together, these promising results suggest that CL2020 may be a possible treatment for subacute stroke.

To date, 6 randomized studies in the subacute phase have been conducted, all with IV infusion of autologous cells,^{18 –23} but only 2 reported positive results in functional outcomes. ^{18 , 22} In an open-label study with 2 injections of autologous MSCs (MSC n = 5; control n = 25), there was a significant improvement of BI at 3 and 6 months, and a tendency for mRS improvement over 12 months that was not statistically significant. ¹⁸ Another open-label, observer-blinded study with 2 injections of autologous MSCs (MSC n = 16; control n = 36) showed a significant increase in the proportion of patients with mRS 0–3 in the MSC group at a median of 3.2 years’ follow-up. ²² In the current study, in addition to a greater proportion of patients achieving mRS ≤2 at week 12, 7 patients in the CL2020 group—but none in the placebo group—achieved mRS 1 (ie, no significant disability), which is rare for patients with an initial mRS 4 or 5 in the subacute phase. By comparison, the percentage of patients achieving mRS 0 or 1 was 2.6% at 90 days with MSCs, ¹⁹ 2% at 180 days with BM-MNCs, ²³ and 6.3% at median follow-up of 3.2 years with MSCs, ²² all with IV autologous cells. Although differences in the use of allogenic/autologous cells, number of injections, and blinding methods make comparison difficult, the percentage of CL2020-treated patients with mRS 1 was 8%, 29%, and 32% at weeks 12, 28, and 52, respectively. Allogenic CL2020 has several advantages over autologous cells: invasive treatments such as BM aspiration can be avoided, and Muse cells are ready for administration without culturing because they are already enriched.

Although improvements in secondary efficacy endpoints, such as SIAS, BI, and the percentage of patients with NIHSS score of ≤1, were not greater with CL2020 compared with placebo, significantly greater improvements in FMMS upper limb and total scores were observed from week 4 to week 52. Such early improvements in upper limb function are difficult to achieve and are directly related to improving mRS to 0 or 1. Previous research suggests that upper limb function is particularly important to “dressing” (changing clothes) as an activity of daily living and is highly correlated with patients becoming independent. ²⁴ Conversely, lower limb function improved equally well in both the CL2020 and placebo groups. This may be related to general effects of rehabilitation therapy and/or compensation from the unaffected side of the body. The lack of effect on NIHSS scores in patients with subacute stroke is not surprising given that the scale primarily measures stroke severity in the acute phase. The BI measures activities of daily living, and improvements may primarily reflect rehabilitation therapy; this could account for the similar improvement in both groups. Moreover, differences in BI between groups may not be detected due to ceiling effects. ²⁵ Quality of life, measured by EQ-5D-5L index and VAS scores, was significantly greater in the CL2020 group than in the placebo group at week 12. Similar to the BI, it is possible that rehabilitation therapy contributed to improvements in quality of life in both groups over time.

As in previous stem cell studies, ⁴ no major safety issues related to CL2020 were observed. Most AEs occurred at similar frequencies in both groups, although treatment-related hair discoloration from gray/white to black was observed in 6 patients in the CL2020 group. Notably, because CL2020 is an enriched Muse cell preparation, the number of cells injected is around 4–20 times lower than used in other cell-based systemic therapies,^{18 –23} thereby lowering the risk of pulmonary embolism. ²⁶ Although there was 1 serious AR of status epilepticus in a CL2020-treated patient, epilepsy is a known complication of stroke, reported in approximately 6%–7% of patients, ^{27 , 28} with an incidence of 2.9% during the first 6 months after stroke. ²⁷ Thus, the single status epilepticus event was not considered unusual, although its relationship to CL2020 could not be ruled out. Given that seizures in a small percentage of patients occurred in other stem cell studies, ^{29 , 30} further research is required to confirm whether these treatments increase the frequency of post-stroke epilepsy.

The main limitation of this exploratory study was that it was conducted at a single medical center, and although the sample size was agreed with the regulatory authority, the PMDA, a larger study conducted at multiple centers is required to confirm the results. We are currently planning a larger phase 3 study, which we expect will also address the slight imbalance in baseline NIHSS scores seen in this phase 2 study.

In conclusion, there is a large unmet medical need for effective, long-lasting treatments for subacute ischemic stroke. This exploratory study suggests that a single IV injection of the clinical-grade allogenic Muse cell product CL2020, without immunosuppressants or HLA matching, may be a safe and effective treatment for subacute stroke, with the potential to make regenerative medicine more accessible.

Supplemental Material

Acknowledgements

The authors thank all study participants and the following collaborators: Dr. Takahiro Morita, Dr. Yoshiharu Takahashi, Dr. Daiki Aburakawa, and Dr. Kanako Sato from the Department of Neurosurgery, Tohoku University, for case registration and clinical evaluation; Dr. Yoshihito Furusawa, Dr. Tatsuma Okazaki, and Dr. Takayuki Mori from the Department of Physical Medicine and Rehabilitation, Tohoku University, for clinical evaluation and patient care; Dr. Takako Ito from the Division of Blood Transfusion and Cell Therapy, Tohoku University Hospital, and Ms Hitomi Okita and Ms Manami Yoshida from the Clinical Research, Innovation and Education Center, Tohoku University Hospital, for preparation and delivery of CL2020 and placebo treatments; and the physical and occupational therapists from the Department of Rehabilitation, Tohoku University Hospital, who were involved in measurement of outcomes. We also thank Mr Makoto Tokuda from Life Science Institute, Inc. and Mr Masakatsu Nomura from EPS Co., Ltd. for statistical analyses.

Footnotes

References

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.