Safety, tolerability, and immune-biomarker profiling for year-long sargramostim treatment of Parkinson's disease

Abstract

Background: Neuroinflammation plays a pathogenic role in Parkinson's disease (PD). Immunotherapies that restore brain homeostasis can mitigate neurodegeneration by transforming T cell phenotypes. Sargramostim has gained considerable attention as an immune transformer through laboratory bench to bedside clinical studies. However, its therapeutic use has been offset by dose-dependent adverse events. Therefore, we performed a reduced drug dose regimen to evaluate safety and to uncover novel disease-linked biomarkers during 5 days/week sargramostim treatments for one year.

Methods: Five PD subjects were enrolled in a Phase 1b, unblinded, open-label study to assess safety and tolerability of 3 μg/kg/day sargramostim. Complete blood counts and chemistry profiles, physical examinations, adverse events (AEs), immune profiling, Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS) scores, T cell phenotypes/function, DNA methylation, and gene and protein patterns were evaluated.

Findings: Sargramostim administered at 3 μg/kg/day significantly reduced numbers and severity of AEs/subject/month compared to 6 μg/kg/day treatment. While MDS-UPDRS Part III score reductions were recorded, peripheral blood immunoregulatory phenotypes and function were elevated. Hypomethylation of upstream FOXP3 DNA elements was also increased.

Interpretation: Long-term sargramostim treatment at 3 μg/kg/day is well-tolerated and effective in restoring immune homeostasis. There were decreased numbers and severity of AEs and restored peripheral immune function coordinate with increased numbers and function of Treg. MDS-UPDRS Part III scores did not worsen. Larger patient numbers need be evaluated to assess conclusive drug efficacy (ClinicalTrials.gov NCT03790670).

Funding: The research was supported by community funds to the University of Nebraska Foundation and federal research support from 5 R01NS034239-25.

Keywords: Granulocyte macrophage-colony stimulating factor; Neuroprotection; Parkinson's disease; Regulatory T cells; Sargramostim; Unified Parkinson's Disease Rating Scale.

Conflict of interest statement

Declaration of Competing Interest The authors declare no conflicts of interest.

Published by Elsevier B.V.

Figures

Fig. 1

Adverse events (AE) comparing two clinical trials of sargramostim. PD subjects were administered sargramostim (Leukine®, human recombinant GM-CSF) in a previous Phase 1a (Ph 1a) (n = 10) and current Phase 1b (Ph 1b) clinical trial (n = 5). Subjects in the Ph 1a trial received 6 μg/kg of sargramostim every day for 2 months. In a proof-of-concept study to attenuate AE frequency and severity and extend administration, subjects in the Ph 1b trial received 3 μg/kg sargramostim on a 5 day on/2 day off regimen for 12 months. (a) Total number of adverse events (AEs) per subject recorded during the 2- and 12- month interventional period (Total) normalized on a monthly basis (Total/Mo). (b) AE severity scored on a scale of 1–3 severity as (1) mild, (2) moderate, or (3) severe. Mild events cause minimal discomfort or concern, may require minimal or no treatment, and do not interfere with daily activities. Moderate events were defined as causing discomfort, inconvenience, or concerns which were ameliorated with simple therapeutic measures. Severe adverse events were defined as causing discomfort or incapacitation that require prescription drug therapy or other treatments or interventions by medical personnel. Differences in means (± SD) for Total AE/Subject, Total AE/Subject/Mo, and Severity of AEs between Ph Ia vs Ph 1b trials were determined by Student's t-test and p values annotated above the pair-wise comparisons. (c) Graphical representation displaying reported AE based on AE severity (y axis), AE category (z axis), and day of treatment (x axis) for the Ph 1a trial. (d) Graphical representation displaying reported AE based on AE severity (y axis), AE category (z axis), and day of treatment (x axis) for the Ph 1b study. (c and d) AE categories are defined in Table 2.

Fig. 2

MDS-Unified Parkinson's Disease Rating Scale (UPDRS) Part III motor assessment before and during sargramostim treatment. Prior to treatment, subjects (n = 5) underwent at least three separate baseline evaluations (at month -4, -3, -2, and/or -1 before initiation) and then began drug administration (3 ug/kg per day, 5 days on/2 days off). After treatment initiation, subjects were evaluated by the study neurologist once/month for 6 months and once/ every 2 months thereafter for 12 months. (a) Raw UPDRS Part III scores over time grouped for individual subjects (2001, 2003, 2004, 2005, 2006). (b) Total mean UPDRS Part III scores grouped by time of treatment. Blue dashed lines indicate mean baseline measurement. (c) Mean UPDRS Part III scores ± SD grouped by combined (All) and individual subjects. Specific p values are indicated above each subject. Baseline values are represented as blue circles and sargramostim treatment is represented as red squares. (d) Change from baseline in UPDRS Part III scores over time grouped for individual subjects. (e) Mean change from baseline ± SD in UPDRS Part III scores grouped by time of treatment. (f) Mean change from baseline in UPDRS Part III scores ± SD grouped by combined (All) and individual subjects. Specific p values are indicated above each subject. Baseline values are represented as blue circles and sargramostim treatment is represented as red squares. Significant differences (± SD) in baseline and treated means were determined by Student's t-test with p values denoted above comparisons. Differences in means ± SD over time were also determined by one-way ANOVA where p ≤ 0.05 compared to baseline (b).

Fig. 3

Flow cytometric analysis of CD4+ peripheral blood populations over time. Quantification of frequencies for the following dependent variables: (a) CD4+ lymphocytes, (b) CD4+CD127lowCD25+ Tregs, (c) CD4+CD127highCD25+ Teffs, (d) CD4+FOXP3+, (e) CD4+FOXP3+HELIOS+, (f) CD4+CD31+, (g) CD4+CTLA+, (h) CD4+ItgB7+, (i) CD4+ItgA4B7+ over the course of treatment. Differences in means (± SD) for each dependent variable grouped by time on treatment were determined by one-way ANOVA and Dunnett's post hoc test where p ≤ 0.05 compared to baseline (*).

Fig. 4

Flow cytometric analysis, immunosuppressive function, and FOXP3 Treg-Specific Demethylated Region (TSDR) methylation status of CD4+CD127lowCD25+ Treg subsets within CD4+ peripheral blood lymphocytes. Quantification for the dependent variables included (a) CD45RA-RO+ Treg, (b) CD31+ Treg, (c) CTLA+ Treg, (d) CD49+ Treg, and (e) ItgB7+ Treg subset frequencies within peripheral blood over time. Difference in means (± SD) for each dependent variable grouped by time of treatment were determined by one-way ANOVA and Dunnett's post hoc test where p ≤ 0.05 compared to baseline (*). (f) Percent demethylation (± SD) within the TSDR of the FOXP3 intron from isolated Tregs before and at 2 and 6 months after initiation of sargramostim treatment. Differences in means (± SD) were determined by one-way ANOVA where p ≤ 0.05 compared to baseline (*). (g) Quantification of Treg-mediated suppression (± SD) of Tresp (CD4+CD25-) proliferation at various Tresp:Treg ratios. Treg-mediated suppression is calculated as % Inhibition = 1 – (% Proliferating Tresp:Treg ÷ % Proliferating Stimulated Tresp Alone) and is reported as percent inhibition. Linear regression analysis indicates r2 ≥ 0.81, p < 0.0001 for all lines and significant elevation (p < 0.0001) from baseline (blue) compared to each month of treatment. (h) Correlation analysis for percent TSDR demethylation and corresponding Treg-mediated inhibition (Treg activity, AUC) at baseline (blue), 2-month (red), and 6-month (green) during sargramostim treatment, indicating a direct correlation of TSDR demethylation and Treg activity with r = 0.3212, p = 0.0004.

Fig. 5

Elevated peripheral blood markers are associated with enhanced Treg function. Correlation analyses are depicted for the dependent variables including AUC for Treg activity and (a) %Integrin B7+CD4+ T cells, (b) %FOXP3+CD4+ T cells, (c) %FAS+CD4+ T cells, (d) %CD45RA-RO+CD4+ T cells, (e) %CD27+CCR7- Treg, (f) %Integrin B7+ Treg, (g) %Integrin A4B7+ Treg, and (h) %CD45RA-CD27+CCR7- Treg. For all correlation analyses, regression bands are indicated by dashed lines that encompass the 95% confidence intervals (red) and 95% prediction values (blue). Pearson r and p values are depicted on individual graphs. Data are displayed as scatter plots using the percentage of peripheral blood marker in either the total CD4 population or the Treg population against Treg activity, AUC. Correlations were determined using Pearson product–moment correlation coefficients, p values determined for correlation coefficients greater than 0.25, and the resulting 16 determinations adjusted for FDR. Best-fit lines were determined by linear regression.

Fig. 6

Elevated peripheral blood markers and enhanced Treg function are associated with decreased UPDRS Part III scores. Correlation analyses for the dependent variables that included UPRS Part III scores and (a) %FAS+ CD4+ T cells, (b) %Integrin B7+ Tregs, (c) %CD45RA+CD27+CCR7- Tregs, (d) %CD27+CCR7- Tregs, and (e) Treg activity as determined by 50% Inhibitory Treg number. For all correlation analyses, regression bands are indicated by dashed lines that encompass the 95% confidence intervals (red) and 95% prediction values (blue). The Pearson r and p values are depicted on individual graphs. Data are displayed as scatter plots using the percentage of peripheral blood marker in total CD4 population, Treg population, or Treg function as determined about 50% inhibitory Treg number against UPDRS Part III scores. Correlations were determined using Pearson product-moment correlation coefficients, p values determined for correlation coefficients greater than 0.25, and the resulting 39 determinations adjusted for FDR. Best-fit lines were determined using linear regression.

Fig. 7

Differential proteomic analysis of peripheral blood lymphocytes at 2 and 6 months after treatment. Volcano plots showing the fold change (treatment versus baseline) plotted against the p value highlighting significantly changed proteins (red – upregulation and green – downregulation; p ≤ 0.05 and an absolute fold change of 2). The vertical lines correspond to the absolute fold change of 2, and the horizontal line represents a p value of 0.05.