Conditions: FSGS MCD Focal Segmental Glomerulosclerosis Minimal Change Disease
Keywords: Kidney Disease Minimal Change Disease Nephrotic Syndrome Focal Segmental Glomerulosclerosis FSGS
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 7
Healthy volunteers: no

Brief summary:
Adalimumab, a treatment which blocks tumor necrosis factor (TNF), was tested to see if it changed levels of urine biomarker levels, tissue inhibitor of metalloprotease-1 (TIMP1), and monocyte chemoattractant protein-1 (MCP1). Results may help develop individualized treatment options for future patients with TNF-driven focal segmental glomerulosclerosis (FSGS) or minimal change disease (MCD).

Detailed description:

Arms:
arm 1: Adalimumab dose every other week (study weeks 0, 2, 4, 6, and 8), subcutaneously

Interventions: intervention 1: adalimumab
Intervention descriptions: intervention 1: Adalimumab will be dosed based on weight

Protocol document:
Prot_000.pdf: 
 
Precision Medicine Proof of Concept for TNF Inhibition in FSGS and Treatment Resistant 
Minimal Change Disease  
 
Principal Investigators: Zubin Modi, MD, MS & Howard Trachtman, MD 
Sponsored by: University of Michigan 
Protocol Version Number:  v. 5.0 
 
IND Number: 143167 
NCT Number: NCT04009668 
 
Protocol Date: 2/15/2019 
Amendment 1: 4/1/2020 
Amendment 2: 12/1/2020 
Amendment 3: 07/28/2021 
Amendment 4: 02/21/2023 
 
 
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023  
 
3 
 
Table of Contents 
LIST OF ABBREVIATIONS ....................................................................................................................................... 1 
STATEMENT OF COMPLIANCE ............................................................................................................................. 1 
PROTOCOL SUMMARY ........................................................................................................................................... 2 
SCHEMATIC OF STUDY DESIGN .......................................................................................................................... 3 
1 
KEY ROLES .................................................................................................................................................... 5 
2  
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ........................ 6 
2.1 
Background Information ...................................................................................................................... 6 
2.2 
Rationale ............................................................................................................................................... 6 
2.3 
Potential Risks and Benefits .............................................................................................................. 9 
2.3.1 
Known Potential Risks ..................................................................................................... 9 
2.3.2 
Potential Benefits ........................................................................................................... 11 
3 
OBJECTIVES AND PURPOSE .................................................................................................................. 11 
4 
STUDY DESIGN AND ENDPOINTS ......................................................................................................... 11 
4.1 
Description of the Study Design ...................................................................................................... 11 
4.2 
ENDPOINTS ....................................................................................................................................... 11 
4.2.1 
Primary Endpoint ............................................................................................................ 11 
4.2.2 
Secondary Endpoints .................................................................................................... 11 
4.2.3 
Exploratory Endpoints ................................................................................................... 11 
5 
STUDY ENROLLMENT AND WITHDRAWAL ......................................................................................... 12 
5.1 
Participant Inclusion Criteria ............................................................................................................ 12 
5.2 
Participant Exclusion Criteria ........................................................................................................... 12 
5.3 
Strategies for Recruitment and Retention ...................................................................................... 13 
5.4 
Participant Withdrawal or termination ............................................................................................. 13 
5.5 
Premature Termination or Suspension of Study ........................................................................... 13 
6 
STUDY AGENT ............................................................................................................................................ 14 
6.1 
Study Agent(s) and Control Description ......................................................................................... 14 
6.1.1 
Acquisition ....................................................................................................................... 14 
6.1.2 
Formulation, Appearance, Packaging, and Labeling ................................................ 14 
6.1.3 
Product Storage and Stability ....................................................................................... 14 
6.1.4 
Dosing 15 
6.1.5 
Route of Administration ................................................................................................. 15 
6.1.6 
Dose Adjustments/Modifications/Delays .................................................................... 15 
6.1.7 
Duration of Therapy ....................................................................................................... 15 
6.2 
Study agent Accountability Procedures .......................................................................................... 15 
6.2.1 
Responsibilities of the Clinical Site Pharmacy ........................................................... 15 
6.2.2 
The Site Investigator Responsibility ............................................................................ 15 
7 
STUDY PROCEDURES AND SCHEDULE .............................................................................................. 15 
7.1 
Study Procedures/Evaluations ......................................................................................................... 15 
7.2 
Study Schedule .................................................................................................................................. 17 
7.2.1 
Screening Visit ................................................................................................................ 17 
7.2.2 
Baseline/Week 0 ............................................................................................................ 18 
7.2.3 
Follow-up ......................................................................................................................... 18 
7.2.4 
Early Termination Visit .................................................................................................. 19 
7.2.5 
Schedule of Events Table ............................................................................................. 19 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023  
 
4 
 
7.3 
Justification for Sensitive Procedures ............................................................................................. 20 
7.4 
Concomitant Medications, Treatments, and Procedures ............................................................. 20 
7.5 
Prohibited Medications, Treatments, and Procedures ................................................................. 20 
7.6 
Participant Access to Study Agent At Study Closure ................................................................... 20 
8 
ASSESSMENT OF SAFETY ...................................................................................................................... 20 
8.1 
Specification of Safety Parameters ................................................................................................. 21 
8.1.1 
Definition of Adverse Events (AE) ............................................................................... 21 
8.1.2 
Definition of Serious Adverse Events (SAE) .............................................................. 21 
8.1.3 
Pregnancy ....................................................................................................................... 22 
8.2 
Classification of an Adverse Event ................................................................................................. 22 
8.2.1 
Severity of Event ............................................................................................................ 22 
8.2.2 
Relationship to Study Agent ......................................................................................... 23 
8.2.3 
Expectedness ................................................................................................................. 23 
8.3 
Time Period and Frequency for Event Assessment and Follow-Up .......................................... 23 
8.4 
Reporting Procedures ....................................................................................................................... 23 
8.4.1 
Adverse Event Reporting .............................................................................................. 23 
8.4.2 
Serious Adverse Event Reporting ............................................................................... 23 
8.5 
Study Halting Rules ........................................................................................................................... 24 
8.6 
Safety Oversight ................................................................................................................................ 24 
9 
CLINICAL MONITORING ............................................................................................................................ 24 
10 
STATISTICAL CONSIDERATIONS .......................................................................................................... 25 
10.1 
Statistical and Analytical Plans ........................................................................................................ 25 
10.2 
Analysis Datasets .............................................................................................................................. 25 
10.3 
Description of Statistical Methods ................................................................................................... 25 
10.3.1 
General Approach .......................................................................................................... 25 
10.3.2 
Analysis of the Primary Efficacy Endpoint(s) ............................................................. 25 
10.3.3 
Analysis of the Secondary Endpoint(s) ....................................................................... 25 
10.3.4 
Safety Analyses .............................................................................................................. 25 
10.3.5 
Adherence and Retention Analyses ............................................................................ 26 
10.3.6  
Baseline Descriptive Statistics ..................................................................................... 26 
10.3.7 
Tabulation of Individual Response Data ..................................................................... 26 
10.3.8 
Exploratory Analyses ..................................................................................................... 26 
10.4 
Sample Size ....................................................................................................................................... 26 
11 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS ................................... 26 
12 
ETHICS/PROTECTION OF HUMAN SUBJECTS ................................................................................... 27 
12.1 
Ethical Standard ................................................................................................................................. 27 
12.2 
Institutional Review Board ................................................................................................................ 27 
12.3 
Informed Consent Process ............................................................................................................... 27 
12.4 
Participant and data Confidentiality ................................................................................................ 27 
12.5 
Future Use of Stored Specimens .................................................................................................... 28 
13 
DATA HANDLING AND RECORD KEEPING .......................................................................................... 28 
13.1 
Data Collection and Management Responsibilities ...................................................................... 28 
13.2 
Study Records Retention .................................................................................................................. 28 
13.3 
Protocol Deviations............................................................................................................................ 28 
13.4 
Publication and Data Sharing Policy ............................................................................................... 28 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023  
 
5 
 
14 
CONFLICT OF INTEREST POLICY .......................................................................................................... 28 
15  
LITERATURE REFERENCES.................................................................................................................... 28 
APPENDIX ................................................................................................................................................................. 29 
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
1 
LIST OF ABBREVIATIONS 
 
DSMB 
Data Safety and Monitoring Board 
EGF 
Epidermal Growth Factor 
ESKD 
End Stage Kidney Disease 
FSGS 
Focal Segmental Glomerulosclerosis 
MCP1 
Monocyte Chemoattractant Protein 1 
TIMP1 
Tissue Inhibitor of Metalloproteinase 1 
MCD 
Minimal Change Disease 
TNF 
Tumor Necrosis Factor 
UPC 
Urine Protein:Creatinine Ratio 
 
STATEMENT OF COMPLIANCE 
This trial will be conducted with Good Clinical Practice (GCP) and in accordance with the Code of Federal 
Regulations on the Protection of Human Subjects (21 CFR Part 50). The Principal Investigator will assure 
that no deviation from, or changes to the protocol will take place without documented approval from 
the applicable Institutional Review Boards (IRBs), except where necessary to eliminate an immediate 
hazard(s) to the trial participants. All personnel involved in the conduct of this study have completed 
Human Subjects Protection Training.  
 
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
2 
 
PROTOCOL SUMMARY 
Title: 
Precision Medicine Proof of Concept for TNF inhibition in FSGS and 
Treatment Resistant Minimal Change Disease 
Précis: 
A Proof of Concept, multi-center, open label, clinical trial addressing the 
treatment of patients with focal segmental glomerulosclerosis (FSGS) or 
treatment resistant minimal change disease (TR-MCD). Up to 8 patients 
with intra-renal tumor necrosis factor (TNF) up-regulation will be treated 
with 8 weeks of adalimumab therapy. The primary assessment will be of 
the change in target engagement biomarker levels of urinary TIMP1/Cr or 
MCP1/Cr from baseline to study week 10. Key secondary outcomes 
include safety measures, serious adverse events, and adverse events.  
 
Objectives: 
 
This study is proposed to test the hypothesis that FSGS and TR-MCD 
patients with up-regulation of the TNF pathway can be identified through 
intra-renal gene expression profiling and assay of urine MCP1/Cr and 
TIMP1/Cr levels by ELISA.  
 
Furthermore, this study will test the hypothesis that TNF inhibition with 
adalimumab (Humira) in patients with up-regulation of the intra-renal TNF 
pathway will result in reduction in urinary MCP1/Cr and/or TIMP1/Cr 
biomarker levels.  
Endpoint 
Primary Endpoint: The change in urine MCP1/Cr and TIMP1/Cr levels from 
baseline to study week 10, i.e. 2 weeks after the final dose of adalimumab  
 
Secondary Endpoints  
(1) Incidence of serious adverse events, adverse events and abnormal 
laboratory tests 
(2) Percent change of eGFR from baseline to week 10  
(3) Percent change in urine protein:creatinine (UPC) from baseline to 
week 10 
(4) Proportion of participants achieving a nadir UPC less than 1.5 g/g 
and at least a 40% reduction from baseline at week 10.   
 
Exploratory Endpoints 
(1) Correlation coefficient between intra-renal gene expression of TNF 
and urinary MCP1/Cr and TIMP1/Cr biomarker levels at baseline 
(2) Time to reduction of urinary MCP1/Cr and TIMP1/Cr biomarkers 
from baseline 
(3) Change in urinary EGF biomarker levels between baseline and 
week 10 
(4) Change in Quality of Life score between baseline and week 10. 
 
Population: 
 
 
 
Up to eight patients with kidney biopsy confirmed FSGS or TR- MCD, 
increased urinary excretion of biomarkers of TNF activation (MCP1/Cr and 
TIMP1/Cr) at study screening, eGFR>30 ml/min/1.73 m2 at enrollment, 
urine protein:creatinine (UPC) ratio ≥1.5 g/g at enrollment, age between 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
3 
6-80 years, inclusive, and stable therapy with angiotensin converting 
enzyme inhibitors, angiotensin receptor blockers, and oral 
immunosuppression agents for at least 30 days prior to enrollment will be 
enrolled and entered into the intervention phase of the study. Additional 
eligibility criteria are listed in the inclusion and exclusion criteria section 
below.  
Phase: 
Proof of Concept/Phase 1b/2a 
 
Number of Sites 
enrolling participants: 
Up to 8 sites in the USA will participate in this study. 
 
Description of Study 
Agent : 
Humira, adalimumab 
Manufactured by Abbvie 
Subcutaneous 
A total of 5 doses will be administered during the trial at weeks 0, 2, 4, 6 
and 8. Dosing will be based on weight: 
Subject Weight 
Adalimumab dose every other week 
15kg to <30kg 
20mg 
>30kg 
40mg 
Study Duration: 
Approximately 14 months  
1. 7 month enrollment phase 
2. 2 week screening, 8 week intervention and 6 week post 
intervention observation period for a total of 14 week 
participation per patient 
3. 3 month analysis phase 
 
Participant Duration: 
16 weeks 
1. 2 week screening period 
2. 8 week intervention period 
3. 6 week follow up post treatment  
 
SCHEMATIC OF STUDY DESIGN 
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
4 
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
5 
 
1 
KEY ROLES 
Administrative Coordinating Center 
University of Michigan 
1500 E. Medical Center Drive 
Ann Arbor, MI 48109 
 
 
 
 
Study Principal Investigator 
Zubin Modi, MD, MS 
Assistant Professor of Pediatrics 
University of Michigan 
1500 E. Medical Center Drive 
Ann Arbor, MI 48109 
Phone 734-232-4830 
Fax 734-232-2353 
Email: modiz@med.umich.edu 
Co-Principal Investigator and Medical Monitor 
Howard Trachtman, MD 
University of Michigan 
Co-Investigator and Medical Monitor 
Karthik Ramani, MD,  
University of Michigan 
 
Director, Biomarker Core 
Wenjen Ju, PhD 
University of Michigan 
 
Director, Gene Expression and Bioinformatics Core 
Matthias Kretzler, MD 
University of Michigan 
Data Coordinating Center 
University of Michigan 
Ann Arbor, MI 48109-2029 
 
Principal Statistician & Director of Data Coordinating Center 
Cathie Spino, D.Sc. 
Research Professor of Biostatistics  
Director, Statistical Analysis of Biomedical 
and Educational Research (SABER) Unit  
School of Public Health  
University of Michigan 
1415 Washington Heights, Rm M4507 
Ann Arbor, MI 48109-2029 
Tel: (734) 615-5469 
Fax: (734) 647-3711 
Email: spino@med.umich.edu 
 
 
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
6 
 
2  
INTRODUCTION: BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE  
 
2.1 
BACKGROUND INFORMATION  
FSGS is a pattern of kidney injury that manifests as isolated, asymptomatic proteinuria or nephrotic 
syndrome. It is a serious condition and may lead to kidney failure in up to 50% of patients over a five to 
ten year period. Although primary FSGS is a rare entity, the incidence of the disease has been steadily 
rising over the last 30 years and it is generally the most common form of primary glomerular disease in 
all patient subgroups. Moreover, it one of the leading causes of chronic and end-stage kidney disease 
(ESKD), accounting for 5% of adult and 12% of pediatric ESRD cases. 
 
The first-line therapy for primary FSGS, especially in those with nephrotic-range proteinuria, is usually 
corticosteroids. In patients who are unresponsive to steroids, calcineurin inhibitors (CNIs) are the 
recommended second-line therapy according to current KDIGO clinical guidelines1. Overall, 
approximately 20% of patients with FSGS will have a favorable response to corticosteroids and up to 
40% will have clinical benefit from treatment with a calcineurin inhibitor. Other agents that have been 
tried with marginal success include mycophenolate mofetil and rituximab. Patients with treatment-
resistant FSGS are at higher risk of progressing to ESKD.  
 
Adjuvant therapies designed to reduce proteinuria in primary FSGS are mainly focused on agents that 
inhibit renin-angiotensin system including angiotensin-converting enzyme inhibitors (ACEIs), angiotensin 
II receptor blockers (ARBs), or aldosterone antagonists. This is based on evidence that reduction in 
proteinuria in response to treatment is renoprotective and an independent predictor of kidney survival. 
These clinical data underscore the limitations of current therapy for FSGS and highlight the need to 
develop new strategies to safely and effectively achieve a reduction in proteinuria, either a partial or 
complete remission, preservation of kidney function and minimization of the adverse effects due to 
reduction of exposure to agents with little expectation of efficacy.  
 
While there are many insults that result in FSGS, molecular profiling can result in identifiable subgroups 
of affected patients with discrete pathophysiological mechanisms that may inform a precision medicine 
driven next generation of treatment strategies for individuals with FSGS and treatment resistant MCD.  
 
2.2 
RATIONALE  
Based on molecular profiling, informed therapy selection targeting the underlying disturbance has been 
used successfully in health care and biomedical research. When successful, this forms the basis of 
precision medicine for the treatment of selected patient populations.     
 
The Nephrotic Syndrome Study Network (NEPTUNE, PI M. Kretzler) study investigators have used 
genome wide expression profiles of renal biopsy specimens available from more than 250 patients to 
identify molecular subgroups of NS2. Three distinct patient subgroups were identified and associated 
with long-term outcomes. Molecular pathway analysis of the subgroups with TR-MCD and FSGS showed 
activation of TNF as defined by causal inference network analysis of established downstream pathway 
members, including TIMP1 and MCP1 in one of the 3 subgroups. These patients presented with higher 
levels of proteinuria, lower levels of GFR, and more rapid progression of their glomerular disease. A TNF 
Z-score was computed for each NEPTUNE participant with MCD and FSGS.  The score is the average Z-
score of gene expression level across 145 TNF pathway genes from the tubulointerstitial compartment 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
7 
of each participant’s renal biopsy.  Urinary MCP1 and TIMP1 were measured by ELISA from their 
baseline study visit and normalized to urine creatinine.  Correlations of TNF gene expression Z-score 
with urinary biomarkers are shown in Figure 1 and distributions of these measures in boxplots from TNF 
z-score < 0 vs > 0 are plotted in Figure 2. 
 
Figure 2:  Scatter plot of TNF z- score and urinary TIMP1/Cr, A: All FSGS and MCD patients (corr = 
0.47, P<0.001) TNF z-score and urinary MCP1/Cr, B: All FSGS and MCD patients (corr = 0.51, 
P<0.001) TNF z-score and urinary TIMP1/Cr ng/mg. (L. Mariani, et al 2017) 
A.                                                                            B.  
 
 
 
Figure 3:  Distribution of biomarker level by TNF Z-score category.  Difference in means was 
statistically significant (TIMP1, p-value 0.009; MCP1 p-value 0.001) (L. Mariani, et al 2017) 
 
 
A logistic model was fit with the outcome of TNF Z-score >0 to assess the predictive ability of urinary 
biomarkers individually and combined.  Results are shown in Table 1 for the subgroup of participants 
with eGFR >45.  Model 3, which included both biomarkers as predictors had the highest positive 
predictive value and correct classification.  Correctly classified was calculated by assigning those with an 
estimated probability of disease >= 50% as having disease and then calculating the % that are correctly 
classified as having and not having disease (true TNF Z-score > vs <= 0) using a patient sample with eGFR 
> 45 and either FSGS or MCD.  
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
8 
Table 1:  Results of Multivariable Logistic Models of TNF Z-score >0 in NEPTUNE FSGS and MCD 
participants with eGFR>45 
Patients with eGFR >45 
(n = 49; 13 Events) 
Model 1 
c-statistic 0.79 
PPV 33% 
correctly classified 69% 
Model 2 
c-statistic 0.79 
PPV 67% 
Correctly classified 78% 
Model 3 
c-statistic 0.82 
PPV 75% 
Correctly Classified 82% 
 
OR 
P-value 
OR 
P-value 
OR 
P-value 
Log2(uTIMP/Cr) 
1.79 
0.017 
-- 
-- 
1.54 
0.07 
Log2(uMCP/Cr) 
-- 
-- 
2.31 
0.008 
1.81 
0.08 
 
Because both biomarkers contributed to greater predictive accuracy, both will be measured for patient 
selection into the clinical trial.  The coefficients from the multivariable model will be used to calculate 
the odds of having a TNF Z-score >0 for each person.  Odds are then converted to probability and 
patients with a probability >= 50% of having a positive TNF Z-score would be eligible for enrollment.   
Mathematically, the following equations will be used to calculate the probabilities for study inclusion: 
1. Constant + [log2(uTIMP1/Cr) * Coef1] + [log2(uMCP1/Cr) * Coef2] = log Odds 
2. Exp(log Odds) = Odds 
3. Probability = Odds / (1 + Odds) 
 
Using the following coefficients and constant from Model 3 in Table 2: 
 
i.e., log Odds = -1.001269 + 0.59214 * log2(uTIMP1/Cr) +0.4325066 * log2(uMCP1/Cr). 
 
In NEPTUNE, these molecular pathway-defined patient subgroups characterized by high intra-renal TNF 
activation have documented persistence in proteinuria and reduced kidney function during prospective 
observation. Past studies of TNF inhibition in unselected treatment resistant FSGS patients 
demonstrated a reduction in proteinuria and preservation of estimated glomerular filtration rate (eGFR) 
in approximately 25% of studied patients3. In the FONT study, there were no clinical or laboratory 
findings that predicted response patterns to TNF-inhibition.  This study also identified the accelerated 
clearance of the TNF monoclonal antibody in this patient population with significant proteinuria 4-6. On 
average the Area Under the Curve (AUC) was 50% lower than that of comparison populations without 
proteinuria. In a parallel study of a small therapeutic compound, similar reduction in AUC in proteinuric 
patients with FSGS was documented suggesting that both large and small molecules are cleared more 
rapidly in patients with proteinuric kidney diseases such as FSGS and TR-MCD7. The recent findings 
within the NEPTUNE investigative community suggest that we may now have a mechanism to identify 
TNF-target relevant patients with primary nephrotic syndrome and use prior evidence to select a likely 
drug dosing regimen for this patient population.  
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
9 
2.3 
POTENTIAL RISKS AND BENEFITS  
 
2.3.1 KNOWN POTENTIAL RISKS  
SERIOUS INFECTIONS 
Some patients treated with Humira (adalimumab) are at increased risk for developing serious infections 
that may lead to hospitalization or death.  Most patients who developed these infections were taking 
concomitant immunosuppressants such as methotrexate or corticosteroids.  
 
Reported infections include: 
Active tuberculosis (TB), including reactivation of latent TB. Patients with TB have frequently presented 
with disseminated or extrapulmonary disease. Study participants will be tested for TB before starting 
Humira.  
 
Invasive fungal infections, including histoplasmosis, coccidioidomycosis, candidiasis, aspergillosis, 
blastomycosis, and pneumocystosis. Patients with histoplasmosis or other invasive fungal infections may 
present with disseminated, rather than localized, disease. Antigen and antibody testing for 
histoplasmosis may be negative in some patients with active infection. Consider empiric anti-fungal 
therapy in patients at risk for invasive fungal infections who develop severe systemic illness. 
Bacterial, viral, and other infections due to opportunistic pathogens, including Legionella and Listeria. 
 
MALIGNANCIES 
Lymphoma and other malignancies, some fatal, have been reported in children and adolescent patients 
treated with TNF blockers, including Humira. Postmarketing cases of hepatosplenic T-cell lymphoma 
(HSTCL), a rare type of T-cell lymphoma, have been reported in patients treated with TNF blockers, 
including Humira. These cases have had a very aggressive disease course and have been fatal. The 
majority of reported TNF blocker cases have occurred in patients with Crohn’s disease or ulcerative 
colitis and the majority were in adolescent and young adult males. Almost all of these patients had 
received treatment with azathioprine or 6-mercaptopurine concomitantly with a TNF blocker at or prior 
to diagnosis. It is uncertain whether the occurrence of HSTCL is related to use of a TNF blocker or a TNF 
blocker in combination with these other immunosuppressants. 
 
HYPERSENSITIVITY 
Anaphylaxis and angioneurotic edema have been reported following Humira administration. If a serious 
allergic reaction occurs, stop Humira and institute appropriate therapy. 
 
HEPATITIS B VIRUS REACTIVATION 
Use of TNF blockers, including Humira, may increase the risk of reactivation of hepatitis B virus (HBV) in 
patients who are chronic carriers. Some cases have been fatal. Study participants will be screened for 
HBV. Those with prior evidence of HBV infection will not be allowed to participate in this study.   
 
NEUROLOGIC REACTIONS 
TNF blockers, including Humira, have been associated with rare cases of new onset or exacerbation of 
central nervous system and peripheral demyelinating diseases, including multiple sclerosis, optic 
neuritis, and Guillain-Barré syndrome. Participants with these disorders will be excluded from the study. 
If any of these disorders develop, Humira will be discontinued.  
 
HEMATOLOGIC REACTIONS 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
10 
Rare reports of pancytopenia, including aplastic anemia, have been reported with TNF blockers. 
Medically significant cytopenia has been infrequently reported with Humira.  
 
CONGESTIVE HEART FAILURE 
Worsening and new onset congestive heart failure (CHF) has been reported with TNF blockers. Cases of 
worsening CHF have been observed with Humira. Participants with known CHF will be excluded from the 
study. 
 
AUTOIMMUNITY 
Treatment with Humira may result in the formation of autoantibodies and, rarely, in development of a 
lupus-like syndrome. Discontinue treatment if symptoms of a lupus-like syndrome develop. 
 
IMMUNIZATIONS 
Patients on Humira should not receive live vaccines. Pediatric patients, if possible, should be brought up 
to date with all immunizations before initiating Humira therapy. Adalimumab is actively transferred 
across the placenta during the third trimester of pregnancy and may affect immune response in the in 
utero exposed infant. The safety of administering live or live-attenuated vaccines in infants exposed to 
Humira in utero is unknown. Risks and benefits should be considered prior to vaccinating (live or live-
attenuated) exposed infants. 
 
ADVERSE REACTIONS 
The most common adverse reactions in Humira clinical trials (>10%) were: infections (e.g., upper 
respiratory, sinusitis), injection site reactions, headache, and rash. 
 
Directly relevant to this protocol, two small studies of TNF-inhibition with monoclonal antibody therapy 
in children and adults with multi-drug resistant FSGS have been reported.  
 
Table 3. FONT trial summary of reported adverse events in 17 treated patients 
Event  
Frequency (% of patients reporting) 
Severe Adverse Events 
 
   Infection with hospitalization 
30% (5/17)  
   Malignancy 
0 
   Death 
0 
Other Adverse Events 
 
   GI 
24% (4/17) 
 
Approaches to reduce risks associated with study drugs 
Due to the frequent and sometimes serious risks associated with the study drug, specific measures have 
been instituted to minimize these risks and improve the risk:benefit ratio for participants. Based on past 
experience with the administration of immunosuppressive therapies to patients with FSGS and TR-MCD, 
the inclusion and exclusion criteria have been selected to enrich for subjects most-likely to benefit from 
therapy (see Inclusion Criteria) and to avoid those subjects who are less-likely to benefit or more likely 
to experience treatment-related toxicity (see Exclusion Criteria).  Pregnancy status will be monitored 
throughout the study. In addition, frequent and routine laboratory monitoring will be carried out 
throughout the study protocol. The Clinical Investigator is responsible for reviewing and acting on 
adverse events in accordance with section 6.1.7.  
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
11 
2.3.2 POTENTIAL BENEFITS  
In previous studies 4 of 17 (24%) of unselected patients with multi-drug resistant FSGS experienced a 
50% reduction in proteinuria and preservation of eGFR with the use of TNF-inhibitor with monoclonal 
antibody therapy over a 16 week period5.  
 
3 
OBJECTIVES AND PURPOSE 
 
The primary purpose of this Proof of Concept study is to test the hypothesis that TNF inhibition with 
adalimumab (Humira) in patients with up-regulation of the intra-renal TNF pathway will result in 
reduction in urinary indicators of TNF pathway activation, specifically urine MCP1/Cr and TIMP1/Cr 
levels.   
 
Furthermore, this study is proposed to test the hypothesis that FSGS patients with up-regulation of the 
TNF pathway can be identified through intra-renal gene expression profiling and assay of urine MCP1/Cr 
and TIMP1/Cr levels by ELISA. 
 
4 
STUDY DESIGN AND ENDPOINTS 
 
4.1 
DESCRIPTION OF THE STUDY DESIGN 
 
This Proof of Concept, multi-center, open label, clinical trial addresses the treatment of patients with 
clinically active FSGS or TR-MCD. Up to eight patients with urinary MCP1/Cr and TIMP1/Cr urinary 
biomarker levels indicating intra-renal tumor necrosis factor (TNF) upregulation will be treated with 8 
weeks of adalimumab therapy to assure a minimum evaluable study sample of six patients. The Humira 
will be dosed based on weight and administered subcutaneously every two weeks for 8 weeks (5 total 
doses). The urinary biomarkers MCP1/Cr and TIMP1/Cr will be assessed throughout the study.  The 
primary assessment will be of the reduction in target engagement biomarker levels of urinary TIMP1/Cr 
or MCP1/Cr from baseline to study week 10. Key secondary outcomes include safety measures, serious 
adverse events, and adverse events.  
 
4.2 
ENDPOINTS 
 
4.2.1 PRIMARY ENDPOINT 
The change in urine MCP1/Cr and TIMP1/Cr levels from baseline to study week 10, i.e. 2 weeks after the 
final dose of adalimumab. 
 
4.2.2 SECONDARY ENDPOINTS 
(1) Incidence of serious adverse events, adverse events and abnormal laboratory tests 
(2) Percent change of eGFR from baseline to week 10  
(3) Percent change in UPC from baseline to week 10 
(4) Proportion of participants achieving a nadir UPC less than 1.5 g/g and at least a 40% reduction 
from baseline at week 10. 
 
4.2.3 EXPLORATORY ENDPOINTS 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
12 
(1) Correlation coefficient between intra-renal gene expression of TNF and urinary MCP1/Cr and 
TIMP1/Cr biomarker levels at baseline 
(2) Time to reduction of urinary MCP1/Cr and TIMP1/Cr biomarkers from baseline 
(3) Change in urinary EGF biomarker levels between baseline and week 10 
(4) Change in Quality of Life score between baseline and week 10. 
 
Endpoint Rationale:  
The target engagement biomarkers, urine MCP1/Cr and TIMP1/Cr, will be the primary endpoints of 
this study as we hypothesize that the levels of this biomarkers at baseline reflect TNF up-regulation 
in the kidney and after TNF inhibition will reflect target engagement. Neither eGFR nor urinary 
protein will be a primary endpoint measure. However, we will measure eGFR and urinary protein to 
provide estimates for future trials 
 
5 
STUDY ENROLLMENT AND WITHDRAWAL 
 
5.1 
PARTICIPANT INCLUSION CRITERIA 
1. Kidney biopsy confirmed FSGS or MCD  
2. For Minimal Change Disease patients only, history of resistance to corticosteroid therapy 
3. Increased urinary excretion of biomarkers of TNF activation (MCP1/Cr and/ or TIMP1/Cr) at 
study screening 
4. eGFR>30 ml/min/1.73 m2 at screening 
5. Urine protein:creatinine ratio ≥1.5 g/g at screening 
6. Age 6-80 years  
7. Weight >15kg 
8. Stable therapy with angiotensin converting enzyme inhibitors, angiotensin receptor blockers, 
and oral immunosuppression agents for at least 30 days prior to enrollment 
9. Birth control use in females of child bearing potential  
10. Informed consent and assent if applicable 
 
5.2 PARTICIPANT EXCLUSION CRITERIA 
1. Kidney or other solid organ or bone marrow transplant recipient 
2. Allergy or intolerance to investigational agent 
3. Secondary FSGS 
4. Severe obesity, i.e., BMI >40 kg/m2  
5. Live virus vaccine in the past 3 months 
6. Malignancy, current or in the past 5 years 
7. Active local or systemic bacterial, fungal or viral infection 
8. Active or latent Hepatitis B, Hepatitis C, HIV, or tuberculosis defined as quantiferon test positive   
9. History of demyelinating disease, e.g. Multiple Sclerosis or Guillain-Barre 
10. History of heart failure 
11. Active liver disease, defined as AST or ALT > 2.5x Upper Limit of Normal  
12. Systemic lupus erythematosus or ANA > 1:80 
13. History of inflammatory bowel disease, e.g. ulcerative colitis or Crohns disease 
14. Cyclophosphamide in past 90 days, Rituximab in the past 180 days 
15. Pregnancy or nursing 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
13 
16. Blood white blood cell count <4,500/mm3; Hg <9 g/dL; Platelet count <150,000/mm3 at 
enrollment. Use of an erythropoiesis stimulating agent will not be an exclusion criterion. 
17. Concurrent use of interleukin-1 antagonist (Anakinra), other TNF blocking agent, methotrexate 
or abatacept 
18. Diabetic nephropathy 
 
5.3 
STRATEGIES FOR RECRUITMENT AND RETENTION 
Patients will be recruited from the active population of patients with FSGS or TR-MCD w at participating 
sites. TR-MCD is defined as persistent proteinuria despite treatment. Following informed consent and 
when appropriate, assent, a screening visit will be conducted. Patients with elevated TIMP1/Cr and 
MCP1/Cr levels and meeting all other eligibility criteria will be scheduled to receive adalimumab every 
two weeks for 8 weeks (5 total doses). It is anticipated that only 25% of screened patients will be eligible 
for study participation based on TIMP1/Cr and MCP1/Cr levels. Eight eligible patients will begin study 
intervention with a goal to have a minimum of 6 patients fully completing the intervention phase and 
therefore contributing to the final analysis of the primary endpoint after 5 doses of adalimumab over an 
8 week period.  
Patients will be compensated for study participation to offset costs related to travel to study visits and 
incidentals.  
 
5.4 
PARTICIPANT WITHDRAWAL OR TERMINATION 
 
5.4.1 
REASONS FOR WITHDRAWAL OR TERMINATION 
Patients participating in this study may withdraw or be terminated for the following anticipated 
reasons.  
● Ongoing participation in this research study is voluntary and patients may leave at any time 
without an identified cause or the need for explanation.  
● Patients experiencing any clinical adverse event (AE), laboratory abnormality, or other 
medical condition or situation such that continued participation in the study would not be in 
the best interest of the participant (as determined by the patient and/or study physician). 
● Pregnancy and/or breast feeding 
● Absolute contraindications to continued therapy with Humira. 
 
5.4.2 
HANDLING OF PARTICIPANT WITHDRAWALS OR TERMINATION 
Should a participant prematurely withdraw or be withdrawn from the study for any reason, he/she will 
be asked to return for key outcome determinations at study week 10 and final safety assessment at 
study week 14.  At the week 10 visit any medication prescribed by their treating physician since leaving 
the study will be recorded, in addition to other required assessments. The participant’s reason for 
withdrawal and their status at the time of withdrawal will be recorded.  Should the participant die, the 
cause of death will be determined and recorded, if possible. This additional data will be utilized in the 
statistical analysis of the primary and secondary study outcomes as defined by the statistical plan. 
 
5.5 
PREMATURE TERMINATION OR SUSPENSION OF STUDY 
 
The investigators and/or other regulatory bodies reserve the right to terminate the study at any time. If 
this becomes necessary, appropriate procedures for continuing long-term follow-up and assuring the 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
14 
adequate treatment and safety of the participating subjects will be arranged after review and approval 
by the Institutional Review Boards. 
 
The DSMB will also provide external oversight concerning the safety and scientific integrity of the study 
for the duration of the clinical trial.  The DSMB will review the progress of the study toward meeting 
enrollment goals, adverse and serious adverse event profiles, at regular intervals to occur at least twice 
annually.  The DSMB may recommend at any time that the study should be terminated due to drug 
toxicity, patient safety, poor compliance and/or futility considerations. In such cases, their 
recommendations will be reviewed and discussed with the Executive Committee, which will make a final 
determination.   
 
6 
STUDY AGENT 
 
6.1 
STUDY AGENT(S) AND CONTROL DESCRIPTION 
 
6.1.1 ACQUISITION 
Each site will be responsible for acquiring and distributing Humira to the study participants in 
accordance with all local institutional policies governing the prescription of drugs for research 
purposes.  
 
6.1.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
Humira is supplied as a sterile, preservative-free solution of adalimumab for subcutaneous 
administration. The drug product is supplied as either a single-use, prefilled pen (Humira Pen), as a 
single-use, 1 mL prefilled glass syringe, or as a single-use institutional use vial. Enclosed within the 
pen is a single-use, 1 mL prefilled glass syringe. The solution of Humira is clear and colorless, with a 
pH of about 5.2. 
 
Formulation and Strength: Any of the commercially available Humira formulations may be used in 
this study:  
●  Each 40 mg/0.4 mL prefilled syringe or prefilled pen delivers 0.4 mL (40 mg) of drug 
product.  
● Each 40 mg/0.8 mL prefilled syringe or prefilled pen delivers 0.8 mL (40 mg) of drug product. 
● Each 20 mg/0.2 mL prefilled syringe delivers 0.2 mL (20 mg) of drug product. 
● Each 20 mg/0.4 mL prefilled syringe delivers 0.4 mL (20 mg) of drug product. 
 
Labeling On-site: At the Clinical Site Research Pharmacy, patient specific labeling will be applied to 
Humira as this study drug will be provided in an open-label format to study participants. In addition 
to patient specific labeling information, the drug name, concentration, and date of preparation will 
be on the label prior to distribution. In accordance with §312.6 the drug will also contain a label with 
the statement “Caution: New Drug – Limited by Federal (or United States) law to investigational 
use.” 
 
6.1.3 PRODUCT STORAGE AND STABILITY 
Humira must be refrigerated at 36°F to 46°F (2°C to 8°C). DO NOT FREEZE. Do not use if frozen even if it 
has been thawed. Store in original carton until time of administration to protect from light. If needed, 
for example when traveling, Humira may be stored at room temperature up to a maximum of 77°F 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
15 
(25°C) for a period of up to 14 days, with protection from light. Humira should be discarded if not used 
within the 14-day period. Do not use beyond the expiration date on the container. Record the date 
when Humira is first removed from the refrigerator in the spaces provided on the carton and dose tray. 
Do not store Humira in extreme heat or cold. 
 
6.1.4 DOSING 
Humira will be dosed based on weight at study weeks 0, 2, 4, 6, and 8. This dose has been selected in 
accordance with the recommended dose of Humira for patients 2 years of age and older with 
polyarticular juvenile idiopathic arthritis.  
 
Subject Weight 
Humira dose (every other week) 
15kg to <30kg 
20mg  
>30kg 
40mg  
 
6.1.5 ROUTE OF ADMINISTRATION 
Humira will be given subcutaneously at every other week for 8 weeks (5 total doses).  
 
6.1.6 DOSE ADJUSTMENTS/MODIFICATIONS/DELAYS 
If there is evidence of toxicity in abnormal laboratory tests or clinical adverse events that, in the 
judgment of the Investigator, could place the subject at increased risk, study drug administration should 
be interrupted and the investigator should notify the trial principal investigator at University of 
Michigan. Subjects may be considered eligible to receive further study medication treatment only after 
discussion with the study principal investigator at the University of Michigan.  
 
6.1.7 DURATION OF THERAPY 
The total duration of therapy will be 8 weeks with a single dose study drug given subcutaneously at 
study weeks 0, 2, 4, 6, and 8. 
 
6.2 
STUDY AGENT ACCOUNTABILITY PROCEDURES 
 
6.2.1 RESPONSIBILITIES OF THE CLINICAL SITE PHARMACY 
The dispensing pharmacy at each site will maintain an independent drug accountability log for all 
Humira that is dispensed for this study. The log must include at a minimum: the date dispensed, drug 
expiration date, lot number or other identifying number, and study participant receiving the drug.  
 
6.2.2 THE SITE INVESTIGATOR RESPONSIBILITY 
The site team will maintain a drug accountability log for each study patient to document subject 
number, visit number, date medication dispensed, and date medication returned. Any unused 
medication should be returned to the study site, documented, and disposed of in accordance with local 
policy.  
 
7 
STUDY PROCEDURES AND SCHEDULE 
 
7.1 
STUDY PROCEDURES/EVALUATIONS 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
16 
Biopsy report: The biopsy report from the qualifying kidney biopsy will have personal identifiers 
redacted, labeled with the study identification number and then submitted into the study database for 
review and confirmation of eligible pathology of FSGS or MCD. 
Archived Kidney Biopsy Tissue: 4 unstained slides from archived paraffin block kidney tissue will be 
submitted for gene expression profiling when available. Patients who have submitted tissue from a 
kidney biopsy as part of NEPTUNE study participation will not be required to have additional tissue 
submitted as part of this study. 
Health Related Quality of Life (HRQOL): The FSGS-MCD Patient Reported Outcome (PRO) assessment 
will be assessed in accordance with the schedule of events table (7.2.5). The FSGS-MCD PRO assessment 
should be completed by the participant at each in-person study visit.  
Adverse events: Adverse event assessments will be done at each study visit as detailed in Section 9.  
Complete medical history: The complete medical history will include a review of pertinent medical 
records covering details related to the patient’s history of FSGS or TR-MCD provided by the patient 
and/or their referring physician(s). 
Physical examination: A physical exam will include a review of systems and current physical findings 
Interval medical history: A review of medications, interval symptoms, and any changes to the patient’s 
medical history will be reviewed and documented.   
Vital signs: Vital signs will include: blood pressure, temperature (oC), height (cm), and weight (kg) will be 
measured. 
 
Pregnancy Testing: For study participants of child-bearing potential, a urine pregnancy test will be 
performed at screening and each study visit prior to adalimumab administration.  
Clinical Laboratories: A comprehensive set of laboratory results will be performed on blood and urine 
samples to evaluate any toxicity or adverse events associated with FSGS or TR-MCD or its treatment 
with the study drug.  
● Comprehensive metabolic panel (Comp): including serum electrolytes, BUN, creatinine, glucose, 
albumin, ALT, AST, alkaline phosphatase, total bilirubin, cholesterol, total protein 
● Complete blood count with differential (CBCD): including WBC, hemoglobin, hematocrit, 
platelet count, and differential cell count.  
● Urine protein/creatinine ratio: 24-hour urine collection preferred, but first morning urine or 
spot urine is acceptable  
 
Assessment of Infections:   
● Hepatitis B: HBsAg, HBcAb IgG testing will be conducted at screening for all potentially eligible 
participants unless hepatitis screening has been conducted clinically within the past 45 days 
with documentation of results.  
● Hepatitis C:  HCAb screen will be conducted at screening for all potentially eligible participants 
unless hepatitis screening has been conducted clinically within the past 45 days with 
documentation of results.  
● HIV -1/HIV-2 antibody differentiation immunoassay will be conducted at screening for all 
potentially eligible participants unless HIV screening has been conducted clinically within the 
past 45 days with documentation of results.  
● Tuberculosis: Quantiferon for tuberculosis should be measured unless tuberculosis screening 
has been conducted clinically within the past 45 days with documentation of results.  
 
Serology: 
● Antinuclear cytoplasmic autoantibody (ANA): should be measured at screening unless 
conducted clinically within the past 45 days with documentation of results. 
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
17 
Urine collection for target engagement biomarkers: A spot urine sample will be collected at each in 
person study visit before the administration of the study drug. 
Optional Biological Specimen Repository: 10ml of Blood (serum, plasma, RNA and DNA (8.5 ml at the 
screening visit only)) and urine samples will be collected at each study visit for storage at the University 
of Michigan Biorepository. The specimens will be made available for approved investigations for future 
kidney disease related research.   
Study drug reconciliation:  Document subject number, visit number, date medication dispensed, and lot 
number at each visit in accordance with section 6.2. 
Study drug administration: At week 0 the study team will administer Humira to the subject, and 
monitor the subject for 15 minutes post injection, recording blood pressure and heart rate at 15 minutes 
post injection. Subject/guardian will be taught how to administer Humira to themselves or their child. At 
week 2, the subject/guardian should administer Humira while study staff observes to ensure proper 
technique. An ice pack may be applied to the injection site to alleviate pain associated with the injection 
as needed.  
 
7.2 
STUDY SCHEDULE 
The complete schedule for study visits is detailed below and summarized in the Study Flow Diagram 
(Figure 1) and Table of Study Visits (see below Schedule of Events, Section 7.2.5) 
 
7.2.1 SCREENING VISIT 
The screening visit will take place preferably within 2 weeks, but no more than 6 weeks prior to the 
baseline visit (Week 0) 
At the screening visit: 
a. Patient/Parent provides written informed consent and HIPAA authorization prior to any 
evaluation or testing. Minor participants should provide assent in accordance with IRB 
guidelines 
b. A review of existing medical records and tests, provided by the patient or referring 
physician, will be performed to document the history of FSGS or TR-MCD and general 
medical history. 
c. Vital signs including height, weight, temperature and blood pressure are obtained. 
d. The patient undergoes a physical examination. 
e. The patient has blood and spot urine collected for monitoring labs, including urine 
pregnancy test (if applicable). 
f. A spot urine sample is obtained for measurement of target engagement biomarkers 
g. If the patient consents, biorepository samples should be obtained 
h. Archived kidney biopsy tissue is requested to be sent [when available and if not already 
submitted to the NEPTUNE study] to the laboratory at the University of Michigan for 
gene expression profiling for mechanisms of disease 
i. 
Archived kidney biopsy report is anonymized and uploaded into the study electronic 
data capture system for central review  
 
At Screening, a preliminary evaluation is performed and once screening is complete, if all inclusion and 
no exclusion criteria are met, the patient is deemed eligible and will be asked to return for the baseline 
visit for study drug initiation.  Subjects who have not met study enrollment criteria will be considered a 
screen failure and their study participation will end. 
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
18 
Repeat Screening. If the patient is found to be ineligible, a repeat screening visit may be performed at 
the local clinician investigator’s discretion.  
 
Once eligibility is confirmed, the patient should be instructed to bring a 24-hour urine specimen to the 
baseline visit.  
 
7.2.2 BASELINE/WEEK 0 
The Baseline visit/Week 0 will occur within 6 weeks of the screening evaluation.  The following 
procedures will take place at the baseline visit: 
a) Interval medical history 
b) Review of concomitant medications 
c) Physical examination  
d) Assessment of adverse events 
e) Vital signs will be obtained including weight, blood pressure, and temperature  
f) Subject to complete HRQOL assessment  
g) 24-hour urine or first morning urine will be collected from the patient, along with blood 
samples for clinical laboratories, including urine pregnancy (if applicable) 
h) Spot urine collection for biomarkers (TIMP1/Cr and MCP1/Cr).   
i) 
Blood collection for the Biological Specimen Repository 
j) 
Humira will be injected subcutaneously, monitor subject for 15 minutes post injection 
k) Record heat rate and blood pressure 15 minutes post injection 
l) 
Teaching on how to administer subcutaneous injections (to self or minor child) 
m) Remind subject to bring a 24-hour urine sample (or first morning urine if 24 hour sample is not 
feasible) to the next study visit 
 
7.2.3 FOLLOW-UP  
Follow-up Visits (Weeks 2 and 8) 
The following procedures will take place at each of the follow up visits 
a) Interval medical history 
b) Review of concomitant medications 
c) Physical examination  
d) Assessment of adverse events 
e) Vital signs obtained including weight, blood pressure, and temperature 
f) Subject to complete the HRQOL assessment  
g) 24-hour urine or first morning urine will be collected from the patient, along with blood 
samples for clinical laboratories, including urine pregnancy (if applicable) 
h) Spot urine collection for biomarkers (TIMP1/Cr and MCP1/Cr) 
i) 
Blood collection for the Biological Specimen Repository 
j) 
Subject to administer Humira to themselves (or child) under the guidance of study staff 
k) Remind subject to bring a 24-hour urine sample (or first morning urine if 24 hour sample is not 
feasible) to the next study visit 
 
Endpoint Visit (Weeks 10) 
The week 10 follow-up visit includes a combination of toxicity monitoring and outcome 
assessments.  
a) Interval medical history 
b) Review of concomitant medications 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
19 
c) Physical examination    
d) Assessment of adverse events 
e) Vital signs obtained including weight, blood pressure, and temperature 
f) Subject to complete the HRQOL assessment  
g) 24-hour urine or first morning urine will be collected from the patient, a fresh spot urine will be 
collected during the visit along with blood samples for clinical laboratories, including urine 
pregnancy (if applicable) 
h) Spot urine collection for biomarkers (TIMP1/Cr and MCP1/Cr)  
i) 
Blood biorepository sample collection  
 
Safety Visit (Week 14) Phone visit or optional in-person study visit may be preformed 
a) Interval medical history 
b) Review of concomitant medications 
c) Assessment of adverse events  
d) If the optional in-person visit is scheduled: 
a. Physical examination 
b. Vital signs obtained including weight, blood pressure, and temperature 
c. Blood sample for clinical laboratories 
d. 24-hour urine or first morning urine will be collected from the patient, a fresh 
spot urine will be collected during the visit along with blood samples for clinical 
laboratories, including urine pregnancy (if applicable) 
      
7.2.4 EARLY TERMINATION VISIT 
Should a participant prematurely withdraw or be withdrawn from the study for any reason he/she will 
be asked to return for the key outcome determinations (week 10 assessments), as detailed above. In 
addition, at that time, any medication prescribed by their treating physician since leaving the study will 
be recorded. The participant will also be asked to participate in additional safety monitoring visits (week 
14) as described in 7.2.3 if at least one dose of the study medication was administered. Should the 
participant die, the cause of death will be determined and recorded, if possible. This additional data will 
be utilized in the statistical analysis of the primary and secondary study outcomes and any missing data 
handled by appropriate statistical methods. 
 
7.2.5 SCHEDULE OF EVENTS TABLE 
 
 
Screening 
Week 0 
Week 2 
Week 8 
Week 10 
Week 14 
Visit window 
+/-2 
DAYS 
+/-2 
DAYS 
+/-2 
DAYS 
+/-2 
DAYS 
+/-2 
DAYS 
+/-4 
DAYS 
Consent 
X 
 
 
 
 
 
Medical History  
X 
X 
X 
X 
X 
X 
Archived Kidney Biopsy Report 
Submission 
X 
 
 
 
 
 
Archived Kidney Tissue 
sections submitted, if 
available* 
 
 
 
 
 
 
Concomitant Medications 
X 
X 
X 
X 
X 
X 
Physical Exam 
X 
X 
X 
X 
X 
X** 
Adverse Events Assessment 
 
X 
X 
X 
X 
X 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
20 
Vital Signs 
X 
X 
X 
X 
X 
X** 
HRQOL assessment 
 
X 
X 
X 
X 
 
Comprehensive Metabolic 
Panel 
X 
X 
X 
X 
X 
X** 
CBC with Platelets and 
Differential 
X 
X 
X 
X 
X 
X** 
Urine Protein:Creatinine [24 
hour sample preferred] 
X 
X 
X 
X 
X 
X** 
Assessment of Infections 
X 
 
 
 
 
 
MCP1/TIMP1 (spot urine) 
X 
X 
X 
X 
X 
 
Pregnancy Test 
X 
X 
X 
X 
 
 
Biorepository specimen 
X*      
X 
X 
X 
X 
 
Adalimumab dosing 
 
X 
X 
X 
 
 
* Do not submit if most recent biopsy specimen has already been submitted to NEPTUNE study; *DNA 
collected at screening only 
** Optional procedures required if study visit is conducted in-person 
 
7.3 
JUSTIFICATION FOR SENSITIVE PROCEDURES 
HIV, hepatitis B and hepatitis C testing is a part of the screening procedures as therapy with adalimumab 
is contraindicated for persons with active infections. Should HIV, hepatitis B, hepatitis C or 
tuberculosis/quantiferon testing be positive, the patient will be referred for clinical care through local 
treatment experts according to local standards of care and excluded from the study.  
7.4 
CONCOMITANT MEDICATIONS, TREATMENTS, AND PROCEDURES  
All concomitant prescription, over-the-counter and non-prescription medications taken during study 
participation will be recorded on the case report forms (CRFs) and checked for compatibility with the 
study drugs as defined in Section 7.5.  
 
7.5 
PROHIBITED MEDICATIONS, TREATMENTS, AND PROCEDURES  
Patients should not receive live vaccines while taking Humira and for 3 months following the final 
adalimumab dose. Participants should not take anakinra, abatacept, or rituximab in combination with 
Humira.  
 
7.6 
PARTICIPANT ACCESS TO STUDY AGENT AT STUDY CLOSURE  
Humira is approved by the FDA for use in other health conditions. Consequently, it may be prescribed 
off label by the patient’s local nephrologist per local clinical decision making. 
 
8 
ASSESSMENT OF SAFETY 
All adverse events, whether serious or non-serious, related or not related, following exposure to 
adalimumab are to be documented by the investigator and recorded in the CRF and in the subject’s 
source records. Investigators must record in the CRF their opinion concerning the relationship of the 
adverse event to the study drug. 
  
All reported serious and non-serious adverse events should be followed up in accordance with clinical 
practice. 
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
21 
8.1 
SPECIFICATION OF SAFETY PARAMETERS 
 
8.1.1 DEFINITION OF ADVERSE EVENTS (AE) 
An adverse event is any untoward medical occurrence in a clinical study subject administered a 
medicinal (investigational or non-investigational) product. An adverse event does not necessarily have a 
causal relationship with the treatment. An adverse event can therefore be any unfavorable or 
unintended sign (including an abnormal finding), symptom, or disease temporally associated with the 
use of a medicinal (investigational or non- investigational) product, whether or not related to that 
medicinal (investigational or non-investigational) product. (Definition per International Conference on 
Harmonisation [ICH]) 
 
This includes any occurrence that is new in onset or aggravated in severity or frequency from the 
baseline condition, or abnormal results of diagnostic procedures, including laboratory test 
abnormalities. 
 
Adverse events may include: 
• Symptoms described by the patient 
• Clinically significant changes in the patient’s physical exam or other signs observed by the investigator 
or medical staff 
• Test abnormalities (laboratory tests, ECG, X-rays, etc.) that reflect a change from baseline and/or that 
may result in changes in administration of investigational product or in an alteration in medical care 
(diagnostic or therapeutic) 
• Conditions present at baseline that have either worsened or recurred following resolution  
 
The patient will be evaluated for new AEs and the status of existing AEs at each study visit, or at any 
time contact is made with the patient outside of a scheduled visit. The Investigator may elicit symptoms 
using an open-ended question, followed by appropriate questions that clarify the patient’s description 
of AEs. 
 
8.1.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE) 
An SAE is an AE that results in any of the following: 
• Death: the patient died as a result of the event. The cause of death of a subject in a study within 30 
days of the last dose of study drug, whether or not the event is expected or associated with the study 
drug, is considered a serious adverse event. 
• Is life-threatening: An AE that places the patient, in the view of the Investigator or sponsor, at 
immediate risk of death from the AE as it occurred, i.e., does not include an AE that had it occurred in a 
more severe form, might have caused death. 
• Requires inpatient hospitalization or prolongation of existing hospitalization: 
o Note: Planned or elective hospital admissions for treatment/procedures for a condition/disease 
that existed prior to signing the informed consent should be recorded in the patient’s screening 
documents and will not be captured as SAEs. If, however, the admission or procedure occurs 
other than as planned due to a worsening of the condition, then the event should be recorded 
as a SAE. Hospitalizations not intended to treat an acute illness or adverse event (e.g., social 
reasons such as pending placement in long-term care facility) should not be recorded as an SAE. 
• Persistent or significant disability/incapacity: An AE that results in a substantial disruption of a person’s 
ability to conduct normal life functions. 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
22 
• Pregnancy, or Congenital anomaly/birth defect: A pregnancy in an individual receiving the 
investigational product or congenital anomaly/birth defect that occurs in the offspring of a patient 
exposed to the investigational product. 
• Other Medically Important Events: An AE that may not result in death, be life threatening, or require 
hospitalization may be considered a serious adverse event when, based upon appropriate medical 
judgment, the event may jeopardize the patient and may require medical or surgical intervention to 
prevent one of the outcomes listed in this definition 
Medical and scientific judgment should be exercised in deciding whether expedited reporting is also 
appropriate in other situations, such as important medical events that may not be immediately life 
threatening or result in death or hospitalization but may jeopardize the subject or may require 
intervention to prevent one of the other outcomes listed in the definition above. These should usually 
be considered serious. 
Any situation that meets the criteria of a serious adverse event should be recorded on a Serious 
Adverse Event Report Form and be reported within 24 hours of becoming aware of the event to the 
IRB (per local policies), and the study Principal Investigator. 
All serious adverse events that have not resolved by the end of the study, or that have not resolved 
upon discontinuation of the subject's participation in the study, must be followed until any of the 
following occurs: 
● The event resolves 
● The event stabilizes 
● The event returns to baseline, if a baseline value/status is available 
● The event can be attributed to agents other than the study drug or to factors unrelated to study 
conduct 
● It becomes unlikely that any additional information can be obtained (subject or health care 
practitioner refusal to provide additional information, lost to follow-up after demonstration of 
due diligence with follow-up efforts) 
 
8.1.3 PREGNANCY 
Females of child bearing potential must use birth control for 30 days prior to the first dose of 
adalimumab through 30 days after the last dose of adalimumab.  
All initial reports of pregnancy must be reported within 24 hours of becoming aware of the event using 
the Serious Adverse Event Form. Abnormal pregnancy outcomes (e.g. spontaneous abortion, fetal death, 
stillbirth, congenital anomaly, ectopic pregnancy) are considered serious adverse events and must be 
reported using the Serious Adverse Event Form.  
Follow-up information regarding the outcome of the pregnancy and any postnatal sequelae in the infant 
will be required. 
 
8.2 
CLASSIFICATION OF AN ADVERSE EVENT 
 
8.2.1 SEVERITY OF EVENT 
Severity indicates the intensity of the event and should not be confused with seriousness (see sections 
8.1.1 and 8.1.2 for assessing seriousness). 
Severity Grading: The Investigator will assess the severity of all AEs/SAEs as Mild, Moderate, or Severe, 
based on the following definitions. 
Definitions: 
• Mild – Awareness of the event but easily tolerated 
• Moderate – Discomfort enough to cause some interference with usual activity 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
23 
• Severe – Inability to carry out usual activity 
 
8.2.2 RELATIONSHIP TO STUDY AGENT 
Assessment of the relationship between the AE and exposure to the investigational product is important 
for regulatory reporting and assists in the overall analysis of the safety information. For each AE/SAE the 
Investigator must determine whether, based on available evidence, there is a reasonable possibility that 
the study drug caused the event. Causal relationship to study drug administration is based on the 
following definitions: 
• None: No relationship between the event and the administration of study drug. The event is related to 
other etiologies, such as concomitant medications or the patient’s clinical state. 
• Unlikely: The current state of knowledge indicates that a relationship to study drug is unlikely or the 
temporal relationship is such that study drug would not have had any reasonable association with the 
observed event. 
• Possible: A reaction that follows a plausible temporal sequence from administration of the study drug 
and follows a known response pattern to the suspected study drug. The reaction might have been 
produced by the patient’s clinical state or other modes of therapy administered to the patient. 
• Related: The administration of study drug and AE were reasonably related in time and the AE was 
more likely explained by exposure to the study drug than by other causes. There is clear evidence to 
suggest a causal relationship, and other possible contributing factors can be ruled out. 
A causality assessment must be provided for each AE/SAE recorded, even if there is only limited 
information at the time. Upon receipt of follow-up information, the Investigator may change his/her 
assessment of causality and amend the AE/SAE report accordingly.  
For the purpose of safety analyses, all AEs that are classified as possible will be considered treatment-
related events. 
8.2.3 EXPECTEDNESS  
An adverse event is considered unexpected if the nature or severity is not consistent with the applicable 
product reference safety information. See Appendix A.  
 
8.3 
TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
Participants should be monitored for adverse events throughout the study with the final assessment 
occurring at week 14.  
 
8.4 
REPORTING PROCEDURES 
 
8.4.1 ADVERSE EVENT REPORTING  
All AEs will be captured from the time informed consent is signed through the week 14 visit. AEs should 
be recorded using appropriate medical terminology. When recording, it is preferable to provide a 
diagnosis. Sufficient information should be sought to assist the Investigator both in determining the 
diagnosis and in making a causality assessment.  
Reporting should not be delayed pending receipt of all required information. If information is 
unavailable at the time of the initial report, the Investigator is expected to follow-up until the required 
information has been obtained 
 
8.4.2 SERIOUS ADVERSE EVENT REPORTING  
All SAEs including pregnancy must be reported to the study Coordinating Center University of Michigan 
through the Electronic Data Capture System, notification of the study Principal Investigator, and the 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
24 
study IRB of record within 24 hours of the Investigator’s first knowledge of the event, regardless of 
causal relationship.  SAEs will be reported to the FDA in accordance with §312.32.  
• A completed Clinical Study SAE Report Form containing a detailed written description of the event 
along with available supporting documents (e.g., discharge summary, autopsy report, diagnostic test 
results, etc.) should be submitted to the data coordinating center 
• Additional information that is not available at the time the initial SAE Report Form was completed, 
must be promptly reviewed and provided to The University of Michigan within 48 hours of receipt. Full 
supporting documentation should be solicited by the investigative site even if the SAE occurred at 
another institution. Such documentation may include copies of relevant patient/hospital records, 
discharge summaries, laboratory/test results or autopsy reports. 
• If, at any time after the patient has completed participation in the study, the Investigator or study staff 
becomes aware of an SAE that they suspect is related to the investigational product, then the event and 
any known details must be reported promptly to the University of Michigan based Principal Investigator 
and coordinating center. 
 
8.5 
STUDY HALTING RULES  
As this study intends to treat a maximum of 8 patients in this study protocol, no halting rules have been 
defined a priori. Routine study safety monitoring will be performed by the study Data Safety and 
Monitoring Board.  
 
8.6 
SAFETY OVERSIGHT 
Safety oversight will be provided by the study DSMB. Due to the small sample size, the DSMB chair will 
be notified of Serious Adverse Events until completion of the trial. The DSMB will review the trial 
protocol prior to enrollment initiation and with any substantial protocol amendments. Safety outcomes 
will be presented at each DSMB meeting. These include number of and proportion of subjects with SAEs 
(including segregation of those involving deaths), treatment emergent AEs, and discontinuation of study 
medication due to AEs. These presentations will be descriptive, with no formal inferential methods used.  
Serious Adverse Events and related or possibly related unexpected adverse events will be reported to 
the study IRB of record according to their reporting requirements. Participating trial sites will be notified 
of these events with an indication if a change in consent form is recommended. 
 
9 
CLINICAL MONITORING 
Monitoring is the act of overseeing the progress of the study and of ensuring that it is conducted, 
recorded and reported in accordance with the protocol, standard operating procedures, good clinical 
practice standards, and the applicable regulatory requirements. Given the sample size, monitoring for 
this study will be done remotely. 
Data entered into the case report forms will be reviewed for completion, integrity, and quality. Data 
reports will be shared with the sites and the steering committee. Data to be reviewed includes: 
enrollment, visit completion, data completeness, outstanding queries, biosample collection and 
shipping. 
The study monitoring staff may conduct site management calls to ensure data compliance and data query 
resolution. Other topics may include: 
• 
Protocol adherence 
• 
Recruitment and retention strategies 
• 
Regulatory document requirements 
• 
Completeness of visits, forms, data, and samples 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
25 
• 
Responses to data queries 
• 
Electronic Case Report Forms (eCRFs) and source documents 
• 
Study-specific training  
 
10 
STATISTICAL CONSIDERATIONS  
 
10.1 
STATISTICAL AND ANALYTICAL PLANS 
This is a pilot, proof of concept investigation to test the proposed biomarkers as an indicator of target 
engagement.  As such, no formal sample size calculations are performed and analyses are primarily 
descriptive in nature.  Our approach will be to assess the biomarker change over time with TNF 
inhibition therapy within each patient and as a group. As the Elisa testing for this indication is new, the 
protocol will use an exploratory approach to assess individual and panel biomarker change and 
correlation with clinical indicators of disease, including proteinuria, eGFR, and serum albumin.  
A statistical analysis plan (SAP) will be written for the study that contains detailed descriptions of the 
analyses to be performed.  The SAP will be finalized prior to database lock. 
 
10.2 
ANALYSIS DATASETS 
All eligible participants who receive at least one dose of adalimumab (as-treated population) will be 
analyzed for primary, secondary and exploratory endpoints.   
 
10.3 
DESCRIPTION OF STATISTICAL METHODS 
 
10.3.1 GENERAL APPROACH 
Analyses are primarily descriptive in nature in this pilot study.   Continuous variables will be summarized 
using descriptive statistics including n, mean, median, standard deviation, interquartile range (i.e., 1st 
and 3rd quartiles), and range (i.e., minimum and maximum). Qualitative variables will be summarized 
using counts and percentages.  Unless otherwise specified, statistical analyses will be performed using 
SAS Version 9 or higher. Where appropriate, statistical tests will be conducted at the 0.05 significance 
level using two‐tailed tests and p‐values will be reported. 
 
10.3.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S) 
The change in urine MCP1/Cr and TIMP1/Cr levels from baseline to study week 10, i.e. 2 weeks after the 
final dose of adalimumab, are the primary endpoints.  We will provide tabular and graphical summaries 
of the changes from baseline to week 10 in MCP1/Cr and in TIMP1/Cr, including summaries at baseline 
and week 10. The changes from baseline to week 10 in urine MCP1/Cr and TIMP1/Cr levels will be tested 
using the paired t-test or Wilcoxon signed-rank test (if distributional assumptions are not met). 
 
10.3.3 ANALYSIS OF THE SECONDARY ENDPOINT(S) 
Discrete outcomes (e.g., AEs, SAEs, proportion of participants achieving a nadir UPC <1.5 g/g and <40% 
from baseline) will be summarized descriptively.  Continuous outcomes (e.g., changes in eGFR, change in 
proteinuria) will be summarized descriptively, including baseline, follow-up and changes from baseline.     
 
10.3.4 SAFETY ANALYSES 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
26 
Safety data, including frequency of events and proportion of participants experiencing events (e.g., 
treatment-emergent AEs and SAEs by body system, severity and relatedness to adalimumab), clinical 
laboratory tests, vital signs, and physical examinations, will be summarized descriptively.   
 
10.3.5 ADHERENCE AND RETENTION ANALYSES 
Subject disposition, including descriptive statistics on-treatment and on-study follow-up, will be 
reported.  Listings of subject disposition, including reasons for early termination, will be reported.   
Other disposition and study conduct information, including major protocol violations, will be 
summarized. 
 
Compliance with study medication will be assessed and summarized, including the proportion of 
participants who adhered to study treatment, the median duration of adherence to study treatment and 
the proportion of participants who permanently discontinued study medication.   
 
10.3.6  BASELINE DESCRIPTIVE STATISTICS 
Demographic data and baseline characteristics will be summarized. 
 
10.3.7 TABULATION OF INDIVIDUAL RESPONSE DATA 
Listings of SAEs, AEs and AEs resulting in treatment discontinuation will be provided. 
 
10.3.8 EXPLORATORY ANALYSES 
Spearman correlations and scatterplots will be provided to assess the correlation of intra-renal gene 
expression of TNF and urine biomarker levels at baseline.  Continuous outcomes (e.g., changes in QOL 
outcomes, EGF biomarker levels) will be summarized descriptively, including baseline, follow-up and 
changes from baseline.   
 
10.4 
SAMPLE SIZE 
No formal sample size calculations were performed (either for estimation or hypothesis testing) because 
this study is a pilot investigation of urine biomarkers for target engagement of TNF inhibition. Rather, 
we assessed the magnitude of effects that can be detected in urine biomarkers using a paired t-test.  
With 6 patients, 80% power, and a two-sided type I error of 10%, we can detect an effect size (change 
from baseline to week 10 / SD) of 1.2 of changes in uTIMP1/Cr and of changes in uMCP1/Cr, with no 
adjustments for multiplicity (EAST 6.2).  
 
11 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS 
Every participating clinical site will maintain appropriate medical and research records for this trial, in 
compliance with GCP, regulatory and institutional requirements for the protection of confidentiality of 
participants. Participating clinical sites will also obtain institutional authorization for external monitoring 
by the Data Coordinating Center to examine (and when permitted by applicable law, to copy) clinical 
records for the purposes of quality assurance reviews, audits, and evaluation of the study safety, 
progress, and data validity.  
 
Source data are defined as all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial. Examples of these 
original documents and data records include, but are not limited to, hospital records, clinical and office 
charts, laboratory notes, memoranda, participant’s memory aids or evaluation checklists, pharmacy 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
27 
dispensing records, recorded audio tapes, recorded data from automated instruments, copies or 
transcriptions certified after verification as being accurate and complete, microfiches, photographic 
negatives, microfilm x-rays, and participant files and records kept at the pharmacy, at the laboratories, 
and medico-technical departments involved in the clinical trial. It is acceptable to use CRFs as source 
documents when the data is collected and recorded there as the primary source of information, but 
CRFs will not constitute the only form of source document information for this trial. 
 
12 
ETHICS/PROTECTION OF HUMAN SUBJECTS  
 
12.1 
ETHICAL STANDARD  
The study will be monitored by clinical study monitors to assess adherence to good clinical practice and 
to this clinical study protocol. 
 
The study may be audited by the regulatory authorities, the IRB, and/or the US Food and Drug 
Administration. Direct access to these documents must be guaranteed by the Investigator, who must 
provide support at all times for these activities. 
 
12.2 
INSTITUTIONAL REVIEW BOARD  
This protocol, investigator’s brochure, informed consent documents, and any other patient facing 
materials must be reviewed and approved in accordance with the IRB of Record’s policies prior to any 
study procedures taking place. 
 
12.3 
INFORMED CONSENT PROCESS  
Each site is responsible for ensuring that informed consent and assent, when applicable, is obtained 
from each patient using a current consent form according to the guidelines of its local IRB and in 
accordance with the Common Rule (45 CFR Part 46 subpart A, Protection of Human Subjects). The 
informed consent (ICF) and assent forms must be signed and dated by the patient or the legal guardian 
if applicable prior to initiation of any study-related activity.  During the consent process, the study team 
will discuss the implications of participation in this study and that the drug dosing under study has not 
been studied previously. All participant questions should be fully answered before the ICF is signed. The 
signed ICF should be placed in the subject’s research file and copies should be provided to the study 
participant as well as placed in the participant’s medical chart according to local regulatory 
requirements.  
 
The ICF as well as any other patient-facing documents, such as the quality of life forms or recruitment 
materials must be approved in accordance with the site’s local IRB requirements. 
Consenting Non-English Speaking Participants: Obtaining consent from non-English speaking participants 
should be conducted in accordance with IRB guidelines.  
 
12.4 
PARTICIPANT AND DATA CONFIDENTIALITY  
Participant identifiers will only be known to the local study team. Any information that leaves the local 
study site should be identified with the participant’s study ID number and no identifying information. 
Data must be kept in a secure location at the local site. The study database will be on a secure University 
of Michigan server.  
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
28 
12.5 
FUTURE USE OF STORED SPECIMENS  
The Steering Committee, including the study PI, PI from each participating site, and DCC PI, oversee all 
requests (both internal and external to the study) to access stored samples and data for ancillary studies 
directed toward the understanding of nephrotic syndrome, kidney disease, and/or the treatment 
protocol. 
 
Once the steering committee for this clinical trial has completed their responsibilities (study closed, 
analysis complete, results reported) the stewardship of the stored biospecimens for future research will 
be under the NIH sponsored Nephrotic Syndrome Study Network (NEPTUNE) ancillary studies 
committee.  
 
13 
DATA HANDLING AND RECORD KEEPING  
 
13.1 
DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
Data will be collected on study Case Report Forms and entered into the study electronic data base.  
Entered data will be assessed for completeness, accuracy, and timeliness. Queries will be distributed 
from the coordinating center to the study site personnel to maximize the data quality and accuracy.  
 
13.2 
STUDY RECORDS RETENTION  
Study records will be maintained for 2 years after completion of the trial. 
 
13.3 
PROTOCOL DEVIATIONS  
Protocol deviations will be reported to the study IRB and the study coordinating center. Every effort will 
be made to avoid protocol deviations except in the setting of patient safety. 
 
13.4 
PUBLICATION AND DATA SHARING POLICY 
This trial is expected to be reported as a scientific publication and will follow the publication and data 
sharing policies of the NEPTUNE study and the Kidney Research Network (KRN). (NEPTUNE-Study.org 
and kidneyresearchnetwork.org) 
14 
CONFLICT OF INTEREST POLICY 
Participating investigators and research personnel are responsible to maintain compliance with local and 
national conflict of interest policies.  
 
15  
LITERATURE REFERENCES  
1. 
KDIGO Board Members. Kidney international supplements. Mar 2012;2(1):3. 
2. 
Gadegbeku CA, Gipson DS, Holzman LB, et al. Design of the Nephrotic Syndrome Study Network 
(NEPTUNE) to evaluate primary glomerular nephropathy by a multidisciplinary approach. Kidney 
international. Apr 2013;83(4):749-756. 
3. 
Trachtman H, Vento S, Herreshoff E, et al. Efficacy of galactose and adalimumab in patients with 
resistant focal segmental glomerulosclerosis: report of the font clinical trial group. BMC 
nephrology. 2015;16:111. 
4. 
Ternant D, Paintaud G, Trachtman H, Gipson DS, Joy MS. A possible influence of age on 
absorption and elimination of adalimumab in focal segmental glomerulosclerosis (FSGS). 
European journal of clinical pharmacology. Feb 2016;72(2):253-255. 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
29 
5. 
Joy MS, Gipson DS, Powell L, et al. Phase 1 trial of adalimumab in Focal Segmental 
Glomerulosclerosis (FSGS): II. Report of the FONT (Novel Therapies for Resistant FSGS) study 
group. American journal of kidney diseases : the official journal of the National Kidney 
Foundation. Jan 2010;55(1):50-60. 
6. 
Roberts BV, Susano I, Gipson DS, Trachtman H, Joy MS. Contribution of renal and non-renal 
clearance on increased total clearance of adalimumab in glomerular disease. Journal of clinical 
pharmacology. Sep 2013;53(9):919-924. 
7. 
Joy MS, Gipson DS, Dike M, et al. Phase I trial of rosiglitazone in FSGS: I. Report of the FONT 
Study Group. Clinical journal of the American Society of Nephrology : CJASN. Jan 2009;4(1):39-
47. 
 
APPENDIX 
 
A. Humira Product Information 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
30 
 
 


Precision Medicine Proof of Concept for TNF Inhibition in FSGS and TR-MCD  
Version 5.0 
      February 21, 2023 
NIH-FDA Clinical Trial Protocol Template – v0.1 20160205  
31 
 
 

SAP_001.pdf: 
Precision Medicine Guided Use of TNF Inhibition in FSGS and Treatment 
Resistant Minimal Change Disease: Proof of Concept Target Engagement 
Study 
Statistical Analysis Plan 
Protocol Version Number: version 5.0, dated 21FEB2023
NCT04009668 
I give my approval for this SAP entitled "Precision Medicine Guided Use ofTNF Inhibition in FSGS 
and Treatment Resistant Minimal Change Disease: Proof of Concept Target Engagement Study 
Statistical Analysis Plan," 31OCT2023. 
Statistical Author: Yan Zhai, MS 
Study Principa ,nvestigator: Zubin Modi, MD, MS 
Co-Principal Investigator: Howard Trachtman, MD 
Co-Investigator: Karthik Ramani, MD 
Statistical Reviewer: Cathie Spino, D.Sc 
Statistical Reviewer: Wei Hao, PhD 
TNF Statistical Analysis Plan 
Version - 2023-10-31 
11/01/2023 
Date: 
11/03/2023 
Date: 
I 
f 
, 
Date: 
Date: 
01NOV2023 
Date: 
11/01/2023 
Date: 
Page 1 of 24 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 2 of 24 
Contents 
Abbreviations and Definitions ....................................................................................................... 4 
1. 
Introduction ............................................................................................................................ 5 
1.1 
Preface .......................................................................................................................................... 5 
1.2 
Scope of the Analyses ................................................................................................................... 5 
2. 
Schematic of Study Design ..................................................................................................... 5 
3. 
Study Aims and Hypotheses ................................................................................................... 6 
3.1 
Primary Aim and Hypothesis ......................................................................................................... 6 
4. 
Endpoints ................................................................................................................................ 6 
4.1 
Primary Endpoints ......................................................................................................................... 6 
4.2 
Secondary Endpoints .................................................................................................................... 7 
4.3 
Exploratory Endpoints ................................................................................................................... 7 
5. 
Study Methods ........................................................................................................................ 8 
5.1 
General Study Plan ........................................................................................................................ 8 
5.2 
Inclusion-Exclusion Criteria and General Study Population .......................................................... 8 
5.2.1 
Participant Inclusion Criteria ................................................................................................. 8 
5.2.2 
Participant Exclusion Criteria ................................................................................................ 9 
5.3 
Randomization and Blinding ......................................................................................................... 9 
5.4 
Study Assessments ........................................................................................................................ 9 
6. 
Outcomes .............................................................................................................................. 10 
6.1 
Primary Outcome Assessments .................................................................................................. 10 
6.2 
Secondary Outcome Assessments .............................................................................................. 10 
6.2.1 
Safety measures .................................................................................................................. 10 
6.2.2 
Laboratory abnormalities .................................................................................................... 11 
6.3 
Preliminary efficacy measures .................................................................................................... 12 
6.4 
Exploratory Outcome Measures ................................................................................................. 12 
7. 
Statistical Considerations ..................................................................................................... 13 
7.1 
General Statistical and Analytical Plans ...................................................................................... 13 
7.2 
Analysis Dataset .......................................................................................................................... 13 
7.3 
Covariates and Subgroups .......................................................................................................... 13 
7.4 
Missing Data ................................................................................................................................ 13 
7.5 
Interim Analyses and Data Monitoring ....................................................................................... 13 
7.6 
Multiple Testing .......................................................................................................................... 13 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 3 of 24 
8. 
Summary of Study Conduct Data ......................................................................................... 14 
8.1 
Subject Disposition ...................................................................................................................... 14 
8.2 
Derived Variables ........................................................................................................................ 14 
8.2.1 
eGFR .................................................................................................................................... 14 
8.2.2 
Blood pressure index .......................................................................................................... 14 
8.2.3 
Laboratory abnormality variables ....................................................................................... 15 
8.3 
Demographic and Baseline Variables.......................................................................................... 15 
8.4 
Treatment Compliance ............................................................................................................... 16 
9. 
Statistical Methods ............................................................................................................... 16 
9.1 
Analyses of Primary Endpoints ................................................................................................... 16 
9.2 
Analyses of Secondary Endpoints ............................................................................................... 16 
9.3 
Tabulation of Individual Response Data ..................................................................................... 16 
9.4 
Exploratory Analyses ................................................................................................................... 17 
9.5 
Sample Size ................................................................................................................................. 17 
10. Summary of Changes to the Protocol and/or SAP .............................................................. 17 
11. References ............................................................................................................................. 20 
12. List of Tables, Listings and Figures ....................................................................................... 20 
12.1 
List of Tables ............................................................................................................................... 20 
12.2 
List of Listings .............................................................................................................................. 22 
12.3 
List of Figures .............................................................................................................................. 22 
 
 
 
 
 
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 4 of 24 
Abbreviations and Definitions 
DSMB  
Data Safety and Monitoring Board  
eGFR 
Estimated Glomerular Filtration Rate 
FSGS  
Focal Segmental Glomerulosclerosis  
LLN 
Lower Limit of Normal 
MCP1  
Monocyte Chemoattractant Protein 1  
NEPTUNE 
Nephrotic Syndrome Study Network 
TIMP1  
Tissue Inhibitor of Metalloproteinase 1  
TNF  
Tumor Necrosis Factor  
TR-MCD  
Treatment Resistant-Minimal Change Disease  
uCr 
Urinary Creatinine 
uEGF  
Urinary Epidermal Growth Factor  
ULN 
Upper Limit of Normal 
UPCR  
Urine Protein to Creatinine Ratio  
 
 
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 5 of 24 
1. Introduction 
1.1 
Preface 
This Statistical Analysis Plan (SAP) describes statistical methods and analyses for the Precision 
Medicine Guided Use of TNF Inhibition in FSGS and Treatment Resistant Minimal Change 
Disease: Proof of Concept Target Engagement Study. This document should be read in tandem 
with the Precision Medicine Guided Use of TNF Inhibition in FSGS and Treatment Resistant 
Minimal Change Disease: Proof of Concept Target Engagement Study Protocol version 5.0, 
dated February 21, 2023. The SAP was finalized prior to the database lock. 
 
The trial proposes to investigate whether urinary markers of intra-renal tumor necrosis factor 
(TNF) pathway activation in patients with focal segmental glomerulosclerosis (FSGS) or 
treatment resistant- minimal change disease (TR-MCD) are reduced with TNF inhibition via 
adalimumab.   
 
This proof-of-concept, multi-center, open-label clinical trial addresses the treatment of patients 
with clinically active FSGS or TR-MCD and urinary biomarkers of intra-renal TNF up-regulation, 
including urinary monocyte chemoattractant protein 1/urinary creatinine (urinary MCP1/uCr) 
and urinary tissue inhibitor of metalloproteinase 1/urinary creatinine (urinary TIMP1/uCr). 
These biomarkers have been shown to indicate intra-renal TNF upregulation. Up to eight 
patients, aged 6-80 years, were treated with 8 weeks of adalimumab therapy to assure a 
minimum evaluable study sample of six patients in this single-arm study. Adalimumab was 
dosed based on weight and administered subcutaneously every two weeks for 8 weeks (5 total 
doses). The urinary biomarkers MCP1/uCr and TIMP1/uCr were assessed throughout the study. 
The primary endpoints are the change in target engagement biomarker levels of urinary 
TIMP1/uCr and MCP1/uCr from baseline to study week 10. Key secondary outcomes include 
safety measures, serious adverse events, adverse events, percent change of estimated 
glomerular filtration rate (eGFR) and urine protein to creatinine ratio (UPCR) from baseline to 
week 10, and proportion of participants achieving a nadir UPCR less than 1.5 g/g and at least a 
40% reduction from baseline at week 10. 
 
1.2 
Scope of the Analyses 
The purpose of this document is to describe the statistical analyses to address the primary, 
secondary, and exploratory objectives to be conducted for this clinical trial.  Ancillary analyses 
are not covered in this document.   
 
2. Schematic of Study Design  
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 6 of 24 
 
 
3. Study Aims and Hypotheses 
3.1 Primary Aim and Hypothesis 
The primary purpose of this proof-of-concept study is to test the hypothesis that TNF inhibition 
with adalimumab (Humira) in patients with up-regulation of the intra-renal TNF pathway results 
in reduction in urinary indicators of TNF pathway activation, specifically urinary MCP1/uCr or 
TIMP1/uCr levels. 
 
Hypothesis:  
TNF inhibition with adalimumab (Humira) in patients with up-regulation of the intra-renal TNF 
pathway results in reduction in urinary MCP1/uCr and/or TIMP1/uCr biomarker levels.  
 
4. Endpoints 
4.1 
Primary Endpoints 
• The change in urinary MCP1/uCr levels from baseline to study week 10, i.e., 2 weeks 
after the final dose of adalimumab 
• The change in urinary TIMP1/uCr levels from baseline to study week 10, i.e., 2 weeks 
after the final dose of adalimumab 
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 7 of 24 
4.2 
Secondary Endpoints 
1. Incidence of serious adverse events, defined as the proportion of participants with 
treatment-emergent serious adverse events and the number of treatment-emergent 
serious adverse events, overall, by system organ classification, by relatedness to study 
medication, and by severity 
2. Incidence of non-serious adverse events, defined as the proportion of participants with 
treatment-emergent non-serious adverse events and the number of treatment-
emergent non-serious adverse events, overall, by system organ classification, by 
relatedness to study medication, and by severity 
3. Incidence of adverse events leading to premature discontinuation of study medication, 
defined as the proportion of participants with treatment-emergent adverse events and 
the number of treatment-emergent adverse events that led to premature 
discontinuation of study medication, overall, by system organ classification, by 
relatedness to study medication, and by severity 
4. Proportion of participants with abnormal laboratory tests for ALT, AST, total bilirubin, 
hemoglobin, hematocrit, and platelet count, proportion of participants with 
temperature ≥38.5˚C (indicating fever), and proportion of participants with any edema 
(over all body locations and by each location) over the course of the 10-week 
intervention period  
5. Percent change in eGFR from baseline to week 10 
6. Percent change in UPCR from baseline to week 10 
7. Proportion of participants achieving a nadir UPCR less than 1.5 g/g and at least a 40% 
reduction from baseline at week 10 
 
4.3 
Exploratory Endpoints 
1. In the subgroup of participants with kidney biopsy tissue collected within 12 months of 
study baseline, correlation coefficient between intra-renal gene expression of TNF (TNF 
activation score) [1] and urinary MCP1/uCr and TIMP1/uCr biomarker levels at baseline 
2. Change from baseline to weeks 2, 4, and 8 in urinary MCP1/uCr 
3. Change from baseline to weeks 2, 4, and 8 in urinary TIMP1/uCr 
4. Percent change from baseline to weeks 2, 4, 8, and 10 in urinary MCP1/uCr 
5. Percent change from baseline to weeks 2, 4,  8, and 10 in urinary TIMP1/uCr 
6. Urinary MCP1/uCr values at baseline and weeks 2, 4, 8, and 10 
7. Urinary TIMP1/uCr values at baseline and weeks 2, 4, 8, and 10 
8. Changes in urinary epidermal growth factor normalized to urinary creatinine (uEGF/uCr) 
biomarker levels from baseline to week 10 
9. Correlation coefficient between uEGF/uCr at baseline with changes and percent changes 
in urinary MCP1/uCr and urinary TIMP1/uCr biomarker levels and in eGFR and UPCR 
values at week 10. 
10. Changes in FSGS-MCD Patient Reported Outcome (PRO) score from baseline to weeks 2, 
4, 8 and 10 
 
Endpoint Rationale: 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 8 of 24 
The target engagement biomarkers, urinary MCP1/uCr and TIMP1/uCr, are the primary 
endpoints of this study as we hypothesize that the levels of this biomarkers at baseline reflect 
TNF up-regulation in the kidney and after TNF inhibition reflect target engagement. Neither 
eGFR nor urinary protein is a primary endpoint measure. However, we measure eGFR and 
urinary protein to provide estimates for future trials. 
 
5. Study Methods 
5.1 
General Study Plan 
This study is designed as a proof-of-concept multi-center, open-label single-arm clinical trial 
addressing the treatment of patients with FSGS or TR-MCD.  Eligibility for the study is assessed 
during a 6-week screening period where bio-samples are used to assess TNF up-regulation and 
other laboratory and medical history criteria.   
 
Patients are recruited from the active population of patients with FSGS or TR-MCD at 
participating sites. TR-MCD is defined as persistent proteinuria despite pre-trial 
immunomodulating treatment. Following informed consent and when appropriate, assent, a 
screening visit is conducted. Patients with elevated urinary TIMP1/uCr and MCP1/uCr levels and 
meeting all other eligibility criteria are scheduled to receive adalimumab every two weeks for 8 
weeks (5 total doses).  
 
A participant is considered to have completed the study if they complete the study through 
week 14 or if they withdraw prior to completion of the final study visit. Participants are 
followed in the study even if study medication is prematurely discontinued unless they 
withdraw their consent to do so.  End of study is constituted by completion of the last visit for 
the final participant in the study protocol.  
 
5.2 
Inclusion-Exclusion Criteria and General Study Population 
5.2.1 Participant Inclusion Criteria 
1. Kidney biopsy confirmed FSGS or MCD  
2. For Minimal Change Disease patients only, history of resistance to corticosteroid 
therapy  
3. Increased urinary excretion of biomarkers of TNF activation (urinary MCP1/uCr and/or 
urinary TIMP1/uCr) at study screening  
4. eGFR>30 ml/min/1.73 m2 at screening  
5. Urine protein to creatinine ratio ≥1.5 g/g at screening  
6. Age 6-80 years  
7. Weight >15kg  
8. Stable therapy with angiotensin converting enzyme inhibitors, angiotensin receptor 
blockers, and oral immunosuppression agents for at least 30 days prior to enrollment  
9. Birth control use in females of child-bearing potential  
10. Informed consent and assent if applicable  
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 9 of 24 
5.2.2 Participant Exclusion Criteria 
1. Kidney or other solid organ or bone marrow transplant recipient  
2. Allergy or intolerance to investigational agent  
3. Secondary FSGS  
4. Severe obesity, i.e., BMI >40 kg/m2  
5. Live virus vaccine in the past 3 months  
6. Malignancy, current or in the past 5 years  
7. Active local or systemic bacterial, fungal or viral infection  
8. Active or latent Hepatitis B, Hepatitis C, HIV, or tuberculosis defined as quantiferon test 
positive  
9. History of demyelinating disease, e.g., Multiple Sclerosis or Guillain-Barre  
10. History of heart failure  
11. Active liver disease, defined as AST or ALT > 2.5x Upper Limit of Normal  
12. Systemic lupus erythematosus or ANA > 1:80  
13. History of inflammatory bowel disease, e.g., ulcerative colitis or Crohns disease  
14. Cyclophosphamide in past 90 days, Rituximab in the past 180 days  
15. Pregnancy or nursing  
16. Blood white blood cell count <4,500/mm3; Hg <9 g/dL; Platelet count <150,000/mm3 at 
enrollment. Use of an erythropoiesis stimulating agent will not be an exclusion criterion. 
17. Concurrent use of interleukin-1 antagonist (Anakinra), other TNF blocking agent, 
methotrexate or abatacept 
18. Diabetic nephropathy 
 
5.3 
Randomization and Blinding 
This is a single-arm study and thus randomization and masking are not possible to minimize 
bias. 
 
5.4 
Study Assessments 
The Schedule of Activities details the study procedures: 
 
Screening 
Week 0 
Week 2 
Week 8 
Week 10 
Week 14 
Visit window 
+/-2 DAYS 
+/-2 DAYS 
+/-2 DAYS 
+/-2 DAYS 
+/-2 DAYS 
+/-4 DAYS 
Consent 
X 
 
 
 
 
 
Medical History  
X 
X 
X 
X 
X 
X 
Archived Kidney Biopsy Report 
Submission 
X 
 
 
 
 
 
Archived Kidney Tissue sections 
submitted, if available* 
 
 
 
 
 
 
Concomitant Medications 
X 
X 
X 
X 
X 
X 
Physical Exam 
X 
X 
X 
X 
X 
X** 
Adverse Events Assessment 
 
X 
X 
X 
X 
X 
Vital Signs 
X 
X 
X 
X 
X 
X** 
HRQOL assessment 
 
X 
X 
X 
X 
 
Comprehensive Metabolic 
Panel 
X 
X 
X 
X 
X 
X** 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 10 of 24 
 
Screening 
Week 0 
Week 2 
Week 8 
Week 10 
Week 14 
CBC with Platelets and 
Differential 
X 
X 
X 
X 
X 
X** 
Urine Protein:Creatinine 
X 
X 
X 
X 
X 
X** 
Assessment of Infections 
X 
 
 
 
 
 
MCP1/TIMP1 (spot urine) 
X 
X 
X 
X 
X 
 
Pregnancy Test 
X 
X 
X 
X 
 
 
Biorepository specimen 
X*      
X 
X 
X 
X 
 
Adalimumab dosing 
 
X 
X 
X 
 
 
* Do not submit if most recent biopsy specimen has already been submitted to NEPTUNE study; *DNA 
collected at screening only 
** Optional procedures required if study visit is conducted in-person 
 
 
6. Outcomes 
6.1 
Primary Outcome Assessments 
Spot urinary biospecimens to assay MCP1 (ng/mL), TIMP1 (ng/mL) and creatinine (mg/mL) are 
collected with spot urine tests at screening and weeks 0, 2, 8, and 10.  Urine creatinine is used 
to normalize the urinary MCP1 and urinary TIMP1 by calculating the ratio of urinary MCP1 and 
urinary creatinine and the ratio of urinary TIMP1 and urinary creatinine.   
 
Urinary MCP1 and Urinary TIMP1 are measured by ELISA methods [2].  There are no published 
reference ranges for the urinary MCP1/uCr nor urinary TIMP1/uCr levels; however, lower 
values for both biomarkers are associated with less intra-renal TNF activation. [1] Thus, either 
absolute or relative changes from baseline to post-treatment time points indicate TNF pathway 
target engagement by adalimumab and support the proof of concept of this trial. 
 
6.2 
Secondary Outcome Assessments 
6.2.1 Safety measures 
An adverse event is any untoward medical occurrence in a clinical study participant 
administered a medicinal (investigational or non-investigational) product. Adverse events, 
whether serious or non-serious, related or not related, are recorded after administration of 
adalimumab (i.e., are treatment-emergent events) until week 14.  All serious adverse events 
that do resolve by the end of the study, or that do not resolve upon discontinuation of the 
subject's participation in the study, are followed until any of the following occurred:  
• The event resolves 
• The event stabilizes  
• The event returns to baseline, if a baseline value/status were available  
• The event can be attributed to agents other than the study drug or to factors unrelated 
to study conduct  
• It becomes unlikely that any additional information can be obtained (participant or 
health care practitioner refusal to provide additional information, or lost to follow-up 
after demonstration of due diligence with follow-up efforts). 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 11 of 24 
 
Adverse events are categorized by severity and by relationship to study medication by the site 
investigators, and by system organ class by the coordinating center investigators. 
 
The status of edema is important in this patient population as a manifestation of disease, so 
any edema (over all body locations and by each location) over the course of the 10-week 
intervention period is evaluated within the adverse event reporting as new or worse than prior 
to study. 
 
As a clinically significant change, any fever event (temperature ≥38.5˚C) is recorded over the 
course of the 10-week intervention period. 
 
6.2.2 Laboratory abnormalities 
For ALT, AST, and total bilirubin, the laboratory result value is abnormal when it is greater than 
2 x upper limit of normal (ULN). Laboratory result values either below the age-matched lower 
limit of normal (LLN) or above the age-matched upper limit of normal is defined as laboratory 
abnormalities for hemoglobin, hematocrit, and platelet count.  The age-matched normal ranges 
of ALT, AST, total bilirubin, hemoglobin, hematocrit, and platelet count are provided below: 
 
 
Age Group 
Male 
Female 
ALT 
> or =6 year 
7-55 U/L 
7-45 U/L 
AST 
6-13 years 
8-60 U/L 
8-50 U/L 
> or =14 years 
8-48 U/L 
8-43 U/L 
Total bilirubin 
6 to 17 years 
0.0-1.0 mg/dL 
0.0-1.0 mg/dL 
Hemoglobin 
6-8 years 
11.5-14.3 g/dL 
11.5-14.3 g/dL 
9-10 years 
11.8-14.7 g/dL 
11.8-14.7 g/dL 
11-14 years 
12.4-15.7 g/dL 
11.9-14.8 g/dL 
15-17 years 
13.3-16.9 g/dL 
11.9-14.8 g/dL 
Adults 
13.2-16.6 g/dL 
11.6-15.0 g/dL 
Hematocrit 
6-7 years 
34-42% 
34-42% 
8-11 years 
35-43% 
35-43% 
12-15 years 
38-47% 
35-43% 
16-17 years 
40-50% 
35-43% 
Adults 
38.3-48.6% 
35.5-44.9% 
Platelet count 
6-9 years 
187-400 x 109/L  
187-400 x 109/L 
10-13 years 
177-381 x 109/L 
177-381 x 109/L 
14-17 years 
139-320 x 109/L 
158-362 x 109/L 
Adults 
135-317 x 109/L 
157-371 x 109/L 
 
We calculate the proportion of participants with laboratory abnormalities at weeks 2, 4, 8 and 
10, and overall (i.e., a participant experienced a lab abnormality at any visit). 
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 12 of 24 
6.3 
Preliminary efficacy measures 
eGFR (mL/min/1.73 m2) is calculated from blood samples at screening and Weeks 0, 2, 8, 10 and 
14 (if the study visit is conducted in person).  Higher values generally indicate better kidney 
function. The calculation of eGFR could be found in Section 8.2.1. 
 
UPCR (mg/mg) is calculated from urinary samples for proteinuria and creatinine at screening 
and Weeks 0, 2, 8, 10 and 14 (if the study visit is conducted in person).  The preferred method 
for collection of urinary samples is through 24-hour urine collection, but first morning urine or 
spot urine is acceptable.  The ratio of urine protein to urine creatinine (UPCR) is the secondary 
outcome measure of interest.  Higher levels of UPCR indicate more significant kidney disease.    
In this pilot, proof-of-concept study, the normal range for UPCR is < 0.3 mg/mg for all ages. 
 
6.4 
Exploratory Outcome Measures 
Intra-renal gene expression of TNF is derived from archived biopsy tissue of study participants 
or from the Nephrotic Syndrome Study Network (NEPTUNE) study data using the NEPTUNE 
study protocol and as published. [3] This parameter was only included for participants with 
biopsies within one year of consent in the TNF pilot study and is generated as a TNF activation 
score.   
 
A TNF activation network was generated from expert curated interactions from NETPro 
annotations in the Genomatix Genome Analyzer database (Precigen Bioinformatics).[1] It was 
based on renal biopsy specimens available from more than 250 NEPTUNE participants with 
MCD and FSGS. From the database, causally downstream genes or proteins that increased 
expression from TNF exposure were used to generate a TNF activation score. Individual gene 
expression values were first Z-transformed. The TNF activation score for each participant was 
the average Z-score of the genes in their kidney RNA-seq profile and defines the intra-renal 
gene expression of TNF measure.    
 
Urinary epidermal growth factor (uEGF, ng/ml) biomarker levels are assessed from spot urine 
specimens collected and normalized by urinary creatinine (mg/ml) at Screening, and Weeks 0, 
2, 8, and 10.  Higher levels of uEGF/uCr (ng/mg) indicate better kidney function. 
 
The FSGS-MCD Patient Reported Outcome (PRO) score is still in development and scoring 
methods have not yet been finalized.  The score will be derived from the FSGS-MCD PRO 
structured questionnaires completed by participants at in-person study visits at Weeks 0, 2, 8 
and 10. The analyses of FSGS-MCD PRO scores will occur when the scoring methods have been 
developed and made available to the study investigators. 
 
Assay Quality Methods: 
Urinary MCP1, TIMP1, creatinine and EGF for all study participants and all time points for were 
assayed on the same plate to reduce batch effects. 
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 13 of 24 
7. Statistical Considerations 
7.1 
General Statistical and Analytical Plans 
This is a pilot, proof-of-concept investigation to test the proposed biomarkers as indicators of 
target engagement. As such, no formal sample size calculations were performed, and analyses 
are primarily descriptive in nature. Our approach is to assess the biomarker change over time 
with TNF inhibition therapy within each patient and as a group. As the ELISA testing for this 
indication is new, the protocol uses an exploratory approach to assess individual and panel 
biomarker changes and correlations with clinical indicators of disease, including proteinuria, 
eGFR, and serum albumin. 
 
This SAP document was finalized and approved prior to the database lock. 
 
7.2 
Analysis Dataset 
All eligible participants who received at least one dose of adalimumab (as-treated population) 
are analyzed for primary, secondary, and exploratory endpoints. 
 
7.3 
Covariates and Subgroups 
No adjustment for covariates is included in the analyses of the primary biomarker endpoints 
because of the limited sample size of this pilot, proof-of-concept study.  No subgroup analyses 
are planned. 
 
7.4 
Missing Data 
For the primary analysis of the primary endpoints, we will impute week 10 biomarker values 
using the week 8 biomarkers values, when needed.  The remainder of our analyses uses a 
complete case approach (i.e., analyzing the observed values with no imputation of missing 
values).  This method is valid under the missing completely at random assumption.  We will 
assess the suitability of this assumption based on the extent and patterns of missingness, as 
well as clinical judgement. 
 
7.5 
Interim Analyses and Data Monitoring 
No formal interim analyses were planned nor carried out for this study.  The study was 
overseen by a Data and Safety Monitoring Board (DSMB) that reviewed the subject disposition, 
study conduct and safety data throughout the trial. 
 
7.6 
Multiple Testing 
Statistical testing is conducted at the 0.05 significance level using two-tailed tests; two-sided p-
values are reported. No adjustments for multiplicity are planned for this pilot study. Rather, 
inferences and decision-making are based primarily on the magnitude of treatment effects. 
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 14 of 24 
8. Summary of Study Conduct Data 
Descriptive summary statistics are tabulated for baseline patient demographics and clinical 
characteristics (overall since this is a single-arm study).  Continuous variables are summarized 
using descriptive statistics including n, mean, median, standard deviation, interquartile range 
(i.e., 1st and 3rd quartiles), and range (i.e., minimum and maximum). Qualitative variables will 
be summarized using counts and percentages. Unless otherwise specified, statistical analyses 
will be performed using SAS Version 9 or higher. Where appropriate, statistical tests will be 
conducted at the 0.05 significance level using two-tailed tests and p-values will be reported. 
 
8.1 
Subject Disposition 
The number and percentage of participants consented, treated and withdrawn, along with 
reasons for withdrawal, are tabulated and summarized in a CONSORT diagram.  Other 
disposition and study conduct information, including major protocol violations are summarized.  
Protocol deviations are provided in a listing. Duration of study follow-up is summarized. 
 
8.2 
Derived Variables 
8.2.1 eGFR 
The formulas used to calculate eGFR in adults and children are given below: 
• CKID U25 age- and sex-dependent equation using serum creatinine (Pierce, 2021) [4] 
for patient whose age is younger than 25 years old: 
eGFR  = k × (height/serum creatinine), with height in meters and serum creatinine in mg/dL 
For males, k is calculated as:   
For 1 to 11 years old:    39.0 × 1.008(age-12) 
For 12 to 17 years old:  39.0 × 1.045(age-12) 
For 18 to 25 years old:   50.8 
For females, k is calculated as:   
For 1 to 11 years old:    36.1 × 1.008(age-12) 
For 12 to 17 years old:  36.1 × 1.023(age-12) 
For 18 to 25 years old:   41.4 
 
• CKD-EPI equation for patient whose age is greater than 25 years old [5]: 
eGFRcr = 142 x min(Scr/κ, 1)α x max(Scr/κ, 1)-1.200 x 0.9938Age x 1.012 [if female] 
where: 
Scr = standardized serum creatinine in mg/dL 
κ = 0.7 (females) or 0.9 (males) 
α = -0.241 (female) or -0.302 (male) 
min(Scr/κ, 1) is the minimum of Scr/κ or 1.0 
max(Scr/κ, 1) is the maximum of Scr/κ or 1.0 
Age (years)  
 
8.2.2 Blood pressure index 
The 2017 ACC/AHA hypertension guideline applied a lower threshold for defining hypertension 
and 130 (mmHg) /80 (mmHg) are the thresholds for hypertension in adult’s guidelines. [6]  
An important update in the 2017 AAP CPG is a change in the definition of hypertension in 
children and adolescents:  In children <13 years of age, hypertension is defined as systolic or 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 15 of 24 
diastolic blood pressure ≥95th percentile on the new sex, age, and height tables. For adolescents 
≥13 years of age, the 95th percentile approximates 130/80 mm Hg. Therefore, the 2017 AAP 
CPG has used the new adult blood pressure cut-points to define hypertension in adolescents 
≥13 years of age. [7]  
 
To allow for comparison across adults and children, systolic and diastolic blood pressure indices 
are calculated with the method below:  
• For the patient whose age was 13 years or greater, the measured systolic blood 
pressure was divided by 130 (and the measured diastolic blood pressure was divided by 
80) 
• For the patient whose age was younger than 13 years, the measured systolic and the 
measured diastolic blood pressure were divided by the sex, age, and height specific 95th 
percentile systolic and diastolic blood pressure, respectively [8] 
 
We use the Childhood Blood Pressure Macro-batch mode to calculate the specific 95th 
percentile blood pressure of the patient whose age is younger than 13 years. [9][10] Then the 
blood pressure index is calculated based on the method above. Blood pressure index ≥1 
indicates blood pressure in the hypertensive range and every 0.1-unit increase represents a 
10% increase in blood pressure above the hypertensive range.  
 
8.2.3 Laboratory abnormality variables 
Elevation of ALT, AST or total bilirubin of > 2x ULN were prespecified as events of interest and 
were coded as 1 for values > 2 x ULN, 0 for values below 2 x ULN. These are calculated for each 
participant at each time point. 
 
Abnormal low values for hemoglobin, hematocrit, and platelet count were specified as events 
of interest and were coded as 1 for values < age-matched LLN and 0 otherwise.  Abnormal high 
values for hemoglobin, hematocrit, and platelet counter were also specified as events of 
interested and were coded 1 for values > age-matched ULN and 0 otherwise.  Any laboratory 
abnormality for these laboratory parameters were coded as 1 if abnormally low or high and 0 
otherwise.  These are calculated for each participant at each time point. 
 
8.3 
Demographic and Baseline Variables 
Demographic and baseline variables for participants include:  age in years at baseline, sex 
(male/female), ethnicity (Hispanic or Latino/not Hispanic or Latino/refused/missing), race 
(White/Black or African American/Asian/American Indian or Alaskan native/native Hawaiian or 
other Pacific Islander/refused/missing), duration of kidney disease (in months), baseline 
medication use (%), weight (kg), edema/no edema status, blood pressure index, eGFR 
(mL/min/1.73 m2), UPCR (mg/mg), albumin (g/dL), hemoglobin (g/dL), WBC (k/mm3), 
cholesterol (mg/dL), AST (U/L), ALT (U/L), total bilirubin (mg/dL), hematocrit (%), platelet count 
(K/uL), urinary MCP/uCr (ng/mg), urinary TIMP1/uCr (ng/mg) , uEGF/uCr (ng/mg), PRO Score, 
and TNF activation score. 
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 16 of 24 
8.4 
Treatment Compliance 
Compliance with study medication is summarized as the proportion of participants who 
received 1, 2, 3, 4, and (all) 5 doses of adalimumab. Descriptive statistics on the duration of 
adherence to study medication is summarized.  The proportion of participants who 
permanently discontinue study medication is summarized with descriptive statistics and a 
listing provided of these participants with their reasons for permanent discontinuation of study 
medication. 
 
9. Statistical Methods 
9.1 
Analyses of Primary Endpoints 
The changes in urinary MCP1/uCr and urinary TIMP1/uCr levels from baseline to study week 10, 
i.e., 2 weeks after the final dose of adalimumab, are the primary endpoints. We provide tabular 
and graphical summaries of the changes from baseline to week 10 in urinary MCP1/uCr and in 
urinary TIMP1/uCr, including summaries at baseline and week 10. The changes from baseline to 
week 10 in urinary MCP1/uCr and urinary TIMP1/uCr levels will be tested using the Wilcoxon 
signed-rank test.  
 
9.2 
Analyses of Secondary Endpoints 
Discrete safety outcomes (AEs, SAEs, AEs leading to premature discontinuation of study 
medication, and proportion of participants with abnormal laboratory tests, fever and new or 
worsening edema compared with baseline over the course of the 10-week treatment period) 
and discrete preliminary efficacy outcomes (proportion of participants achieving a nadir UPCR 
<1.5 g/g and at least a 40% reduction from baseline at week 10) are summarized descriptively.  
 
Percent change in eGFR from baseline to week 10 and percent change in UPCR from baseline to 
week 10 are summarized descriptively in tabular and graphical formats, including baseline, 
follow-up and changes from baseline. Graphical summaries include spaghetti plots of 
participants’ changes over time.   
 
Clinical laboratory tests as described in Section 6.2.2 are summarized descriptively in tables and 
graphically with individual plots of laboratory value by visit with a reference line for LLN, ULN 
and 2xULN for those participants who have at least one abnormal value during the course of 
the study. A shift table (normal and abnormal results for screening and post-treatment values 
at each time point) for each clinical laboratory test of interest will be presented. 
 
For discrete safety outcomes, frequency of events and proportion of participants experiencing 
events (i.e., treatment-emergent AEs and SAEs by body system, severity, and relatedness to 
adalimumab) will be summarized descriptively.   
 
9.3 
Tabulation of Individual Response Data 
Listings of SAEs, AEs and AEs resulting in treatment discontinuation will be provided. 
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 17 of 24 
9.4 
Exploratory Analyses 
Spearman correlations and scatterplots are provided to assess the correlation of TNF activation 
score and urinary MCP1/uCr and urinary TIMP1/uCr biomarker levels at baseline, and the 
correlation of uEGF/uCr at baseline with changes and percent changes in urinary MCP1/uCr and 
urinary TIMP1/uCr biomarker levels and in eGFR and UPCR values at week 10.  
 
Continuous outcomes (e.g., eGFR, uEGF/uCr biomarker levels) will be summarized descriptively, 
including baseline, follow-up and changes from baseline and graphically as spaghetti plots of 
participants’ changes (absolute and relative) over time and values over time.  
 
9.5 
Sample Size 
No formal sample size calculations were performed (either for estimation or hypothesis testing) 
because this study is a pilot investigation of urine biomarkers for target engagement of TNF 
inhibition. Rather, we assessed the magnitude of effects that can be detected in urine 
biomarkers using a paired t-test. With 7 patients, 80% power, and a two-sided type I error of 
10%, we can detect an effect size (change from baseline to week 10 / SD) of 1.2 of changes in 
urinary TIMP1/uCr and of changes in urinary MCP1/uCr, with no adjustments for multiplicity 
(EAST 6.2). 
 
10. Summary of Changes to the Protocol and/or SAP 
The changes from the protocol-specified definitions of aims, outcomes and statistical analytic 
approaches are outlined below.  These changes reflect advances in our knowledge of FSGS and 
MCD since the design of the study that were not incorporated as protocol amendments but 
were discussed during the formation of the Statistical Analysis Plan.  These changes are 
documented herein and represent changes made prior to the database lock. 
 
1. Change in the primary aim and hypothesis 
Exclusion of the hypothesis that FSGS patients with up-regulation of the TNF pathway can be 
identified through intra-renal gene expression profiling and assay of urinary MCP1/uCr and 
urinary TIMP1/uCr levels by ELISA. This is demonstrated by the successful completion of the 
screening protocol that determined if they were included/eligible for drug. 
 
Protocol: 3. OBJECTIVES AND PURPOSE 
The primary purpose of this Proof of Concept study is to test the hypothesis that TNF inhibition 
with adalimumab (Humira) in patients with up-regulation of the intra-renal TNF pathway will 
result in reduction in urinary indicators of TNF pathway activation, specifically urinary 
MCP1/uCr and TIMP1/uCr levels. 
 
Furthermore, this study is proposed to test the hypothesis that FSGS patients with up-
regulation of the TNF pathway can be identified through intra-renal gene expression profiling 
and assay of urinary MCP1/uCr and TIMP1/Cur levels by ELISA. 
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 18 of 24 
SAP:  
Section 1.1 Preface  
This proof-of-concept, multi-center, open-label clinical trial addresses the treatment of patients 
with clinically active FSGS or TR-MCD and urinary biomarkers of intra-renal TNF up-regulation, 
including urinary monocyte chemoattractant protein 1/urinary creatinine (urinary MCP1/uCr) 
and urinary tissue inhibitor of metalloproteinase 1/urinary creatinine (urinary TIMP1/uCr). 
 
Section 3.1 Primary Aim and Hypothesis. 
The primary purpose of this proof-of-concept study is to test the hypothesis that TNF inhibition 
with adalimumab (Humira) in patients with up-regulation of the intra-renal TNF pathway results 
in reduction in urinary indicators of TNF pathway activation, specifically urinary MCP1/uCr or 
TIMP1/uCr levels. 
Hypothesis:  
TNF inhibition with adalimumab (Humira) in patients with up-regulation of the intra-renal TNF 
pathway will result in reduction in urinary MCP1/uCr and/or urinary TIMP1/uCr biomarker 
levels. 
 
2. Inclusion of additional safety endpoints 
Additional safety endpoints were added to the analysis that were not described in the protocol:  
AEs leading to premature discontinuation of study medication, proportion of participants with 
temperature ≥38.5˚C (indicating fever), and proportion of participants with new or worsening 
edema compared to baseline.  They are standard safety parameters in this population. 
 
Protocol:  None 
 
SAP:  Section 4.2.  Secondary Endpoints.   
3. Incidence of adverse events leading to premature discontinuation of study medication, 
defined as the proportion of participants with treatment-emergent adverse events and 
the number of treatment-emergent adverse events that led to premature 
discontinuation of study medication, overall, by system organ classification, by 
relatedness to study medication, and by severity 
4. Proportion of participants with abnormal laboratory tests for ALT, AST, total bilirubin, 
hemoglobin, hematocrit, and platelet count, proportion of participants with 
temperature ≥38.5˚C (indicating fever), and proportion of participants with new or 
worsening edema (over all body locations and by each location) over the course of the 
10-week intervention period compared to baseline 
 
3. Clarification of exploratory endpoints 
The protocol included time to reduction in the two biomarkers, urinary MCP1/uCr and urinary 
TIMP1/uCr.  The SAP clarifies that the trajectory of the biomarkers over the course of the study 
will provide the evaluation of the time to reduction. 
 
Protocol:  Section 4.2.3 EXPLORATORY ENDPOINTS.   
Time to reduction of urinary MCP1/uCr and urinary TIMP1/uCr biomarkers from baseline 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 19 of 24 
 
SAP:  Section 4.3 
2. Change from baseline to weeks 2, 4, and 8 in MCP1/uCr 
3. Change from baseline to weeks 2, 4, and 8 in TIMP1/uCr 
4. Percent change from baseline to weeks 2, 4, 8 and 10 in urinary MCP1/uCr 
5. Percent change from baseline to weeks 2, 4, 8 and 10 in urinary TIMP1/uCr 
6. Urinary MCP1/uCr values at baseline and weeks 2, 4, 8 and 10 
7. Urinary TIMP1/uCr values at baseline and weeks 2, 4, 8 and 10 
9. Correlation coefficient between uEGF/uCr at baseline with changes and percent changes 
in urinary MCP1/uCr and urinary TIMP1/uCr biomarker levels and in eGFR and UPCR 
values at week 10. 
 
4. Change in analysis of the primary endpoints 
The nonparametric approach was considered more appropriate for the small sample size. 
 
Protocol:  Section 10.3.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT(S).  
The changes from baseline to week 10 in urinary MCP1/uCr and TIMP1/uCr levels will be tested 
using the paired t-test or Wilcoxon signed-rank test (if distributional assumptions are not met). 
 
SAP:  Section 9.1 
Analyses of Primary Endpoints.  
The changes from baseline to week 10 in urinary MCP1/uCr and urinary TIMP1/uCr levels will 
be tested using the Wilcoxon signed-rank test. 
 
 
 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 20 of 24 
11. References 
1. Mariani LH, Eddy S, AlAkwaa FM, McCown PJ, Harder JL, Nair V, et al. Precision 
nephrology identified tumor necrosis factor activation variability in minimal change 
disease and focal segmental glomerulosclerosis. Kidney Int. 2023;103(3):565-579  
2. Aydin S. A short history, principles, and types of ELISA, and our laboratory experience 
with peptide/protein analyses using ELISA. Peptides. 2015;72:4-15. 
3. Gadegbeku CA, Gipson DS, Holzman LB,  Ojo AO,  Song PXK, Barisoni L, et al. Design of 
the Nephrotic Syndrome Study Network (NEPTUNE) to evaluate primary glomerular 
nephropathy by a multidisciplinary approach. Kidney international. 2013;83(4):749-756. 
4. Pierce CB, Muñoz A, Ng DK, Warady BA, Furth SL, Schwartz GJ. Age- and sex-dependent 
clinical equations to estimate glomerular filtration rates in children and young adults 
with chronic kidney disease. Kidney Int. 2021 Apr;99(4):948-956.  
5. Inker LA, Eneanya ND, Coresh J, et al. New creatinine- and cystatin c-based equations to 
estimate GFR without race. N Engl J Med. 2021;385(19):1737–1749.   
6. Whelton PK, Carey RM, Aronow WS, Casey DE Jr, CollinsKJ, Dennison Himmelfarb C, et 
al.2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA   Guideline   for 
the Prevention, Detection, Evaluation, and Management of High Blood Pressure in 
Adults: a report of the American College of Cardiology/American Heart Association Task 
Force on Clinical Practice Guidelines.  Hypertension. 2018;71:e13–e115 
7. Flynn JT, Kaelber DC, Baker-Smith CM, Blowery D, Carroll AE, Daniels SR, et al. Clinical 
Practice Guideline for Screening and Management of High Blood Pressure in Children 
and Adolescents. Pediatrics. 2017;140:pii: e20171904. 
8. Sethna CB, Meyers KEC, Mariani LH, Psoter KJ, Gadegbeku CA, Gibson KL, et al. Blood 
Pressure and Visit-to-Visit Blood Pressure Variability among Individuals with Primary 
Proteinuric Glomerulopathies. Hypertension. 2017;70(2):315-323. 
9. Rosner B, Cook N, Portman R, Daniels S, Falkner B. Determination of blood pressure 
percentiles in normal-weight children: some methodological issues. American Journal of 
Epidemiology. 2008; 167:653–666. 
10. Rosner B (2023, October 18). Childhood Blood Pressure Macro-batch mode. Pediatric 
Blood Pressure. 
https://sites.google.com/a/channing.harvard.edu/bernardrosner/pediatric-blood-
press/childhood-blood-pressure.  
 
12. List of Tables, Listings and Figures 
All tables, listings and figures are saved in a separate file as supplementary material.  
12.1 List of Tables 
• Table 1.  Subject Disposition Summary. All Subjects. 
• Table 2.  Reasons for Early Study Discontinuation. All Subjects. 
• Table 3.  Duration of Follow-Up Period. All Subjects. 
• Table 4.  Number of Enrolled Subjects by Site. All Subjects. 
• Table 5.  Proportion of Participants Receiving 1, 2, 3, 4 and 5 (all) Doses of Adalimumab.  
As-Treated Population. 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 21 of 24 
• Table 6.  Duration of Adherence to Study Medication Summary. As-Treated Population 
• Table 7.  Demographic and Baseline Characteristics.  As-Treated Population. 
• Table 8.  Change in urinary MCP1/uCr levels from Baseline to Week 10. As-Treated 
Population. 
• Table 9.  Change in urinary TIMP1/uCr levels from Baseline to Week 10. As-Treated 
Population. 
• Table 10.1.  Treatment-Emergent Serious Adverse Event (SAE) Summary. As-Treated 
Population. 
• Table 10.2.  Treatment-Emergent Serious Adverse Event (SAE) by System Organ Class 
and by Severity. As-Treated Population. 
• Table 10.3. Treatment-Emergent Serious Adverse Events (SAE) by System Organ Class 
and by Relatedness to Study Medication. As-Treated Population. 
• Table 11.1.  Non-Serious Treatment-Emergent Adverse Event (AE) Summary. As-Treated 
Population. 
• Table 11.2.  Non-Serious Treatment-Emergent Adverse Events (AE) by System Organ 
Class and by Severity. As-Treated Population. 
• Table 11.3.  Non-Serious Treatment-Emergent Adverse Events (AE) by System Organ 
Class and by Relatedness to Study Medication. As-Treated Population. 
• Table 12.1.  Treatment-Emergent Adverse Events Leading to Premature Discontinuation 
of Study Medication Summary. As-Treated Population. 
• Table 12.2.  Treatment-Emergent Adverse Events Leading to Premature Discontinuation 
of Study Medication by System Organ Class and by Severity. As-Treated Population. 
• Table 12.3.  Non-Serious Treatment-Emergent Adverse Events Leading to Premature 
Discontinuation of Study Medication by System Organ Class and by Relatedness to Study 
Medication. As-Treated Population. 
• Table 13. Proportion of participants with temperature ≥38.5˚C (indicating fever). As-
Treated Population. 
• Table 14. Proportion of participants with new or worsening edema (over all body 
locations and by each location) over the course of the 10-week intervention period 
compared to baseline. As-Treated Population. 
• Table 15. Percent change in eGFR from baseline to week 10. As-Treated Population. 
• Table 16. Percent change in UPCR from baseline to week 10. As-Treated Population. 
• Table 17. Proportion of participants achieving a nadir UPCR <1.5 g/g and at least a 40% 
reduction from baseline at week 10. As-Treated Population. 
• Table 18.1. Proportion of Participants with Abnormal Laboratory Tests for ALT, AST, 
Total Bilirubin, Hemoglobin, Hematocrit, and Platelet Count Over the Course of the 10-
Week Intervention Period. As-Treated Population. 
• Table 18.2.  Shift Table for Normal and Abnormal Laboratory Test Changes from 
Screening to Weeks 0, 2, 8, 10, and 14 in ALT, AST, Total Bilirubin, Hemoglobin, 
Hematocrit, and Platelet Count. As-Treated Population.  
• Table 19.  Correlations between TNF activation score and urinary MCP1/uCr and 
TIMP1/uCr biomarker levels at baseline.  As-Treated Population with Kidney Biopsy 
Tissue Collected within 12 Months of Study Baseline. 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 22 of 24 
• Table 20.1.  Change from baseline to weeks 2, 4, and 8 in urinary MCP1/uCr. As-Treated 
Population. 
• Table 20.2.  Percent change from baseline to weeks 2, 4, 8, and 10 in urinary MCP1/uCr. 
As-Treated Population. 
• Table 20.3.  Urinary MCP1/uCr values at baseline and weeks 2, 4, 8, and 10.  As-Treated 
Population. 
• Table 21.1.  Change from baseline to weeks 2, 4, and 8 in urinary TIMP1/uCr. As-Treated 
Population. 
• Table 21.2.  Percent change from baseline to weeks 2, 4, 8, and 10 in urinary TIMP1/uCr. 
As-Treated Population. 
• Table 21.3.  Urinary TIMP1/uCr values at baseline and weeks 2, 4, 8, and 10. As-Treated 
Population. 
• Table 22.  Change in uEGF/uCr levels from baseline to weeks 2, 4, 8, and 10. As-Treated 
Population. 
• Table 23.  Correlation coefficient between uEGF/uCr at baseline with changes and 
percent changes in urinary MCP1/uCr and urinary TIMP1/uCr biomarker levels and in 
eGFR and UPCR values at week 10. As-Treated Population. 
• Table 24.  Changes in FSGS-MCD Patient Reported Outcome (PRO) score from baseline 
to weeks 2, 4, 8 and 10. As-Treated Population. 
 
12.2 List of Listings 
• Listing 1.  Dosing Data. As-Treated Population. 
• Listing 2.  Reasons for Permanent Discontinuation of Study medication.  As-Treated 
Population. 
• Listing 3.  Serious Adverse Events (SAEs). As-Treated Population. 
• Listing 4.  Non-Serious Adverse Events (AEs).  As-Treated Population. 
• Listing 5.  Deaths. As-Treated Population. 
• Listing 6.  Adverse Events Leading to Premature Discontinuation of Study Medication. 
As-Treated Population. 
• Listing 7.  Protocol Deviations.  All Subjects. 
 
12.3 List of Figures 
• Figure 1. CONSORT diagram. All Subjects. 
• Figure 2.1. Spaghetti plot of changes from baseline to weeks 2, 4, 8, and 10 in urinary 
MCP1/uCr. As-Treated Population. 
• Figure 2.2. Spaghetti plot of percent changes from baseline to weeks 2, 4, 8, and 10 in 
urinary MCP1/uCr. As-Treated Population. 
• Figure 2.3. Spaghetti plot of urinary MCP1/uCr at baseline and weeks 0, 2, 4, 8, and 10. 
As-Treated Population. 
• Figure 3.1. Spaghetti plot of changes from baseline to weeks 2, 4, 8, and 10 in urinary 
TIMP1/uCr. As-Treated Population. 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 23 of 24 
• Figure 3.2. Spaghetti plot of percent changes from baseline to weeks 2, 4, 8, and 10 in 
urinary TIMP1/uCr. As-Treated Population. 
• Figure 3.3. Spaghetti plot of urinary TIMP1/uCr at baseline and weeks 2, 4, 8, and 10. As-
Treated Population. 
• Figure 4.1.  Spaghetti plot of changes from baseline to weeks 2, 4, 8, and 10 in eGFR. As-
Treated Population. 
• Figure 4.2.  Spaghetti plot of percent changes from baseline to weeks 2, 4, 8, and 10 in 
eGFR. As-Treated Population. 
• Figure 4.3.  Spaghetti plot of eGFR at baseline and weeks 2, 4, 8, and 10. As-Treated 
Population. 
• Figure 5.1. Spaghetti plot of changes from baseline to weeks 2, 4, 8, and 10 in UPCR. As-
Treated Population. 
• Figure 5.2. Spaghetti plot of percent changes from baseline to weeks 2, 4, 8, and 10 in 
UPCR. As-Treated Population. 
• Figure 5.3. Spaghetti plot of UPCR at baseline and weeks 2, 4, 8, and 10. As-Treated 
Population. 
• Figure 6.1.  Spaghetti plot of participants with at least one abnormal laboratory value:  
ALT.  As-Treated Population. 
• Figure 6.2.  Spaghetti plot of participants with at least one abnormal laboratory value:  
AST.  As-Treated Population. 
• Figure 6.3.  Spaghetti plot of participants with at least one abnormal laboratory value:  
Total Bilirubin.  As-Treated Population. 
• Figure 6.4.  Spaghetti plot of participants with at least one abnormal laboratory value:  
Hemoglobin.  As-Treated Population. 
• Figure 6.5.  Spaghetti plot of participants with at least one abnormal laboratory value:  
Hematocrit.  As-Treated Population. 
• Figure 6.6.  Spaghetti plot of participants with at least one abnormal laboratory value:  
Platelet Count.  As-Treated Population. 
• Figure 7.1.  Scatterplot of TNF activation score and baseline urinary MCP1/uCr. As-
Treated Population. 
• Figure 7.2.  Scatterplot of TNF activation score and baseline urinary TIMP1/uCr. As-
Treated Population. 
• Figure 8. Spaghetti plot of participants’ uEGF/uCr from baseline to week 10.  As-Treated 
Population. 
• Figure9.1. Scatterplot of baseline uEGF/uCr and change from baseline to week 10 in 
urinary MCP1/uCr.  As-Treated Population. 
• Figure 9.2. Scatterplot of baseline uEGF/uCr and percent change from baseline to week 
10 in urinary MCP1/uCr.  As-Treated Population. 
• Figure 10.1. Scatterplot of baseline uEGF/uCr and change from baseline to week 10 in 
urinary TMP1/uCr.  As-Treated Population. 
• Figure 10.2. Scatterplot of baseline uEGF/uCr and percent change from baseline to week 
10 in urinary TMP1/uCr.  As-Treated Population. 


TNF Statistical Analysis Plan  
Version – 2023-10-31 
 
Page 24 of 24 
• Figure 11.1. Scatterplot of baseline uEGF/uCr and change from baseline to week 2, 8, 
and 10 in eGFR.  As-Treated Population. 
• Figure 11.2. Scatterplot of baseline uEGF/uCr and percent change from baseline to week 
2, 8, and 10 in eGFR.  As-Treated Population. 
• Figure 12.1. Scatterplot of baseline uEGF/uCr and change from baseline to week 2, 8, 
and 10 in UPCR.  As-Treated Population. 
• Figure 12.3. Scatterplot of baseline uEGF/uCr and percent change from baseline to week 
2, 8, and 10 in UPCR.  As-Treated Population.