A multiyear follow-on study examining safety, immunity, and clinical outcomes
for sargramostim therapy in Parkinson’s disease

doi:10.21203/rs.3.rs-1957469/v1

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A multiyear follow-on study examining safety, immunity, and clinical outcomes for sargramostim therapy in Parkinson’s disease

https://doi.org/10.21203/rs.3.rs-1957469/v1

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Safety, biomarker, and motor outcomes in Parkinson’s disease (PD) were evaluated during extended sargramostim (Leukine®, granulocyte-macrophage colony-stimulating factor) therapy. Hematologic, metabolic, regulatory T cell (Treg) numbers and function, motor, and behavioral functions were scored in five PD patients during a 5-day on, 2-day off sargramostim regimen administered at 3 µg/kg/dose over the 33-month study. Adverse events for sargramostim included injection site reactions, increased total white cell counts, and bone pain. Treg numbers and functional improvements were sustained throughout the study. After 24 months on treatment regimen, drug use was discontinued for three months, and followed by an additional six months of treatment. No significant changes in adverse events were discernible after treatment pause or re-initiation; however, UPDRS scores increased during the pause and decreased after initiation of treatment. Taken together, the data affirmed long-term safety, Treg functional stability, and clinical stability of sargramostim in the PD population. Confirmation requiring larger numbers of enrolled patients is planned in a phase II evaluation.

Parkinson’s disease

granulocyte-macrophage colony-stimulating factor

Unified Parkinson’s Disease Rating Scale

regulatory T cells

neuroprotection

sargramostim

Globally, Parkinson’s disease (PD) disabilities and related death are increasing at rates faster than any other neurodegenerative disorder, with more than 8.5 million affected worldwide. The characteristic signs and symptoms are tremor, muscle stiffness, limited movement, impaired balance, and poor coordination. Such signs and symptoms parallel nigrostriatal degeneration with losses in nigrostriatal dopamine and dopaminergic neuron loss with extension to other parts of the neural network¹. To date, therapy is nearly exclusively devoted to symptomatic control of disease manifestations focused on the uses of levodopa, dopamine agonists, and monoamine oxidase-B inhibitors². Supportive therapies include the management of diet and exercise together with physical and occupational therapies providing and improvements for a general sense of well-being, control of blood pressure, relief of constipation, weight stability and alleviating commonly observed dysphagia, stiffness, and limited coordination³. While research efforts focus on a better understanding of disease mechanisms to date novel therapies have not substantively affected disease outcomes as available medicines are for the most part palliative. Current evidence supports the notion that PD is a multifactorial disease that occurs due to an interplay between genetic and environmental factors⁴. Animal models, epidemiologic, neuropathologic, and cellular-based research support the notion that inflammation and mitochondrial dysfunction both play central roles in PD pathophysiology^5–7. Both lead to deficits in bioenergetics, the generation of reactive oxygen species, aberrant immune homeostasis, and adaptive immune imbalance which ultimately affect the pathways of neuronal cell death in PD. Each in the presence of familial PD-linked genes and environmental factors affects brain homeostasis and neural cell integrity. Our prior works have been devoted towards discovery of both innate and adaptive immune pathways that affect nigrostriatal degeneration and as a result are keys to novel therapeutic intervention in PD by transforming neurotoxic immunity to a neuroprotective outcome^8–15. In support of this notion Lewy bodies, containing aggregated and nitrated α-synuclein (N-α-syn), released into the extraneuronal environment affect the emergence of effector T cell (Teff) populations which exacerbate disease outcomes^16,17.

Sargramostim (Partner Therapeutics, Inc., Lexington, MA), a human recombinant granulocyte-macrophage colony-stimulating factor, is known to affect myeloid recovery in bone marrow transplantation or chemotherapy during cancer therapy¹⁸. Its abilities to transform Teff to regulatory T cells (Treg) immune responses have been demonstrated to lead to nigrostriatal neuroprotection in a broad range of animal models^{8,11,14,19−23}. The current report is a continuation of an early safety, biomarker, and clinical drug evaluations^24,25. We now show that 33 months of treatment results in stable hematologic and metabolic profiling. Immune transformation is highlighted by sustained numbers and function of Treg which have been shown to be neuroprotective in many models²⁶. Notably, albeit considering limited patient numbers, both motor and behavioral functions proved stable in a 5-day on, 2-day off treatment regimen at 3 µg/kg/dose. Adverse events were limited to injection site reactions, increased total white cell counts, and mild bone pain. Drug discontinuation proved to exacerbate disease signs and symptoms, while drug restoration improved disease signs and symptoms. These data further support the drug’s use as an adjunctive therapy for PD. A planned phase II study is in the offering to substantiate the early treatment outcomes.

Safety, tolerability, and adverse event profiles

Decreasing sargramostim dosage from 6 µg/kg daily to 3 µg/kg for 5 days on/2days off and prolonging treatment over the 33-month study was found to be safe and generally well-tolerated in PD subjects^24,27. However, 100% of subjects reported at least one adverse event during extended treatment, and the average likelihood that adverse events were study drug-related was scored less than possible. More succinctly, 56% (112/200) of recorded adverse events were classed as unrelated or unlikely to be related to drug treatment, whereas 37.5% (75/200) were deemed probably or definitely related to drug; the remainder (13/200, 6.5%) were scored as possibly related to drug treatment. The reported adverse events included elevated WBC counts (5/5; 100%), injection site reactions (4/5, 80%), falls (3/5, 60%), non-infectious skin lesions (4/5, 80%), GI tract problems/nausea (3/5, 60%), neurological dyskinesias (3/5, 60%), and ophthalmological issues (3/5, 60%) (Table 1). Less frequently reported adverse events included chest pain, pain in the upper torso/extremities, pain other than extremities, headache, cardiovascular issues, sleep anomalies, and neoplasms for 1/5 subjects (20%) as well as infection, muscle soreness, and weight loss for 2/5 subjects (40%). Treatment resulted in one serious adverse event and one severe adverse event. The severe adverse event involved leg cramping that was scored as possibly related to drug therapy, and the serious adverse event was a thromboembolic event that was unlikely related to drug therapy. However, neither of these adverse events led to drug withdrawal. Additionally, and as expected, sargramostim treatment significantly increased levels of white blood cells, including eosinophils, neutrophils, monocytes, and basophils in peripheral blood (Supplemental Table 1). Additionally, evaluation of blood chemistry revealed a relatively normal comprehensive metabolic profile over time (Supplemental Table 2) further validating the safety of an extended low-dose therapy. Treatment resulted in a significant decrease from baseline in aspartate transaminase (AST), total protein, and albumin levels. However, the alteration was deemed safe and non-concerning by the study neurologist. Absolute T cell profiles and ratios also remained unchanged during treatment (Supplemental Table 3).

Unified Parkinson’s Disease Rating Scale (UPDRS) scores

UPDRS Part II and III scores were monitored over three months prior to initiating treatment to establish baseline motor function for assessment of disease progression. No worsening of motor function scores measured either in UPDRS Part III (Fig. 1a) or as combined Parts II and III (Fig. 1b) was observed while on sargramostim during the course of treatment compared to baseline values. Indeed, sargramostim treatment resulted in sustained decreases in UPDRS scores for Part III and combined Parts II and III by three months (Fig. 1a and 1b). Average decreases from baseline of five points were observed and maintained for 24 months while on sargramostim (Fig. 1c and 1d). After initiation of a 3-month drug intermission at 24 months, UPDRS Part III and combined Parts II and III scores returned to baseline, but were not significantly elevated above pretreatment baseline levels measured 24 months prior to intermission (Fig. 1a-d). Additionally, upon re-introduction of sargramostim, subjects experienced another diminution of motor scores below pretreatment baseline that resulted in significant changes below baseline levels by six months after reinitiation of sargramostim.

Treg Function and Phenotype

Evaluation of peripherally isolated Treg function revealed a significant increase in immunosuppressive capacity following initiation of sargramostim at all times measured during treatment and including one month after drug cessation (Fig. 2a and 2b). However, quantification of Treg activity using area under the curve (AUC) for percentage inhibition of Tresp proliferation as a function of Treg number reveals a significant elevation in function at all time points during sargramostim treatment compared to the mean AUC for pretreatment baseline measurements (Fig. 2c). Additionally, determination of Treg activity utilizing the number of cells required to inhibit 50% proliferation indicates that sargramostim treatment induces a Treg population with 75% greater mean capacity to significantly inhibit Tresp proliferation compared to Treg isolates tested before sargramostim treatment (Fig. 2d). This increased Treg activity was maintained for at least one month after treatment cessation. Correlation analyses of Treg activity with combined UPDRS Parts II and III motor scores indicate that increasing motor scores correlated with decreasing Treg activity measured either as AUC (Fig. 2e) or as the number of Tregs necessary to attain 50% inhibition of T cell proliferation (Fig. 2f). Furthermore, long-term treatment with sargramostim resulted in a non-significant elevation in CD4 + T lymphocytes and CD4 + CD25 + CD127high Teff over time (Fig. 3a and 3b). However, CD4 + CD25 + CD127low Treg frequencies were significantly increased and sustained for 24 months of treatment (Fig. 3c). Following drug cessation, Treg levels returned to baseline, but upon re-introduction of drug, Treg levels reached another significant elevation within two months. Flow cytometric evaluation of Treg immunosuppressive and migratory markers also revealed a sustained increase in FOXP3+, CTLA+, ItgB7+, CD31 + and CD45RO + Treg populations over time (Fig. 3d-3h). Increased Treg markers are congruent with Treg function data that show sargramostim induces a population of Tregs with higher overall function (Fig. 2a-d), which may be due in part to increases in Treg subset frequencies, suppressive capabilities, or both.

Effect of Neutralizing anti-sargramostim antibodies on Treg number, Treg function, or UPDRS scores

Presence of neutralizing anti-sargramostim antibodies was evaluated in serum isolated from peripheral blood of individual subjects before and during the first 12 months of treatment. Four of the initial five subjects developed anti-drug antibodies (ADAs) within the first four months of treatment (Fig. 4a). However, correlation analyses assessing change in Treg frequency or change in UPDRS Part III motor scores with ADA titers revealed that increased Treg frequency and improved motor function were not stunted by the presence of neutralizing antibodies (Fig. 4b and 4c). This affirmed an earlier clinical trial showed that anti-sargramostim antibodies produced little or no untoward effects as significant titers of neutralizing antibodies were present at times during sargramostim treatment when UPDRS Part III scores were below pretreatment levels, and Treg frequencies and function were significantly increased compared to placebo controls²⁷.

Standard approaches for PD treatment centers on pharmacologic dopamine replacement using either carbidopa/levodopa, dopamine agonists, or medicines that prolong the actions of endogenous dopamine². However, such a therapeutic intervention deals only with symptoms and does not consider the broader aspects of what is certainly a multifaceted disease⁴. Additionally, deep brain stimulation surgery is utilized when anti-parkinsonian medication responses can no longer affect disease signs and symptoms²⁸. Alternative medicine and integrative medicine approaches can improve the sense of well-being as well as brain and overall health. Exercise, diet, and behavioral interventions have also proven to be neuroprotective leading to improvements in the quality-of-life^29–31. Previously, we have shown that sargramostim stimulates peripheral T cell and monocyte responses in vivo that are shown to affect reactive oxygen species, autophagy, and anti-inflammatory responses are linked to neuroprotective outcomes^24,25,27. Data provided in the current report support this notion by clear demonstration of sustained effects on immune function that occurs in a safe, and well-tolerated therapeutic setting.

In the current report, an extended therapeutic regimen of sargramostim was demonstrated to be safe for 33 months of administration in PD subjects. Expected adverse events included increased WBC, injection site reactions, and bone and chest pain that have been previously reported with sargramostim treatment^18,24,27. Hematologic, metabolic, immune, motor, and behavioral functions were stable or had improved during a 5-day on, 2-day off treatment regimen. Potential efficacy was supported during three months of drug discontinuation followed by re-initiation. Taken together, the data affirmed clinical stability of the treatment regimen. The study serves to support a body of accumulating research highlighting a prominent role of the innate and adaptive immune system in both the development and progression of PD along with many other nervous system pathologies^7,32−36. Specifically, a body of pre-clinical and translational studies demonstrate that Teff responses affect disease onset and progression by exacerbating innate microglial inflammation³⁷. In contrast, Tregs have been shown to suppress adaptive and innate effector populations³⁸. Such results were demonstrated in diverse neurodegenerative disorders that include multiple sclerosis (MS), Guillain-Barre syndrome (GBS), neuropathic pain, traumatic brain injuries, stroke, amyotrophic lateral sclerosis (ALS), PD, and Alzheimer’s disease (AD)^24,39−48.

Treg hold significant promise as candidates to affect immune transformation and develop novel therapeutics in multiple clinical settings. For instance, transient depletion of Treg was demonstrated to facilitate AD cognitive decline and linked to diminished microglial clearance of amyloid⁴⁵. On the other hand, restoration of Treg numbers and function increased microglia plaque clearance and improved cognitive functions. Reductions in Treg numbers and suppressive function parallel AD clinical progression. However, following Treg expansion, cell function was restored including control of pro-inflammatory macrophage activities. Each support the notion that restoration of Treg function can serve to restore brain homeostasis by reductions in the inflammatory disease state. Parallel findings have been observed in a spectrum of autoimmune and degenerative diseases of the nervous system where disease severity was found to be associated more with changes in T cell numbers and function than with age, onset, duration, and/or progression^7,32−36. Taken together, each of these findings serve to support the importance in transforming Treg function, as supported in the current report, to control immune responses and to demonstrate a sustained multiyear neuroprotective outcome for halting disease progression and maintaining homeostatic control.

Tregs serve as a subpopulation of immunosuppressive T cells that serve to sustain immune homeostasis. They do so by maintaining self-tolerance serving as negative regulators of inflammation during autoimmune and degenerative diseases whose functions are altered in nervous system pathologies^49–51. Tregs are reduced in number and function including the cell’s abilities to suppress activated pro-inflammatory macrophage. When function is restored, Tregs display increased expression of factors such as FOXP3, IL2Ra (CD25), NT5E (CD73), IL10, IL13, CTLA4, PDCD1 (PD1), and GRZMB⁵². There is a shift towards a pro-inflammatory peripheral immune response in PD with the loss of Treg suppressive functions affecting disease typified by a systemic pro-inflammatory response^24,27,53. The restoration and enhancement of Treg suppressive functions in this study underlies the importance of control of adaptive immune activities as a therapeutic approach for PD⁵⁴. Long-term sargramostim therapy resulted in a sustained increase in peripheral Treg frequency displaying a stable immunosuppressive phenotype and elevated expression of surface markers required for migration to sites of inflammation. Additionally, presence of this population was correlated with clinical enhancement, indicating their potential role in affecting disease course. Lastly, although neutralizing ADAs were developed in some subjects, it is not likely that these antibodies had a detrimental effect on Treg number, function, or motor improvements. Likewise, while the sample size is small and larger clinical studies are needed to confirm the data sets, it is clear that therapeutic interventions targeting broad disease components of PD are needed to slow progression and lead to a viable outcome to those affected individuals.

Study Design and Subject Enrollment

This report served to evaluate the safety and tolerability of sargramostim (Partners Therapeutics, Inc., Lexington, MA) administered subcutaneously at a dose of 3 µg/kg for 33 months with a 5-days on and 2-days off regimen. Five subjects who met the study inclusion criteria were recruited. Patients were evaluated for three months to assess baseline immune, hematological, and metabolic profiles that followed treatment initiation. The study evaluation continued for 33 months. At two years of continuous treatment, drug use was discontinued for three months. This was then followed by an additional six months where treatment was reinstated. One of four subjects halted study after 25 months due to deep brain surgery.

Ethics

The research study protocol was continuously approved by the UNMC Institutional Review Board during the entire course of study (IRB Protocol 839 − 18). Subjects were referred to the Clinical Research Center (CRC) by their primary care physician or the study neurologist. Subjects were enrolled after informed consent was obtained by the study physician following Good Clinical Practice standards. No randomization or blinding was performed, as all study subjects were provided treatment. The trial is registered at ClinicalTrials.gov, identifier: NCT03790670.

Procedures

The current study is a continuation of our previous year-long evaluation in which the same subjects and study protocol was followed²⁴. PD subjects underwent three baseline appointments to determine initial immune, hematologic, and metabolic profiles (Supplemental Tables baseline column). After baseline assessment, subjects initiated self-administration of sargramostim (Leukine, hu-reGM-CSF) at 3 ug/kg/day (five days on, two days off) subcutaneously for 24 months, returning for clinical assessments every four weeks or eight weeks. After 24 months, drug cessation and wash out occurred for three months, followed by re-introduction of sargramostim for an additional six months. At each clinical visit, peripheral blood samples, physical examinations, and motor assessments were completed. Anti-parkinsonian therapies (carbidopa-levodopa) were not altered during the course of study for 4/5 subjects. The study neurologist performed UPDRS assessments in an “on” anti-parkinsonian drug state, noted observable clinical adverse events, and determined their likelihood of relationship to treatment. In between clinical visits, subjects were provided an “adverse event log” that was discussed and recorded during scheduled clinical evaluations. WBC counts with differentials, comprehensive blood chemistry profiles, and CD4 and CD8 T cell percentages and ratios were monitored for safety. Additionally, peripheral blood was stained with fluorescently-conjugated monoclonal antibodies against CD4 (FITC or AF700), CD127 (PerCP-Cy5.5), CD25 (PE), FOXP3 (AF647), Helios (AF-488), CD152/CTLA-4 and/or iCTLA-4 (APC), CD95/FAS/Apo1 (APC), CD39 (APC), CD31 (AF647), CD27 (APC), CD45RA (AF700), CD45RO (APC), CCR7 (PE-Cy7), Integrin β7 (APC) (all from BD Biosciences, San Jose, CA), and CD49d (PE-Cy7) (BioLegend Inc., San Diego, CA), with isotype-matched antibodies serving as negative controls. Populations were gated as previously described²⁴. Fluorescent labels were then examined with an LSR II flow cytometer (BD Biosciences) and analyzed using BD FACSDiva software. Immunosuppressive function, cellular assays, and quantification of anti-sargramostim antibodies was done as previously described^24,27.

Outcomes

The primary study endpoint was drug safety and tolerability assessed by complete blood counts with differential, comprehensive blood chemistry profiles, physical examination, and changes in UPDRS scores. Hematological and blood chemistry profiles were performed by a clinical diagnostics laboratory, and one neurologist performed all clinical examinations. Adverse events were recorded and scored based on event severity as mild (1), moderate (2), or severe (3). Events were also scored in relation to drug treatment as unrelated (1), unlikely (2), possible (3), probable (4), or definitely related (5), as previously described²⁴. No adverse events required interruption of treatment, and all safety events were evaluated and monitored by a data and safety monitoring board consisting of UNMC physicians and faculty during the course of study. Secondary outcomes were peripheral blood immune profiles, cellular function, and presence of anti-drug antibodies over time.

Statistical Analysis

Sample size estimates of five PD subjects were determined to provide 80% power and to afford an increased score of 1·63 (32%) in baseline immune response using a two-sided Wilcoxon test assuming normal distribution. Statistical analysis was performed using GraphPad Prism 8.0 software (La Jolla, CA) and Statistica v13.3 (Tibco Software, Palo Alto, CA). All values are expressed as mean ± SD. Differences in between-group means were analyzed using one-way ANOVA and p values for multiple comparisons with baseline were adjusted by Dunnett’s post hoc test and for false discovery rate (FDR) at 5% using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli⁵⁵. Significant differences for these studies were selected at p < 0·05. All correlation analyses were determined using Pearson product-moment correlation coefficients, best-fit lines were determined using linear regression, p values were determined for r values.

Role Of Funding Source

The funding sources had no role in the study design or data collection, analysis, interpretation, or writing of the manuscript. The corresponding author had access to all data sets and takes complete responsibility for the published manuscript.

Author contributions

K.E.O.: execution of the study, drafting the manuscript, study review, and data analysis. K.L.N., Y.L., M.M.A.: data collection, study execution, and manuscript preparation. H.O., P.S.: subject recruitment, study execution, data collection, execution of the research project. R.L.M., H.E.G.: conception and organization of the research project, design and organization of the research, statistical analysis, review, and critique of manuscript. All authors critically read and approved the final manuscript.

Data availability

The datasets generated for this study and full study protocol are available upon request from the corresponding author.

Competing interests

The authors declare no conflicts of interest.

Acknowledgements

The authors thank the participants and family members for their willingness to participate to the study as well as those who supported our research efforts including investigational pharmacy services, the Great Plains Center for Clinical and Translational Research, and our data safety monitoring board. The authors would also like to thank the UNMC Flow Cytometry Research Facility for exceptional flow cytometric analysis and support, the INBRE grant from NIH (2P20GM103427) for supporting a site license to EndNote software. Lastly, we express our gratitude to the funding sources of the University of Nebraska Foundation, which includes community donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, the Eisenberg Parkinson's Research Fund, and the Frances and Louie Blumkin and Harriet Singer Research Foundations. We also thank the Vice Chancellor's Office of the University of Nebraska Medical Center for Core Facility support and Partner Therapeutics who provided medicines for study and vibrant discussions in study design.

Funding: The study was supported by private support from the University of Nebraska Foundation and National Institute of Neurological Disorders and Stroke grant 5 R01NS034239-25.

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A multiyear follow-on study examining safety, immunity, and clinical outcomes for sargramostim therapy in Parkinson’s disease

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Abstract

Figures

Introduction

Results

Safety, tolerability, and adverse event profiles

Unified Parkinson’s Disease Rating Scale (UPDRS) scores

Treg Function and Phenotype

Effect of Neutralizing anti-sargramostim antibodies on Treg number, Treg function, or UPDRS scores

Discussion

Materials And Methods

Study Design and Subject Enrollment

Ethics

Procedures

Outcomes

Statistical Analysis

Role Of Funding Source

Declarations

References

Table

Additional Declarations

Supplementary Files

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