Combined Regulatory T-Lymphocyte and IL-2 Treatment Is Safe, Tolerable, and Biologically Active for 1 Year in Persons With Amyotrophic Lateral Sclerosis

Abstract

Background and objectives: In a phase 1 amyotrophic lateral sclerosis (ALS) study, autologous infusions of expanded regulatory T-lymphocytes (Tregs) combined with subcutaneous interleukin (IL)-2 were safe and well tolerated. Treg suppressive function increased and disease progression stabilized during the study. The present study was conducted to confirm the reliability of these results.

Methods: Participants with ALS underwent leukapheresis, and their Tregs were isolated and expanded in a current Good Manufacturing Practice facility. Seven participants were randomly assigned in a 1:1 ratio to receive Treg infusions (1 × 10⁶ cells/kg) IV every 4 weeks and IL-2 (2 × 10⁵ IU/m²) injections 3 times/wk or matching placebo in a 24-week randomized controlled trial (RCT). Six participants proceeded into a 24-week dose-escalation open-label extension (OLE). Two additional participants entered directly into the OLE. The OLE included dose escalation of Treg infusions to 2 × 10⁶ cells/kg and 3 × 10⁶ cells/kg at 4-week intervals.

Results: The Treg/IL-2 treatments were safe and well tolerated, and Treg suppressive function was higher in the active group of the RCT. A meaningful evaluation of progression rates in the RCT between the placebo and active groups was not possible due to the limited number of enrolled participants aggravated by the COVID-19 pandemic. In the 24-week OLE, the Treg/IL-2 treatments were also safe and well tolerated in 8 participants who completed the escalating doses. Treg suppressive function and numbers were increased compared with baseline. Six of 8 participants changed by an average of -2.7 points per the ALS Functional Rating Scale-Revised, whereas the other 2 changed by an average of -10.5 points. Elevated levels of 2 markers of peripheral inflammation (IL-17C and IL-17F) and 2 markers of oxidative stress (oxidized low-density lipoprotein receptor 1 and oxidized LDL) were present in the 2 rapidly progressing participants but not in the slower progressing group.

Discussion: Treg/IL-2 treatments were safe and well tolerated in the RCT and OLE with higher Treg suppressive function. During the OLE, 6 of 8 participants showed slow to no progression. The 2 of 8 rapid progressors had elevated markers of oxidative stress and inflammation, which may help delineate responsiveness to therapy. Whether Treg/IL-2 treatments can slow disease progression requires a larger clinical study (ClinicalTrials.gov number, NCT04055623).

Classification of evidence: This study provides Class IV evidence that Treg infusions and IL-2 injections are safe and effective for patients with ALS.

Figure 1. Study Design

Seven participants with rapidly progressing ALS were enrolled and underwent leukapheresis. The participants were randomized at a 1:1 ratio to active drug or placebo. Participants received IL-2 or placebo subcutaneous injections between 2 and 4 weeks before week 0. In the RCT, placebo or Treg infusions were administered every 4 weeks beginning week 0 for a total of 6 infusions. The final assessment of the RCT was performed on week 24. Six participants from the RCT entered the open-label extension (OLE). Two additional participants were directly enrolled into the OLE. All 8 participants received IL-2 beginning 2–4 weeks before the first Treg infusion. Treg infusions were administered every 4 weeks in the OLE beginning week 26 (week 0 for 2 participants who entered directly into the OLE) for a total of 6 infusions. The first 2 infusions were a 1× dose of Tregs, the next 2 infusions were a 2× dose, and the final 2 infusions were a 3× dose. The final assessment of the OLE was performed on week 50, and the final study visit occurred on week 54. IL = interleukin; RCT = randomized controlled trial; Treg = regulatory T-lymphocyte.

Figure 2. Treg Suppressive Function and Numbers in the Randomized Controlled Trial

(A) Treg suppressive function was assessed in each participant in the placebo and Treg/IL-2 treatment groups at screening and week 24 (4 weeks after the last infusion of the RCT). (B) Treg numbers were assessed in each participant in the placebo and Treg/IL-2 treatment groups at screening and week 24 (4 weeks after the last infusion of the RCT). Data were depicted as visit-specific estimates ±standard error and were compared for progression of continuous end points using a shared-baseline, linear mixed model. A p < 0.05 was considered significant. IL = interleukin; RCT = randomized controlled trial; Treg = regulatory T-lymphocyte.

Figure 3. Disease Progression During the RCT and OLE

(A) Four participants were randomized to the placebo group and received infusions every 4 weeks as depicted by the vertical dotted lines. Infusions #1–6 were placebo infusions in the RCT, and #7–12 were Treg infusions in the OLE. (B) Three participants were randomized to the active group (Treg/IL-2 treatment) and received infusions every 4 weeks. Infusions #1–6 were Treg infusions in the RCT, and #7–12 were Treg infusions in the OLE. Disease progression was documented in each participant by the ALSFRS-R over the duration of the 54-week study including the screening period. Placebo or IL-2 subcutaneous injections were administered beginning 2–4 weeks before the first infusion at week 0 for the RCT and 2 weeks before the 7th infusion at week 26 for the OLE. (C) Two participants were directly enrolled into the OLE and received Treg infusions every 4 weeks as depicted by the vertical dotted lines. Disease progression was documented in each participant by the ALSFRS-R over the duration of the 28-week study including the screening period. IL-2 subcutaneous injections were administered beginning 4 weeks before the first infusion at week 0. ALSFRS-R = ALS Functional Rating Scale–Revised; IL = interleukin; OLE = open-label extension; RCT = randomized controlled trial; Treg = regulatory T-lymphocyte.

Figure 4. Treg Suppressive Function and Numbers in the Open-Label Extension (OLE)

(A) Treg suppressive function was assessed in each participant at screening, 4 weeks after the second infusion of a 1× dose of Tregs (week 34), 4 weeks after the second infusion of a 2× dose (week 42), and 4 weeks after the second infusion of a 3× dose (week 50). (B) Treg numbers were assessed in each participant at screening, 4 weeks after the second infusion of a 1× dose of Tregs (week 34), 4 weeks after the second infusion of a 2× dose (week 42), and 4 weeks after the second infusion of the 3× dose (week 50). Data were depicted as visit-specific estimates ±standard error and were compared for progression of continuous end points using a shared-baseline, linear mixed model. A p < 0.05 was considered significant. Treg = regulatory T-lymphocyte.

Figure 5. Disease Progression of Participants During the Open-Label Extension (OLE)

Disease progression was documented in each participant by the ALSFRS-R over the duration of the 24-week OLE. The timing of the Treg infusions was depicted by the vertical dotted lines with the corresponding Treg dosages. The baseline ALSFRS-R value for the OLE was taken at week 26 for the 6 participants who went through the RCT and week 0 for the 2 participants who entered directly into the OLE. The total change in the ALSFRS-R was calculated after 24 weeks. Six of the 8 participants showed intermediate to no progression of the ALSFRS-R (average of −2.7 points). Two of the 8 participants showed rapid progression (average of −10.5 points). ALSFRS-R = ALS Functional Rating Scale–Revised; Treg = regulatory T-lymphocyte.

Figure 6. Peripheral Markers of Inflammation and Oxidative Stress During the Open-Label Extension (OLE)

Sera from all 8 participants were collected during the OLE and assayed through an Olink proteomic study for markers of inflammation and oxidative stress. Two markers of inflammation, (A) IL-17F and (B) IL-17C, were higher in the 2 participants who progressed rapidly during the OLE compared with the 6 participants who showed intermediate to no progression. One marker of oxidative stress, (C) oxidized low-density lipoprotein receptor 1 (OLR1), was elevated in the 2 rapidly progressing participants compared with the other 6 participants. An ELISA for another marker of oxidative stress, (D) oxidized low-density lipoprotein (ox-LDL), showed higher levels in the 2 rapidly progressing participants compared with the other 6 participants. Levels of IL-17F, IL-17C, and OLR1 in sera from 9 healthy controls were depicted as shaded areas of the mean ± SD. Levels of ox-LDL in sera from 26 healthy controls were depicted as a shaded area of the mean ± SD. The levels of ox-LDL in the participants were standardized to the control levels. These data were not statistically analyzed as there were only 2 participants in the rapidly progressive group. IL = interleukin.