Phase II Trial to Evaluate Safety and Efficacy of GM-CSF/Sargramostim in Alzheimer's Disease (SESAD)
Phase: PHASE2
Conditions: Alzheimer Disease
Sponsor: University of Colorado, Denver

Clinical Study Protocol 
 
COMIRB# 19-2727 
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      Confidential 
 
 
 
 
 
 
 
 
Study Protocol Number: COMIRB # 19-2727 
 
Study Protocol Title:  Phase II Trial to Evaluate Safety and Efficacy of GM-
CSF/Sargramostim in Alzheimer’s Disease (SESAD) 
 
Sponsor(s): Huntington Potter, PhD 
Department of Neurology 
University of Colorado School of Medicine 
 
Safety Monitor Vijaya Knight, MD, PhD 
  
Lead Medical Sub-
Investigator 
Gemlyn George, MD 
  
Investigational Product 
Name: 
Sargramostim  
 
  
Indication: Alzheimer’s Disease 
 
Phase: 2 
 
Version Date: 17JUL2025 
IND Number 152023 
 
Confidentiality 
Statement: 
This document, and the information contained within is 
confidential.  It contains proprietary information of the 
sponsor.  Any disclosure of such information that is not 
authorized in writing by the sponsor is prohibited.  The 
information contained in this protocol may be only used 
for the purpose of reviewing and/or conduct of this 
study. 
 
 

Clinical Study Protocol 
 
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TABLE OF CONTENTS 
1 
Protocol Synopsis 
4 
2 
Schedule of Events 
9 
3 
List of Abbreviations 
14 
4 
Introduction 
16 
 
4.1 
Introductory Statement and General Investigational Plan 
16 
 
4.2 
Background and Research Rationale 
16 
 
4.3 
Preliminary Preclinical Results 
17 
 
4.4 
Preliminary Clinical Results 
18 
 
 
4.4.1 
Sargramostim effects in chemotherapy-induced cognitive impairment 
18 
 
 
4.4.2 
Safety and Efficacy Trial at CU-Anschutz and USF 
18 
 
4.5 
Specific Objectives 
19 
 
4.6 
Benefits and Risks Assessment 
23 
 
 
4.6.1 
Benefits 
24 
 
 
4.6.2 
Risks 
24 
5 
Study Design and Methods 
25 
 
5.1 
Overall Design 
25 
 
 
5.1.1 
Investigational Product 
25 
 
 
5.1.2 
Justification for Dose 
25 
 
5.2 
Recruitment of Participants 
26 
 
5.3 
Screening Period (Screening Visit) 
26 
 
 
5.3.1 
Screening Procedures 
26 
 
 
 
Tier 0.5 Screening Procedures 
26 
 
 
 
Tier 1 Screening Procedures 
27 
 
 
 
Tier 2 Screening Procedures 
28 
 
 
 
Tier 3 Screening Procedures 
28 
 
 
 
Tier 4 (Optional Substudy) 
29 
 
 
5.3.2 
Additional Screening Procedures and Considerations 
30 
 
 
 
5.3.2.1 Screening Flow Chart 
30 
 
5.4 
Double-Blind Treatment Period (Baseline Visit through Visit 24) 
30 
 
 
5.4.1 
Double-Blind Treatment Procedures 
31 
 
 
 
5.4.1.1 Baseline Visit Procedures 
31 
 
 
5.4.2 
Additional Double-Blind Treatment Study Procedures 
32 
 
 
 
5.4.2.1 Double-Blind Treatment Labs 
32 
 
 
 
5.4.2.2 Double-Blind Treatment Cognitive Assessment 
33 
 
 
 
5.4.2.3 Double-Blind Treatment Imaging 
33 
 
 
 
5.4.2.4 Double-Blind Treatment Lumbar Puncture and blood draw 
33 
 
5.5 
Follow-Up Visit 
33 
 
5.6 
End of Treatment / End of Study Definitions 
33 
 
5.7 
At-Home Visits 
33 
 
5.7.1 
Narrative Summary of Home Visits 
34 
 
5.7.2 
Summary of Traveling Nurse Duties During at-Home Visits 
34 
 
5.7.3 
Logistics Associated with Traveling Nurses 
34 
 
5.7.4 
Drug Handling Details 
 
34 
6 
Study Population 
36 
 
6.1 
Inclusion and Exclusion Criteria 
36 
 
 
6.1.1 
Inclusion Criteria 
36 
 
 
6.1.2 
Exclusion Criteria 
37 
 
6.2 
Lifestyle Restriction 
40 
 
6.3 
Screen Failures 
41 
 
6.4 
Withdrawal from Study 
41 
 
 
6.4.1 
Withdrawal Procedures 
41 
7 
Treatments 
42 

Clinical Study Protocol 
 
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7.1 
Treatment Administered 
42 
 
7.2 
Method of Treatment Assignment 
42 
 
7.3 
Blinding 
43 
 
7.4 
Dosage Modification 
43 
 
7.5 
Interruption of Treatment 
45 
 
7.6 
Treatment Compliance 
46 
 
7.7 
Concomitant Therapy 
46 
8 
Study Assessments and Procedures 
47 
 
8.0 
Screening Assessments 
47 
 
8.1 
Efficacy Assessment 
48 
 
8.1.1 
Functional Ability Assessments 
49 
 
8.2 
Laboratory Assessments 
50 
 
8.3 
Biomarkers 
51 
 
 
8.3.1 
Serum Anti-Drug Antibody (ADA) Testing 
51 
 
 
8.3.2 
MRI 
52 
 
 
8.3.3 
PET 
53 
 
8.4 
Safety Events Monitoring 
57 
 
 
8.4.1  
Physical and Neurological Examinations 
57 
 
 
8.4.2 
Electrocardiograms (ECG) 
57 
 
 
8.4.3 
Vital Signs 
57 
 
 
8.4.4 
Additional Safety Assessments 
57 
 
 
8.4.5 
Adverse Events, Serious Adverse Events and Monitoring 
57 
 
 
 
8.4.5.1  Serious Adverse Events 
58 
 
 
 
8.4.5.2 Protection of Participants 
58 
 
 
 
8.4.5.3 Data Monitoring Plan 
58 
9 
Statistical, Data and Other Considerations 
63 
 
9.1 
Sample Size Considerations 
63 
 
9.2  
Data Analysis Plan 
63 
 
 
9.2.1 
Primary Objective 
63 
 
 
9.2.2 
Secondary Objectives 
64 
 
 
9.2.3 
Exploratory Objectives 
64 
 
9.3 
Additional Statistical Considerations 
67 
 
 
9.3.1 
Full Statistical Analysis Plan 
67 
 
9.4 
Interim Analyses 
67 
 
9.5 
Data Storage 
67 
 
9.6 
Specimen Storage 
67 
 
9.7 
Control of Conflict of Interest 
67 
10 
Appendices 
 
 
Appendix A: References Cited 
69 
 
Appendix B: GM-CSF Upregulated in Rheumatoid Arthritis Reverses Cognitive Impairment and 
Amyloidosis in Alzheimer Mice (Boyd et al., 2010). 
 
Appendix C: Granulocyte Macrophage Colony Stimulating Factor Treatment is Associated with 
Improved Cognition in Cancer Patients (Jim et al., 2012).  
 
Appendix D: Briefing Package – FDA Advisory Committee Meeting May 3, 2013; Study Drug: 
Leukine® (Sargramostim). 
Appendix E: Leukine® (Sargramostim) Package Insert and Full Prescribing Information 
 
Appendix F: Common Terminology Criteria for Adverse Events (CTCAE) v5.0. 
 
Appendix G: Evaluation of the safety and immunomodulatory effects of sargramostim in a 
randomized, double-blind phase 1 clinical Parkinson’s disease trial (Gendelman et al., 2017) 
 
Appendix H: List of prohibited concomitant drugs 
 
Appendix I: DSMB membership 
 
Appendix J: Available Resources 
 
 
 

Clinical Study Protocol 
 
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1  Protocol Synopsis 
 
Compound Name: Sargramostim 
 
Active Ingredient: Recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (GM-
CSF) 
 
Study Protocol Title: Phase II Trial to Evaluate Safety and Efficacy of GM-CSF/Sargramostim in 
Alzheimer’s Disease (SESAD) 
 
Principal Investigator: Huntington Potter, PhD 
 
Site: Anschutz Medical Campus 
Department of Neurology 
University of Colorado School of Medicine 
 
Study Period: For an individual participant, the total study period is up to 12 weeks of 
screening, 24 weeks of double-blind placebo controlled treatment, and ~6 weeks 
(45 days) of follow-up, totaling approximately 38 weeks. 
 
Objectives  
 
Primary Objective: 
• 
To assess long-term tolerability and safety of sargramostim in 
individuals with mild-to-moderate Alzheimer’s Disease (AD). 
 
Secondary Objective: 
• 
To test the hypothesis that sargramostim treatment, as compared to 
placebo, slows, or halts cognitive decline, or improves cognitive 
function, in individuals with mild-to-moderate AD. 
 
Exploratory Objectives 
• 
To assess the effect of sargramostim treatment, as compared to placebo, 
on clinical progression in individuals with mild-to-moderate AD. 
 
 
• 
To assess the effect of sargramostim treatment, as compared to placebo, 
on metabolic activity in brain regions known to be affected by AD, in 
individuals with mild-to-moderate AD. 
 
 
• 
To assess the effect of sargramostim treatment, as compared to placebo, 
on brain amyloid deposition in individuals with mild-to-moderate AD. 
 
 
• 
To assess the effect of sargramostim treatment, as compared to placebo, 
on AD related changes in peripheral blood and CSF biomarkers in 
individuals with mild-to-moderate AD. 
 
• 
To assess the effect of sargramostim treatment, as compared to placebo, 
on AD related changes in blood-brain barrier permeability in individuals 
with mild-to-moderate AD. 
 
 
 
 
 

Clinical Study Protocol 
 
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1  Protocol Synopsis (Continued) 
 
 
• 
To assess the effect that Apolipoprotein E genotype may have upon 
predicting sargramostim treatment outcomes in individuals with mild-to-
moderate AD. 
 
• 
To assess the effect of sargramostim treatment, as compared to placebo, 
on inhibiting, reducing or slowing medial temporal atrophy (MTA) in 
individuals with mild-to-moderate AD. 
 
• 
To assess the effect of sargramostim treatment, as compared to placebo, 
on changes in microstructural white matter measurements, in regions 
known to be affected by AD, in individuals with mild-to-moderate AD. 
 
• 
To assess the effect of sargramostim treatment, as compared to placebo, 
on changes in sleep-related measures in individuals with mild-to-
moderate AD. 
 
• 
To assess the effect of sargramostim treatment, as compared to placebo, 
on changes in speech measures in individuals with mild-to-moderate 
AD. 
 
Study Design: 
The study is double-blind, placebo-controlled in the Alzheimer’s Disease 
population, and will include individuals with mild AD and moderate AD, 
consistent with Montreal Cognitive Assessment (MoCA) score range of 4-24 
at time of screening.  Individuals who meet inclusion/exclusion criteria will 
be randomized in a double-blind manner, to receive either sargramostim 
(178.57 µg/m2/day subcutaneously 7days per week for 24 weeks) or placebo 
(7 days per week) in an approximate 2:1 randomization ratio.  
 
The study population will be recruited from the Memory Disorders Clinic at 
the University of Colorado Hospital (UCH) as well as from the community 
at large.  
 
The estimated maximum duration of participation for each individual 
participant is approximately 10.5 months (12 week screening phase, 24 week 
post-randomization double blind treatment phase, and ~6 week (45 day) 
post-treatment follow-up phase. 
 
Number of Study 
Participants 
Approximately 150 individuals and their study partners will be screened, 
~47 participants randomized, with an estimated 42 individuals completing 
the treatment and follow-up for full analysis (~28 individuals to the 
sargramostim 178.57 µg/m2/day subcutaneously arm of the study (1250 
µg/m2 per week) and ~ 14 individuals to the placebo control arm of the 
study.)   
 
 
 

Clinical Study Protocol 
 
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Protocol Synopsis (Continued) 
 
Key Inclusion Criteria 
Individuals between age 60 and 85 years, inclusive, at time of consent who 
meet criteria for probable AD dementia according to the National Institute of 
Aging – Alzheimer’s Association (NIA-AA) research framework criteria 
(2018), and have a diagnosis of mild or moderate AD, or a study MD 
provisional diagnosis consistent with mild-to-moderate AD, with a screening 
MoCA score between 4-24, inclusive, have (optional per PI judgment of 
necessity) positive plasma biomarker for Alzheimer’s disease, and have 
positive biomarker for brain amyloid pathology by CSF assay, obtained via 
screening lumbar puncture, or a positive amyloid PET scan. 
 
Key Exclusion Criteria 
Any individuals with a first degree relative diagnosed with AD before 55 years 
of age, have contraindication to lumbar dural puncture, PET or MRI, have 
other neurological or psychiatric condition (other than AD) that can impact 
cognition or procedure adherence, a BMI ≥35, active cancer or malignant 
neoplasm within 5 years of screening other than non-melanoma skin cancers, 
have had prior treatment with an investigational anti-amyloid or anti-
tauopathy therapy, or AD vaccine, a history of latex or yeast allergy or has a 
history of, or treatment for, Rheumatoid Arthritis. 
 
Full Inclusion / Exclusion Criteria is presented later in this protocol. 
 
Study Treatment 
Sargramostim is commercially available from the manufacturer, Partner 
Therapeutics, and will be obtained by the sponsor.  Seven doses of 
sargramostim (178.57 µg/m2or placebo doses that look identical, will be 
prepared weekly by an unblinded pharmacist into small syringes for injection 
at home by the participant / caregiver.  Doses are to be administered at 
approximately the same time every scheduled administration day. First dose 
will be administered at the Baseline visit overseen by trained staff at the 
CTRC, with training provided for at home dosing to the participant and study 
partner.   
 
 
 
 

Clinical Study Protocol 
 
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1  Protocol Synopsis (Continued) 
 
Data Safety 
Monitoring Board 
An independent Data Safety Monitoring Board (DSMB) will be chartered for 
this trial to review safety and other relevant data. The DSMB will convene at 
regular, predetermined time points to monitor the overall safety of the study, 
and to make recommendations, when necessary, related to the safety of the 
study, and the ability of the study to continue unchanged or with 
modifications, to the Sponsor.  The study will proceed with regular order 
during DSMB safety reviews.  
 
Assessments 
 
Efficacy Assessments 
The Mini-Mental State Examination (MMSE), Montreal Cognitive 
Assessment (MoCA), Alzheimer’s Disease Assessment Scale – Cognitive 
(ADAS-Cog), Clinical Dementia Rating (CDR), Alzheimer’s Disease 
Cooperative Study – Activities of Daily Living (ADCS-ADL), Trail Making 
Test –A (TMT-A), lexical and semantic fluency, speech and verbal 
performance measures, and the Neuropsychiatric Inventory (NPI) are well 
validated and reliable instruments for measurement of cognitive 
performance.  Selected cognitive assessments will be performed during 
Screening for study inclusion, and at Baseline, week 12, End of Treatment 
(EOT), and at the follow-up visit. 
 
Blood and CSF 
Biomarker 
Assessments 
Per PI judgement, plasma may be collected via venipuncture during screening 
to assess for AD biomarkers (such as Aβ(42)/ Aβ(40) ratio or pTau 217).  If 
drawn, the findings will be confirmed via Cerebrospinal Fluid (CSF) collected 
via lumbar puncture (LP) later in screening, and CSF will be assayed for Aβ(1-
42) and tau:Aβ(1-42) ratios. (Note: Participants could opt for Amyloid PET in 
lieu of LP for screening confirmation). If the LP option is chosen, a 
venipuncture blood collection will occur at the same visit as the LP procedure 
and assayed in conjunction with CSF serum and CSF albumin levels to 
determine an albumin index for assessing AD-related changes in blood brain 
barrier permeability. 
 
If opting for LP, randomized participants will also have CSF collected via LP, 
and a venipuncture blood collection, after the final dose of treatment article, 
post visit week 24. The LP and venipuncture procedures will occur on the 
same day, which will be at least 2 days after and up to 10 days following the 
last day of treatment article injection.  Exploratory biomarker analyses will 
assess individual changes in measurements between each participant’s EOT 
blood samples compared to their baseline samples and between each 
participant’s EOT CSF samples compared to their screening samples, as well 
as determining group differences from sargramostim treatment, as compared 
to placebo treatment. Changes in the albumin index will be used as  
 
 

Clinical Study Protocol 
 
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1  Protocol Synopsis (Continued) 
 
Blood and CSF 
Biomarker 
Assessments 
(Continued) 
an indication for treatment-related effects on blood brain barrier permeability 
and as a correlate with MRI analyses for any incidence of vasogenic edema or 
microhemorrhage. CSF and plasma will be assayed for Aβ(1-42), Aβ(1-40), t-
tau, p-tau, tau:Aβ(1-42) ratios, NfL, GFAP, UCHL1, and panels of cytokines, 
chemokines, and other circulating factors to determine any treatment-related 
effects that might correlate with cognitive and activities of daily living 
changes.  
 
Serum Anti-Drug Antibody (ADA) Testing for Anti-GM-CSF Antibody will 
be assayed from serum collected by venipuncture blood draws at baseline, 
week 3, week 12, week 18, EOT, and follow-up visits. 
 
Apolipoprotein E (ApoE) Genetic Status will be determined via blood sample 
collected at Baseline visit. 
Imaging Assessments 
Fluorodeoxyglucose F18 (FDG) PET scan will be completed as part of 
baseline and EOT visit. 
 
MRIs of the brain will be conducted throughout the study period, including 
during screening period, week 6 visit, week 12 visit, week 18 visit, EOT visit, 
and at the follow-up visit. 
 
Amyloid PET utilizing an approved radiotracer (such as Vizamyl 
(flutemetamol F18)) will be done as confirmation of elevated amyloid, or as an 
optional sub-study if LP is completed as confirmation.  If participant consents, 
the PET will occur during Screening and at the follow-up visit. 
 
Safety Assessments 
Blood work including CBCs with differential and comprehensive metabolic 
panels (CMPs) will be collected at baseline visit, for the remainder of the 
treatment period, CBCs with differential twice a week and CMP once a week. 
At the End of Treatment Visit and follow-up visits, CBC with differential only. 
CBC will be repeated approximately 2 days and 9 days after last dose. 
 
Physical exams by study physician or qualified designee will be conducted at 
screening visit, Baseline, week 12, EOT, and follow-up visits. 
 
Electrocardiograms (ECGs) will be conducted at screening visit, week 12, and 
EOT visits. 
 
MRIs of the brain will be conducted throughout the study period, including 
during screening (Tier 2), week 6 visit, week 12 visit, week 18 visit, EOT visit, 
and at the follow-up visit. 
 
Vital signs and adverse events (AEs) will be monitored weekly. 
 
C-SSRS assessment of suicidality will be conducted at Screening, Baseline 
week 12 and EOT visits. 
 
 

Clinical Study Protocol 
 
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2  Schedule of Events 
 
Schedule of Events - Screening Period 
 
Visit: 
Screening Visit 
End of week relative to study randomization day1: 
Week -12 to Week -1 
Tolerance Interval for Visit (in days)1 
84 
Screening Visit Procedures (By Tier)  
 
Tier 0.5 
 
Screening Procedures 
 
Mini Informed Consent 
X 
Demographics 
X 
Cognitive Health Review Battery13 
X 
Optional blood draw for biomarker AD evaluation (such as Aβ(42)/Aβ(40) ratio or 
pTau 21714 
X 
Tier 1  
 
Screening Procedures 
 
Full Informed consent (participant and study partner)2 
X 
Demographics 
X 
Medical History 
X 
Previous / Concomitant Meds 
X 
Physical / Neurological Exam 
X 
Diagnostic assessment for mild-to-moderate AD (as necessary)12 
X 
Screening Entry Diagnostics 
 
MoCA 
X 
Vital signs (i.e. BP, HR, Temperature, Respirations)3 
X 
Height and Weight 
X 
ECG 
X 
GDS4 
X 
GAD-75 
X 
C-SSRS6 
X 
Screening Laboratory Diagnostics 
 
Urinalysis 
X 
 
Prohibited drugs 
X 
 
Pregnancy (if applicable) 
X 
CBC+ Diff, CMP, PT/INR 
X 
Blood draw for counter-indicated conditions7 
X 
Tier 2 
 
Screening Imaging Diagnostics 
 
MRI Scan8 
X 
Tier 3 
 
Screening Laboratory Diagnostics 
 
Lumbar Puncture for Aβ CSF Assay (if selected for entry criteria)9 
X 
Amyloid PET10 (if selected for entry criteria) 
X 
Blood draw for serum/CSF Albumin index, CSF and Serum Glucose, CSF Protein 
& Total cell count (if LP conducted) 
X 
Vital signs prior to Lumbar Puncture (i.e. BP, HR, Temperature, Respirations) if 
LP conducted)3 
X 

Clinical Study Protocol 
 
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Review of NIA-AA Criteria 
X 
Review of Inclusion/Exclusion Criteria11 
X 
Tier 4 (Optional) 
 
Screening Laboratory  and Imaging Diagnostics 
 
Amyloid PET10 (if selected for optional substudy) 
X 
Lumbar Puncture for Aβ CSF Assay (if selected for optional substudy)9 
X 
1The interval between the screening visit and the baseline visit is a maximum of 84 days to allow for completion of all screening 
procedures, assessments and evaluations.  The visit is not considered complete until all procedure to determine eligibility (Tiers 
0.5-3) are completed and reviewed.  Screening visit can take place over multiple days, with expectation that each separate Tier 
will be completed within a 1 day visit, with an expected 3 mandatory site visits and 1 optional visit.  
2The participant and the study partner must have a completed, signed and dated informed consent before any data is collected. 
3Blood pressure and pulse should be measured in the sitting position only after sitting for 5 minutes. Refer to section 8.4.5.3 Data 
Monitoring Plan for acceptable BP parameters.  
4The Geriatric Depression Scale (GDS) will be administered at screening.  Participants who score 20 or higher at screening, 
indicating severe depression, will be excluded from the study. 
5The Generalized Anxiety Disorder (GAD-7) will be administered at screening.  Participants who score 15 or higher at screening, 
indicating severe anxiety, will be excluded from the study.  
6The baseline version of the C-SSRS will be administered at screening.  Participants at higher imminent risk (‘yes’ to question 4 
or 5 on the C-SSRS) at screening, will be excluded from the study. 
7The blood draw will be examined for hepatitis B, hepatitis C, HIV, and/or spirochetal (syphilis) infections. Positive results of 
any of these tests will be reported to the state or local public health agency according to CDC or other governmental guidelines. 
8A screening MRI will be performed as part of the screening visit eligibility criteria.  The MRI and/or MRI report will be 
reviewed by the Principal Investigator or qualified sub-Investigator designee for inclusion.   
9If selected for entry criteria or as optional substudy, the lumbar puncture will collect a sample of CSF for assay to determine the 
presence of elevated amyloid burden in the brain.  The assay will examine Aβ(1-42) and tau:Aβ (1-42) ratios (ADmark® 
Phospho-Tau/Total-Tau/ABeta42; Athena Diagnostics).  The participant should meet all other eligibility criteria before the LP is 
conducted. Platelet and INR results to be reviewed prior to procedures, and must be within preceding 30 days of LP. 
10An Amyloid PET scan will be conducted within the screening period if the participant consents to the optional sub-study.  If 
chosen as inclusion criteria for amyloid burden,, participant must meet all other inclusion criteria before the Amyloid PET will be 
conducted.  The Amyloid PET will be reviewed by the Principal Investigator or qualified designee. 
11A final review of all inclusion and exclusion criteria will be conducted and given to the Principal Investigator or qualified sub-
Investigator for approval before the participant can move to the baseline visit for randomization. 
12 A diagnostic assessment may be conducted by a study doctor, as necessary, to confirm probable mild or moderate AD in a 
participant who otherwise meets criteria to begin screening, but may not have a neurologist verified Dx of mild-to-moderate AD. 
It will consist of a clinical interview and measures of functioning such as the DSRS and/or FAQ, and consultation and consensus 
with one additional study doctor.  
13 The Cognitive Health Review Information System Battery is designed around standardized diagnostic criteria and practice 
guidelines to identify common mimics of Alzheimer’s disease, some of which can co-exist with Alzheimer’s disease pathology.  
Questionnaires include Everyday Assessment of Cognition (ECog), Neuropsychiatric Symptoms Questionnaire (NPI-Q), 
Dementia Cognitive Fluctuation Scale, Early screen from the Columbia Suicidality Scale, Epworth Sleepiness Scale, Lewy Body 
Composite Risk Score, Functional Activities Questionnaire (FAQ), a Dementia Severity Rating Scale (DSRS), an assessment of 
activities of daily living, and the Revised Self-Monitoring Scale – Expressive Score (RSMS-Ex). Depending on answers given, 
additional questionnaires may include the RSMS, Behavior Inhibition Scale, Interpersonal Reactivity Index, and Unified 
Parkinson's Disease Rating Scale–II (UPDRS-II).  Other questions were created specifically for the purpose of more specific 
diagnosis or ensuring best management according to recommended clinical practice guidelines.  Answers are provided on a 
secure Qualtrics system,and provided by caregivers. The questionnaires take approximately one hour to fill out. 
14Any assay such as the plasma assay for Aβ(42)/ Aβ(40) ratio or pTau 217 will be a clinically validated plasma assay to 
determine levels of circulating Aβ(42) and Aβ(40) or pTau, which has been normed against CSF derived levels.   

Clinical Study Protocol 
 
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STUDY SCHEDULE OF EVENTS 
 
DOUBLE BLIND RANDOMIZATION PERIOD 
 
WEEK of Visit 
Visits 
Baseline 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
EOTl 
F/U 
Tolerance Interval for Visit (days) 
0 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+2-10 
+/-7 
Concomitant Med 
Review 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical / Neuro Exam 
Xb 
 
 
 
 
 
 
 
 
 
 
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
Xb 
Xb 
Contact pharmacy to 
dispense study drugc 
Xc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Study Drug Injection 
Training 
Xd 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study Medication 
Administration 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
Xe 
 
 
Assessment of 
compliance 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Cognitive Assessmentsf 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MMSEf  
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
MoCAf  
-X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
ADAS-Cog13f 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
TMT Af 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
ADCS-ADLf 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
CDRf 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
NPIf 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Verbal Fluencyf  
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Sleep Assessmentsg 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Speech and Verbal 
Performance Measuresg 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Safety Assessments 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
C-SSRS, GDS, GAD-7h 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Adverse Events  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Injection site reviewi 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Vital Signsj 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Weight 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum Anti-Drug 
Antibody (ADA) 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X 
ECG 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 

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WEEK of Visit 
Visits 
Baseline 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
18 
19 
20 
21 
22 
23 
24 
EOTl 
F/U 
Tolerance Interval for Visit (days) 
0 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+/-3 
+2-10 
+/-7 
Safety Assessments 
(Continued) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MRI 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
X n 
Safety blood drawsk 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Xp 
X 
PT/INR 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Enhanced safety blood 
draw-cytokine panelo 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
Enhanced safety blood 
draw-t-cello 
X 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Additional Assessments 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
LP and blood draw for 
Aβ CSF assay, albumin 
index, total cell count 
and biomarkers (if opted 
in)O 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Xl 
 
Blood Draw 
(biomarkers) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Xl 
Xm 
FDG PET 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Xm 
 
Amyloid PET (if opted 
in) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
ApoE Genotyping 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
aFinal review to confirm participant has met all screening eligibility criteria and can be randomized 
bNeuro and physical examinations are to be conducted by the Principal Investigator or a qualified designee. 
cThe unblinded pharmacist will used pre-approved method of assigning participants into either the study drug or placebo controlled arm, and continue to dispense the study 
drug/placebo throughout the study treatment period. 
dTraining on how to inject the study drug will be provided to the participant and study partner, or other designated caregiver if not the study partner, for administering the daily 
injections. Training will include methods of administration as well as how to fill out the injection site diary. Training will be refreshed on an as needed basis. 
eClinic injections to be completed once weekly, for the first 3 weeks, by study partner, LAR, or caregiver with observation by study nurse or qualified designee for  appropriate 
injection technique with additional observation at in person visits (Wk 6, Wk 12, Wk 18) and ongoing education as needed. If they are unable to administer the once weekly dose at 
the in-person visit with study staff, a study nurse may administer. Home injection administration to be completed by study partner and logged in injection diary.  
fAt Visits where the cognitive and functional assessments will be conducted, they should be administered before any medical procedures that could be stressful for the participant.  
All attempts should be made to administer the assessments at the same time of day, and with the same individual, designated by the site, as the administrator.    
gThe Insomnia Severity Index (ISI), and Pittsburgh Sleep Quality Index (PSQI) sleep scales will be administered and the Speech and verbal performance measures will be 
performed and recorded for the Baseline, Week 12, EOT and Follow-up visits. 
hThe “Since last visit” where assessed version of the C-SSRS will be utilized for the Baseline, Week 12, EOT and Follow-up visits. Standard forms for GDS and GAD-7 will be 
used for the Baseline, Week 12, EOT and Follow-up visits. 
iStudy nurse will assess the injection site(s) at each in-person visit for injection site reaction.  If necessary, and in consultation with the Principal Investigator or appropriate sub-
investigator or designee, a topical agent can be prescribed to address any swelling or redness at the injection site. 
jTemperature, respirations, and seated blood-pressure and pulse after sitting for 5 minutes. Refer to section 8.4.5.3 Data Monitoring Plan for acceptable BP parameters.  
kSafety blood draws will include CMP (once per week) and CBC + diff (twice per week). EOT and FU visits will not have a CMP, only one CBC draw will be collected for these 
visits.  

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lThe LP (if opted in) and blood draw for end of treatment should be conducted at least 2 days after and within 10 days following the final treatment injection, and both performed 
on the same day with the LP procedure performed first. PT/INR and platelets must have been performed within 3 months of LP procedure, refer to manual of procedures for LP 
parameters. 
mThe blood draw for follow-up visit, FDG PET, and Amyloid PET (if opted in) should be conducted within ± 7 days of anticipated visit. 
nThe MRI for follow-up visit is only scheduled for participants who participate in the optional Amyloid PET procedure. If the LP is done as a substudy, not for confirmation of the 
plasma results Aβ(42)/ Aβ(40) ratio, it will not be tested at outside lab as inclusion LP is for confirmatory results.   
OThe Enhanced safety blood draws will consist of a cytokines panel (IL 1 beta, IL 2, IL 6, TNF alpha such as from CHCO Lab 10055) recommended by study immunologist and 
safety monitor at baseline, week 6, 12, 18 and EOT, and lab (such as CHCO LAB9496) testing characterization of activated, senescent, and exhausted T cells done at visits 
(Baseline, Week 12 and EOT visits) 
pThe CBC w/ diff will be repeated approximately 2 days and 9 days after last participant dose at end of treatment or withdrawal from study. 
 
 
LP/PET sequences based on which is used for study inclusion: 
 
 
If Choose Lumbar Puncture for Study Inclusion (with OPTIONAL AMYLOID PET SUBSTUDY) 
 
Screening Tier 3 
Visit 
Screening Tier 
4 Visit 
Baseline 
Visit 
End of Treatment 
Visit 
Follow-up 
Visit 
Lumbar Puncture (Mandatory) 
X 
 
 
X 
 
FDG PET (Mandatory) 
 
 
X 
X 
 
Amyloid PET (Optional) 
 
X 
 
 
X 
 
If Choose Amyloid PET for Study Inclusion (with OPTIONAL LUMBAR PUNCTURE SUBSTUDY) 
 
Screening Tier 3 
Visit 
Screening Tier 
4 Visit 
Baseline 
Visit 
End of Treatment 
Visit 
Follow-up 
Visit 
Amyloid PET (Mandatory) 
X 
 
 
 
X 
FDG PET (Mandatory) 
 
 
X 
X 
 
Lumbar Puncture (Optional) 
 
X 
 
X 
 

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3  List of Abbreviations 
 
Abbreviation 
Term 
Aβ 
amyloid beta 
Aβ(1-x) 
 
ADCC 
ADRC 
amyloid beta (1-x) (i.e., the amyloid beta monomer from amino acid 1 to 
x [e.g. 1-42]) 
Alzheimer’s Disease Core Center 
Alzheimer’s Disease Research Center 
ADCS-ADL 
Alzheimer’s Disease Cooperative Study – Activities of Daily Living 
AChEI 
acetylcholinesterase inhibitor 
AD 
ADA 
Alzheimer’s disease 
Anti-Drug Antibodies 
ADAS-Cog  
Alzheimer’s Disease Assessment Scale - cognitive subscale 
AE 
AI 
anti-HCV 
anti-HIV 
adverse event 
albumin index 
Hepatitis C antibodies 
Human Immunodeficiency Virus antibodies 
ApoE  
Apolipoprotein E 
APP 
amyloid precursor protein 
BP 
blood pressure 
BIC 
Brain Imaging Center 
CBC 
complete blood count 
CDR 
Clinical Dementia Rating 
CDR-SB  
CIT 
Clinical Dementia Rating -Sum Of Boxes 
Cognitive Interference Task 
CMP 
Comprehensive Metabolic Panel  
CNS 
COMIRB  
central nervous system 
Colorado Multiple Institutional Review Board 
CRF  
case report form 
CSF  
cerebrospinal fluid 
C-SSRS  
CUACC 
CU-AMC 
CU-RIC 
Columbia Suicide Severity Rating Scale 
University of Colorado Alzheimer’s and Cognition Center 
University of Colorado – Anschutz Medical Campus 
University of Colorado Research Imaging Center 
DSMB  
DTI 
data safety monitoring board 
diffusion tensor imaging 
ECG  
Electrocardiogram 
eCRF  
electronic case report form 
ED 
EDTA 
EOT 
FA  
early discontinuation 
Ethylenediaminetetraacetic acid 
End of Treatment 
fractional anisotropy 
FDA  
Food and Drug Administration 
FDG PET 
GAD 
Fluorodeoxyglucose (18F-FDG) positron emission tomography 
Generalized Anxiety Disorder 
GCP 
G-CSF 
GDS  
GFAP 
GFR 
GM-CSF 
Good Clinical Practice 
Granulocyte Colony-Stimulating Factor 
Geriatric Depression Scale 
glial fibrillary acidic protein 
glomerular filtration rate 
Granulocyte-Macrophage Colony-Stimulating Factor 

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HBsAg 
HIMSR 
HIPPA 
HR 
Hepatitis B surface antigen 
Human Immune Monitoring Shared Resource 
Health Insurance Portability and Accountability Act of 1996 
heart rate 
ICF 
IND 
IP  
Informed Consent Form 
Investigational New Drug 
investigational product 
IRB  
ISI 
Institutional Review Board 
Insomnia Severity Index 
LP 
MD 
lumbar puncture 
mean diffusivity 
MMSE  
Mini-Mental State Examination 
MoCA 
Montreal Cognitive Assessment 
MRI  
magnetic resonance imaging 
MTA 
NACC 
NDI 
medial temporal atrophy 
National Alzheimer’s Coordinating Center 
neurite density index 
NIA-AA  
National Institute of Aging-Alzheimer’s Association 
NfL  
neurofilament light 
NFTs  
NODDI 
NREM 
ODI 
neurofibrillary tangles 
neurite orientation and dispersion density imaging 
orientation dispersion index 
non-rapid eye movement 
PD  
pharmacodynamic(s) 
PET  
positron emission tomography 
PK  
pharmacokinetic(s) 
PRN 
PSQI  
pro re nata 
Pittsburgh Sleep Quality Index 
p-Tau  
phosphorylated-Tau 
QTc  
corrected QT interval 
QTcF  
REDCap 
QTc interval calculated using Fridericia’s formula 
Research Electronic Data Capture 
SAE 
SAP 
SOP 
SST 
Serious adverse event 
Statistical analysis plan 
standard operating procedure 
serum separation tube 
SUSAR  
SWA 
suspected unexpected serious adverse reaction 
slow wave activity 
TEAE  
treatment-emergent adverse event 
TEMAV  
treatment-emergent markedly abnormal laboratory values 
TIA  
transient ischemic attacks 
t-Tau 
UCH 
UCHL1  
total Tau 
UCHealth 
Ubiquitin C-terminal hydrolase L1 
vMRI  
volumetric magnetic resonance imaging 
WBC  
WM 
white blood cell (count) 
white matter 
 
 
 
 
 
 

Clinical Study Protocol 
 
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4  Introduction 
 
4.1 
Introductory Statement and General Investigational Plan 
This trial protocol is designed to evaluate primarily whether the long-term use of sargramostim 
(recombinant human GM-CSF), administered five days per week for six consecutive months (24 weeks), 
will be tolerated by and safe for use in participants with mild-to-moderate AD, secondarily whether 
sargramostim can slow, halt, or reverse cognitive decline, and exploratory whether sargramostim can 
slow, halt, or reverse decline in activities of daily living, reverse or improve several biomarkers associated 
with AD, as evaluated by multimodal neuroimaging techniques and blood and cerebrospinal fluid 
analyses.  This trial extends the safety results from recently completed Phase 2 double-blind, placebo-
controlled clinical trial in mild-to-moderate AD participants (NCT01409915, COMIRB#12-1273), using 
sargramostim that was administered five days per week for three consecutive weeks and in which there 
were no incidence of drug-related serious adverse events (SAEs). This concluded in December 2019, and 
although the trial only treated a small number of participants for a very short treatment phase and was not 
powered to adequately evaluate efficacy measures, an interim analysis has hinted at potential 
improvements in some secondary and exploratory efficacy measures. Thus, this 6-month trial protocol 
aims to evaluate sargramostim treatment in participants with mild-to-moderate AD over an extended 
period of time and determine whether sargramostim can indeed safely ameliorate cognitive decline and 
reverse pathological biomarkers of AD, in a similar fashion to our preclinical studies showing GM-CSF’s 
effects in transgenic animal models of AD (1, attached as Appendix B).  
 
4.2 
Background and Research Rationale 
As the sixth leading cause of death and a major cause of disability in the U.S., AD affects around 12% of 
those over age 65 and around 40–50% of those over age 85, for a total of about 5.8 million people in the 
U.S. and more than 35 million people worldwide. Without significant medical breakthroughs, the number 
of people age 65+ with AD in the U.S. is projected to reach about 14 million by 2050. Beyond its toll on 
affected individuals, AD is also extremely costly to society, with an estimated annual cost of $290 billion 
in the U.S. alone, which is expected to quadruple by 2050. Although treatments slowing the clinical course 
of the disease, such as cholinesterase inhibitors or a glutamate receptor antagonist are available, their 
benefits are minor and they do not attack the origin of the AD pathogenic pathway. It is therefore imperative 
that new pharmacological interventions be developed that can prevent or reverse the disease and the 
associated cognitive decline. 
 
Most AD treatments that are currently under investigation are designed to attack the formation of the Aβ 
peptide or its polymerization into neurotoxic oligomers/fibrils and subsequent amyloid plaque deposition. 
Anti-amyloid immunotherapy strategies have provided encouraging results in transgenic animal models of 
AD. However, the human trials have thus far not been very successful and have been plagued with serious 
adverse events (SAEs) defined as amyloid-related imaging abnormalities (ARIAs), including cerebral 
microhemorrhage and vasogenic edema 2. Evidently, a completely new therapeutic approach to AD may be 
necessary 3. In particular, the essential role of neuroinflammation in AD has remained relatively unexplored 
as a potential therapeutic target.  
 
Amyloid deposition and cognitive decline in transgenic animals is fundamentally different from that which 
leads to AD in humans, perhaps because it develops over a relatively short period of time and in the absence 
of clear neuronal cell death, and this concern always overshadows the use of animal models for developing 
AD therapies. We therefore sought a different approach to identify a new mechanism that would provide 
protection against AD. Our investigation is based on epidemiological evidence that rheumatoid arthritis 
(RA) patients have about an 8-fold reduced risk of developing AD 4, motivating us to determine the 
underlying basis for their AD resistance. 
 

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RA is an autoimmune disease in which inflamed synovial tissue and highly vascularized pannus form and 
irreparably damage the cartilage and bone. In this inflammatory pannus, leukocyte populations are greatly 
expanded, perhaps as an endogenous, but ineffective attempt to remove the autoantibody-mediated 
inflammatory insult. As a result, many other proinflammatory factors are produced that work together in 
feed-forward mechanisms to further increase leukocytosis, cytokine/chemokine release, osteoclastogenesis, 
angiogenesis, and continued autoantibody production (rheumatoid factors and anti-citrullinated protein 
antibodies) 5, 6. Additionally, the adaptive immune system presents a Th17 phenotype within CD4+ 
lymphocytes, with ultimate production of interleukin 17 (IL-17), which is then responsible for inducing 
much of the pro-inflammatory effects 7, 8, concurrently with the reduction in the number of and function of 
T regulatory lymphocytes 9-11. Further enhancements of leukocyte populations come from increased 
expression of structurally unrelated hematopoietic colony-stimulating factors (CSFs), namely M-CSF 
(macrophage colony-stimulating factor), G-CSF (granulocyte colony-stimulating factor), and GM-CSF 
(granulocyte-macrophage colony-stimulating factor) 12-15. 
 
While it has been commonly assumed that RA patients’ usage of non-steroidal anti-inflammatory drugs 
(NSAIDs) was protective against the onset and progression of AD 4, clinical trials with NSAIDs have 
proven unsuccessful in AD patients, and, in some cases, such as the trial with mild cognitive impairment 
(MCI) subjects, the NSAIDs accelerated the progression to AD 16. We took a reverse approach and 
developed and investigated the new hypothesis that intrinsic factors within RA pathogenesis itself may 
underlie the protective effect(s) of RA. After researching the literature regarding the potential interplay 
between the innate immune system and AD 17-20, we focused on the hematopoietic colony-stimulating 
factors that stimulate production of their respective innate immune system leukocytes, which we postulated  
might traffic to the brain to prevent AD onset or to reverse AD pathogenesis.  
 
4.3 
Preliminary Preclinical Results 
Intrahippocampal injections of colony-stimulating factors: In our first experiments, we performed bolus 
injections of either M-CSF, G-CSF, or GM-CSF into the hippocampus of one (ipsilateral) brain hemisphere, 
and with vehicle (i.e., artificial cerebrospinal fluid), injected into the contralateral hippocampus as a control, 
into cohorts of aged cognitively-impaired transgenic AD (PS/APP) mice 1. The mice were then sacrificed 
one week later and analyzed. Whereas M-CSF injection resulted in significant hyperplasia to the treatment 
hemisphere and no effect on amyloid deposition, G-CSF showed a modest reduction in amyloid deposition 
in the injected side. This outcome was later confirmed by colleagues using daily peripheral G-CSF 
injections, resulting in reduced amyloidosis and cognitive deficits in the AD mice 21 and leading to a small 
clinical trial in mild-to-moderate AD participants (NCT01617577), which found Filgrastim to be safe and 
well tolerated in people with AD but efficaciously inconclusive 22. In contrast to the mild effect observed 
with G-CSF in AD mice, our intrahippocampal GM-CSF injections resulted in pronounced effects of over 
40% reductions in amyloid deposition, relative to the negative control hemispheres 1.  
 
Daily subcutaneous injection of GM-CSF: Based on the remarkably positive results of GM-CSF at only 
7 days post-injection, we next investigated the effects of a daily subcutaneous (SC) GM-CSF injection on 
AD pathology and cognitive function. Both APPswe transgenic (Tg) mice and non-transgenic control (NT) 
mice were used in these experiments. After confirming cognitive impairment of Tg mice using a widely-
used radial arm water maze (RAWM) task of working memory, Tg and NT mice were each sub-divided 
into two cognitively balanced groups. The mice then received 20 days of a daily SC injection of either GM-
CSF or saline control, and ending behavioral testing consisting of RAWM testing during injection days 10-
14 and a double RAWM task (i.e. cognitive interference task (CIT)) performed during injection days 16-
20, after which the animals were sacrificed and tissues analyzed for AD pathology. Surprisingly, the GM-
CSF-treated Tg mice performed as well as or better than saline-treated NT mice at the end of only 14 days, 
which was confirmed by the CIT results at 20 days. Interestingly, some blocks of testing also showed that 
GM-CSF-treated NT mice exhibited improved cognitive performance as compared to saline-treated NT 
mice. After sacrifice, histochemical analyses of GM-CSF-treated Tg mice showed over 50% less amyloid 

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plaque deposition as compared to saline-treated Tg mice, which correlated with increased neuronal synaptic 
area and microglial densities in the GM-CSF-treated Tg mice 1.  
 
After these findings that only 14 days of daily subcutaneous GM-CSF injections completely reversed the 
cognitive impairment of the transgenic AD mice and that 20 days of GM-CSF reduced over half of their 
cerebral amyloid deposition, we researched the literature for GM-CSF’s effects in other neurological 
diseases. There is an accumulating body of research showing that GM-CSF has beneficial effects for stroke 
23-32, spinal cord injury 33-42, traumatic brain injury 43, 44, retinal degeneration 45-47, and Parkinson’s disease 
(PD) 48-51, including in a recent PD clinical trial using sargramostim for 56 consecutive days and that was 
successful in achieving its primary endpoint 52. GM-CSF is also anti-apoptotic, neuroprotective, and a 
growth factor for neural stems cells 53-58, plays a major role in neuronal plasticity critical for learning and 
memory 59, and has also recently been shown to improve the function of mesenchymal stem cells 60, 61. GM-
CSF is being investigated as a treatment for other inflammatory conditions 62-65, as well as recent studies 
that support or have replicated our work for GM-CSF to treat AD 66-68 or to improve cognition in general 
69. 
  
4.4 
Preliminary Clinical Results 
4.4.1 
Sargramostim effects in chemotherapy-induced cognitive impairment  
Together with Dr. Heather Jim of the Moffitt Cancer Center, Tampa, FL, we performed a retrospective 
analysis of the data that were gathered from a study assessing cognition in bone marrow transplant patients, 
who acquired cognitive deficits from the chemotherapy or irradiation procedures they underwent (70, 
attached as Appendix C; 71). Their cognitive function was measured before treatments and then six months 
and 12 months after hematopoietic cell transplantation (HCT) and associated treatment with a combination 
of GM-CSF plus G-CSF or treatment with G-CSF alone.  
 
The neuropsychological measures used included some of the cognitive measures that are used routinely to 
assess AD patients. Total neuropsychological performance z scores (TNP) were calculated by summarizing 
the cognitive domains of memory, executive functioning (i.e., complex cognition), and attention. Scores 
indicate change in TNP from pre-transplant baseline. Kruskal-Wallis one-way analyses of variance were 
conducted at six months and 12 months after baseline assessment and HCT, using all available data to 
compare between-group changes in TNP by receipt of GM-CSF. Wilcoxon signed rank tests were 
conducted using all available data to examine within-group changes in TNP by receipt of GM-CSF. Despite 
having a high level of education (average of 13.89 years), the subjects displayed a statistically significant 
cognitive deficit at baseline. Our preliminary results show that patients who received GM-CSF plus G-CSF 
improved their cognitive functions (neuro minus motor features) between baseline and six months, whereas 
subjects receiving only G-CSF did not improve. This result validates in humans that GM-CSF can improve 
cognition in an at-risk population using the standard recommended, FDA-approved dosage that we also 
propose to use in this new protocol. This human study also showed that a small number of subjects (less 
than 20) who received GM-CSF plus G-CSF was sufficient to reveal that GM-CSF led to improved 
cognition at six months after treatment, compared to the much larger group who received G-CSF alone.  
 
4.4.2 
Safety and Efficacy Trial at CU-Anschutz and USF (COMIRB# 12-1273):  
After GM-CSF’s preclinical results described above in models of AD and other neurodegenerative diseases 
and the association of GM-CSF with improved cognition in bone marrow transplant patients, the pilot safety 
and efficacy trial was initiated at the University of South Florida (USF), in which sargramostim/Leukine® 
was administered to mild-to-moderate AD participants for 15 days over a 19-day period (NCT01409915). 
At USF, the first 14 participants completed the study given placebo or sargramostim at a dosage of 125 
µg/m2/day SC, which is half of the FDA-recommended dose. The Data Safety Monitoring Board (DSMB) 
met and determined that sargramostim was safe, and approved subsequent participants to receive treatment 
dosage of sargramostim at the full FDA-recommended dose of 250 µg/m2/day SC. Then with the relocation 
of Drs. H. Potter and T. Boyd to Colorado, the trial was halted and amended to increase new enrollment to 

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40 participants (i.e. 20 sargramostim-treatment, 20 placebo treatment). All participants (3 at USF and 37 at 
CU-AMC) completed the trial either with placebo or at the full FDA-recommended dose of 250 µg/m2/day 
SC. The only AEs reported have been mild expected reactions to sargramostim, including injection site 
reactions, such as redness, swelling, itching, and bruising. No drug-related SAEs were reported, including 
no incidence of amyloid-related imaging abnormalities (ARIAs), indicating vasogenic edema (ARIA-E) 
and/or microhemorrhage (ARIA-H), which have been observed in all other trials testing anti-amyloid 
immunotherapy drugs in AD participants.   
 
Final analysis of 40 participants showed a statistically significant treatment effect of sargramostim as 
assessed by MMSE, but none for the other assessments (ADAS-cog, CDR-SB, ADCS-ADL, TRAILS A). 
 
 
4.5 
Specific Objectives 
The specific objectives address two hypotheses: The primary hypothesis is that long-term administration of 
sargramostim (recombinant human GM-CSF) will be tolerated by and safe for use in participants with mild-
to-moderate AD; and the secondary and exploratory hypotheses that sargramostim treatment in participants 
with mild-to-moderate AD will slow or halt cognitive decline and/or activities of daily living, or improve 
cognitive functioning and/or activities of daily living,, reduce amyloid load, inhibit progression of medial 
temporal atrophy (MTA), improve AD-related microstructural white matter changes, improve brain 
metabolic activity, improve sleep-related measures, improve indices of semantic verbal fluency, and induce 
changes in peripheral blood and cerebral spinal fluid (CSF) biomarkers toward levels measured in 
 
Improved MMSE change scores in rHuGM-CSF/sargramostim-treated participants compared to placebo-
treated participants. Mixed model analysis of MMSE data of the completed study of 20 sargramostim-treated 
participants and 20 placebo-treated participants shows a statistically significant improvement in the sargramostim-
treated group at the EOT visit (a) compared to baseline (p=0.074) and to the placebo-treated group (p=0.037), and at 
the first follow-up visit (45 days post-EOT) compared to the placebo-treated group (p=0.0272) (b). (B) Distribution of 
delta MMSE scores at the EOT for random groups of 10 sargramostim-treated participants and 10 placebo-
treated participants. The 20 sargramostim-treated participants and 20 placebo-treated participants were divided into 
10,000 random groups of 10 participants each, and the mean Mini-Mental State Exam (MMSE) scores were calculated 
and graphed as a delta distribution. The mean and median delta was 1.8, as was the delta of the entire 20 sargramostim-
treated participants and 20 placebo-treated participants analyzed in (A) showing that the statistically significant 
difference between the sargramostim-treated participants and the placebo-treated participants at the EOT reflected all 
parts of the trial. 
A
B

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individuals without dementia. These aims are expressed through primary, secondary and exploratory 
objectives with related endpoints. 
 
Primary Objective 
Primary Endpoint 
To assess long-term 
tolerability and safety of 
sargramostim in individuals 
with mild-to-moderate 
Alzheimer’s Disease (AD). 
Effects of sargramostim on white blood cell mobilization will be 
determined by CBCs with differential. Comprehensive metabolic 
panels (CMPs), electrocardiograms (ECGs), serology testing for anti-
drug antibodies (ADAs), physical exams, and high-resolution MRI 
scans to identify amyloid-related imaging abnormalities (ARIAs) will 
be performed, and vital signs and adverse events (AEs) will be 
monitored. 
Secondary Objective: 
Secondary Endpoint 
To test the hypothesis that 
sargramostim treatment, as 
compared to placebo, slows or 
halts cognitive decline, or 
improves cognitive 
functioning in individuals with 
mild-to-moderate AD. 
 
Change in cognition from Baseline to End of Treatment (EOT) visit as 
measured by: 
• 
The Mini-Mental State Examination(MMSE) 
 
Exploratory Objectives 
Exploratory Endpoints 
To assess the effect of 
sargramostim treatment, as 
compared to placebo, on 
clinical progression in 
individuals with mild-to-
moderate AD. 
Change in cognition from Baseline to EOT visit as measured by: 
• 
the change in the MoCA (Montreal Cognitive Assessment) 
score 
• 
the change in the ADAS-cog13 (Alzheimer's Disease 
Assessment Scale-cognitive subscale) score 
• 
the change in the Clinical Dementia Rating Scale Sum of Boxes 
(CDR-SB) score 
• 
the change in the Trail Making Test A (TMT A) score 
• 
the change in the ADCS-ADL (Alzheimer's Disease 
Cooperative Study Activities of Daily Living Inventory) score 
• 
the change in the Neuropsychiatric Inventory Score (NPI) 
• 
the change in tests of Verbal Fluency (Semantic and Lexical) 
scores 
 
To assess the effect of 
sargramostim treatment, as 
compared to placebo, on 
metabolic activity in different 
brain regions in individuals 
with mild-to-moderate AD. 
 
Change from Baseline to EOT visit in FDG-PET assessment of 
metabolic activity, overall, as well in different brain regions, 
particularly in the medial temporal lobe in potential correlation with 
changes in MTA 
 
To assess the effect of 
sargramostim treatment, as 
compared to placebo, on brain 
amyloid deposition in 
individuals with mild-to-
moderate AD. 
CSF assay will be performed examining Aβ(1-42) and tau:Aβ(1-42) 
ratios to test the hypothesis that sargramostim treatment of mild-to-
moderate AD participants reduces cerebral amyloid load.  If 
participant opts in, the CSF assay will be conducted as part of 
screening and at the end of treatment.  Additionally, there is an option 
utilizing Amyloid PET scans, performed using Vizamyl, produced by 
the Pharmalogic cyclotron laboratory located adjacent to the CU 
Research Imaging Center (CU-RIC), which allows for amyloid-beta 

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neuritic plaque density imaging.  If the participant opts in, the PET 
scans will be performed during screening and at the follow-up visit, or 
within a week thereof depending upon scheduling availability, to 
assess for treatment-related changes in neuritic plaque density, as well 
as to correlate with any changes in tau:Aβ(1-42) ratios. 
  
To assess the effect of 
sargramostim treatment, as 
compared to placebo, on AD 
related changes in peripheral 
blood and CSF biomarkers in 
individuals with mild-to-
moderate AD. 
Biomarkers from peripheral blood and CSF collection will include 
various forms of the hallmark proteins that form amyloid plaques in 
AD (i.e., amyloid-beta [Aβ] peptides), as well as other proteins whose 
levels are known to be altered in AD (e.g., phosphorylated Tau 
(p-Tau), Total Tau (t-Tau), and various AD- and inflammation-
associated cytokines, chemokines, and other factors, for example, NfL, 
UCHL1, GFAP, IL-6, BDNF, etc.). Blood for these biomarker 
purposes will be collected at baseline, and at the EOT visit. CSF will 
be collected during screening, and at the EOT visit.  
 
To assess the effect of 
sargramostim treatment, as 
compared to placebo, on AD 
related changes in blood-brain 
barrier permeability in 
individuals with mild-to-
moderate AD. 
CSF will be collected from participants choosing CSF as confirmation 
for inclusion, or as an optional substudy, during screening, and at the 
EOT visit. A venipuncture blood draw will be performed at the same 
visit of each LP procedures to obtain serum. Albumin levels will be 
quantitated within CSF (in mg/dL) and serum (in g/dL) to calculate an 
Albumin Index (AI). Because albumin is neither synthesized nor 
metabolized intrathecally, albumin that is found within CSF that is free 
of blood contamination indicates that the albumin must have come 
from the plasma through the blood-brain barrier (BBB). An AI value < 
9 indicates an intact BBB, values 9-14 as slight impairment, values 14-
30 as moderate impairment, values 30-100 as severe impairment, and 
values > 100 as complete breakdown of the BBB 72. AI values will be 
calculated at screening and at EOT visit to assess any treatment-related 
changes in BBB permeability and to correlate values with any 
potential trial emergent ARIA events. 
 
To assess the effect that ApoE 
genotype may have upon 
predicting sargramostim 
treatment outcomes in 
individuals with mild-to-
moderate AD. 
ApoE genotyping will be performed using blood samples collected at 
Baseline visit of this trial for each participant. ApoE genotype will be 
determined by UC Health lab using established methods. Outcomes in 
the context of ApoE genotype will be compared. The genetic testing 
will be carried out at the end of the trial from stored blood, and 
participants will not be notified of any genetic testing results. 
 
To assess the effect of 
sargramostim treatment, as 
compared to placebo, on 
inhibiting MTA in individuals 
with mild-to-moderate AD. 
Atrophy in the entorhinal cortex (ERC), parahippocampal gyrus, and 
hippocampus (HPC), measured on high-resolution MRI scans, predict 
future cognitive decline and conversion to AD among individuals with 
mild cognitive impairment (MCI). Severity of MTA, assessed with 
high-resolution MRI scans, is strongly associated with severity of 
medial temporal lobe degenerative pathology, especially the severity 
of neurofibrillary pathology at autopsy. High-resolution MRI scans 
will be performed at screening, EOT visit, (and among participants in 
the optional amyloid PET procedure, at follow-up (45 days post-
treatment)). 
 

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To assess the effect of 
sargramostim treatment, as 
compared to placebo, on AD-
related changes in 
microstructural white matter 
measures in individuals with 
mild-to-moderate AD. 
Numerous neuroimaging studies have shown that white matter (WM) 
tracts are associated with cognitive function, and that microstructural 
abnormalities are found in AD and other neurodegenerative diseases, 
as determined from high-resolution MRI diffusion tensor imaging 
(DTI) techniques 73-77. DTI metrics have been proposed as sensitive 
biomarkers for disease progression over short periods of time 78, which 
makes it useful for assessing any WM changes in response to 
treatments, and specific DTI patterns have been shown to help 
distinguish between AD and other comorbid neurodegenerative 
diseases 79, 80, to correlate with AD-related verbal fluency impairments 
81, and with using neurite orientation dispersion and density imaging 
(NODDI) sequences of DTI, changes in neurite density can be 
approximated 82, 83. High-resolution MRI with DTI and NODDI 
sequences will be performed at screening, weeks 6, 12, 18, and EOT, 
(and among participants in the optional amyloid PET procedure, at 
follow-up (45 days post-treatment)) and metrics of DTI (e.g. FA, MD, 
NDI, ODI, etc.) will be monitored for any treatment-related changes. 
 
To assess the effect of 
sargramostim treatment, as 
compared to placebo, on 
changes in sleep-related 
measures in individuals with 
mild-to-moderate AD. 
Changes in quality of sleep have been long reported in onset and 
progression of AD 84-86, and decreases in non-rapid eye movement 
(NREM) slow wave activity (SWA) is known to be associated with Aβ 
deposition 87. Our preclinical work and others have revealed that GM-
CSF quickly removes amyloid in AD models 1, 68, of which it is not 
known whether quality of sleep improves with reductions in amyloid. 
For assessing any changes in qualitative measures of sleep in 
participants, the participant and the study partner/caregiver interview 
will be given the ISI and PSQI assessments at baseline, weeks 12, 
EOT, and at follow-up (45 days post-treatment) visits to assess any 
changes in participant sleep-related measures. Mechanistically, 
melatonin and its signaling have been shown to be decreased in AD 
progression 88-92, and GM-CSF has also been reported to increase 
melatonin secretion in preclinical studies 93. Melatonin is made and 
secreted by the pineal gland, and calcification of the pineal gland 
increases with AD progression 94-97. High-resolution MRI scans, using 
susceptibility weighted imaging (SWI) sequence, will be performed at 
screening, weeks 3, 12, 18, EOT, (and among participants in the 
optional amyloid PET procedure, at follow-up (45 days post-
treatment)) to assess changes in pineal calcification measurements. 
 
To assess the effect of 
sargramostim treatment, as 
compared to placebo, on 
changes in speech measures in 
individuals with mild-to-
moderate AD. 
AD is known to adversely impact temporal characteristics of 
spontaneous speech, such as speech tempo, number of pauses in 
speech, and their length, with speech alterations often noticed years 
before other cognitive deficits become apparent 98, 99. Among the 
neuropsychological measures frequently used to assess semantic 
memory impairment in AD are verbal fluency tests, or speeded word-
list generation, which consists of two types: phonemic (letter) fluency 
tests and category fluency tests. Fluency performance involves 
multiple brain processes and regions that are adversely impacted by 
AD pathology, and both phonemic and category fluency performances 
reveal declines in AD 100, 101. Word retrieval in connected speech is 
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descriptions, the total word output, percentages of content words, 
percentages of nouns, and percentages of pronouns out of all words, 
type-token ratio of all words and type-token ratio of nouns alone, 
mean frequency of all words and mean frequency of nouns alone, and 
mean word length can be analyzed 102. A study participant interview 
will be given at baseline, week 12, EOT, and at follow-up (45 days 
post-treatment) visits to assess changes in verbal fluency and word 
retrieval measures.  
 
 
4.6 
Benefits and Risks Assessment 
4.6.1 
Benefits 
There is a tremendous unmet medical need for treatments to slow, halt, or reverse AD pathogenesis and 
cognitive decline.  Animal models, as shown above, suggest that GM-CSF-treated Tg mice performed 
equally well or better than NT control mice during individual blocks and overall.  The reversal of cognitive 
dysfunction and reduced cortical amyloidosis of GM-CSF-treated Tg mice were paralleled by increased 
synaptic area and microglial density as compared to saline-treated Tg mice, as well as increasing cognitive 
function in NT control mice 1, and of which effects have subsequently been replicated by others 68, 69.   
 
In human preliminary clinical results, a retrospective analysis on data assessing cognition among bone 
marrow transplant patients, found that patients receiving GM-CSF plus G-CSF had improved cognitive 
function between baseline and at six and twelve months, as well as being significantly improved over 
G-CSF alone at 6 months 70.   
 
Further, recombinant human GM-CSF (Leukine®/sargramostim) has been FDA-approved and safely used 
worldwide since 1991 for leukopenia, to shorten time to neutrophil recovery and to reduce the incidence of 
severe and life-threatening infections. Specifically, recombinant human GM-CSF (sargramostim) is a 
leukocyte growth factor indicated for use following induction chemotherapy in AML, for use in 
mobilization and following transplantation of autologous peripheral blood progenitor cells, for use in 
myeloid reconstitution after autologous bone marrow transplantation, for use in myeloid reconstitution after 
allogeneic bone marrow transplantation, and for use in bone marrow transplantation failure or engraftment 
delay. Any adverse events (AEs) associated with sargramostim are usually rare mild-to-moderate pyrogenic 
effects that subside upon reducing the dosage of sargramostim by half or by halting administration. 
Sargramostim has not been withdrawn from investigation or marketing in any country for any reason related 
to safety or effectiveness. In fact, a briefing package composed by Genzyme Corporation in May 2013 for 
an FDA Advisory Committee Meeting states that “a large body of safety data exists with Leukine®, in both 
approved and investigational therapeutic settings, representing over 21 years of post-marketing 
experience,” that “approximately 470,000 patients have received Leukine® treatment in the post-marketing 
setting from the time of product launch in March 1991 through December 2012,” and that “the safety profile 
observed to date has been consistent and predictable across multiple indications and across special patient 
populations (healthy volunteers, pediatric and geriatric subjects)” (103, attached as Appendix D). As such, 
sargramostim has an excellent safety profile for use among many human populations, and current results 
from our ongoing clinical trial within mild-to-moderate AD participants (NCT01409915) have revealed no 
drug-related SAEs, including no incidence of ARIAs that have plagued all other anti-amyloid 
immunotherapy trials.  
 
 
 

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4.6.2 
Risks 
Potential risks for GM-CSF (sargramostim) include reported side effects including “first-dose effect," 
wherein the first dose of sargramostim may cause a person to experience low blood pressure, fast heart 
rate, flushing, lightheadedness or feeling faint, and tends not to happen with future doses. As well, some 
individuals have reported diarrhea, local reactions at the injection site (i.e. swelling, redness and/or 
tenderness), and feelings of weakness and fatigue.  Less common potential side effects include mild flu-
like syndrome (i.e. fever, headache, generalized aches and pains, weakness and fatigue) and swelling in 
hands and feet.  A rare (<1%) side effect is a problem with blood clots. There is a potential risk related to 
immunosuppression (neutropenia or leukopenia) that may occur in select individuals taking sargramostim, 
which may increase risk of infection. There is also a risk of infection independent of low neutrophil count 
which could range from mild to life-threatening. This does not appear to have been previously reported in 
other use of sargramostim.  
 
In addition to the potential drug-related risks, there are study procedure-related risks. All procedures have 
varying degrees of risk, ranging from very low risk (ECG) to higher risk (lumbar puncture), and are all 
standard clinical assessments.  All procedures have limited associated risks for the participant and are 
described in the respective parts of this clinical study protocol (e.g. associated risk from low to higher - 
ECG, blood draw, MRI, and PET, lumbar puncture). Please see section 8.2 for more discussion on 
specific procedural risks. 
 
An independent DSMB will be chartered for this study to review safety and other relevant data on an 
ongoing basis. 
 
A placebo arm is necessary to allow for an accurate assessment of safety and tolerability, as well as the 
potential beneficial effect on cognition and function. Because there is currently limited treatments 
available for participants at risk for Alzheimer’s disease dementia, the use of a placebo is warranted. 
Initiation of approved symptomatic treatment is possible during the course of the study, if that is deemed 
necessary by the participant and the respective treating physician.  
 
 
 

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5  Study Design and Methods 
 
5.1 
Overall Design 
The study is a Phase 2, double-blind, placebo-controlled study of sargramostim in the Alzheimer’s disease 
population.  It will include individuals diagnosed with mild AD or moderate AD (or meeting criteria 
consistent with a provisional research diagnosis), consistent with MoCA score range of 4-24 (137, 
138,139)at time of screening and NIA-AA (2018) research criteria.  The study includes a screening period 
of up to 12-weeks, a treatment period of 24-weeks, and an approximately 6-week follow-up.  Individuals 
who meet inclusion/exclusion criteria will be randomized in a double-blind manner, in an approximate 
2:1 randomization ratio to one of the following treatments: 
 
• 
Sargramostim 178.57 µg/m2/day subcutaneously (7 days per week) (This is adjustment from 
original 250 µg/m2/day subcutaneously (5 days per week). Any participant on the original dosing 
schedule will be changed to the new dosing schedule the first refill after amendment is approved) 
• 
Placebo equivalent volume subcutaneously (7 days per week). 
 
The estimated maximum main study visits for each individual participant is approximately 55, including 4 
screening visits (Tier .5-3), 48 treatment period visits (i.e. 4 in-person clinic visits, 20 at home visits, and 
24 weekly blood draw at central laboratory for 2nd safety CBC w/diff), 1 End of Treatment visit, 1 FDG 
PET visit, and 1 visit in the post-treatment follow-up phase.  Participants who opt to participate in the 
optional PET substudy or optional LP substudy would have 2 additional visits, 1 during screening, and 1 
during either EOT (LP) or follow-up (PET).  All participants will have the option of visits to happen at 
the main study site when the home nursing visit option is available. 
 
5.1.1 
Investigational Product: 
Recombinant 
human 
GM-CSF 
is 
commercially 
available 
from 
Partner 
Therapeutics 
as 
Leukine®/sargramostim, which is FDA-approved for use in elderly patients (> 55 years old) and has a long 
and well documented safety record. The investigational product will be obtained by the study Sponsor for 
use in this clinical trial, and prepared by an unblinded pharmacist for administration to the randomized 
participant. 
 
5.1.2 
Justification for Dose 
Recombinant human GM-CSF (Leukine®/sargramostim) is currently marketed in the U.S. by Partner 
Therapeutics. Leukine® has been FDA-approved since 1991 and safely used worldwide for leukopenia, in 
multiple stem cell transplantation settings, and following induction chemotherapy in patients 55 years and 
older with acute myelogenous leukemia (AML) to shorten time to neutrophil recovery and to reduce the 
incidence of severe and life-threatening infections. Specifically, sargramostim is a leukocyte growth factor 
indicated for use following induction chemotherapy in AML, for use in mobilization and following 
transplantation of autologous peripheral blood progenitor cells, for use in myeloid reconstitution after 
autologous bone marrow transplantation, for use in myeloid reconstitution after allogeneic bone marrow 
transplantation, and for use in bone marrow transplantation failure or engraftment delay (104, attached as 
Appendix E). The FDA-approved recommended dosage is 250 µg/m2/day SC for up to 42 days or until 
absolute neutrophil count (ANC) reaches 1,500 cells/mm3 for three consecutive days. Although it is not 
possible to directly translate the dosage used in mice to that to be used in humans for cognitive 
enhancement, the dosage of GM-CSF administered to transgenic AD mice that resulted in cognitive 
improvement and amyloid plaque reduction was about twice the recommended dosage of sargramostim 
(~500 µg/m2/day SC or ~167 µg/kg/day) for a total of 20 injections (cumulative amount per mouse = 100 
µg). However, several clinical trials with sargramostim have shown no serious AEs at dosages of >500 
µg/m2/day 105-107, and the maximum amount of sargramostim that can safely be administered by single or 
multiple dose has yet to be determined. Doses up to 100 µg/kg/day (4,000 µg/m2/day or 16 times the 

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recommended dose) were administered to four patients in a Phase I uncontrolled study by continuous 
intravenous infusion for 7 to 18 days and with WBC reaching up to 200,000 cells/mm3. All AEs (dyspnea, 
malaise, nausea, fever, rash, sinus tachycardia, headache and chills) were reversible upon discontinuation 
of the drug, as discussed above (104, attached as Appendix E). These and other results, from the 
approximately 470,000 patients that have received sargramostim during its post-marketing setting (103; 
attached as Appendix D), indicate that sargramostim has an extremely good safety profile.  
 
Notably, the Moffitt Cancer Center study, described in the Preliminary Clinical Results section above, used 
the recommended FDA-approved dosage (250 µg/m2/day SC) and found highly significant (p < 0.01) 
cognitive enhancement in patients treated with GM-CSF plus G-CSF compared to those treated with G-CSF 
alone (70, attached as Appendix C). And, most importantly, in the recently completed pilot safety and 
efficacy trial (described above, NCT01409915) in which sargramostim (250 μg/m2/day SC) was 
administered to mild-to-moderate AD participants for 15 days over a 19-day period, the randomized 
participants have completed the trial with no drug-related serious AEs, including no evidence of ARIAs. 
Therefore, randomized participants will be started at the full FDA-approved recommended sargramostim 
dose.  
 
During the course of this clinical investigation, the study team has determined that full dosing at 250 
μg/m2/day SC for the full 6 month treatment period of the study may have unknown risks related to immune 
functioning of participants, and the decision was made to change the dosing schedule from all 24 weeks at 
250 μg/m2/day SC five days a week, to 178.57 μg/m2/day SC seven days a week. This maintains total 
weekly dosing while reducing total daily dosing. 
 
5.2 
Recruitment of Participants 
This phase 2 clinical trial of sargramostim in AD will recruit participants with mild-to-moderate AD.  At 
CU-AMC, Individuals who are patients in the Memory Disorders Clinic at the University of Colorado 
Hospital (UCH), will be recruited by study personnel.  Recruitment of participants will also take place 
within the larger community surrounding CU-AMC.  To recruit from the community, study flyers will be 
posted on the CU-AMC campus and social media may be utilized.  Additionally, Clinicaltrials.gov, the CU-
AMC Clinical Trials Website, and sites such as TrialMatch.alz.org will be utilized. The recruitment 
communication will emphasize the voluntary nature of the research study, and the fact that declining 
participation will not alter clinical care. To help defray the costs of participation, participants will be 
informed that, if randomized, they will be receiving a $75.00 gift card at the end of the 24-week treatment 
period, even if they cannot or choose not to complete the study.  They will also receive a $25.00 gift card 
after the follow-up visit (45-days post-treatment), for a subtotal of $100.00. Additionally, the participants 
will receive a $75.00 gift card after receiving each LP procedure, for a subtotal of $150.00, and a $125.00 
gift card after undergoing each Amyloid PET procedure, for a subtotal of $250.00, which all add up to an 
overall trial total maximum of $500.00.   
 
5.3 
Screening Period (Screening Visit) 
At the first contact for the Screening Visit, the study will be explained to the participant and the study 
partner and legally authorized representative (LAR) if applicable and a different individual than the study 
partner, by the study Principal Investigator or delegated sub-investigator.  Informed consent must be 
obtained before any study procedures are conducted.  The Screening Visit consists of four Tiers (0.5-3) of 
events (with an additional Tier for participant in optional amyloid PET substudy or LP substudy) that must 
be completed within 84 days (day -84 to day 0).  The Screening study events and assessments, and their 
related Tiers, are shown in in the Schedule of Events section (Section 2). Informed consent discussion for 
Tier 0.5 procedures may occur remotely or in person, with the remote taking place via HIPAA-compliant 
platform, in which case e-consent will be obtained using 21 CFR Part 11 compliant version of eSignature 
system. If e-consent is not possible, potential participants may scan or fax the signed consent form to the 
study team, or send them a picture, and will be asked to bring the original signed version to the first in-

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person study visit, should one occur. Decisional capacity will be assessed by a medically qualified and 
delegated investigator at all relevant time points (ie Tier .5 and Tier 1 of screening) using a standardized 
checklist. 
 
5.3.1  Screening Procedures 
Screening procedures are listed below and shown in Schedule of Events section (Section 2).  Assessments 
and procedures used throughout the study are described in more detail in Section 8.  If any results of the 
screening procedures warrant concern for the participant’s well-being and/or additional care, the participant 
will either be referred to their appropriate medical provider or if urgent, escorted or transferred to the 
Emergency Department for follow-up care. 
 
All mandatory Tier 0.5 screening procedures and assessments must be completed and evaluated by the 
PI/sub-I before any Tier 1 procedures and assessments are initiated. All Tier 1 screening procedures and 
assessments must be completed and evaluated by the PI/sub-I before any Tier 2 procedures and assessments 
are initiated.  Similarly, all Tier 2 assessments and procedures (Screening MRI) must be completed and 
evaluated by the PI/sub-I before any Tier 3 procedures (Screening LP or Screening Amyloid PET).  The 
optional amyloid PET or optional LP substudy procedures (Tier 4) will be only be conducted after results 
of the Tier 3 entry criteria are evaluated and the participant meets inclusion/exclusion criteria. 
 
• 
Tier 0.5 
o Mini-Informed Consent 
o Demographics 
o Cognitive Health Review Battery* 
§ 
*At study physician discretion, the cognitive health review battery can be minimized 
to included only the FAQ and DSRS based upon available medical history. 
o Optional (per PI/sub-I discretion for clarity on eligibility for screening) Blood draw for 
screening biomarker for Alzheimer’s disease, such as plasma Aβ(42)/ Aβ(40) ratio or pTau 
217 may be conducted 
 
• 
Tier 1 
o Full Informed Consent 
o Physical and neurological exam  
o Montreal Cognitive Assessment (MoCA): After informed consent is obtained, the cognitive 
entry criteria, the MoCA, will be one of the first assessments or procedures, performed by a 
trained cognitive rater. Participants who do not meet the MoCA screening criteria are not to 
have any further screening procedures performed, and participants who qualify based on the 
MoCA entry criteria will then proceed to the remaining screening criteria.   
o Medical history review 
o Previous and concomitant medications 
o Vital signs (i.e. BP, HR, Temperature, Respirations) Blood pressure and pulse should be 
measured in the sitting position only. Refer to section 8.4.5.3 Data Monitoring Plan for 
acceptable BP parameters.  
o Height and weight 
o ECG 
o Diagnostic assessment for mild-to-moderate Alzheimer’s disease: In the case of a potential 
participant who otherwise meets criteria for inclusion in the study, has a confirmed diagnosis 
of mild cognitive impairment, but does not have a confirmed diagnosis of mild or moderate 
AD, the PI or sub-I may conduct an evaluation as part of screening utilizing clinical interview 
and validated measures of functioning related to disease severity (such as the Functional Ability 
Questionnaire (FAQ) and/or the Dementia Severity Rating Scale (DSRS) to create a research 
provisional diagnosis of mild or moderate AD. NOTE: The PI or sub-I who conducts the 

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diagnostic assessment will meet with at least one additional study doctor or qualified sub-
Investigator to arrive at consensus of the research provisional diagnosis. 
o Safety evaluations (C-SSRS, GDS, GAD-7) 
§ 
Columbia Suicide Severity Rating Scale (C-SSRS): The C-SSRS will be 
administered at the Screening Visit to assess psychological health.  It was designed to 
capture the occurrence, severity and frequency of suicide related thoughts and 
behaviors during the defined assessment period.  At screening, the C-SSRS version to 
be administered will be the “Baseline” version, with individuals who score at high risk 
for suicidality (positive response to items 4 and/or 5) will be excluded.  If any results 
of the C-SSRS warrant concern for the participant’s well-being, the participant will 
either be referred to their appropriate medical provider or if urgent, escorted or 
transferred to the UCH Emergency Department for follow-up care. 
§ 
Geriatric Depression Scale (GDS): The GDS is a simple 30-item measure used to 
identify depression in elderly individuals. The simplicity of the GDS (questions are 
answered only by “yes” or “no”) enables the scale to be used with ill or moderately 
cognitively impaired individuals. One point is assigned to each answer and the 
cumulative score is rated on a scoring grid. The grid sets a range of 0 to 9 as ‘normal’, 
10 to 19 as ‘mildly depressed’, and 20 to 30 as ‘severely depressed’.  
§ 
Generalized Anxiety Disorder (GAD)-7:  The GAD-7 is a simple seven item 
questionnaire used to identify anxiety levels in individuals. The GAD-7 is calculated 
by assigning scores of 0, 1, 2, and 3, to the response categories of “not at all,”  “several 
days,” “more than half the days,” and “nearly every day,” respectively. GAD-7 total 
score for the seven items ranges from 0 to 21. Scores of 5, 10, and 15 represent cut-
points for mild, moderate, and severe anxiety, respectively. Though designed primarily 
as a screening and severity measure for generalized anxiety disorder, the GAD-7 also 
has moderately good operating characteristics for three other common anxiety 
disorders: panic disorder, social anxiety disorder, and post-traumatic stress disorder. 
o Screening Laboratory Assessments 
§ 
Approximately 16 ml of blood will be drawn for a CBC with differential and CMP, PT 
INR, and for serology evaluation of treponema, HBsAg and anti-HCV, anti-HIV1, and 
anti-HIV2 antibodies.  
§ 
The creatinine level from the CMP will be used to calculate the estimated GFR to 
assess kidney function if not provided by the central laboratory. 
§ 
Urine for exclusionary substances.  
§ 
For women who cannot confirm they are post-menopausal per clinician satisfaction, a 
urine pregnancy test will be performed.  
 
• 
Tier 2 
o Screening MRI: A screening MRI will be performed as part of eligibility to assess for evidence 
of prior or existing neurological disease (e.g. stroke, microhemorrhage, cancer, etc.), to exclude 
other potential causes of dementia, and to assess for MTA. Aside from the Tier 3 procedure, 
the participant should have met all other screening inclusion criteria before the MRI is 
performed. The MRI scan will be reviewed by a Radiologist and then results and/or the scan 
will be reviewed by the Principal Investigator to determine eligibility. The PI will share any 
medically relevant information that might indicate a health risk with the participant and LAR 
that is in the MRI report.   
 
• 
Tier 3 
o  
o Participant may choose either as entry criteria to confirm plasma Aβ(42)/ Aβ(40) ratio from 
Tier .5 

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§ 
Screening Lumbar Puncture (LP) for CSF Assay: A lumbar puncture (LP) will be 
performed to obtain cerebrospinal fluid (CSF) and each participant’s amyloid status 
will be evaluated at a central screening laboratory via CSF Aβ1-42 and p-tau:Aβ(1-42) 
ratios using validated assay methods demonstrating high sensitivity and specificity 
(e.g. Athena Diagnostics; ADmark® Phospho-Tau/Total-Tau/A Beta42). These assays 
will be performed at a central laboratory (i.e. Athena Diagnostics) with the p-tau:Aβ(1-
42) ratio results being reported to the investigator for eligibility determination. The 
accepted reference values in non-AD individuals is Aβ(1-42) >1026 pg/mL and p-
tau/Aβ(1-42) ratio ≤0.023. For inclusion criteria consistent with the presence of 
pathological changes associated with AD, the values for Aβ(1-42) should be <1026 
pg/mL and the values for p-tau/Aβ(1-42) ratio >0.023. CSF will also be assessed for 
glucose and protein levels to ensure safety.  
§ 
Additionally at this Tier 3 visit, approximately 4ml of blood will be drawn at the same 
visit to be used in conjunction with the CSF to quantitate both serum and CSF albumin 
levels and calculate an albumin index (AI) for assessment of AD-related permeability 
changes in the blood brain barrier 108.  
• 
CSF total protein 
• 
CSF Total cell count 
• 
CSF and serum glucose 
• 
Extra CSF sample that is not used for these assays will be aliquoted and stored 
for biomarker assays (e.g. 5.3.1.1 below), which are dependent on the 
participant meeting all inclusion criteria and being enrolled into the trial. If the 
participant does not enroll into the trial, the extra CSF samples will be removed 
from storage, discarded, and not used for any biomarker assays, unless the 
participant consents for these samples to be banked and used in further 
research studies. 
• 
It is anticipated that results of the CSF assay will be available within the 84 
day screening period.  However, it will not be considered a protocol violation 
should the assay results not be available until after the 84 day screening period 
has concluded.  It will be allowable for the screening period to be extended if 
the CSF assay results are not available within the 84 day screening window. 
• 
Vital signs including BP, pulse, respirations, and temperature will be collected 
prior to the LP procedure, if performed. 
 
§ 
Amyloid PET scan: An Amyloid PET scan will be performed using an FDA approved 
ligand Vizamyl, produced at Pharmalogic, which allows for amyloid-beta neuritic 
plaque density imaging, and will be used to explore the hypothesis that sargramostim 
treatment of individuals with mild-to-moderate AD reduces cerebral amyloid load.  
The Amyloid PET scans will be performed during the screening period and at the 
follow-up visit, or within a week thereof depending upon scheduling availability.  The 
radiologists who read the MRI and PET imaging scans will remain blinded to the 
participant’s identity and test article treatment group. Vizamyl is FDA approved for 
research and clinical use. 
 
• 
Tier 4 (Optional) 
o After either the screening Amyloid PET scan or the LP results have been received, participants 
have an option to participate in an optional substudy for either LP or amyloid PET scan.  
• 
If receiving an LP for entry, and consenting to an optional Amyloid PET scan it will 
be conducted using Vizamyl, Pharmalogic, which allows for amyloid-beta neuritic 
plaque density imaging, and will be used to explore the hypothesis that sargramostim 

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treatment of individuals with mild-to-moderate AD reduces cerebral amyloid load and 
will follow the same schedule of events as listed for using the Amyloid PET used for 
entry criteria.   
• 
If receiving an amyloid PET scan for entry, and consenting to an optional LP, it will 
follow the same schedule of events as listed for using the LP procedure used for entry 
criteria.  
 
o Participation in, or result of, either the optional Amyloid PET or optional LP are not required 
for entry to the study. 
 
5.3.2 
Additional Screening Procedures and Considerations 
Participants will also have to meet the additional inclusion/exclusion criteria (such as, but not limited to, 
stability on medications per appendix H). As well, in regards to hematologic values from screening visit 
labs, if screening ANC>70% or 7.7X103/mm3 or WBC>11,500/mm3 and if this is sustained for > two weeks 
on recheck, a medical evaluation and clearance for underlying conditions, as well as return to normal prior 
limits, must be met prior to consideration for further enrollment procedures.) 
 
To determine whether inclusion/exclusion criteria are met, a review committee consisting of the principal 
investigator or delegated sub-Investigator and the trial coordinator will review all screening data points. If 
all inclusion/exclusion criteria are met, with results reviewed by the principal investigator or qualified sub-
investigator, the principal investigator or delegated sub-investigator will provide documented confirmation 
that the participant is eligible to be randomized.  After that confirmation, the participant may complete their 
optional Tier 4 Screening Visit or Baseline Visit.  More detailed information of this process can be found 
in a separate document, the Manual of Procedures.  
 
5.3.2.1 Screening Flow Chart 
 
 
 
5.4 
Double Blind Treatment Period (Baseline Visit through Visit 24) 
The treatment period is double-blinded, beginning at the Baseline Visit.  At the Baseline Visit, appointments 
for all remaining visits should be made and scheduled as close as possible to the targeted date, based on the 
Baseline Visit. Visits done outside the allowable window will be considered a protocol deviation.  
Participants who meet the entry criteria for the clinical trial will be enrolled, randomized, and receive 24 
weeks of treatment with either sargramostim or placebo.   
 
The participant will follow the schedule of 7 days (Sunday through Saturday) of subcutaneous injection 
(178.57 µg/m2/day sargramostim or placebo) over the course of 24 weeks, for a total maximum of 168 

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treatment days. The first injection will be administered at the time of baseline (randomization) visit in the 
site’s designated facility. The participant and study partner will receive training and a schedule on how to 
administer additional doses at home.  For the first 3 weeks, the participant will receive a weekly dose 
administered by their caregiver with observation by a study nurse for correct injection technique. After the 
first 3 weeks, the injection will be observed by a study nurse at in-person visits at the site (Wk 6, Wk 12, 
Wk 18). Ongoing injection technique education will be provided as needed. If the caregiver is unable to 
administer the in-person visit injection, the injection will instead be administered by a trained medical 
professional.  The investigational product will be distributed weekly to the participant either at their in-
person clinic visit (Baseline, week 6, week 12, week 18) or at-home visit for subsequent subcutaneous 
injection at home, along with a new injection diary. An empty sharps container will be provided at the 
baseline visit and be replaced as needed for participants/caregivers to place used syringes into at home.  
 
At the weekly double-blind treatment period site visits (Visit 2- through Visit 24), delegated study personnel 
will review and record changes to conmeds, adverse events, compliance and vital signs.  As well, qualified 
staff will review inject sites to assess for possible adverse reactions.   
 
5.4.1  Double-Blind Treatment Procedures 
All treatment visit administrative procedures, cognitive assessment, safety assessments and laboratory 
assessments are to be done before administration of the investigational product. 
 
5.4.1.1 Baseline Visit Procedures 
• 
Randomization 
• 
Concomitant medication and AE review 
• 
Physical/neurological exam 
• 
Vital sign / weight 
• 
Cognitive assessments and other measures 
Participant 
o Mini-Mental State Examination (MMSE) 
o Alzheimer’s Disease Assessment Scale – cognitive subscales (ADAS-Cog13) 
o Trail Making Test-A (TMT-A) 
o Verbal Fluency (Lexical and Semantic Fluency) 
o MoCA 
o Clinical Dementia Rating Scale (CDR) 
o GDS 
o GAD-7 
o C-SSRS 
Study Partner 
o CDR 
o Alzheimer’s Disease Cooperative Study Activities of Daily Living Inventory (ADCS-ADL) 
o Neuropsychiatric Inventory (NPI) 
o Sleep assessments (ISI, PSIQ) 
• 
Laboratory assessments 
• 
FDG PET 
• 
IP administration training 
• 
Administration of first dose 
• 
Dispensing of IP 
Note:  It is acceptable to perform the required procedures of the baseline visit over more than one visit to 
accommodate the participants and study site scheduling. 
 
At the Baseline Visit, the pharmacy will be contacted to perform participant randomization (this may be 

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done in advance of the visit to allow for study drug / placebo preparation by the pharmacy), a physical exam 
is performed, vital signs are recorded, approximately 52 ml of blood will be drawn for a CBC with 
differential, a CMP, ADA evaluation (anti-sargramostim antibodies), and for biomarker assays (e.g. NfL, 
UCHL1, GFAP, IL-6, BDNF, etc.).  As well, a sample will be obtained and stored for later ApoE 
genotyping. 
 
Baseline assessments of cognition will be conducted by trained personnel, including the, MMSE, MoCA, 
ADAS-cog13, ADCS-ADL, Trail Making Test-A, CDR, NPI, and the verbal fluency measures (lexical and 
semantic). (Note that the qualified clinical rater who administers the CDR and ADCS-ADL will not be the 
same individual who administers the remainder of the cognitive assessment, thereby ensuring that CDR 
raters are blind to other clinical data.)  Additionally, safety assessments, (i.e. C-SSRS, GDS and GAD-7) 
will be administered.  More information about these testing assessment procedures can be found in a 
separate document, the Manual of Assessments. 
 
The investigational product will be prepared by the unblinded pharmacist for acceptance by study 
personnel, with the initial dose administered by the study partner or designated caregiver after injection 
technique education by a study nurse with study nurse oversight, or by a study nurse if needed.  
Investigational product administration training will be given, with injection diary, to the participant and 
study partner. The remaining injections will be administered by or under the supervision of the study partner 
or other designated caregivers in the home setting until the next in-person visit.  More detailed information 
can be found in a separate document, the Manual of Procedures. 
 
5.4.2  Additional Double Blind Treatment Study Procedures 
In addition to the regularly scheduled collection of vital signs, conmed and AE review, labs, cognitive 
assessments, imaging and a physical/neurological exam will be completed at visits 12 and EOT. More 
detailed information can be found in a separate document, the Manual of Procedures. 
 
Pharmacy orders will be submitted using the most recent weight documented for each participant, and 
coordinators will ensure the weight is within 3-weeks of the order submission.  If a new weight is 
documented after order submission and by noon the day before the pharmacy dispenses, the coordinator 
will verify if the weight has changed by more than 3%.  If the change is greater than this amount, a new 
pharmacy order will be submitted with the updated weight.  If the change is less than this amount, the order 
will proceed with the original submission.   
 
5.4.2.1 Double-Blind Treatment Labs – Visit 2 through 24 
Prior to investigational product administration at weekly visits (Visit 2 through Visit 24), either at-home 
visits or research site visits, qualified medical staff will draw blood for safety labs and metabolic functioning 
(approximately 8 ml for CBC with differential and CMP) with two CBC with differential and one CMP per 
study week. Additionally, prior to administration of investigational product, on specific visits (Visit 6, 12, 
18 and 24), there will be an additional blood draw for ADA testing (approximately 9 ml). If there are 
outstanding clinically significant lab values after CMP during week 24, additional CMP lab draw(s) may 
take place to resolve these clinically significant values, or the participant referred to their health care 
provider for clinical care follow-up, based upon the judgement of the PI. 
 
An additional, weekly, safety blood draw (i.e. CBC with differential) will be completed. Depending on the 
participant’s preference, the participant can either visit the CU -AMC site for the blood draw or the 
participant can travel to a local contracted lab site and have the safety draw (i.e. central contracted 
laboratory with phlebotomy services, as specified in Manual of Procedures).  Lab results will be forwarded 
to study staff for review by the study physician. The EOT and FU Visits will comprise of a safety blood 
draw CBC w/differential and other specified labs per the schedule of events). 
 

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Additionally, a PT INR will also be drawn at Visit 24 in preparation for review prior to the EOT.  
 
5.4.2.2 Double-Blind Treatment Cognitive Assessment – Visits 12 and End of Treatment (EOT)  
At Visit 12 and EOT Visit, Cognitive assessments will be conducted by trained personnel, including the 
MMSE, MoCA, ADAS-cog13, ADCS-ADL, Trail Making Test-A, CDR, NPI, verbal fluency measures 
(lexical and semantic), , and sleep-related measures (i.e. ISI and PSQI). As mentioned above, there will be 
blinding between the cognitive and the clinical rater. As well, all attempts are to be made to schedule the 
assessments at approximately the same time of day, with the same rater to reduce variability. 
 
Additionally, safety C-SSRS, GDS and GAD-7 assessments will be administered by qualified personnel at 
Visit 12 and EOT Visit. 
 
5.4.2.3 Double-Blind Treatment Imaging 
High-resolution MRI scans will be completed at Visit 6, Visit 12, Visit 18 and EOT Visit. 
 
In addition to the baseline FDG PET Scan, an End of Treatment FDG PET Scan will be completed at EOT 
Visit. 
 
5.4.2.4 EOT / Double-Blind Treatment blood draw and Lumbar Puncture (if applicable) 
Occurring within 10 days, and at least 2 days following the final treatment day of week 24, a lumbar 
puncture will be conducted. Vital signs and weight will be collected at lumbar puncture visits.  CSF for 
assays including Aβ(1-42) and  tau:Aβ(1-42) ratios, total cell count, and for biomarker assays (e.g. NfL, 
UCHL1, GFAP, IL-6, BDNF, etc.) will be collected. At the same visit as the LP procedure has obtained 
CSF, a blood draw of approximately 44 ml will be obtained for biomarkers assays (e.g. NfL, UCHL1, 
GFAP, IL-6, BDNF, etc.).  Additionally, both blood and CSF samples will be analyzed by UC Health to 
determine a serum/CSF albumin index.  
 
5.5 
Follow-up Visit 
After the 24-week double-blind treatment period, all participants will be monitored during a 45-day follow-
up period.  At this visit, a physical/neurological exam will be performed, vital signs recorded, draw 
approximately 52 ml of blood for a CBC with differential, ADA analysis, and biomarker assays, and 
administer cognitive tests, including the MoCA, MMSE, ADAS-cog13, the ADCS-ADL scale, Trail Making 
Test A, CDR, NPI, verbal fluency measures (lexical and semantic), and sleep-related measures (i.e. ISI, 
PSQI). 
 
If the participant opted to engage in the Amyloid PET scan (either as entry criteria or optional), the PET 
will be performed using Vizamyl, and an MRI will be performed.   
 
5.6 
End of Treatment / End of Study Definitions 
End of Treatment is the date of the last Visit or scheduled procedure for a participant, as listed in the 
Schedule of Events (Section 2) 
 
End of Study is the date of the last Visit or scheduled procedure for the last participant randomized, as listed 
in the Schedule of Events (Section 2) 
 
Every attempt should be made to follow participants through their final visit of the double-blind treatment 
phase and follow-up visit. If a participant discontinues the study prematurely for any reason, all reasonable 
attempts will be made to complete an Early Termination Visit, which will consist of the events listed for 
the End of Treatment Visit. 
 
5.7 
At-Home Visits 

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As noted above, when the option becomes available to participants, nurses, contracted through an accredited 
traveling or in-home nursing organization, will be utilized to provide at-home visits during the treatment 
period of the clinical trial. The nurse(s) will visit the participant’s home one day per week (Monday through 
Friday, as their selected day as designated by study staff) during weeks 2–24 (excepting weeks with in-
clinic visits, week 6, week 12, and week 18, as described above) to verify delivery/presence of the weekly 
doses of the study drug, record vital signs, perform injection sites review, record conmed changes, record 
adverse events, and draw blood. Participants will also be given the option to come to the study site for their 
weekly visits, rather than have home visits. Except under extenuating circumstances, participants will be 
discouraged from switching the location of the weekly visits.   
 
5.7.1 
Narrative Summary of Home Visits: 
Each participant’s first injection will be administered at the time of baseline (randomization) visit in the 
Clinical and Translational Research Center (CTRC) at the CU-AMC campus. As noted above, injections 
for the first 3 weeks, at in-person visits either at home, or in-clinic (select visits) will be administered by 
the study partner under observation by a trained (blinded) study nurse after training by study staff, and 
under observation at Wk 6, Wk 12, and Wk 18 on site study visits, with refresher training as necessary.  At 
the Home visits (weeks 2-5, 7-11, 13-17, 19-24), the nurse will verify delivery of the study drug (by a 
contracted delivery service), collect vital signs, perform an injection site review, and record adverse events.  
As well, the nurse will perform a blood draw of approximately 4 mL for CBC plus differential on the day 
of each home visit for safety labs, and deliver to the contracted central laboratory.  
 
5.7.2 
Summary of Traveling Nurse Duties During at Home Visits:  
• 
Verification of delivery study drug to home of participant 
• 
Perform and record vital signs 
• 
Injection sites review 
• 
Record and monitor Adverse Events. 
• 
Record conmed changes, if any 
• 
Blood draw (CBC + Diff,) at each home visit;,; deliver blood samples to contracted laboratory 
drop-off site within two hours of collection. 
• 
Oversee administration, by study partner, of study drug injection until Week 3 and as needed 
thereafter. 
• 
Provide injection training refresher, as necessary. 
• 
Return injection diary and all other study visit related paperwork to study staff.  
 
5.7.3 
Logistics Associated with Traveling Nurses: 
Day before/day of visit: The study drug will be collected from the CU Outpatient Research Pharmacy by 
for delivery to the participants residence. The study drug syringes will be placed in hard cases, in an 
insulated bag with a cold pack for transport.  Refrigeration required for drug: drug to be placed in the 
refrigerator at the participant’s residence, with a recording thermometer (supplied by the study).  
 
Day of Visit: Nurse will travel to participant’s residence to perform the required duties (verify delivery of 
drug, injection site review, check vital signs, monitor adverse events, oversee injection of study drug by 
study partner as needed, draw blood, complete source documentation, injection site diary, drop off blood 
samples to lab). Submit data updates daily/ every-other-day (unless urgent) to study team.  
 
At Home Visit Nurse requirements: 
• 
Current nursing license with the State of Colorado (LPN or higher) 
• 
Current driver’s license with no outstanding citations.  
• 
Reliable vehicle 
• 
Coordinated back up from employer  

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5.7.4 
Drug Handling Details (when performed by courier service for at home visits): 
Study drug will be provided with a temperature monitoring device, which will record the temperature and 
record an excursion if the temperature is outside of the established range for the study drug when stored in 
the participant’s home refrigerator. On the designated day for the home visit (or day before if able to be 
appropriately stored), the study drug will be picked up by an authorized staff member at CUAMC Research 
Pharmacy, and either deliver it to the participant’s home, or delivery will pick up from AMC staff and 
deliver it to the participants home,and the study partner will placed in the refrigerator. During transit from 
CUAMC to the participant’s home, the pre-filled syringes will be maintained in a temperature-controlled 
container in a hard case designed to prevent any inadvertent changes to the syringes. As well, a temperature-
monitoring device will be placed in the container with the drug to ensure proper temperature exposure and 
provide a read-out of temperature reading if an excursion is noted.  The temperature-monitoring device will 
be left in the participant refrigerator with the study drug and reviewed at in-person visits by the study team.  

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6  Study Population 
 
6.1 
Inclusion and Exclusion Criteria 
 
6.1.1  Inclusion Criteria 
All participants must meet the following criteria to be included into the study: 
 
Inclusion Criteria 
 
 
Participant Characteristics 
1 
Males or females between age 60 and 85 years, inclusive, at time of consent. 
 
2 
Have a dedicated partner/caregiver informant who is in the company of the 
participant at least 12 hours a week, who can accompany them to scheduled visits, 
and who is able to provide accurate reporting upon the behavioral, cognitive and 
functional abilities of the participant. Additionally, the study partner (or LAR or 
caregiver, if different) must be willing and able to administer the treatment 
injections. If a study partner must withdrawal from study participation, a 
replacement study partner may be allowed, at the investigators discretion.  The 
replacement study partner, would need to agree to and sign a separate informed 
consent on the first visit that he/she accompanies the participant to participate. 
 
3 
Be physically able to participate by medical history, clinical exam, and other 
testing, with adequate visual acuity and auditory discrimination. 
 
4 
Be willing / able to provide written informed consent or assent.  If assent is 
provided, consent must be provided by a legally authorized representative (LAR) 
or proxy decision maker, who may or may not be the dedicated study partner / 
caregiver.  Documentation of LAR or proxy decision maker status will follow local 
laws and regulations. 
 
Note: Individuals utilizing a proxy decision maker will not be able to participate in 
optional sub-studies as a proxy decision maker cannot provide consent for optional 
non-therapeutic interventions. 
 
5 
Must reside within a proximity of the study site that will not preclude their 
regularly-scheduled participation in the trial, as well as a catchment area for local 
lab blood draws (i.e. central contracted laboratory). 
 
 
Disease Characteristics 
6 
Meet criteria for probable AD dementia according to the National Institute of Aging 
– Alzheimer’s Association (NIA-AA) 2018 core research criteria109, and have the 
following at screening: 
• 
A diagnosis of mild AD or moderate AD, or  
• 
A provisional research diagnosis consistent with probable mild AD or moderate 
AD, and 
• 
MoCA score of 4-24 inclusive. 
7 
Have positive biomarker for brain amyloid pathology as shown by: 

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• 
(Optional, per PI judgment for clarification on appropriateness for 
screening) Positive plasma assay for AD biomarkers (such as Aβ(42)/ 
Aβ(40) ratio or pTau217) AND/OR 
• 
Either positive CSF assay for AD assessment (Aβ(1-42) is <1026 pg/mL, 
p-tau:Aβ(1-42) ratio is >0.023) or positive amyloid PET, per PI read. 
 
8 
If receiving anti-dementia treatment (i.e., cholinesterase inhibitor and/or 
Memantine), be on stable treatment for at least 60 days before initial screening visit 
and up to the baseline visit. 
 
 
Be stable on all other medications for at least 30 days prior to initial screening visit. 
 
 
9 
Have had a dental exam within 6 months of date of screening. 
 
6.1.2  Exclusion Criteria 
Individuals who meet any of the following criteria will be excluded from this study: 
 
Exclusion Criteria 
 
Participant Characteristics Exclusions 
1 
Individuals with a first degree relative diagnosed with AD before 55 years of age. 
 
2 
BMI ≥35. 
 
3 
Is unable to read/write at an appropriate level to reliably participate in clinical trial 
psychometric assessments. 
 
4 
Is a prisoner. 
 
 
Medical Condition Exclusions 
 
 
5 
Other neurological or psychiatric condition (other than AD) that can impact 
cognition, as well as atypical presentations of AD and AD related dementias, 
including logopenic primary progressive aphasia (PPA), or posterior cortical 
atrophy (PCA); or, CT/MRI evidence of potentially significant intracranial 
abnormalities not related to AD (e.g., evidence of major stroke or lacune in an area 
critical to cognition, infections, cancer, hydrocephalus, multiple sclerosis, etc.); or 
abnormal CSF not consistent with AD. 
 
6 
Presence of current, serious mood or anxiety disorder, and/or a psychotic disorder, 
and/or a substance-related disorder according to Diagnostic and Statistical Manual 
of Psychiatric Disorders, Edition IV, text revision (DSM-IV-TR) or DSM-V that, 
in the opinion of the Principal Investigator, might impact cognitive assessment, 
affect participants ability to complete the study, or confound interpretation of the 
study drug effect; or is considered suicidal or shows suicidal ideation as assessed 
by the study physician, including, but not limited to: 
• 
Endorsing positive answer to C-SSRS suicidal ideation (items 4 or 5) or is 
positive for any suicidal behaviors within 6 months of screening, or has 
been hospitalized or treated for suicidal behaviors within past 5 years 
before screening. 

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• 
Severe depression as indicated by a GDS score of 20 or higher. 
 
• 
Severe anxiety as indicated by a GAD-7 score of 15 or higher. 
• 
Recent history (within 2 years of Screening) of alcohol or substance abuse 
or dependence, as determined by DSM-V criteria and in the judgement of 
the investigator. 
 
• 
Positive urine drug panel (due to non-prescription drug) at Screening, or 
use of cannabinoids (prescription or recreational). 
 
7 
History of unprovoked deep vein thrombosis, pulmonary embolism, familial 
predisposition for deep vein thrombosis, or pulmonary embolism, per study doctor 
judgement. 
 
8 
Active cancer / malignant neoplasm within 5 years of screening other than non-
melanoma skin cancers (e.g. Basal cell or squamous cell). Previous diagnosis of 
Leukemia, despite remission state or length of time, is considered exclusionary.  
 
9 
History of a latex or yeast allergy. 
 
10 
Presence/history of drug hypersensitivity; or known hypersensitivity to 
sargramostim, yeast-derived products, any other component of the product, or 
benzyl alcohol (present in bacteriostatic water or saline for injection). 
 
11 
History of asplenia, hyposplenia, or splenectomy (for any indication). 
 
12 
History of, or treatment for, an autoimmune disease (e.g. Rheumatoid Arthritis, 
Multiple Sclerosis, Myasthenia Gravis, etc.). 
 
 
13 
Untreated or unstable medical condition that could interfere with the study 
assessments in the opinion of the study physician, or may require use of excluded 
medications or treatments as specified in Appendix H.  
 
14 
History of seizures (except infant febrile seizures). 
 
15 
Pregnant or breastfeeding female, or female of childbearing potential and not 
protected by highly effective contraceptive method of birth control (i.e., oral or 
depot contraceptives or intrauterine device (IUD) or participant was surgically 
sterilized) and/or unwilling or unable to be tested for pregnancy; Male refusing to 
use condoms, if partner can get pregnant. 
 
 
Lumbar Puncture, MRI, PET, Vital Signs, ECG, Laboratory Tests and 
Physical Examination Exclusions 
16 
MRI evidence of >4 micro-hemorrhages; participants who may be prone to 
spontaneous ARIA-H and/or may be more susceptible to adverse effects of the 
ARIA-H. 
 
17 
Laboratory results that are, in the judgement of the investigator, indicative of an 
untreated medical or hematologic condition that could increase risk or interfere with 
study assessments, including untreated hypo- or hyperthyroidism, vitamin B12 

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deficiency, hyperleukocytic syndrome (including, but not restricted to, chronic 
myelogenous leukemia, Hodgkin and non-Hodgkin lymphoma), monoclonal 
gammopathy, and/or thrombocythemia. (Regarding hematologic values, if 
screening ANC>70% or 7.7X103/mm3 or WBC>11,500/mm3 and if this is 
sustained for > two weeks on recheck, a medical evaluation and clearance for 
underlying conditions, as well as return to normal prior limits, must be met prior to 
consideration for further enrollment procedures.) 
 
18 
Evidence of: 
• 
Clinically significant pre-existing fluid retention (clinical or radiological); 
• 
respiratory symptoms (e.g., dyspnea), moderate-to–severe lung disease 
(e.g. COPD, pulmonary infiltrates); 
• 
cardiovascular symptoms or electrocardiographic evidence of cardiac 
disease that warrant therapeutic intervention (e.g., congestive heart failure, 
supraventricular arrhythmia, heart block, uncontrolled atrial fibrillation, 
etc.) 
• 
a resting pulse less than 50, as reviewed by the study physician; 
• 
prolonged QTc interval >470 ms in females, 450 ms in males). 
• 
screening blood pressure measurement of greater than 160 systolic and/or 
95 diastolic. If initial screening blood pressure is outside these parameters, 
BP will be taken in triplicate over the course of the visit with average BP 
to be evaluated by the delegated clinical sub-I. 
 
19 
Known renal dysfunction or serum creatinine >150 μmol/L, or Glomerular 
Filtration Rate (GFR) less than 55 ml/min. 
 
20 
Known hepatic dysfunction (apart from Gilbert’s syndrome) or serum ALT ≥3 
times the upper limit of normal (ULN). 
 
21 
Positive serology for hepatitis B surface antigen (HBs Ag), anti-hepatitis C virus 
(anti-HCV), anti-human immunodeficiency virus 1 and 2 antibodies (anti-HIV1 and 
anti-HIV2 Ab) or spirochetal infection (e.g. syphilis).  
 
 
 
23 
Contraindication or inability to complete magnetic resonance imaging (e.g., cardiac 
pacemaker/defibrillator, ferromagnetic metal implants) or PET scan. 
 
24 
Sensitivity to fluorodeoxyglucose F 18 
 
25 
Having past or planned exposure to ionizing radiation that would, together with the 
radiation resulting from the administrations of the PET tracer(s) used in this study, 
exceed applicable institutional, local, or national recommendations for annual or 
lifetime exposure. 
 
26 
Poor venous access. 
 
27 
History of chronic or recurrent bacterial infections, at the discretion of the PI or 
delegated sub-I. 
 
Prior / Concomitant Therapy Exclusions 

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28 
 
 
 
 
Is taking any prohibited medication or therapy (see 7.7 Concomitant Therapy 
section and Appendix H). 
 
 
 
Prior / Concurrent Clinical Trial Experience 
29 
Be the recipient of an investigational drug within 60 days of screening, or within 5 
times the elimination half-life of that drug, whichever is the longest. 
 
30 
Prior treatment with an investigational anti-amyloid or anti-tauopathy therapy, or 
AD vaccine, unless it can be documented that they were on placebo. 
 
31 
Participation in the treatment phase of an investigational sargramostim clinical trial  
within 6-months of screening. 
 
32 
Have previously withdrawn from this study (This exclusion does not apply to 
participants who are eligible to rescreen before randomization in this study) 
 
 
Other Exclusions 
33 
Any interested participant who:  
a. Is in the judgement of the Principal Investigator likely to be non-compliant with 
study protocol, including, but not limited to, leaving the area of the study for 
any extended period; or separate from the designated caregiver/informant, 
without acceptable replacement, for any of the scheduled assessment visits 
during the study. 
b. Is unable to cooperate because of a language problem or because of a 
developmental disability. 
c. Oversees or implements any aspect of the study, or is employed by Partner 
Therapeutics or its affiliates or subsidiaries, or is an employee of the University 
of Colorado Alzheimer’s and Cognition Center and is engaged in the conduct 
of the study, or first degree relative of such. 
 
34 
Any technical/administrative reason that makes it impossible to randomize the 
participant in the study (e.g. kyphosis or other physical condition that prevents 
neuroimaging acquisition or other examinations, conflicts in participant schedules, 
etc.). 
 
NOTE: Investigators will ensure that all study enrollment criteria have been met at screening. If a 
participant’s status changes (including laboratory results or receipt of additional medical records) after 
screening but before the first dose of study drug is given such that he or she no longer meets all eligibility 
criteria, then the participant will be excluded from participation in the study. 
 
6.2 
Lifestyle Restrictions 
1. Participants should refrain from donating blood or blood products from the time of the screening 
visit until 6 months following last dose of the study drug. 
2. Participants should avoid excessive consumption of alcohol from screening visit until after follow-
up visit is completed.  Excessive consumption for this study is defined as more than 1 drink per day 
for female participants, or more than 2 drinks a day for male participants, on average. 
3. Avoidance of products containing tetrahydrocannabinol (THC) and other recreational drugs.  
 

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6.3 
Screen Failures 
Screen failures are defined as participants who sign an informed consent to participate, but fail to meet 
eligibility criteria. A minimal set of data from screen fails is required to be maintained, including basic 
demographics, reason for screen failure, and any serious adverse events that occurred between the signing 
of the consent and time of screen failure. 
 
Individuals who do not meet criteria for study participation (screen failure) may not be rescreened if the 
screen failure is due to non-eligibility due to plasma Aβ(42)/ Aβ(40) ratio assay, pTau217 assay, 
confirmatory CSF assay, or confirmatory amyloid PET scan.  If the screen failure is due to not meeting the 
cognitive inclusion criteria (MoCA out of range), one rescreen may be allowed, at the investigators 
discretion, after 6 months if the MoCA score is above the cut-off, or if previous score is within range of 
amended MoCA range. If the screen failure is due to prohibited treatments (see Appendix H), or changes 
in medication, the individual may be rescreened if they subsequently discontinue use of the prohibited 
drug(s) and are then stable on all other medications for at least 30 days prior to rescreening, and previously 
collected data such as LP and/or MRI scans may be used for rescreening if the visit is within two months 
of said procedure. Other participants who fail screening procedures may be rescreened at the discretion of 
the investigator. 
 
6.4 
Withdrawal from Study 
A withdrawal from the study shall be defined as withdrawing any time after being randomized into the 
double-blind treatment period, and before completing the events of the End of Treatment Visit.  
Participants who permanently discontinue use of the investigational product will be considered to be 
withdrawn from the study intervention, but may, if willing, complete other timepoint visits (such as EOT 
or F/U visits . 
 
Participants may withdraw from the study at any time and for any reason. The investigator (or qualified 
designee) must document the reason for withdrawal in the participant file. If the participant withdraws 
consent for the continued use of data, biological or genetic samples, or imaging, the request must be 
received by the Sponsor, in writing, from the participant or legally authorized representative. 
 
6.4.1 
Withdrawal Procedures 
If a participant prematurely discontinues treatment with the investigational product, the investigator must 
make every reasonable effort to perform the evaluations scheduled for the End of Treatment Visit as an 
Early Termination Visit. In the case where the participant permanently discontinues study medication 
between scheduled clinic visits, he/she should be requested to visit the clinic as soon as possible for the 
Early Termination Visit. 
 
If a participant is lost to follow-up, every reasonable effort must be made by study personnel to contact 
the participant and study partner to inquire about the reason for discontinuation/withdrawal, and follow up 
with any unresolved AEs/SAEs. A minimum of 3 attempts at contact should be made, with all measures 
taken to contact the participant and information received during those attempts documented. 

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7  Treatments 
 
7.1 
Treatment Administered 
The investigational product (IP) in this study is defined as recombinant human GM-CSF (sargramostim) 
administered at a dosage of 178.57 µg/m2/day by subcutaneous injection and its matching placebo.  
Sargramostim is commercially available from Partner Therapeutics, and will be obtained by the study 
Sponsor for this clinical trial. The sargramostim injections and placebo injections will be prepared by an 
unblinded UCH pharmacist or other qualified pharmacy personnel delivered to the blinded staff at 
location of the clinical visit. The IP and placebo will indistinguishable from each other. 
 
The investigational product for the double-blind treatment period will be supplied as individual syringes 
sufficient for weekly dosing until their next scheduled dosing visit. In-clinic and at-home visit injections 
will be administered by the study partner with observation by a blinded study nurse or qualified designee 
for appropriate injection technique for the first 3 weeks, and at on site visits (Wk 6, Wk 12, Wk 18), with 
ongoing education as needed. If the study partner is unable to administer the once weekly in-person dose, 
qualified personnel may administer. Home injection administration to be completed by trained study 
partner with take home syringes for home administration. The IP will be placed into a hard-case for travel 
and storage and then within a travel container that will be monitored and that is designed to ensure 
temperature compliance.  In conjunction with the take home syringes, an empty sharps container will be 
provided for the take home syringes to be placed into after they have been administered each treatment 
day. If sharps containers are filled they may be returned to the site and exchanged for empty containers as 
needed, otherwise the sharps container should be retuned at the end of the treatment. This cycle will be 
repeated every week for the 24 week double-blind treatment period. Additional details of these 
procedures are found in the Manual of Procedures. 
 
The investigational product is to be stored in the home refrigerator of the participant.  A temperature 
monitor will be provided, which will be reviewed every week to check for extreme temperature 
excursions. At the end of each dosing period (the projected number of dosing days between deliveries), if 
there are unused doses due to IP being held or form not being administered for any other reason, the 
participant and study partner will be instructed to dispose of the syringes in the sharps container, even if 
they are not expired, and to continue dosing only with the newly delivered doses.   
 
The investigator or qualified designee is responsible for the following: 
• 
explaining and training of the correct storage and use of the investigational product to the 
participant and her/his legal representative and her/his study partner (if different from LAR). 
• 
verifying that the instructions are being properly followed 
• 
maintaining accurate records of all investigational product dispensing and compliance. 
 
For dosing modifications or titration, please refer to Section 7.4. 
 
7.2  
Method of Treatment Assignment 
Participants who meet all criteria for enrollment will be randomized into sargramostim or placebo groups 
at Baseline Visit, at a 2:1 ratio. This enrollment ratio was determined using the sample size power analysis, 
as well as to maximize recruitment and minimize attrition.  The randomization schedule will be enacted 
and maintained at the CU Research Pharmacy (CURP), which has policies and procedures in place that 
describe randomization and blinding. Briefly, the pharmacy has built and implemented an Excel spreadsheet 
that produces random number sets that may be used for simple, weighted, block (two-, four-, or six-sized), 
and stratified randomization. For blinding, the pharmacy staff includes the pharmacist manager and 
technician. The pharmacy staff has been involved in several clinical trials that require double blinding, and 
double dummy. The pharmacy is an independent entity, and has the ability to ensure and maintain blinding 

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in any regard so that the staff at the clinical site will not know which study participants are receiving the 
study drug or the placebo. 
 
 

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7.3 
Blinding 
As this is a double-blind study, to preserve the blind, a minimal number of study administration personnel 
will have access to the randomization table and treatment assignments before the study is deemed complete.  
These individuals will be designated by the Sponsor, Huntington Potter, PhD, and kept in a log.  The Data 
Safety Monitoring Board (DSMB) may be unblinded for safety evaluations or dose reduction decisions 
related to safety. 
 
Additionally, preselected key personnel may have access to unblinded data to conduct pre-planned interim 
analysis for safety or for pre-planning activities for future clinical trials.  Related, an external, independent 
Steering Committee will be established by the Sponsor to review data from the interim analysis to provide 
guidance on future trial planning. 
 
Emergency unblinding for AEs may be performed through communication with the Investigational 
Pharmacy.  This option can ONLY be used if the participant’s well-being requires knowledge of the 
treatment assignment.  The principal investigator holds the sole responsibility for determination of 
unblinding is warranted for medical management of the event.  In such cases, the safety of the participant 
must always be the primary consideration for such determinations.  
 
If an emergency unblinding occurs, the investigator must promptly document the decision and rationale, 
and notify the Sponsor and DSMB at earliest opportunity. 
 
Decision to either discontinue the participant or continue with the study will rest with the Sponsor after 
consultation with the investigator and/or with the DSMB. 
 
A list of all those who are unblinded to study treatment as the study progresses will be maintained and 
stored with the final documentation of the study. 
 
7.4 
Dosage Modification 
Note: As the safety of the participant is paramount, the participants and their study partners will be told at 
the time of the informed consent that the study team will be monitoring their health and well-being through 
review of adverse events, laboratory results, and examinations performed during the trial.  To help minimize 
the possibility of any unblinding of participants or study partners within any of these scenarios listed below, 
all participants will be informed at their time of consent that they may be asked to do one of three things if 
they are having adverse effects or abnormal laboratory results including: stopping treatment; receiving new, 
replacement syringes; or continuing treatment with the same syringes.  Additionally, they will be told that 
the study team will continuously monitor their lab results, but they may not be told their specific results.  
Additionally, they will be informed that the trial physician or clinician designee will take the opportunity 
to examine them for clinical condition as a general safety measure if they are ever instructed to return to 
the CU-AMC site for an unscheduled visit.  
 
The greatest safety concern noted with the investigational product is excessive leukocytosis. As advised by 
the product safety instructions, a CBC with differential, including examination for the presence of blast 
cells, will be competed twice weekly during the 24-week-long treatment phase. Additionally the product 
insert states that “If the ANC exceeds 20,000 cells/mm3 or if the platelet count exceeds 500,000/mm3, 
LEUKINE administration should be interrupted or the dose reduced by half. The decision to reduce the 
dose or interrupt treatment should be based on the clinical condition of the patient.”  If a participant has 
CBC with differential results which indicate that their ANC has exceeded 20,000 cells/mm3, their white 
blood cell count has exceeded 50,000 cells/mm3, or their platelet count has exceeded 500,000/mm3, the 
participant and the study partner will be immediately contacted, asked whether the participant is 
experiencing any adverse health effects, AND instructed NOT to administer any additional study product 

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injections. Whether the participant is experiencing any adverse health effects or not, treatments will be 
interrupted until the following steps are completed and the Principal Investigator or qualified medical 
designee has determined whether to resume treatments at full dosage (178.57 µg/m2/day) or 50% reduced 
dosage (89.29 µg/m2/day): 
 
• 
If there are adverse health effects occurring, the participant and study partner will be instructed to 
immediately hang up and dial 911 if deemed an emergency or, if not deemed an emergency, to as 
quickly as possible return to the CU-AMC site to be examined by the trial physician or qualified 
clinician designee AND to bring all unused study product/syringes with them. (Note: At the time of 
initial contact with the participant and/or study partner, the trial personnel have the capability to 
immediately contact 911 emergency services if they believe the participant needs immediate 
attention.) 
 
OR 
 
• If there are not any adverse effects occurring (other than the blood value), the participant and study 
partner will be instructed to, as soon as possible, either return to the CU-AMC clinic or to a central 
contracted laboratory with phlebotomy services, whichever is quickest, to have an unscheduled blood 
draw performed “due to potentially abnormal or inconclusive lab results that need to be confirmed 
(exact phrasing may vary as deemed necessary by study personnel in their best attempts to prevent 
any un-blinding to the participant and study partner).”  At this unscheduled visit to the CU-AMC site 
or central contracted laboratory, a follow-up CBC with differential will be performed and results 
obtained as quickly as possible.  
• The ensuing steps will be followed pending results of this follow-up CBC with differential:  
 
o If the results indicate that the ANC levels are now less than 20,000 cells/mm3, white blood 
cell count is less than 50,000 cells/mm3, and the platelet count is less than 500,000/mm3, 
and if determined appropriate by the trial’s lead clinical sub-Investigator or delegated study 
clinician, the participant and study partner will be contacted and instructed to resume any 
additional study product injections and actions according to their regular daily scheduled 
times, NOT to administer any of the syringes that were missed during the days in which 
the unscheduled CBC with differential was performed and analyzed, if out of date, and to 
return any used AND unused syringes as normally done for each treatment week when they 
have their next study visit.  
 
OR 
 
o Although rarely expected based upon the experiences of the completed Leukine-AD trial 
(NCT01409915), the dosage may be reduced by half (i.e. 89.29 µg/m2/day subcutaneous 
injection) if the ANC exceeds 20,000 cells/mm3, the white blood cell count exceeds 50,000 
cells/mm3, or the platelet count exceeds 500,000/mm3 in the most recent CBC, and if the 
trial’s delegated sub-Investigator has determined, perhaps in consultation with a 
hematologist, oncologist or other qualified designee of the Sponsor, that the clinical 
condition of the participant warrants changing the dosage or continued interruption of 
treatment. If the results from the unscheduled follow-up CBC with differential has 
indicated that the ANC levels are still greater than 20,000 cells/mm3, and/or the white blood 
cell count is greater than 50,000 cells/mm3, and/or the platelet count is more than 
500,000/mm3, the participant and the study partner will be immediately contacted again 
and instructed not to administer any additional study product injections AND to bring all 
unused and used study product/syringes to the clinic as soon as possible, so that they can 

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be assessed for clinical condition by a trial physician or qualified clinician designee AND 
to then potentially receive new syringes of the study product, which will be provided by 
the Research Pharmacy (prescribed by the lead clinical sub-Investigator) using procedures 
designed specifically for these types of unusual circumstances.  In contacting and 
interacting with the study partner and participant for such a scenario, extreme care will be 
taken to not inadvertently unblind the participant, study partner, or any trial personnel. If 
unblinding is suspected, provisions described in prior section 7.3 will be followed and a 
decision will be made by the Sponsor regarding whether to discontinue the participant or 
to continue with the study.  
 
 
 
 
 

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In addition to these key blood values above, the responsible clinician will review the absolute eosinophil 
value, with a critical value, determined by the hematology specialist,  set at ≥1.5x10E3/uL as a potential 
adverse event/clinically significant lab value. Management of this value will be as ongoing AE until the 
value returns to < 1.5x10E3/uL and is evaluated by the responsible clinician as not clinically 
significant.  Once the AE is resolved, a subsequent occurrence of an absolute eosinophil ≥1.5x10E3/uL will 
be classified as a new AE.  For each new adverse event due to absolute eosinophil values, a symptom check 
will be conducted using guidelines developed by the hematology specialist.  If they are free of symptoms, 
injections will continue on the regular schedule.  If the participant is positive for any symptoms, they will 
be instructed to hold the injections pending further follow-up by the responsible sub-I.  Within each ongoing 
AE, symptom checks will occur each time the participant's absolute eosinophil count is above the previous 
high value for that AE.   
7.5 
Interruption of Treatment 
Temporary interruption of treatment is allowed if a short-term treatment with an excluded medication is 
necessary, secondary to hospitalization, personal circumstances, or to evaluate the study drug impact on an 
uncertain AE, including but not limited to abnormal CBC with differential results (see Section 7.4), Grade 
2 or higher AEs, and Grade 2 or higher abnormal laboratory values. Interruption of treatment should only 
be limited to significant events. If temporary interruption is due to an AE, it will be reported to the DSMB 
as part of the regularly scheduled aggregate reports. In such cases as above, reinitiation of treatment is at 
the trial physician(s) or qualified delegated clinician(s)  judgement. 
 
If a participant has to interrupt treatment for an extended period of time due to a significant medical 
condition or life event (such as greater than 7 dosing days off drug), it is possible to re-initiate treatment, 
and restarting the treatment will be at the trial physician’s discretion, though it is suggested that if deemed 
necessary, consultation with the Sponsor may be done. Treatment re-initiation should occur as soon as 
possible and has to be confirmed by trial physician. Re-initiation of treatment will include a general safety 
examination by qualified medical personnel and safety labs. Restarting treatment after an interruption 
period that is greater than 10% of the trial length (i.e. 18 consecutive days) will be discussed between 
sponsor and trial physician before a final decision is made to re-initiate treatment at the discretion of the 
Sponsor. All information relating to interruption of treatment and re-initiation will be documented. Missed 
doses from the above category will not be considered in determining non-compliance. 
 
7.5.1 
Interruption of Treatment not related to short term treatment with excluded medication, 
hospitalization, personal circumstance or AE. 
At the discretion of Principal Investigator, participants may request an interruption of treatment due to 
commitments such as travel obligations, for one block of time, up to 4 weeks in duration (28 dosage days).  
The study recording of interruption of treatment will allow for the addition of the time taken off to the study 
treatment period, up to the predetermined 28 dosage days. This interruption will not be factored into non-
compliance, and a safety examination by study clinician is not required prior to resuming treatment. 
 
7.6 
Treatment Compliance 
Compliance will be assessed at study visits via participant diary when the materials are returned at each 
weekly study visit at the clinic or by the traveling nurse if the weekly visit occurs at home.  When possible, 
clinic injections will be performed by caregivers with delegated study staff observation for confirmation of 
correct injection technique. Ongoing injection technique education will be provided as needed. Delegated 
study staff will perform clinic injections as needed at visits if caregivers are unable to administer the 
investigational product or placebo. Non-compliance is generally defined as a willful disregard for following 
the treatment plan by the participant or study partner).  
 
Only participants who use 90% (151 out of 168 administration days) of the prescribed daily dosage will be 
considered compliant (missed doses due to circumstances described in Sections 7.5 and 7.5.1 are not 
factored into treatment compliance).  Participants deemed non-compliant may be, at the discretion of the 

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investigator, in consultation with the Sponsor, discontinued from the study.  If a participant is deemed non-
compliant within the first 3 weeks of the trial, a discussion at the next in-person clinical visit or via phone 
prior to or during the next in-person visit with the PI or sub-I or other delegated study staff and retraining 
on treatment administration and compliance will occur to attempt to increase compliance. 
 
As stated above, reduction in dosage (i.e. reduction from 178.57 µg/m2/day to 89.29 µg/m2/day) OR doses 
withheld due to AEs or other factors relevant, as determined and approved by the PI, and Sponsor, will not 
be factored into non-compliance of a participant.   
 
7.7 
Concomitant Therapy 
All concomitant medications taken during the study are to be recorded on the concomitant therapy log 
maintained by the study personnel.  The participant and study partner will be asked at each weekly visit if 
there have changes to the concomitant medications, and if changes occur, they will be recorded and 
reviewed by the investigator.   
 
Participants and/or their study partner will be instructed to consult the investigator or other qualified study 
personnel before initiation of new medications or changes to existing medications or supplements. 
 
Use of approved standard of care medications for AD is permitted during the study with PI/Sponsor 
approval, as long as the duration meets the inclusion criteria, and there is no unapproved change through 
the double-blind treatment period. Participants and/or study partners are instructed to notify the study team 
as soon as they are aware of any medication changes.  If unplanned initiation, change or stopping of standard 
of care medication occurs prior to the EOT Visit without express approval for continued participation 
okayed by the PI prior to medication changes, the Sponsor should be consulted to determine if the 
participant should continue in the study. 
 
See Appendix H for list of prohibited concomitant therapies. 
 
 
 

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8  Study Assessments and Procedures 
 
The Schedule of Events (Section 2) and Section 5 outline the study procedures and timing. 
 
8.0 
Screening Assessments (from Tier .5) 
The Cognitive Health Review Information System Batter is Battery is designed around standardized 
diagnostic criteria and practice guidelines to identify common mimics of Alzheimer’s disease, some of 
which can co-exist with Alzheimer’s disease pathology.  Questionnaires may include: 
 
FAQ (Functional Activities Questionnaire)- Will be included 
The FAQ is a well validated instrument used in both clinical and research settings to measure instrumental 
activities of daily living (iADL), providing useful information in determination of level of ADLs to help 
qualified professionals stage impairment related to dementia. It has been widely used as part of the National 
Alzheimer’s Coordinating Center (NACC) longitudinal research initiatives.  
 
DSRS (Dementia Severity Rating Scale) – Will be included 
The DSRS is a functional evaluation tool that is a commonly used instrument in staging dementia, and 
utilizes a semi-structured interview format to collect information about an individual’s ability to function 
in various domains, allowing a trained clinician stage the current severity of impairment displayed by an 
individual.  
 
ECog (Everyday Assessment of Cognition) – At study physician discretion 
The ECog is an carefully validated assessment of cognition filled out by a care partner that reflects basic 
mental functioning, and is widely used within clinical practice. 
 
NPI Q (Neuropsychiatric Symptoms Questionnaire) - At study physician discretion 
The NPI-Q is a study partner/caregiver self administered questionnaire to assess any changes in 
neuropsychiatric status in such domains as hallucinations, delusions, agitation, depression, anxiety, 
disinhibition, apathy and aberrant motor behaviors. 
 
DCFS (Dementia Cognitive Fluctuation Scale) - At study physician discretion 
The DCFS is a scale developed to distinguish levels of cognitive fluctuation among patients with 
dementia, and help provide distinguishing characteristics between various types of dementia.  It is widely 
used in clinical trials and clinical practice. 
 
Early screen from the Columbia Suicidality Scale - At study physician discretion 
The early screen from the Columbia Suicidality scale is a study partner answered questionnaire about past 
and current suicidal ideation and behaviors. It is a validated and reliable scale that is widely used across 
clinical practice and clinical research trials. 
 
ESS (Epworth Sleepiness Scale) - At study physician discretion 
The ESS is a wall validated and widely used instrument across clinical practice and clinical research to 
identify issues with sleep and daytime sleepiness that often is apparent within dementia populations. 
 
Lewy Body Composite Risk Score - At study physician discretion 
The Lewy Body Composite Risk score is a 10 item questionnaire designed to capture signs and symptoms 
associated with Lewy Body pathology, and help distinguish from other forms of dementia.  It is widely 
used across clinical practice and clinical trials. 
 
 
RSMS-Ex (Revised Self-Monitoring Scale – Expressive Score) - At study physician discretion 

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The RSMS-Ex is a scale developed to detect socioemotional deficits in functioning, and effectively 
distinguish neurodegenerative diseases from psychiatric expressions that might be similar.  It is widely 
used in clinical practice to aid in the diagnosis of such diseases. 
 
Depending on study physician discretion, and potential answers given, additional questionnaires may 
include the full RSMS, Behavior Inhibition Scale, Interpersonal Reactivity Index, and Unified Parkinson's 
Disease Rating Scale–II (UPDRS-II)  for diagnostic clarification, all instruments used within both clinical 
practice and clinical trials to gain more information regarding cognitive and functional abilities.  The 
questionnaires are provided on a secure Qualtrics system, or RedCap (with the option for paper and pen if 
potential participant does not have access to a computer or internet access, and answers provided by 
caregivers. The questionnaires take, at maximum, approximately one hour to fill out.  
 
8.1 
Efficacy Assessments 
Cognitive and clinical testing for each participant should be performed at approximately the same time on 
each assessment day to reduce potential variability.  As previously noted, the CDR and the ADCS-ADL 
should be administered by a different rater than the cognitive scales (i.e. MoCA, MMSE, ADAS-Cog, TMT 
A, tests of verbal fluency), and all attempts made to maintain these two raters administering the same scales 
with the same participant throughout the study.  The principal investigator holds the responsibilities for 
selection of the raters who will administer the scales, and verifying that they have met all training 
requirements. 
 
On visits when administered, the cognitive and clinical assessments should be completed before medical 
procedures that may be stressful to the participant (for example blood draws, ECG), and that some 
procedures (such as MRI) can be conducted on other days within the visit window, if necessary. 
 
MMSE (Mini-Mental State Exam)  
The Folstein Mini-Mental State Exam is a brief psychometric instrument developed to assess cognitive 
function in elderly populations.  It is a standard assessment used by all NIH Alzheimer’s Disease Centers 
(ADCCs and ADRCs) to identify and monitor individuals with AD.  The range for scores in the MMSE is 
from 0 to 30, with lower scores indicating greater impairment. 
 
MoCA (Montreal Cognitive Assessment) 
The MoCA is a brief, assessment developed for detection and tracking of cognitive impairment and is 
sensitive for detecting Alzheimer’s disease. Measuring multiple domains, it is commonly used in both 
clinical and research settings, and is well validated, with a range of scores from 0-30. 
 
ADAS-Cog13 (Alzheimer’s Disease Assessment Scale-cognitive subscale)  
ADAS was designed to measure the severity of the most important symptoms of AD. Its subscale ADAS‐
Cog13 is the most popular cognitive testing instrument used in clinical trials of nootropics (drugs or agents 
that improve cognitive function). It consists of 13 tasks measuring the disturbances of memory, language, 
praxis, attention and other cognitive abilities, which are often referred to as the core symptoms of AD.  
 
ADCS-ADL (Alzheimer’s Disease Cooperative Study Activities of Daily Living Inventory)  
ADCS-ADL is a caregiver/study partner rated questionnaire of 23 items, with possible scores over a range 
of 0–78, where 78 implies full functioning with no impairment. The ADCS-ADL assesses functional 
capacity across a wide spectrum of severity and will be the primary tool for collecting ADL data. 
 
TRAIL MAKING TEST-A  
Psychomotor speed will be assessed by the Trail Making Test-A, a timed test in which participants must 
connect a series of numbers randomly placed on a page. 
 

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CDR (Clinical Dementia Rating) 
The CDR is a study partner/caregiver and participant based interview to assess changes in domains such as 
memory, orientation, judgment and problem solving, community affairs, home and hobbies, and personal 
care. Each domain is rated as 0 (no dementia), 0.5 (uncertain dementia), 1 (mild dementia), 2 (moderate 
dementia), or 3 (severe dementia). Further, the Sum of Boxes score (CDR-SB) shall be tallied for each 
administration of the instrument All raters in this trial will have completed the Washington University 
Alzheimer’s Disease Research Center CDR Training. 
 
Tests of Verbal Fluency 
Tests of verbal fluency test are assessments where the participant is asked to produce, as fast as they can, 
all the words that can think of in 60 seconds.  The two areas are Semantic fluency (producing words that 
belong in a category, such as animals, also known as category fluency) and Lexical fluency (producing 
words that start with a specific letter of the alphabet, also known as letter fluency).  The more correct words 
that a participant produces, the higher the score. 
 
Neuropsychiatric Inventory (NPI) 
The NPI is a study partner/caregiver interview to assess any changes in neuropsychiatric status in such 
domains as hallucinations, delusions, agitation, depression, anxiety, disinhibition, apathy and aberrant 
motor behaviors.  
 
Sleep Quality Assessments 
Changes in quality of sleep have been well documented in onset and progression of AD 84-86. The Pittsburgh 
Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI), will be administered to both the participants 
and their partner/caregiver at Baseline, Week 12, EOT, and Follow-up (45 days post-treatment) visits to 
assess measures of sleep quality and any changes that may occur throughout the duration of the trial. 
 
 
8.1.1  Functional Ability Assessments (if needed by study clinician for diagnostic clarity) 
Functional Activities Questionnaire (FAQ) 
The FAQ is a well validated instrument used in both clinical and research settings to measure instrumental 
activities of daily living (iADL), providing useful information in determination of level of ADLs to help 
qualified professionals stage impairment related to dementia. It has been widely used as part of the National 
Alzheimer’s Coordinating Center (NACC) longitudinal research initiatives.  
 
Dementia Severity Rating Scale (DSRS) 
The DSRS is a functional evaluation tool that is a commonly used instrument in staging dementia, and 
utilizes a semi-structured interview format to collect information about an individual’s ability to function 
in various domains, allowing a trained clinician stage the current severity of impairment displayed by an 
individual.  
 
8.2 
LABORATORY ASSESSMENTS: 
During the Screening Visit, and at in-person (clinic or at home) research weekly visits (Baseline through 
Visit 24), phlebotomy will be performed by trained personnel of the UCH CTRC or other qualified study 
personnel, such as a contracted nurse. These qualified personnel may perform additional blood draws, if 
necessary, at unscheduled visits.   
 
During the double-blind treatment period of the study, a second weekly blood draw / phlebotomy will be 
performed at a local central contracted laboratory site with phlebotomy services by trained staff.  If 
preferred by the participant, the second weekly blood draw can be scheduled for the main study site (i.e. 
CU-AMC).  
 

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Drawing blood may cause mild pain at the site of needle insertion and occasionally local ecchymosis 
formation. With phlebotomy, rarely people may experience vasovagal reactions that are typically limited 
and resolve spontaneously. The total amount of blood (cumulative amount for research and clinical 
procedures) drawn will not exceed five percent of estimated total blood volume over a 24-hour time period. 
 
Complete blood count (CBC) with differential and complete metabolic panel (CMP):  
These labs will be processed by the central contracted laboratory (see Manual of Procedures). Blood 
samples will be delivered to a local contracted central laboratory drop –off location by designated staff 
personnel within an acceptable time frame to maintain viability of sample. The CBC and CMP results will 
be reported by the contracted central laboratory to the Study Coordinator or qualified designee and 
presented to qualified medical designee to evaluation.  
 
Lumbar puncture  
A lumbar puncture (LP or “spinal tap”), in which 25 to 30 ml (approximately 2 tablespoons) of spinal fluid 
is removed, will be performed for either entry criteria or as an optional sub-study. The LP procedure will 
use an atraumatic needle that is designed to reduce the risk of post-dural-puncture headache. The CSF will 
be used to measure a number of biomarkers, including amyloid and tau protein, which are markers for 
Alzheimer’s disease (AD) progression, as well as for measuring albumin levels in correlation with blood 
serum albumin levels, in order to assess AD-associated changes in blood brain barrier permeability. The 
procedure will involve the insertion of a very thin needle into the participant’s lower back in order to reach 
the spinal canal. The procedure will be done by a licensed medical provider and under sterile conditions 
using local anesthetic in the area in which the needle is inserted. The participant will rest for at least one 
half \ hour after this procedure. Each participant will be screened by history, and if there is suspicion of 
increased risk associated with the LP, additional assessment will be done before the procedure. Individuals 
on blood thinners including but not limited to: warfarin (Coumadin), rivaroxaban (Xarelto), apixaban 
(Eliquis), and dabigatran (Pradaxa) will be excluded from this procedure, and excluded from study entry. 
Those taking PRN regimens of ibuprofen, or other NSAIDs will be considered if they agree to stop said 
regiment for 7 days prior to the procedure. For those taking regimens of daily 81 mg aspirin, the dose should 
be held for 24 hours prior to the procedure.  Additionally, individuals with history of spinal fusions (most 
often in the lumbar region) or other major back procedures will be excluded from the procedure unless 
imaging can be provided demonstrating that their anatomy will not increase the chances of an unsuccessful 
LP.  
 
The following risks exist related to lumbar puncture: pain, bleeding, infection and headache. To limit 
discomfort, the skin, surrounding tissue and underlying muscle will be numbed with lidocaine. During the 
LP, participants may experience brief pain lasting seconds in the lower back during insertion of the 
needle, especially if the needle contacts bone. This occurs about 1 in 5 times. Less than 1 in 10 patients 
experience a brief pain shooting down the leg if the LP needle touches a nerve root. Less than 1 in 100 
patients are allergic to the iodine solution used to clean their back and will develop rash. After LP, 5 to 15 
out of 100 individuals develop a headache that is worse when standing and disappears when lying down. 
It typically begins 6-48 hours after the LP, and may last 1 to 6 days. The research team will instruct the 
participants to lie flat and take caffeine (via coffee consumption or via prescription caffeine sodium 
benzoate, according to PI or qualified designee discretion) if by chance this does occur.  LP induced 
headaches may also be treated with oral hydration, analgesics such as acetaminophen, or ibuprofen and 
caffeine.  
 
If a participant’s headache persists for more than 7 days, an epidural blood patch may be advised for 
relief. Post-lumbar puncture headache is thought to be due to continued leakage of spinal fluid out of a 
hole made by the spinal needle in the membrane sac that holds the spinal fluid. An epidural blood patch 
involves injection of some of the patient’s own blood in the area of the spinal puncture, with the local 
blood clot then ‘plugging’ this hole. This procedure is effective in eliminating the headache in about 90 

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percent of people with post spinal tap headache. The cost of this procedure would not be covered by the 
study.   
 
Sharing of optional LP Information with Participant and/or Providers: 
The study team does not plan to release (optional) LP results to study participants, as they are used for study 
analysis, not diagnostic or entry criteria. 
 
8.3 
Biomarkers 
Whole blood and CSF samples will be received in the CU-AMC Neurology laboratory under the 
administration of the Sponsor, Huntington Potter, PhD, and processed as quickly as possible to maintain 
viability of the samples using SOPs that were developed to conform to NACC guidelines for processing 
Alzheimer’s disease biospecimens. CSF samples will be centrifuged, any leukocyte pellets will be 
cryopreserved, and the CSF will be aliquoted into storage tubes. The whole blood samples that are collected 
in EDTA vacutainer tubes will be processed for storage aliquots of plasma, cryopreserved buffy coat, and 
erythrocytes, whereas whole blood samples collected in serum separator tubes (SSTs) will be processed 
only for aliquots of serum. All sample aliquots will be immediately placed for storage within -80ºC freezers 
or in liquid nitrogen storage within the University of Colorado Alzheimer’s and Cognition Center 
(CUACC)’s human biorepository until samples are needed for testing. Testing of these biomarkers will 
occur using core facilities at CU-AMC (i.e. HIMSR and/or the Neurology Translational Core), and 
biomarker results will be analyzed by the trial’s biostatistician, who has no direct contact with trial 
participants, and who will receive the treatment code from the UCH pharmacist. However, biomarker 
evaluation will only occur after completion of the trial or at predetermined interim analysis time-points, of 
which results would only be disclosed for select individuals, for example in pursuit of grants or other 
funding opportunities. 
 
8.3.1 
Serum Anti-Drug Antibody (ADA) Testing: Anti-GM-CSF Antibody  
Serum samples collected from blood draws in visits at baseline, weeks 6, 12, 18, EOT, and follow-up will 
be screened using a bridging electrochemiluminescence assay for anti-sargramostim antibodies by a 
dedicated research laboratory associated with Partner Therapeutics. Each participant’s baseline ADA values 
will be determined from serum that was obtained at their baseline time point blood draw. Any treatment-
related ADA response (positive, negative, or inconclusive) will be assessed by comparing ADA values 
from the treatment phase visits and the follow-up visit to baseline values. All individual data, including 
rechecked values, will be recorded. Serum samples will be shipped in batches to the designated laboratory 
after a participant has completed all study visits. 
 
On visit days that the participants receive their test article injection, the participants will be monitored by 
the study physician(s), or other clinicians or qualified study personnel after the test article injection for any 
symptoms of acute anaphylaxis reaction. Each participant’s study partner will be trained for symptoms of 
anaphylaxis and for immediate emergency procedures that they must perform if anaphylaxis occurs, for the 
days in which the study partners are administering the test article to the participants at home.  However, 
anaphylaxis reactions or any other SAEs due to ADA responses are not expected. In a recent clinical trial 
within participants having Parkinson’s disease, full FDA-approved dosage of sargramostim was 
administered to participants for 56 consecutive days (52; attached in Appendix G) The authors stated that 
“Serum anti-sargramostim antibodies were detected in the drug group by week 4 of treatment (visit 5), but 
diminished by week 8 (visit 7). Antibody levels were marginal at 4 weeks after drug cessation,” and no 
participants had to discontinue the trial due to ADA response.   
 
8.3.2 
MRI  
Atrophy in the entorhinal cortex (ERC), parahippocampal gyrus, and hippocampus (HPC), measured on 
MRI scans, predicts future cognitive decline and conversion among individuals with mild cognitive 
impairment (MCI) to AD 110, 111. Severity of MTA, assessed with MRI scans, is strongly associated with 

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severity of medial temporal lobe degenerative pathology, especially the severity of neurofibrillary 
pathology, at autopsy 112. Because of the high prevalence of AD in the elderly, 85–90% of all degenerative 
pathology in the medial temporal lobe, either alone or in combination with other diseases, is AD pathology 
113. 
 
An automated rating system to grade the severity of atrophy in the entire medial temporal region (MTA) 
will be employed using the FreeSurfer software automated rating system of individual medial temporal 
structures, i.e., the HPC, ERC, parahippocampal gyrus and perirhinal cortex (PRC), which are specific 
regions of interest (ROIs) in AD pathogenesis. It has been shown that these individual and summed MTA 
scores distinguish AD and acute MCI individuals from normal elderly control individuals and predict 
progression from acute MCI to dementia/AD 114, 115.  
 
Numerous neuroimaging studies have shown that white matter (WM) tracts are associated with cognitive 
function, and that microstructural abnormalities are found in AD and other neurodegenerative diseases, as 
determined from high-resolution MRI diffusion tensor imaging (DTI) techniques 73-77. DTI metrics have 
been proposed as sensitive biomarkers for disease progression over short periods of time 78, which makes 
it useful for assessing any WM changes in response to treatments, and specific DTI patterns have been 
shown to help distinguish between AD and other comorbid neurodegenerative diseases 79, 80, to correlate 
with AD-related verbal fluency impairments 81, and with using neurite orientation dispersion and density 
imaging (NODDI) sequences of DTI, changes in neurite density can be approximated 82, 83. Because GM-
CSF has been shown to be a growth factor for oligodendrocytes 116, induce oligodendrocyte progenitor cells 
concurrent with amelioration of experimental spinal cord injury 34, 117, and increase synaptic area concurrent 
with reversing cognitive impairment and pathology in transgenic AD models 1, 68, high-resolution MRI with 
DTI and NODDI sequences will be performed at screening, weeks 6, 12, 18, EOT, and among participants 
of the optional amyloid PET procedure, at follow-up (45 days post-treatment) and metrics of DTI (e.g. FA, 
MD, NDI, ODI, etc.) will be monitored for any treatment-related changes. 
 
Melatonin and its signaling have been shown to be decreased in AD progression 88-92, and GM-CSF has 
also been reported to increase melatonin secretion in preclinical studies 93. Melatonin is made and secreted 
by the pineal gland, and calcification of the pineal gland increases with AD progression 94-97. High-
resolution MRI scans, using susceptibility weighted imaging (SWI) sequence, will be performed at 
screening and EOT to assess changes in pineal calcification measurements. 
 
Brain MRI scans will be obtained on a 3.0-tesla MRI machine using proprietary three-dimensional 
magnetization-prepared rapid-acquisition gradient echo or the three-dimensional spoiled gradient recalled 
echo sequences; MRI scans will be acquired with isotropic resolution, and contiguous slices with thickness 
of 1.5 mm or less will be reconstructed. Multiple MRI scans without contrast are safe as they do not involve 
exposure to radiation. Some individuals may have difficulty lying motionless during the scan or may 
experience anxiety while in the scanner. These issues can be mitigated by administering a sedative, 
prescribed by the study physician(s), prior to scanning. If necessary for the procedure, prescription/use of 
the prescription (e.g. lorazepam) will be documented by the study team, and the study will reimburse the 
participant for the cost of the prescription. If such a prescribed drug is used in the conduct of an MRI, the 
participant will need a driver (friend or family) to provide transportation for the visit.  As well, any risks 
associated with the administration or use will be described to the study participant. 
 
Brain MRI scans will be performed in Building 400 in the BIC or in Biosciences 3 on the CU-AMC campus, 
which is part of the AMC’s comprehensive CU-Research Imaging Center. A separate Radiologist, blinded 
to participant identity and test article group, will examine each MRI scan for any evidence of ARIAs (i.e. 
vasogenic edema and microhemorrhage), and results, including any incidental findings, will be reported to 
the Study Coordinator and Study Physician. Another Radiologist or designate, also blinded to participant 
identity and test article group, will perform the FreeSurfer analyses.  

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Brain MRIs will be conducted at the Screening Visit, and Visit 6, 12, 18, EOT and, for individuals 
participating in the optional amyloid PET, at Follow-up. 
 
8.3.3 
PET  
Fluorodeoxyglucose F18 (FDG)  
The FDG PET procedure will utilize the CU-RIC research PET scanner for human imaging studies. The 
Philips Gemini 64TF PET/CT imaging system is located in Biosciences 3 on the Anschutz Medical Campus 
at the University of Colorado. An additional GE Signa PET/MR imaging system is also located within the 
CU-RIC for use as necessary.  
 
Fluorodeoxyglucose F18 (FDG) is an FDA approved radiotracer that has been used extensively to evaluate 
glucose metabolism within many organs within the body, and in relation to the brain, FDG PET has been 
at the forefront of functional neuroimaging over the past 3 decades, used to measure local cerebral metabolic 
rate of glucose that is indicative of synaptic activity in the brain, and contributing to the understanding of 
cognitive functions in healthy humans and how these functional patterns change with cognitive alterations. 
FDG has also been approved by the FDA for the identification of regions in human brain of abnormal 
glucose metabolism associated with foci of epileptic seizures, as well as being used as a useful biomarker 
of the regional reductions in glucose metabolism associated with the different forms of dementia. 
Evaluation of regional brain metabolism with FDG, might also prove useful or necessary to make a 
diagnosis, particularly in difficult or atypical cases. Perhaps FDG’s best use may be in combination with 
other neuroimaging modalities, such as with structural MRI (sMRI) analyses and in relation to AD, to help 
predict conversion of mild cognitive impairment (MCI) to AD. The FDG PET scans will be performed at 
baseline and at EOT Visit. 
 
FDG PET Imaging Procedure 
Prior to the visit, participants will be asked to abstain from food or drinks for 4 hours before the procedure. 
Each study visit will take approximately 2 hours. A 5-10 mCi intravenous injection of fluorodeoxyglucose 
F18 will be administered. After the fluorodeoxyglucose F18 dose is injected, a 3-5 minute head CT scan 
will be performed to aid in alignment and calibration, and adjust for attenuation effects. Dynamic images 
will be acquired for approximately 20-30 minutes. The IV catheter will be removed at the conclusion of the 
scan and materials will be disposed of in biohazard containers or decay chambers until they can be safely 
disposed.  
 
Indications and Usage 
FDG is a PET radiopharmaceutical that was first administered to human normal volunteers in 1976, and 
that has since been used in oncology imaging since the 1980s, in order to determine abnormal glucose 
metabolism and to assist in evaluating malignancy in patients with abnormalities found by other testing 
modalities or in patients with an existing diagnosis of cancer. FDG is indicated in PET for diagnostic use 
in patients for the evaluation of pulmonary nodules to distinguish benign from malignant and the evaluation 
of non-small cell and small cell lung cancers for staging and restaging and for the evaluation of colorectal 
cancer for recurrence, restaging, and distant metastases. For lung cancer evaluation, certain thoracic area 
non-cancerous lesions may show FDG uptake including acute and chronic infections (such as abscesses, 
tuberculosis and histoplasmosis), and inflammatory / granulomatous conditions (such as sarcoidosis, 
bronchiectasis, or post radiotherapy sites) that could mimic tumor accumulation. Absent or less intense 
relative uptake of FDG may be observed in specific lesions including bronchoalveolar, mucinous and 
lobular carcinoma as well as carcinoid and fibroadenoma sites. For colorectal cancer evaluation, certain 
abdominal / pelvic area non-cancerous lesions may show FDG uptake including sites of post radiation or 
post-surgical inflammatory response, lesion site flare following chemotherapy, colonic adenomas and 
bladder diverticula that could mimic tumor accumulation. Absent or less intense relative uptake of FDG 
may be observed in specific lesions including mucinous carcinoma. Lesion size may also affect detectability 

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based on relative FDG accumulation and PET imaging system resolution, as it has been shown that FDG 
PET imaging may have a lower sensitivity in evaluating lesion sizes of less than 1 cm.  
 
Mechanism of Action 
Fluorodeoxyglucose F 18 is a glucose analog that concentrates in cells that rely upon glucose as an energy 
source, or in cells whose dependence on glucose increases under pathophysiological conditions. 
Fluorodeoxyglucose F 18 is transported through the cell membrane by facilitative glucose transporter 
proteins and is phosphorylated within the cell to [18F] FDG-6-phosphate by the enzyme hexokinase. Once 
phosphorylated it cannot exit until it is dephosphorylated by glucose-6-phosphatase. Therefore, within a 
given tissue or pathophysiological process, the retention and clearance of Fluorodeoxyglucose F 18 reflect 
a balance involving glucose transporter, hexokinase and glucose-6-phosphatase activities. When allowance 
is made for the kinetic differences between glucose and Fluorodeoxyglucose F 18 transport and 
phosphorylation (expressed as the ''lumped constant'' ratio), Fluorodeoxyglucose F 18 is used to assess 
glucose metabolism. In comparison to background activity of the specific organ or tissue type, regions of 
decreased or absent uptake of Fluorodeoxyglucose F 18 reflect the decrease or absence of glucose 
metabolism. Regions of increased uptake of Fluorodeoxyglucose F 18 reflect greater than normal rates of 
glucose metabolism. 
 
Radiation Exposure 
Any exposure to radiation carries a very small risk of causing damage to tissues and the possibility of 
triggering a new cancer. However, the amount of radiation that each participant will be exposed to per 
(Fluorodeoxyglucose F18) PET scan is similar to the amount received from natural sources, such as the sun 
(background radiation) over the course of living in Denver for approximately 1.5 years. The FDG PET 
imaging will be performed at the Baseline visit and at EOT Visit. The administered dose of 
(Fluorodeoxyglucose F18) will be 5-10 mCi. For a (Fluorodeoxyglucose F18) dose of 10-15 mCi (185-370 
MBq), the expected whole body effective dose is 6.3 mSv. In addition, the effective dose for a low dose 
head CT performed for localization and attenuation correction as part of the standard brain PET/CT protocol 
is 0.6 mSv. Thus, it is expected the trial participants will be exposed to about 7 mSv per fluorodeoxyglucose 
F18 scan, and when added to expected radiation exposure from flutemetamol F18 (Vizamyl, Optional), will 
remain well below the 50 mSv maximum annual dose of radiation exposure. On the pre-screening medical 
history form, participants and/or the study partner/caregiver will be asked to provide information whether 
the participant has been exposed to radiation during the past year (e.g. prior X-ray, CT, PET scans, etc.), so 
that the Study Physician and/or the Nuclear Medicine Imaging Professional can determine whether there 
will be a risk of the participant exceeding maximum allowed levels of radiation exposure.  
 
Amyloid PET  
The amyloid PET scan, for either entry criteria or as an optional sub-study, will use a common radiotracer 
(such as flutemetamol F18 (Vizamyl) which is a commonly used radiotracer that binds to β-amyloid plaques 
in the brain, as a biological marker of Alzheimer’s disease.  For evidence of β-amyloid neuritic plaques, if 
a participant opts to take part in the sub-study, they will receive a screening \ PET scan before the baseline 
visit, and a follow-up scan at the post-treatment follow-up visit, that will be assessed by a Nuclear Medicine 
Radiologist within the University of Colorado School of Medicine. The Nuclear Medicine Radiologist will 
remain blinded to participant identity and randomized test article group, and qualitative results, including 
any incidental findings, will reported to the Study Coordinator and Study Physician or qualified designee. 
If the PET scan is performed on the same day as the MRI scan, the PET scan will be performed after the 
MRI scan. The PET scans will be performed at an outpatient visit at the CU-RIC at CU-AMC. For 
quantification of the brain amyloid load, assessments will evaluate the screening PET scan before the 
baseline visit and another PET scan within +/- 7 days of the Follow-Up Visit approximately 45-days after 
the end of treatment with sargramostim or placebo. The change from the screening PET scan’s SUVR to 
follow-up SUVR will be analyzed by a qualified nuclear medicine radiologist within the University of 
Colorado Hospital - Department of Radiology or by a contracted central reading facility (i.e., BioClinica, a 

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specialty clinical trial services provider) using the participant's MRI to reduce white matter contamination, 
thereby increasing the SUVR signal change by eliminating counts in the region that do not change (white 
matter). The amyloid PET scans and accompanying MRI scans will be stripped of any personal identifying 
information, containing only the trial’s randomized ID number and date. The results of the PET quantitative 
analyses will not be used to make any treatment decisions. 
 
Amyloid  PET Imaging  
The amyloid PET procedure will utilize the CU-RIC research PET scanner for human imaging studies. The 
Philips Gemini 64TF imaging system is located in Biosciences 3 building on the Anschutz Medical Campus 
at the University of Colorado.  
 
Each study amyloid PET will take approximately 2 hours. A preliminary, 3-5 minute head CT scan will 
precede the administration of the radiotracer to aid in alignment and calibration, and adjust for attenuation 
effects. Immediately following, the participant will be comfortably positioned in a chair. An intravenous 
catheter will be inserted using standard aseptic technique. A 5 mCi (Vizamyl) intravenous injection will be 
administered. The participant will be placed on the scanning table and moved into the scanner. Dynamic 
images will be acquired for approximately 70 minutes. The IV catheter will be removed at the conclusion 
of the study and materials will be disposed of in biohazard containers or decay chambers until they can be 
safely disposed.  
 
Indications and Usage 
Vizamyl (flutemetamol F18) is a radioactive imaging agent for Positron Emission Tomography (PET) 
imaging of the brain to estimate β-amyloid neuritic plaque density in adult patients with cognitive 
impairment who are being evaluated for Alzheimer’s Disease (AD) and other causes of cognitive decline. 
A negative amyloid scan indicates sparse to no neuritic plaques, and is inconsistent with a neuropathological 
diagnosis of AD at the time of image acquisition; a negative scan result reduces the likelihood that a 
patient’s cognitive impairment is due to AD. A positive amyloid scan indicates moderate to frequent 
amyloid neuritic plaques; neuropathological examination has shown this amount of amyloid neuritic plaque 
is present in patients with AD, but may also be present in patients with other types of neurologic conditions 
as well as older people with normal cognition. The use radiotracers such as Vizamyl has become a widely 
accepted amyloid imaging method in human subject research. Vizamyl received initial U.S. approval for 
use in research in 2013 and is in wide using in clinical trials and clinical practice. 
 
Mechanism of Action 
The radiotracer Vizamyl (flutemetamol F18) is a F18 -labeled positron emission tomography (PET) imaging 
agent that binds with high affinity to the amyloid-β (Aβ) peptide fibrils that constitute amyloid plaques, and 
maps on to amyloid deposition in post-mortem studies 119. Vizamyl contains flutemetamol F 18, a molecular 
imaging agent that binds to β-amyloid aggregates and is intended for use with PET imaging of the brain. 
Chemically, flutemetamol F 18, is described as 2-[3-[18F]fluoro-4-(methylamino) phenyl]-6-
benzothiazolol. It has the molecular formula C H 18FN OS.  . 
 
Radiation Exposure 
Any exposure to radiation carries a very small risk of causing damage to tissues and the possibility of 
triggering a new cancer. However, the amount of radiation that each participant will be exposed to per  PET 
scan is similar to the amount received from natural sources, such as the sun (background radiation) over the 
course of living in Denver for approximately 1-2 years for Vizamyl. The optional amyloid PET imaging 
will occur at the Screening Visit and at the Follow-Up visit, or within a week thereof, depending upon 
scheduling availability. Administered dose of Vizamyl will be 5 mCi. For a Vizamyl dose of 5 mCi (185 
MBq), the expected whole body effective dose is 5.92 mSv. In addition, the effective dose for a low dose 
head CT performed for localization and attenuation correction as part of the standard brain PET/CT protocol 
is 0.6 mSv. Thus, with two  scans, it is expected the trial participants to be exposed to about 13 mSv 

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(Vizamyl) and when added to expected radiation exposure from FDG-PET, will remain well below the 50 
mSv maximum annual dose of radiation exposure. On the pre-screening medical history form, participants 
and/or the participant’s study partner/caregiver will be asked to provide information whether the participant 
has been exposed to radiation during the past year (e.g. prior X-ray, CT, PET scans, etc.), so that the Study 
Physician and/or the Nuclear Medicine Imaging Professional can determine whether there will be a risk of 
the participant exceeding maximum allowed levels of radiation exposure.  
 
PET Safety Monitoring 
All participants will be monitored carefully from injection until the conclusion of the imaging study. The 
PI and/or qualified designee will be available to consult if any questions arise during the PET scan. The 
research coordinator and Nuclear Medicine Technologist will be present through the entire duration of the 
PET scan, consistent with CU-RIC PET center guidelines. Each study participant will be contacted by 
phone approximately 2 (+/- 1) business days after they were injected with the specific PET ligand to confirm 
their well-being and query them about any new adverse events. If any AE’s are present they will be 
reviewed by the PI. 
 
Sharing of optional Amyloid PET Information with Participant and/or Providers: 
The study team does not plan to release (optional) amyloid scan results to study participants, as they are 
used for study analysis, not diagnostic or entry criteria. 
 
8.4 
Safety Monitoring 
8.4.1 
Physical and Neurological Examinations 
Physical and neurological examinations will be performed at the Screening Visit, Baseline Visit, Visit 12, 
EOT Visit, and the Follow-up Visit. 
 
8.4.2 
Electrocardiograms (ECG) 
For each participant, an ECG will be conducted at the Screening Visit, Visit 12 and EOT Visit.  The ECG 
will be conducted by qualified study personnel, and be interpreted by a qualified physician or other 
designated qualified medical personnel as soon as possible after the administration. 
 
8.4.3 
Vital Signs 
Vital signs including blood pressure, heart rate, temperature and respirations will be measured at all at-
home and in-clinic visits.  Blood pressure and pulse should be measured with participant in sitting position 
for at least 5 minutes. Height will be measured at screening.  Body weight will also be measured according 
to the schedule of events table in protocol section 2. 
 
8.4.4 
Additional Safety Assessments 
C-SSRS 
Consistent with FDA regulatory guidance, any occurrence of suicide related thoughts or behaviors will be 
assessed per the Schedule of Events (Section 2).  The C-SSRS is an instrument that collects the 
occurrence, severity and frequency of suicide related thoughts or behaviors.  The “Baseline” version will 
be used for the initial screening, and the “Since Last Visit” when assessed shall be used at following 
visits, when scheduled for administration. 
 
Geriatric Depression Scale (GDS) 
The GDS is a simple 30-item measure used to identify depression in elderly individuals. The simplicity of 
the GDS (questions are answered only by “yes” or “no”) enables the scale to be used with ill or moderately 
cognitively impaired individuals. One point is assigned to each answer and the cumulative score is rated on 
a scoring grid. The grid sets a range of 0 to 9 as ‘normal’, 10 to 19 as ‘mildly depressed’, and 20 to 30 as 
‘severely depressed’.  
 

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Generalized Anxiety Disorder (GAD)-7 
The GAD-7 is a simple seven item questionnaire used to identify anxiety levels in individuals. The 
GAD-7 is calculated by assigning scores of 0, 1, 2, and 3, to the response categories of “not at all,”  
“several days,” “more than half the days,” and “nearly every day,” respectively. GAD-7 total score for the 
seven items ranges from 0 to 21. Scores of 5, 10, and 15 represent cut-points for mild, moderate, and 
severe anxiety, respectively. Though designed primarily as a screening and severity measure for 
generalized anxiety disorder, the GAD-7 also has moderately good operating characteristics for three 
other common anxiety disorders: panic disorder, social anxiety disorder, and post-traumatic stress 
disorder. 
 
8.4.5 
Adverse Event(s), Serious Adverse Events and Monitoring 
The principal investigator is responsible for monitoring the safety of participants in the trial, and, with 
medically qualified sub-Investigators, will oversee and document any event that seems unusual, and 
document the review of each laboratory safety report.  The investigators are responsible for the follow-up 
for all AEs that are considered serious or otherwise medically important, considered related to the 
investigational product or procedures in the trial, or that caused the participant to discontinue the 
investigational product before the recognized end of study treatment.  The frequency of follow-up 
evaluations is at the discretion of the investigator.   
 
After the informed consent is signed, designated study staff will document and report the occurrence and 
nature of any new medical conditions or events since the informed consent signing as an adverse event.  
For each AE, the investigator will evaluate relatedness to study participation, severity, and expectedness, 
taking into account issues including AD pathology, concomitant treatments, and medical history. 
 
A safety medical monitor will advise and consult with the Sponsor, PI, sub-Investigator(s) and study staff 
on the following matters: Review protocol halting rules; Advise protocol team on safety oversight; Evaluate 
adverse events/SAEs and reviews safety reports once quarterly or more often if deemed necessary. This 
individual will be independent of the study team, and properly credentialed. 
 
As a note: If a participant receives two injections in one day, the study team will report this as a protocol 
deviation.  If the team learns of this at the time of the injections, the participant will be instructed to skip 
the dose on the following day.   As determined by Gemlyn George, M.D., hematologist and SESAD lead 
Medical Sub-I, in consideration of the phase 1 trial that administered sargramostim at 16 times the 
recommended dosage without causing harm, instances of two doses in one day for this trial does not 
constitute patient harm. 
 
 
8.4.5.1 Serious Adverse Events 
A serious adverse event, in relation to this clinical trial, is defined as something that results in: 
• 
death. 
• 
initial or prolonged inpatient hospitalization. 
• 
a life-threatening event. 
• 
persistent or significant disability. 
• 
congenital birth defect. 
• 
important medical events that might not be immediately life-threatening, or result in 
hospitalization, or death, but may require intervention to prevent such, including serious allergic 
reaction. 
 
All AEs since the signing of the informed consent are to be assessed for seriousness, documented, and, if a 
serious adverse event, reported to the Sponsor within 24 hours of becoming known to the principal 

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investigator or qualified sub-Investigator.  As well, the Data Safety Monitoring Board will monitor reported 
AEs and SAEs as a part of their role overseeing the protection of human participants. 
 
8.4.5.2 Protection of Participants 
Because of the extensive safety history of sargramostim in elderly patients, serious safety issues are not 
anticipated, however, vigilance in assessing safety is required. A review of published AEs for GM-CSF and 
the Leukine® Product Insert and the papers it references will be used to guide the safety monitoring protocol 
(attached as Appendix E). The greatest safety concern noted with sargramostim is excessive leukocytosis. 
Therefore, as advised by the product insert, a CBC with differential, including analysis for blast cells, will 
be conducted at two time-points every week during the 24- week-long treatment phase.  Refer to section 
7.4 for details on dosage modification guidelines. In addition, the DSMB (described below) will monitor 
laboratory results, the MRI studies, and the cognitive assessments for any indications of toxicity. Following 
are the abbreviated categories of the National Cancer Institute’s (NCI) graded toxicity protocol used by 
Daud et al., (2008) for assessing safety of Leukine® 123, although the latest version of the NCI-CTCAE 
(Version 5.00: November 27, 2017) will also be referenced for all AEs (attached as Appendix F): 
 
Anemia  
Lymphopenia  
Pain  
Fever  
Flu-like symptoms  
Arthralgia/myalgia  
Injection-site reaction  
Fatigue  
Abdominal cramps  
GERD/indigestion  
Insomnia  
Cough  
Sweating 
 
Full description of the DSMB will be provided in the charter.  The DSMB will be comprised of members 
of relevant disciplines to overseeing conduct of a clinical trial for AD. The DSMB will be charged with 
overseeing participant safety. An initial meeting of the DSMB will be held before enrollment occurs in 
order for the members to review the study protocol and the study/participant termination guidelines. The 
DSMB will be required to meet at a minimum of one time per year. Data will be reviewed according to the 
DSMB mandated timeframe  if a treatment related serious AE (SAE) or serious unexpected AE (SUAE) is 
reported. As mentioned above, SAEs and SUAEs will be reported per DSMB timeframes to the DSMB to 
discuss the SAEs/SUAEs and to decide whether the study should continue. Of note, if SAEs of Grade 3 or 
4 occur in 20% of the initial 14 participants following sargramostim (or placebo) administration, the study 
will be halted while the Sponsor consults with the DSMB for final determination of study status. On the 
other hand, if the trial data indeed demonstrate clear safety and positive efficacy results from administration 
of sargramostim into AD participants at the end of the trial analyses, the DSMB may ethically consider 
and provide recommendations for open label extension study for trial participants, in which case the 
Sponsor will seek guidance from COMIRB and the FDA regarding how to proceed with these 
recommendations. All DSMB meetings will be documented and submitted to SARC.  Once chartered, the 
DSMB membership will be listed in Appendix I.    
 
8.4.5.3 Data Monitoring Plan: 
Data Recording: 

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These functions will be carried out by the study staff and transcribed into the case report forms (adverse 
events page). The principal investigator or qualified medical designee will review, edit, and sign off on the 
AE form, and the AE form filed by the PI or designated staff. Laboratory results and data from case report 
forms will be entered into a computerized data registry.  
 
Data Verification: The PI or designated qualified sub-Investigator will discuss the AEs with the study 
coordinator(s), and will verify AE data entry. 
 
Data to be monitored, assessed, and reported to/by the DSMB: (From Product insert) 
Below is a list of possible data to be monitored, assessed and potentially reported to the DSMB.  This list 
is not exhaustive, but comprised of information found in other studies of the investigational product. 
 
Common but non-life threatening side effects include: 
• 
Fatigue or asthenia  
• 
Malaise  
• 
Rash  
• 
Itching (pruritus)  
• 
Vomiting 
• 
Diarrhea  
• 
Abdominal pain  
• 
Sore throat (pharyngitis)  
• 
Chills  
• 
Bone pains  
• 
Joint pains  
• 
Weight loss or gain 
• 
Elevations in kidney or liver enzymes  
• 
Fluid retention (peripheral edema)  
• 
Injection site irritation 
• 
Insomnia 
• 
Headaches 
• 
Transient Fevers 
 
 
Less common but potentially serious side effects include: 
• 
Bleeding into the eye  
• 
Allergic reactions 
• 
Excessive increase in white blood cell count  
• 
Fluid retention in the lung lining (pleura) and in the heart lining (pericardium) 
• 
Shortness of breath 
• 
Occasional transient heart rhythm changes (supraventricular tachycardia) 
• 
Sweating, tachycardia, claustrophobia, or anxiety induced by the MRI procedure 
 
Potential side effects of unknown frequency: 
• 
Leukopenia (low white blood cell count) 
• 
Neutropenia (low neutrophil count) 
 
 
Adverse event of special interest (AESI) include: 
An AESI is an AE (serious or non-serious) of scientific and medical concern specific to the program, for 

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which ongoing monitoring and immediate notification to the DSMB is required. Such events may require 
further investigation in order to characterize and understand them. Adverse events of special interest may 
be added or removed during a study by protocol amendment.  
• 
ADA 
• 
Acute hypersensitivity/anaphylaxis 
• 
Leukopenia (low white blood cell count) 
• 
Neutropenia (low neutrophil count) 
 
Report of Unexpected Problems to the DSMB: The study coordinator will notify the PI or qualified sub-
Investigator of any unexpected problems. The PI will contact the chair of the DSMB when unexpected 
problems arise that require immediate action.  
 
Criteria for Action: 
Specific triggers that will dictate when a specified action (such as reporting or stopping) is required. 
Any SAE or unexpected problem that meets the COMIRB criteria of reporting will be reported per 
COMIRB guidelines. Any serious AEs meeting the FDA reporting criteria, the FDA will also be notified 
according to their reporting guidelines. 
 
Stopping rules:  
Study: If treatment related serious AEs (SAEs) of Grade 3 or 4 occur in 20% of participants following 
sargramostim administration, the study will be stopped (Appendix F).  
 
Participant: For individual participants, the PI or Sub-I may discontinue any participant if they reach 
grade 3 or 4 in any category, if laboratory results indicate blast cell appearance, or if they worsen in any of 
the cognitive tests by more than two standard deviations than would be normally expected from someone 
at their baseline measurements, or if they have any values that have exceeded greater than two times upper 
limits of normal for any trial measures. Participants are, of course, free to withdraw from the study at any 
time for any or no reason. 
 
Specific participant stopping criteria for Absolute Neutrophil (ANC) count values are as follows: 
• 
If a participants Absolute Neutrophil Count (ANC) is less than 0.5x10E3/uL (500 cells per 
microliter) at any CBC with differential safety blood draw post randomization, the participant will 
be monitored for potential infections over the course of 1 week until another CBC with differential 
is completed.  If the ANC value does not demonstrate improvement (returned to a value above 
0.5x10E3/uL), administration of the study  drug to the participant should be discontinued. 
 
Participants will be followed-up according to the study procedures as specified in this protocol up to the 
scheduled date of study completion, or up to recovery or stabilization of any AE to be followed-up as 
specified in this protocol, whichever comes last. If possible, and after the permanent discontinuation of 
treatment, the participants will be assessed using the procedures normally planned for the EOT visit with 
the treatment/placebo, including, but not limited to, LP (if chosen), imaging assessments and cognitive 
assessments. All cases of permanent treatment discontinuation should be recorded after confirmation. 
 
The PI may also determine that a trial participant should no longer remain in the trial if there is evidence 
of uncontrolled hypertension. For an individual who has consented to volunteer for the trial, meets 
inclusion criteria and is enrolled, then begins the treatment phase of the trial, but who subsequently is 
determined to exhibit uncontrolled hypertension for more than 2 weeks (14 treatment days), the DSMB 
will be notified, within accepted timeframe, adverse events reporting protocols will be followed, and the 
following criteria will be used to determine whether the participant needs to be removed from the trial: 
 

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Inclusion Screening Criteria for hypertension: 
• 
The screening BP will be no greater than 160 systolic or 95 diastolic.  If the initial BP reading 
is greater than 160 systolic or 95 diastolic, the BP will be measured in triplicate over the 
course of the visit, with the average BP calculated. (This limit will apply to all and any BP 
measurements during the screening process.)  
 
Management Criteria during the treatment phase of the study: 
• 
Should blood pressure rise above 165 systolic or 95 diastolic, indicating uncontrolled 
hypertension, the participant will have treatment interrupted and may be removed from the 
study. Determination of uncontrolled hypertension will be assessed through taking of BP in 
triplicate during the visit in which the average criteria of greater than 165 systolic or 95 
diastolic is noted.  
• 
If the participant displays symptoms related to, or meets clinical criteria of, hypertensive 
urgency or emergency, treatment will be interrupted and the participant referred to emergency 
services by the PI, clinical sub-Investigator or other qualified designee. 
• 
The participant’s hypertensive regimen may be adjusted by the primary care provider during 
the study as needed. 
• 
If any participant, who has been interrupted from treatment for uncontrolled hypertension and 
depending upon the period of time taken for their BP to be controlled below 160 systolic or 
95 diastolic, the trial physician, in consultation with the Sponsor, may decide for re-initiation 
of treatment (see Section 7.5 Interruption of Treatment).   
 
Reporting: 
Timeframe for reporting: Any Grade 2 AEs and abnormal Grade 2 laboratory results, including CBC 
with differential results, which require temporary interruption of treatment (Sections 7.4 - 7.5), will be 
reported to the DSMB for review within the standard DSMB aggregate report. Serious AEs (SAE) and 
unexpected SAEs related to the trial will  be reported within five business days to the DSMB for review, 
and to COMIRB within their reporting guidelines according to their policies and procedures. The FDA will 
be notified by written report within 15 days of the Sponsor determining that the SAE or suspected adverse 
reaction meets FDA criteria for reporting.  
 
Reporting mechanisms: AE forms will be completed and submitted on-line using the COMIRB AE 
reporting system. The report will include a description of the event, judgment as to whether or not it was 
related to study medication, and the outcome of the AE. All non-serious AEs, and those that are not 
unexpected or that are normal to the study medication, will be reported to the DSMB at the time in which 
the rest of data from an aggregate block of participants is submitted, or if the DSMB requests the 
information for their analysis in another trial-related matter.   
 
Responsibility for preparing and submitting reports: The study coordinator will prepare the AE report 
and the PI or qualified sub-Investigator will review/edit as appropriate and the PI, study coordinator or 
other designated staff will submit the AE.  
 
Confidentiality Procedures:  
The meetings of the DSMB will be closed to respect the privacy of the research participants. Data will be 
kept in case report binders in a locked office when not in use. Data entered into the computerized data 
registry will only be accessible to those members of the research team. 
 
 
 

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9  Statistical, Data, and Other Considerations 
 
9.1 
Sample size considerations 
As the primary goal of this clinical trial is the safety and tolerability of long-term use of sargramostim in 
participants with mild-to-moderate Alzheimer’s disease, a sample size of 42 participants (28 sargramostim-
treated [1250 mcg/m2/ week subcutaneously], and 14 placebo equivalent) is considered sufficient to meet 
the endpoint of safety and is consistent with the lack of drug related SAEs in our recently-completed 
randomized, double blind, placebo-controlled trial of sargramostim in 40 (20 sargramostim-treated:20 
placebo-treated) subjects with mild-to-moderate Alzheimer’s disease. 
Based on results for 40 participants at the EOT, 45-day follow-up, and 90-day follow-up in our recently 
completed sargramostim trial in participants with mild-to-moderate Alzheimer’s disease, an effect size of 
around 2–4 was predicted by the MMSE. We believe that the 42 participants (28 sargramostim-treated 
[1250 µg/m2/week (178.57 µg/m2/day subcutaneously seven days a week], and 14 placebo equivalent) who 
will be recruited for this study will provide sufficient power to allow valid analyses of the secondary and 
exploratory endpoints.  Therefore, at least 42 individuals with mild-to-moderate Alzheimer’s disease, age 
60–85, of either sex, will be randomized (28 sargramostim participants and 14 placebo participants) for this 
proposed placebo-controlled study. For effect sizes of 2, 4, 6, or 8 (the expected treatment group differences 
in change between baseline and follow-up for MMSE total score), Table 1 shows the number of participants 
required to achieve 91–97% power. Recruitment and treatment initiation will be timed to allow for the 
interim assessments before further participants are treated, thus assuring maximum early knowledge of any 
safety concerns.   
 
The study will have two arms with participants assigned in permuted blocks examining placebo and full 
FDA-recommended sargramostim dose. As mentioned above, in the ongoing trial with participants who 
have thus far received sargramostim, there have been no serious AEs reported by participants receiving 
either the half dose or full dose sargramostim using the same methods proposed by this protocol. Therefore, 
participants will be started at the adjusted weekly dose (178.57 µg/m2/day SC, seven days per week, 1250 
µg/m2/week equivalent.)  
 
9.2 
Data Analysis Plan 
 
9.2.1 
Primary Objective: To assess long-term tolerability and safety of sargramostim in individuals 
with mild-to-moderate Alzheimer’s Disease 
Generally, descriptive statistics will be utilized to examine safety of participants who received 
sargramostim injections compared to placebo injections. Safety will be assessed by measuring the toxicity 
grades of AEs. These toxicity grades have been standardized by the National Cancer Institute (NCI) 
 
Mean Treatment 
Difference in Change from 
Baseline to End of 
Treatment (Day 18) 
Total 
Number of 
Patients 
Number of 
Sargramostim 
Patients 
Number 
of 
Placebo 
Patients 
Alpha 
Power 
2 
102 
68 
34 
0.05 
0.91 
4 
27 
18 
9 
0.05 
0.92 
6 
12 
8 
4 
0.05 
0.92 
8 
9 
6 
3 
0.05 
0.97 
 
Table 1. Model for MMSE Total Score, Assumes no Treatment Effect at Baseline (successful 
randomization). 

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Common Toxicity Criteria for Adverse Events (CTCAE Version 5.00: November 27, 2017, attached as 
Appendix F). Differences in toxicity grades will be compared between treatments using a Wilcoxon test for 
ordered outcome. MRI scans will be assessed to identify ARIAs, including vasogenic edema and 
microhemorrhage. Additional safety assessments will include neurological and physical examinations, vital 
signs and suicidality evaluations. The current study will utilize a Data Safety Monitoring Board (DSMB) 
to review the clinical data and ensure participant safety within the parameters of the trial. 
 
9.2.2 
Secondary Objective: To test the hypothesis that sargramostim treatment, as compared to 
placebo, slows or halts cognitive decline or improves cognitive function in individuals with mild-to-
moderate AD. 
The foremost measure used for this secondary endpoint of assessing cognitive function will be the MMSE, 
as it has been reported to be most sensitive to cognitive changes over time due to treatments in people 
ranging from late mild cognitive impairment to AD in clinical trials up to 2 years in length 124. The cognitive 
assessment will be based on the participants’ MMSE total score changes from Baseline to End of Treatment. 
 
9.2.3 
Exploratory Objectives 
To assess the effect of sargramostim treatment, as compared to placebo, on clinical progression of 
in individuals with mild-to-moderate AD. 
Well validated neuropsychological assessments will be administered and scored using the standardized 
scoring methods associated with each of the neuropsychological tests. Analysis of the change in cognition 
from Baseline to EOT will be measured by the change in scores for the MoCA, ADAS-cog13 total scores, 
CDR-SB, TMT A, ADCS-ADL score, measures of verbal fluency (lexical and semantic) and the NPI score.  
Due to the multitude of both the neuropsychological tests and the different time points that these tests are 
administered, a correction method will be used to counteract the problem of multiple comparisons. 
 
To assess the effect of sargramostim treatment, as compared to placebo, on metabolic activity in 
different brain regions in individuals with mild-to-moderate AD. 
FDG-PET will be used to assess overall metabolic activity in different brain regions, particularly in the 
medial temporal lobe in potential correlation with changes in MTA, at baseline and at EOT. Standard 
protocols for 18F-FDG PET imaging studies in conjunction with amyloid PET imaging studies, will be 
used 125, 126.  
 
To assess the effect of sargramostim treatment, as compared to placebo, on brain amyloid deposition 
in individuals with mild-to-moderate AD. 
CSF assay may be performed examining Aβ(1-42) and t-tau:Aβ (1-42) ratio.  The assay will be conducted 
during of screening and at the End of Treatment).  Additionally, there will be an option utilizing Amyloid 
PET scans, performed using Vizamyl, produced at the CU-AMC / Pharmalogic cyclotron laboratory, 
which allows for amyloid-beta neuritic plaque density imaging.  The Amyloid PET scans may be 
performed at Screening Visit and at the Follow-up Visit. Vizamyl is approved by the FDA for use in 
research and clinical application. For this protocol, the change from the screening scan’s standardized 
uptake value ratio (SUVR) to follow-up SUVR will be similarly analyzed by a contracted central reading 
facility, using the participant's MRI to reduce white matter contamination, thereby increasing the SUVR 
signal change by eliminating counts in the region that do not change (i.e. white matter). The PET scans 
and accompanying MRI scans will be stripped of any personal identifying information, containing only 
the trial’s randomized subject ID number and date. The results of the PET quantitative analyses will not 
be used to make any treatment decisions.   
 
To assess the effect of sargramostim treatment, as compared to placebo, on AD related changes in 
peripheral blood and CSF biomarkers in individuals with mild-to-moderate AD. 
Levels of AD-associated biomarkers in peripheral blood samples (collected at Baseline, 12, EOT and 
follow-up) and CSF samples (collected at Screening Visit and EOT) will be compared between placebo 

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controls and sargramostim treatment groups. The identification of circulating biomarkers in AD is a 
relatively new field, with inconsistent biomarker level results reported as increased, decreased, or 
unchanged between studies 127. These mixed results mean that standardized levels for AD-associated 
circulating biomarkers have yet to be established. Therefore, we will compare observed biomarker levels 
from our project to other published levels to explore whether any similarities are observed. This 
comparison, as well as our own results, will add to the AD-associated blood and CSF biomarker fields of 
literature. 
 
To assess the effect that ApoE genotype may have upon predicting sargramostim treatment outcomes 
in individuals with mild-to-moderate AD. 
ApoE genotyping will be performed using blood samples collected on Day 1 of this trial for each participant. 
ApoE genotype will be determined either by Athena Diagnostics, or in the University of Colorado 
laboratory under the direction of Huntington Potter, PhD, using established methods. Outcomes in the 
context of ApoE genotype will be compared. The genetic testing will be carried out at the end of the trial, 
and participants will not be notified of any genetic testing results. 
 
To assess the effect of sargramostim treatment, as compared to placebo, on inhibiting MTA in 
individuals with mild-to-moderate AD.  
MTA will be assessed using an automated rating system to grade the severity of atrophy in the entire medial 
temporal region will be employed using the FreeSurfer software automated rating system of individual 
medial temporal structures, i.e., the hippocampus (HPC), entorhinal cortex (ERC) and perirhinal cortex 
(PRC), which are specific regions of interest (ROIs) in AD pathogenesis. FreeSurfer automatically 
computes cortical reconstruction and volumetric segmentation, including segmentation of the subcortical 
white matter and deep gray matter volumetric structures 128 and parcellation of the cortical surface 129 
according to a previously published parcellation scheme 130. This labels cortical sulci and gyri, and thickness 
values are calculated in the ROIs. 
 
Group comparisons of changes in regional volumes and thicknesses will be analyzed using a series of one-
way analyses of variance (ANOVAs). The Scheffé post hoc procedure will be used to examine differences 
between means, and Pearson product–moment correlation coefficients will be used to evaluate the strength 
of relationships between changes in regional volumes and thicknesses and cognitive measures. Receiver 
operator curve analyses will determine sensitivity and specificity of various MTA cut-off points for 
distinguishing between diagnostic groups. Hazard ratios (HRs) for specific predictors of MTA progression 
and regression will be calculated using a three-state Markov model in continuous time. The proportional 
intensity model will be used to analyze effects of covariates on HRs. 
 
To assess the effect of sargramostim treatment, as compared to placebo, on AD related changes in 
blood brain barrier permeability in individuals with mild-to-moderate AD. 
CSF may be collected during screening, and at the EOT visit (i.e. within 2-10 days following the final 
treatment article injection) for a subset of participants for entry into the study or as part of an optional 
substudy. A venipuncture blood draw will be performed at each LP procedure visit to obtain serum. 
Albumin levels will be quantitated within CSF (in mg/dL) and serum (in g/dL) to calculate an Albumin 
Index (AI). Because albumin is neither synthesized nor metabolized intrathecally, albumin that is found 
within CSF that is free of blood contamination indicates that the albumin must have come from the 
plasma through the blood brain barrier (BBB). An AI value < 9 indicates an intact BBB, values 9-14 as 
slight impairment, values 14-30 as moderate impairment, values 30-100 as severe impairment, and values 
> 100 as complete breakdown of the BBB 72. The BBB is known to be impaired in AD 131-133, and cerebral 
amyloid angiopathy (CAA), characterized by Aβ deposition on vessel walls, has been found in over 90% 
of AD cases 134. In immunotherapy trials targeting amyloid in the parenchyma and cerebral vascular, the 
removal of pre-existing vascular amyloid has been attributed to onset of ARIA events (i.e. vasogenic 
edema and microhemorrhage) 135, and while we and others have found GM-CSF to quickly reduce 

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amyloid in transgenic AD models 1, 68, no incidences of ARIAs have been observed in the current ongoing 
sargramostim-AD trial (NCT01409915). GM-CSF is known to induce angiogenesis and restore cerebral 
blood flow following experimental stroke 23, 24, 27, 28. However, if sargramostim does indeed cause 
reductions in amyloid deposition over 24 weeks of administration in mild-to-moderate AD participants, 
evaluating treatment effects on BBB permeability in correlation with MRI safety measures is warranted. 
Thus, AI values will be calculated at screening and at EOT to assess any treatment-related changes in 
BBB permeability and to correlate values with any potential trial emergent ARIA events. 
 
To assess the effect of sargramostim treatment, as compared to placebo, on AD-related changes in 
microstructural white matter measures in individuals with mild-to-moderate AD. 
Numerous neuroimaging studies have shown that white matter (WM) tracts are associated with cognitive 
function, and that microstructural abnormalities are found in AD and other neurodegenerative diseases, as 
determined from high-resolution MRI diffusion tensor imaging (DTI) techniques 73-77. DTI metrics have 
been proposed as sensitive biomarkers for disease progression over short periods of time 78, which makes 
it useful for assessing any WM changes in response to treatments, and specific DTI patterns have been 
shown to help distinguish between AD and other comorbid neurodegenerative diseases 79, 80, to correlate 
with AD-related verbal fluency impairments 81, and with using neurite orientation dispersion and density 
imaging (NODDI) sequences of DTI, changes in neurite density can be approximated 82, 83. Because GM-
CSF has been shown to be a growth factor for oligodendrocytes 116, induce oligodendrocyte progenitor cells 
concurrent with amelioration of experimental spinal cord injury 34, 117, and increase synaptic area concurrent 
with reversing cognitive impairment and pathology in transgenic AD models 1, 68, high-resolution MRI with 
DTI and NODDI sequences will be performed at screening, weeks 3, 12, 18, EOT and metrics of DTI (e.g. 
FA, MD, NDI, ODI, etc.) will be monitored for any treatment-related changes. 
 
To assess the effect of sargramostim treatment, as compared to placebo, on changes in sleep-related 
measures in individuals with mild-to-moderate AD. 
Changes in quality of sleep have been long reported in onset and progression of AD 84-86, and decreases in 
non-rapid eye movement (NREM) slow wave activity (SWA) is known to be associated with Aβ 
deposition 87. Our preclinical work and others have revealed that GM-CSF quickly removes amyloid in 
AD models 1, 68, of which it is not known whether quality of sleep improves with reductions in amyloid. 
For assessing any changes in qualitative measures of sleep in the participants, two sleep assessments (i.e. 
ISI, and PSQI) will be administered to both them and their study partner/caregivers at baseline, week 12, 
EOT, and at follow-up (45 days post-treatment) visits to assess quality of sleep measures and any changes 
that may occur throughout the trial. Mechanistically, melatonin and its signaling have been shown to be 
decreased in AD progression 88-92, and GM-CSF has also been reported to increase melatonin secretion in 
preclinical studies 93. Melatonin is made and secreted by the pineal gland, and calcification of the pineal 
gland increases with AD progression 94-97. High-resolution MRI scans, using susceptibility weighted 
imaging (SWI) sequence, will be performed at screening, weeks 3, 12, 18, EOT, and at follow-up (45 
days post-treatment) to assess changes in pineal calcification measurements. 
 
To assess the effect of sargramostim treatment, as compared to placebo, on changes in speech 
measures in individuals with mild-to-moderate AD. 
AD is known to adversely impact temporal characteristics of spontaneous speech, such as speech tempo, 
number of pauses in speech, and their length, with speech alterations often noticed years before other 
cognitive deficits become apparent 98, 99. Among the neuropsychological measures frequently used to 
assess semantic memory impairment in AD are verbal fluency tests, or speeded word-list generation, 
which consists of two types: phonemic (letter) fluency tests and category fluency tests. Fluency 
performance involves multiple brain processes and regions that are adversely impacted by AD pathology, 
and both phonemic and category fluency performances reveal declines in AD 100, 101. Word retrieval in 
connected speech is also impaired in AD, and by using picture naming and picture descriptions, the total 
word output, percentages of content words, percentages of nouns, and percentages of pronouns out of all 

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words, type-token ratio of all words and type-token ratio of nouns alone, mean frequency of all words and 
mean frequency of nouns alone, and mean word length can be analyzed 102. A study participant interview 
will be given at baseline, week 12, EOT, and at follow-up (45 days post-treatment) visits to assess 
changes in speech fluency and word retrieval measures.  
 
9.3 
Additional Statistical Consideration 
Additional statistical tests that may be utilized include: independent t-tests to examine differences 
between means; the Chi-square test to assess association between variables; logistical regression to 
calculate the ratio of the odds of an event occurring in one group compared to another group, and 
longitudinal regression to analyze the data over time. 
 
9.3.1 
Full Statistical Analysis Plan 
A full statistical analysis plan will be developed and approved prior to final database lock of participant 
data and will be outlined in a separate Statistical Analysis Plan (SAP) document.  
 
9.4 
Interim Analyses 
No interim analyses will be conducted as a part of this trial.   
 
9.5 
Data Storage:  
All data collected at the study site will be stored in REDCap, a HIPPA compliant database. Data collected 
will include general participant information (e.g., age, phone, address, etc.), background health information, 
and research outcomes. The Principal Investigator is responsible for the individually identifiable private 
health information. Access to the REDCap database key will be restricted to the research team approved on 
the application in order to minimize risk of unintentional disclosure. All affiliated/local principal 
investigators, co-investigators, and research coordinators involved in consenting and carrying out the 
proposed study will complete Collaborative Institutional Training Initiative, an online basic Course in 
Human Subject Protections, and the HIPAA research course prior to submitting a protocol. Source data will 
be collected and stored in participant binders, OnCore CTMS, Epic EMR, and the CU_AMC computer 
system. 
 
For the general manipulation of data, the clinical data will be transformed into a de-identified data set using 
the unique study IDs. Any paper record of the data will be kept under lock and key with the Principal 
Investigator.  
 
9.6 
Specimen Storage:  
Samples will be stored for at least 10 years, or the entire course of this study. Excess samples may also be 
added to the University of Colorado Alzheimer’s and Cognition Center (CUACC) Biobank for future 
internal or external studies, if given consent by the participant. If a participant withdraws from the study, 
the samples will be discarded at the written request of the participant or appropriate consenting party.  
 
9.7 
Control of Conflict of Interest: 
The Sponsor and Principal Investigator, Huntington Potter, PhD, was one of the conceivers of the 
Arthritis/GM-CSF/G-CSF approach to AD therapy and is one of the inventors on several University of 
South Florida issued patents on the use of GM-CSF to improve cognition and/or to reduce amyloid 
deposition in AD and/or in chemobrain. These patents have never been licensed and are of no commercial 
value presently. To eliminate any real or even perceived conflict of interest, Dr. Potter has relinquished any 
and all personal rights to shared royalties from USF patents and has instructed the USF Technology Transfer 
Office to transfer any such income that might come in the future to the University of Colorado Foundation 
to support research by the Potter Alzheimer Dementia Fund. Furthermore, the following steps will be used 
to help further isolate Dr. Potter from potential influence related to the outcomes of the trial and to ensure 
that the trial is carried out with the highest level of scientific integrity: 

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1) All investigators who interact with participants directly and all investigators who collect and record 
data will have zero financial interest in the outcome of the study and will be blinded to the treatment 
group. 
2) Endpoints are quantitative and objective (i.e., assessments of cognition using a battery of 
neuropsychological and clinical assessments used in the UCH clinic, brain imaging, and measures of 
blood biomarkers), and the investigators are blinded to the treatment group. 
3) With regard to potential financial interests of any investigators with pending patent applications at 
CU Anschutz, no such pending patent applications have been licensed and they have no current 
commercial value. 
4) An independent DSMB has been established to oversee participant safety. 
5) Notification of any financial interests of Dr. Potter or of other investigators in the study that may 
arise in the future will be made to the CU Research Compliance Office and appropriately managed, 
to editors of journals publishing study-related results and to the public in the context of 
communication of any research results. 
 
 
 

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Appendix B: GM-CSF Upregulated in Rheumatoid Arthritis Reverses Cognitive Impairment and 
Amyloidosis in Alzheimer Mice (Boyd et al., 2010).  
Appendix C: Granulocyte Macrophage Colony Stimulating Factor Treatment is Associated with Improved 
Cognition in Cancer Patients (Jim et al., 2012). 
Appendix D: Briefing Package – FDA Advisory Committee Meeting May 3, 2013; Study Drug: Leukine® 
(Sargramostim). 
Appendix E: Leukine® (Sargramostim) Package Insert and Full Prescribing Information. 
Appendix F: Common Terminology Criteria for Adverse Events (CTCAE) v5.0. 
• 
https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/CTCAE_v5_
Quick_Reference_5x7.pdf  
Appendix G: Evaluation of the safety and immunomodulatory effects of sargramostim in a randomized, 
double-blind phase 1 clinical Parkinson’s disease trial (Gendelman et al., 2017). 
 
 
 
 
 
 
 
 
 
 

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Appendix H 
 
General Rules for Excluded Concomitant Medications 
This list is intended to be comprehensive, but not exhaustive.  If a concomitant medication is in doubt, the 
decision of the Principal Investigator (PI), or Sponsor as specified, shall be the final determinant for 
allowance or not.    
 
The PI will review all prior and concomitant medications, and review concomitant medication changes on 
an ongoing basis. If disallowed concomitant medications with exceptions for PRN use exceed frequencies 
as specified, the PI will be consulted prior to participant continuation in the study.  
 
The PI and/or Sponsor may allow a participant to continue in the study while taking an excluded 
concomitant medication (or at an excluded frequency) on a case by case basis to ensure the safety of 
participants and the scientific validity of study data. Exceptions to excluded concomitant medications may 
be needed for situations such as treatment of an adverse event. Any exceptions to excluded concomitant 
medications or frequencies will result in a protocol deviation.  
. 
EXCLUDED CONCOMITANT MEDICATIONS1. Participation in any other investigational drug 
study within 4 weeks of screening (individuals may not participate in any other drug study while 
participating in this protocol). 
• 
Any prior use of study drug that was investigated for anti-amyloid or anti-tauopothy effects, or 
AD vaccine, unless it can be documented that the participant was on placebo. 
• 
Any prior use of sargramostim within 6 months of screening. 
 
2. Diuretic drugs should not be started or discontinued within 4 weeks prior to screening.  Any change in 
diuretic medication during the study should be reported. 
 
3. As a class, chronic use of systemic corticosteroids are prohibited from within 4 weeks of screening 
through the follow-up visit due to possible risk of myeloproliferation. 
 
4. Lithium is prohibited from within 4 weeks of screening through the follow-up visit due to possible risk 
of myeloproliferation. 
 
5. Systemic immunosuppressive or immunomodulatory agents/drugs (other than corticosteroids, see 
below) are not permitted for a period of 60 days before screening.  Rules for PRN use of select drugs that 
have a systemic effect, such as corticosteriods, are listed below. 
 
6. Immunoglobulin therapy and biologic drugs are not permitted within 6 months before screening. 
 
7. Off label or non-standard of care usage of leukotriene inhibitors, such as montelukast, are not allowed 
from before screening until after the final study visit. 
 
8. AChEI and/or memantine therapy should follow standard of care parameters, and be a stable dosage for 
60 days prior to and during screening.  Initiation of, or changes to, AChEI or memantine therapy is 
strongly discouraged during the treatment period, and must be approved by the PI prior to change for 
participant continuation in the study. If changes occur without prior PI approval, the Sponsor must be 
consulted to approve of continuation in the study. 
 
9. If a medication is found in more than one category, the rule for the most strict category will be used for 
use/exclusion. 
 

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ANTICOAGULATION THERAPY:  Not allowed within 4 weeks prior to screening through follow-
up visit (with exception of 81 mg aspirin daily therapy) 
GENERIC NAME 
BRAND NAME 
Apixaban 
Eliquis 
Dabigatran 
Pradaxa 
Edoxaban 
Savaysa 
Enoxaparin 
Lovenox 
Fondaparinux 
Arixtra 
Heparin 
 
Rivaroxaban 
Xarelto 
Warfarin 
Coumadin 
 
 
NARCOTIC ANALGESICS:  Chronic use not allowed within 4 weeks prior to screening through 
follow-up visit PRN prescribed narcotic analgesic use is acceptable (Note definition of chronic use is 
weekly usage of a narcotic analgesic drug for three or more times per week AND for two or more weeks 
within any four-week period) 
 
GENERIC NAME 
BRAND NAME 
Hydromorphone 
Dilaudid 
Oxycodone/Acetaminophen 
Percocet 
Oxycodone/Aspirin 
Percodan 
Propoxyphene/Darvon and its variations 
 
Narcotics that contain codeine or morphine 
 
 
NEUROLEPTICS: Not allowed within 4 weeks prior to screening through follow-up visit 
GENERIC NAME 
BRAND NAME 
Chlorpromazine 
Thorazine 
Fluphenazine  
Prolixin 
Loxapine  
Loxitane 
Perphenazine 
Etrafon, Trilafon 
Thioridazine  
Mellaril 
Thiothixene 
Navane 
Trifluoperazine 
Stelazine 
Clozapine 
Clozaril 
Haloperidol 
Olanzapine 
Quetiapine 
Haldol 
Zyprexa 
Seroquel 
 
NEUROLEPTICS: Use of the following is permitted if dose is stable for 4 weeks prior to screening: 
GENERIC NAME 
BRAND NAME 
Aripiprazole 
Abilify 
Risperidone 
Risperdal (up to 2mg/day) 
Ziprasidone 
Geodon 
 
ANTIPARKINSONIAN MEDICATIONS:  Not allowed within 4 weeks prior of screening through 
follow-up visit 
GENERIC NAME 
BRAND NAME 
Amantadine 
Symmetrel 

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Bromocriptine 
Parlodel 
Deprenyl/Selegilene 
Eldepry 
Levodopa 
Sinemet 
Pergolide 
Permax 
Pramipexole 
Mirapex 
 
SEDATIVES/BENZODIAZEPINES: Chronic use allowed if on stable doses 4 weeks prior to 
screening or PRN use if prescribed for a specific procedure. 
GENERIC NAME 
BRAND NAME 
Alprazolam 
Xanax 
Buspirone 
Buspar 
Chloral Hydrate 
Noctec 
Chlordiazepoxide 
Librium 
Clonazepam 
Klonopin 
Diazepam 
Valium 
Flurazepam 
Dalmane 
Lorazepam 
Ativan 
Meprobamate 
Miltown 
Oxazepam 
Serax 
Temazepam 
Restoril 
Trazodone 
Desyrel 
Trizolam 
Halcion 
Zaleplon 
Sonata 
Zolpidem 
Ambien 
 
RHEUMATOID ARTHRITIS MEDICATIONS:  Not allowed within 4 weeks of screening through 
follow-up visit 
GENERIC NAME 
BRAND NAME 
Abatacept 
Orencia 
Adalimumab 
Humira 
Anakirna 
Kineret 
Barictinib 
Olumiant 
Betamethasone 
Celestone 
Certolizmab 
Cimzia 
Etanercept 
Enbrel 
Golimumab 
Simponi 
Hydroxychloroquine sulfate 
Plaquenil 
Infliximab-dyyb 
Inflectra, Remicade 
Leflunomide 
Arava 
Methotrexate 
Rheumatrex/Trexall 
Prednisone 
 
Rituximab 
Rituxan 
Sarilumab 
Kevzara 
Tocilizumab 
Actemra 
Tofacitinib 
Xeljanz 
 
ANTIHYPERTENSIVE AGENTS WITH FREQUENT CNS SIDE-EFFECTS: Not allowed within 
4 weeks of screening through follow-up visit 
Clonidine 
Catapres 
 

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SYSTEMIC CORTICOSTEROIDS: Not allowed within 4 weeks of screening through follow-up 
visit excepting PRN or short course therapy as a necessary prescribed therapeutic for an acute 
condition. PI must be notified of use. (Note definition of chronic use is weekly usage of a systemic 
corticosteriod drug for three or more times per week AND for two or more weeks within any four-week 
period) 
GENERIC NAME 
BRAND NAME 
Cortisone 
Cortone Acetate 
Prednisone 
Rayos, Sterapred, Deltasone 
Prednisolone 
Flo-Pred, Millipred, Orapred, Pediapred, Veripred 
20, Prelone, Hydeltra, Hydeltrsol, Key-Pred, 
Predacort, Predalone, Predate, Predaject, 
Medicort, Pri-Cortin, Predcor, AsmalPred 
Methylprednisolone 
Medrol, MethylPREDNIsolone 
Dexamethasone 
Baycadron, Decadron, Dexamethasone, Intensol, 
DexPak, TaperDex, Zema-Pak, ZoDex, Zonacort 
Betamethasone 
Celestone Soluspan, Pod-Care 
hydrocortisone 
Cortef 
 
OTHER SYSTEMIC IMMUNOSUPPRESSANTS AND/OR IMMUNOMODULATORS NOT  
LISTED ELSEWHERE:  Not allowed within 60 days of screening through follow-up visit.   
GENERIC NAME 
BRAND NAME 
Cyclosporine 
Neoral, Sandimmune, SangCya 
Tacrolimus 
Astagraf XL, Envarsus XR, Prograf 
Sirolimus 
Rapamune 
Everolimus 
Afinitor, Zortress 
Azathioprine 
Azasan, Imuran 
Lefunomide 
Avara 
Mycophenolate 
CellCept, Myfortic 
Ixekizumab 
Taltz 
Natalizumab 
Tysabi 
Secukinumab 
Consentyx 
Ustekinumab 
Stelara 
Vedolizumab 
Entyvio 
Basiliximab 
Simulect 
Daclizumab 
Zinbryta 
 
 

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Appendix I 
The DSMB will be chartered previous to the initiation of enrollment activities of the clinical trial.   
The Colorado Clinical and Translational Sciences Institute (CCTSI) will set up and manage an independent 
DSMB for this study. No staff at CCTSI work as personnel on this study, but they will recruit qualified 
scientific and medical professionals from inside and outside of the University of Colorado scientific 
community to review the patient safety data generated by this study. The appropriate DSMB member 
oversight will be managed by the leader of the CCTSI DSMB. The DSMB will meet at a regularly scheduled 
interval (every six months) to review participant safety data. 
 
Assurance from Conflicts of Interest (COI): If recruited from the at-large CU-AMC professional 
community, members of the DSMB will be employed within different departments on the CU Anschutz 
Medical Campus, and no members have any real or perceived conflicts.  All other members will be recruited 
from outside the University of Colorado scientific community.  In addition, DSMB members will provide 
annual CU-mandated disclosures of any perceived or real COIs, and will notify fellow members of the 
DSMB, the trial’s PI and/or Sponsor of any potential COIs that may arise during the trial.  
 
 
 

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Appendix J 
AVAILABLE RESOURCES:  
 
CU-AMC and UCH: 
 
Research Clinic – UCH: The Clinical and Translational Research Center (CTRC) Outpatient Clinic on the 
3rd floor of the UCH Leprino Building, and Building 400 will be the location for participant evaluation in 
private rooms. The CTRC clinic contains 11 Exam Rooms, three Metabolic Testing Rooms, an 
Echocardiogram Testing Room with ECG services, a Cognitive Testing Room, a 5-Chair Infusion Center, 
and a Phlebotomy Room. There is also a patient registration area and waiting room. Building 400 has an 
examinination room available that may be used for procedures. 
 
The University of Colorado Alzheimer’s and Cognition Center (CUACC) Memory Disorders Clinic: 
The Memory Disorders Clinic includes examination rooms, procedure and consultation rooms, and four 
neuropsychometric testing rooms. Participants will be recruited from the Memory Disorders Clinic, but the 
informed consent, screening, and study-related tests will be performed at the CTRC Outpatient Clinic or 
Building 400.  
 
The University of Colorado Research Imaging Center (CU-RIC)  
and Brain Imaging Center (BIC): The CU-RIC in building Biosciences 3houses the research PET/CT, 
PET/MRI and cyclotron laboratory managed by Pharmalogic.  The BIC houses a research MRI in Building 
400.. 
 
CUACC Laboratory:  Located in the Research Center 2 (RC2) building, a short walk from the clinic. Two 
-80 degree freezers located within Dr. Huntington Potter’s laboratory will be utilized for banking of blood 
samples. Biomarker freezers are connected to continuous power supplies. Available cores include a state-
of-the-art microscopy core with stereologer, fluorescent, confocal, and near IR microscopes, flow 
cytometry, qRT-PCR, ultra centrifuges, cryostat, flow cytometer/cell sorter, plate washer, dark room and 
developer, gel doc system, Meso Scale Discovery (MSD) Biomarker System, and Fuidigm Cell Sorting and 
Analysis System. 
 
Data Management: Includes REDCap, a secure, HIPPA compliant database, database software (FileMaker 
Pro), PrismGraphPad, SPS, SAS, AnyMaze, MetaMorph, and FreeSurfer and NeuroQuant MRI analysis 
programs are all available. University of Pittsburgh collaborators have agreed to share their PET imaging 
analysis software. 
 
Computer: The laboratories and clinic are adequately equipped with a PC and Apple computers with access 
to the Internet and literature search programs. All staff members also have computers running Windows 
XP or more recent, with necessary software, printer and internet access. The server is administered and 
backed-up daily by UCH IT. 
 
 
Office: The Sponsor, Huntington Potter, PhD, and the PI each have 100–200 sq. ft. offices with telephone, 
fax access, and voice mail. The study coordinator(s) has office space in Building 400 of the CU-AMC, and 
the psychometrist will utilize space in the Cognitive Testing Room of the CTRC Outpatient Clinic and 
Building 400. Within each office or adjacent are PC’s, telephones, fax access, and voice mail. Additional 
office space is available on the CU-AMC campus as needed.  Various large and small conference/lecture 
rooms are available for reservation for meetings, seminars, and outreach events. All conference space is 
equipped with tele-communication equipment for tele-conferencing and audio visual equipment and marker 
boards for presentations.