Conditions: Diabetes Mellitus, Type 1
Keywords: Type 1 Diabetes TrialNet
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 89
Healthy volunteers: no

Brief summary:
This is a three-arm, 1:1:1 randomized, placebo controlled, double- blinded trial in which at least 28 subjects will receive active Anti-Thymocyte Globulin and Granulocyte colony-stimulating factor (ATG-GCSF), at least 28 subjects will receive ATG alone and at least 28 subjects will receive placebo alone within 100 days from diagnosis of Type 1 Diabetes (T1D).

The primary objective of the study will be to determine the safety and ability of low dose ATG plus GCSF and low dose ATG alone to retain/enhance C-peptide production in new onset T1D patients demonstrating residual beta cell function.

Detailed description:
The primary statistical hypothesis to be assessed in the study is whether the 2 hour area under the curve (change in baseline to 12 months) in residual beta cell function (C-peptide) will differ between those treated with ATG and GCSF or ATG alone as compared with placebo.

The study will also examine the effect of the proposed treatments on surrogate markers for immunologic and metabolic outcomes.

Arms:
arm 1: Anti-Thymocyte Globulin (ATG)/Placebo: Anti-Thymocyte Globulin (ATG) will be administered at a dose of 2.5mg/kg as two divided IV infusions of 0.5mg/kg and 2mg/kg. First dose (0.5mg/kg) will be infused over a minimum of 12 hours, and the second dose (2mg/kg) over a minimum of 8 hours. The second dose should be given no less than 12 and no more than 24 hours after the previous dose.

Placebo(for GCSF) treatment will begin 6 hours after completion of the ATG. Placebo will be given subcutaneously every 2 weeks for a total of 6 doses arm 2: Granulocyte colony stimulating factor (GCSF) is supplied in 0.6 mL prefilled syringes for subcutaneous injection. Each syringe contains 6 mg GCSF (based on protein weight), in a sterile, clear, colorless, preservative-free solution (pH 4.0) containing acetate (0.35 mg), sorbitol (30.0 mg), polysorbate 20 (0.02 mg), and sodium (0.02 mg) in water for injection, U.S. Pharmacopeial Convention (USP). The standard 6mg dose will be given with the exception of subjects who weigh less than 45 kg.

GCSF treatment will begin 6 hours after completion of the ATG / Placebo. GCSF will be given subcutaneously every 2 weeks for a total of 6 doses arm 3: Placebo for ATG will be administered by IV infusion in 2 doses. Placebo for GCSF will be administered subcutaneously every 2 weeks for a total of 6 doses

Interventions: intervention 1: Anti-Thymocyte Globulin (ATG) intervention 2: Granulocyte colony stimulating factor (GCSF) intervention 3: Placebo (for ATG) intervention 4: Placebo (for GCSF)
Intervention descriptions: intervention 1: Thymoglobulin intervention 2: Granulocyte colony stimulating factor (GCSF) intervention 3: Normal saline administered by IV infusion to mimic ATG intervention 4: Placebo prepared to mimic 6mg subcutaneous injection of GCSF

Protocol document:
SAP_002.pdf: 
1 
 
 
Statistical Analysis Plan 
 
 
TRIAL FULL TITLE 
 
Anti-Thymocyte Globulin (Thymoglobulin®) and Pegylated 
GCSF (Neulasta®)  
in New Onset Type 1 Diabetes (Protocol TN-19) 
 
EUDRACT NUMBER 
Phase 
Phase IIb 
SAP VERSION 
  
ISRCTN NUMBER 
 
SAP VERSION DATE 
August 30, 2017 
TRIAL STATISTICIAN 
Brian Bundy 
TRIAL CHIEF 
INVESTIGATOR 
Michael Haller 
SAP AUTHOR 
Jeffrey Krischer  
  


2 
 
Table of Contents 
1. 
Abbreviations and Definitions............................................................................................................. 3 
2. 
Introduction .............................................................................................................................................. 3 
2.1       Preface ............................................................................................................................................... 3 
2.2       Purpose of the analyses ............................................................................................................... 3 
2.3       Primary Outcome ............................................................................................................................ 3 
3. 
Primary analysis ..................................................................................................................................... 4 
4.       Secondary Outcomes and Analyses ................................................................................................. 4 
5.       Sample Size and Power Calculations ................................................................................................ 7 
6.       Interim Monitoring Plan ......................................................................................................................... 8 
7.        General Considerations ...................................................................................................................... 11 
7.1 
Timing of Analyses ........................................................................................................................ 11 
7.2 
Analysis Populations .................................................................................................................... 11 
7.2.1 
The Intention to Treat Population (ITT) ............................................................................ 11 
7.2.2 
Full Analysis Population ...................................................................................................... 12 
7.2.3 
Per Protocol Population ....................................................................................................... 12 
7.2.4 
Safety Population .................................................................................................................. 12 
7.2.5 
Adjustment of Confidence Intervals and p-values ........................................................ 12 
8. 
Safety Analyses .................................................................................................................................... 12 
8.1 
Adverse Events ............................................................................................................................... 12 
8.2 
Deaths, Serious Adverse Events and other Significant Adverse Events ........................ 13 
9. 
Reporting Conventions ....................................................................................................................... 13 
11. 
Technical Details .................................................................................................................................. 13 
12. 
References ............................................................................................................................................. 14 
 
 


3 
 
1. 
Abbreviations and Definitions 
 
 
AE 
Adverse Event 
ADA 
American Diabetes Association 
ANCOVA 
Analysis of Covariance 
AUC 
Area Under the Curve 
BMI 
Body Mass Index 
DSMB 
Data Safety Monitoring Committee 
ITT 
Intent to Treat 
MMTT 
Mixed Meal Tolerance Test 
PH 
Proportional hazards 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
T1D 
Type 1 Diabetes 
 
 
 
 
 
2. 
Introduction 
 
2.1 Preface 
 
A phase 2 study in humans with new-onset T1D and funded by the Immune Tolerance Network 
(ITN) was completed to determine if ATG (6.5mg/kg) could preserve C-peptide in new onset 
T1D. The study tested the hypothesis that selective deletion of lymphocytes would reset the 
immunologic rheostat, effect dynamic immune regulation and perhaps induce and maintain 
tolerance in T1D. While this study helped to establish the relative safety of ATG in humans with 
T1D, the study failed to demonstrate benefit (1). Post-hoc analyses have suggested that an 
initial decline in beta cell function amongst those who were treated with active drug was 
followed by a relative preservation of beta cell function. We hypothesize that the initial decline in 
beta cell function was related to the severity of cytokine release syndrome and serum sickness. 
Furthermore, we hypothesize that the currently proposed protocol (utilizing ~1/3 of the ATG 
dose in combination with GCSF) will promote a more tolergenic state while inducing less severe 
cytokine release/serum sickness and, as is supported by our preliminary data, 
preservation/improvement in beta cell function 12 months after therapy. 
 
2.2 Purpose of the analyses 
 
Analyses of study data will be conducted to address all objectives and other interrelationships 
among elements of study data of interest to the investigators and of relevance to the objectives 
of the study. Analyses by sex and race/ethnicity, as appropriate, are also planned.  
All primary analyses will be conducted under the intention-to-treat principle whereby all outcome 
data in all randomized subjects will be included, regardless of treatment compliance.  
 
2.3 Primary Outcome 
The primary outcome of each participant is the area under the stimulated C-peptide curve 


4 
 
 (
AUC
Y
) over the first 2 hours of a mixed meal glucose tolerance test conducted at the one-year 
visit.  The 
AUC
Y
 is computed using the trapezoidal rule which translates into a weighted sum of 
the timed C-peptide values over the 120 minute MMTT.  The more appealing quantity is the AUC 
mean which is 120
AUC
Y
 in nmol/L and will be denoted as 
Cp
Y
.   
3. 
Primary analysis 
Let  
/
,
, and 
ATG GCSF
ATG
Control
Cp
Cp
Cp
Y
Y
Y
 represent the C-peptide AUC mean for study patients receiving 
both ATG and GCSF, receiving ATG alone, and those receiving placebo, respectively. Likewise, 
let 
/
,
, and 
ATG GCSF
ATG
Control
Cp
Cp
Cp



 represent the population mean of C-Peptide for these groups at 
one year, respectively. 
The primary statistical hypotheses to be assessed in the study are:  
/
/
0
H : 
 versus H :  
ATG GCSF
Control
ATG GCSF
Control
Cp
Cp
a
Cp
Cp






 
 
0
H : 
 versus H :  
ATG
Control
ATG
Control
Cp
Cp
a
Cp
Cp






 
The primary analysis will be conducted on a transformed scale using the function log(
1)
Cp
Y

  .  
This provides better normal distributional behavior by the test statistic.  The comparison 
between any two treatment arms will be based on a Wald test of treatment effect in an ANCOVA 
model adjusting for gender, baseline age, and baseline (from the adjusted linear model) at the 
0.025 level (one-sided). Thus the overall Type I error for both tests is approximately 0.05.  
 
Using a step-up procedure additional covariates will be tested and included in the model only if 
they improve the log-likelihood at 0.05 level (2-sided).  This will be accomplished with the 
treatment assignment variable included but the inclusion/exclusion of the candidate covariate 
will be completely independent of the treatment variable’s impact on the model.  The Wald test 
associated with treatment variable in the full adjusted model will be used only as confirmation of 
the hypothesis test of treatment described in the previous paragraph. Thus the adjustment of 
the significance level is superfluous.   
 
 
4.  Secondary Outcomes and Analyses  
 
Additional analyses of the primary outcome to determine the effect of ATG with and 
without GCSF include: 
 
C-peptide AUC mean at 3, 6, 9, 12, 18 and 24 months using the ANCOVA 
mentioned above. 
 


5 
 
 
The hazard rate of C-peptide failure (C-peptide failure defined as the first occurrence 
at which the 2 hour peak C-peptide < 0.2 nmol/L during a MMTT) using the 
proportional hazards model while adjusting for baseline level of C-peptide, gender and 
baseline age. 
 
 
Longitudinal analyses of C-Peptide AUC means 3, 6, 9, 12, 18 and 24 months using 
a mixed effects model with a random intercept and slope by subject, adjusted for 
the baseline level of C-peptide, sex and baseline age. The mean intercept and slope 
will be compared between treatment groups. 
 
 
Treatment interactions with the covariates baseline C-peptide, sex and baseline age 
will be analyzed with a homogeneity test categorizing the continuous variables into 
3 approximately equal groups [could substitute age groupings recommended by 
investigators]; ladder plots will be constructed. Other variables to be tested for 
treatment interactions are HbA1c levels, HLA, other genotype and immune 
phenotypes, and race/ethnicity, as appropriate. 
 
Additional secondary objectives to determine the effect ATG with and without GCSF has on 
the following: 
 
 
HbA1c, Insulin dose (units/kg) and blood glucose over time by treatment group 
using ANCOVA. 
 
Adverse events 
o Number and severity. 
o The rates of severe adverse events will be computed (total number of 
events divided by total subject years of follow-up). 
 
Hypoglycemia 
o Number of major hypoglycemic events (defined as loss of consciousness, 
seizure, or requiring assistance from another person because of altered 
state of consciousness). 
o Reported hypoglycemic events confirmed with capillary blood glucose 
measurement less than 70 mg/dl. 
o The rates of severe hypoglycemic will be computed (total number of  events 
divided by total subject years of follow-up). 
 
Additional Outcomes and Analyses 
A mechanistic analysis plan will explore the following hypotheses: 
 
Effector T cell phenotypes are depleted upon treatment 
 
Treg phenotypes are partially spared upon treatment 
 
Hypo-responsive T cell phenotypes are increased upon treatment 
 
Tolerant/regulatory APC phenotypes correlate with kinetic changes in tolerance T-cell 
phenotypes 
 
Favorable changes in one or a combination of the above may correlate with treatments 
success (clinical outcome TBD). 
 


6 
 
To evaluate these hypotheses, the ITN will perform an extensive phenotypic characterization of 
T and APC cells using already developed flow panels to evaluate the impact of ATG+GCSF v 
ATG treatments on their relative frequencies and their activation status in this trial.  These same 
panels were previously tested in T1DAL and START (T cell panel) and AbATE (T cell and APC 
panels) trials; ITN will also be able to compare T cell and APC responses across therapeutic 
strategies in these T1D trials. 
Flow Panels: 
Modified X-trial T cell panel 
APC panel 
CD56 
CD19 
Granzyme B 
CD274 (PD-L1) 
L/D 
CD141 (BDCA-3) 
CD8 
CD3 
CD57 
CD304 (BDCA-4) 
Eomes 
HLA-DR 
CD3 
CD123 
FOXP3 
CD11c 
CD279 (PD-1) 
CD1c 
TIGIT 
CD16 
CD197 (CCR7) 
CD4 
CD127 (IL-7Ra) 
CD14 
CD45RA 
CD86 
KLRG1 
CD56 
CD45RO 
 
CD4 
 
 
A minimum of 5 million cryopreserved PBMC will be provided from randomized study 
participants at study weeks 0, 2, 10, and months 6 and 12 for longitudinal batched assessment. 
Based on the clinical outcome of the trial and/or successful identification if immune biomarkers 
of treatment and/or response outcome from this initial flow cytometric analysis, future 
immunophenotyping experiments with additional study time points may be proposed to further 
characterize cell populations of interest, an explore the kinetics of immune signatures of 
treatment and/or clinical response. 
Given the unknown expectations and interpretive power of the proposed analyses, the study 
team understands that the results may be somewhat limited; however, early positive findings 
would reinforce the primary publication so the team agreed that this the most practical way to 
move forward.  
Additional analyses will compare the results in this trial to other trials using ATG and GCSF 
and other TrialNet studies.  Data in this trial will be used in conjunction with other TrialNet 
data for exploratory analysis. 
 
 


7 
 
5.  Sample Size and Power Calculations 
The primary analysis will compare the difference in C-Peptide between experimental and 
placebo treatment groups at 12 months using log(
1)
Cp
Y

 transformation and ANCOVA model 
adjusting for sex, baseline age and the baseline value of log(
1)
Cp
Y

. Estimates of the mean 
and standard deviation of log(
1)
Cp
Y

 (expressed algebraically as: 
log(
1)
ˆ
Cp
Y

 and 
log(
1)
ˆ
Cp
Y

) in 
the placebo group were derived from the last four TrialNet studies of early onset disease. The 
90% confidence bound estimates were used to provide good confidence that this trial’s study 
population characteristics fall within these limits to assure the advertised statistical power; the 
estimates are 
log(
1)
ˆ
0.360
Cp
Y


 and 
log(
1)|
ˆ
0.167
Cp
Y
X



 (i.e., the residual mean squared error 
regressing on sex, baseline age and C-peptide). The geometric-like mean of 
Cp
Y
 for the placebo 
group is exp(0.341) 1
0.406

nmol/L. 
Using standard equations for the comparison of two means and a 1:1:1 allocation, a sample 
size of 78 participants (26 per group) with complete data would provide power of 85% to detect 
a 50% increase in the geometric-like mean in either experimental treatment group (compared to 
the control) using the Wald test (from the adjusted linear model) at the 0.025 level (one-sided).  
Thus the overall Type I error for both tests is approximately 0.05. 
Assuming that 10% of the participants will have missing data (one-year MMTT was not done or 
subject withdrew prior to the one-year assessment), the sample size goal for this study will be 
set at 84 participants (28 per group).  
The study will be closed to additional participants when the total number then randomized plus a 
fraction of those in screening (i.e., screening for eligibility) is expected to provide the proper 
number of eligible participants.  Participants who had already conducted the initial screening 
visit at that time will be allowed to complete screening and be randomized if both consenting 
and eligible. 
In the situation where both hypotheses are rejected in favor of the experimental regimen, then it 
is appropriate to decide which experimental regimen should be considered first for additional 
clinical trials.  If ATG and ATG+GCSF truly differ in efficacy, it is not expected that the difference 
would be as large as the effect size used in this design.  Thus requiring the difference to reach 
statistical significance would be too stringent due to the lack of statistical power.  The plan is to 
employ the method of Simon2 to select the treatment with the largest geometric-like mean in C-
Peptide regardless of how small the difference is over the other experimental regimen. Formally, 
the decision rule is to select the experimental regimen with highest predicted mean based on 
the fitted linear regression model of C-Peptide used in the formal hypothesis tests.  Given the 
one experimental treatment is associated with 25% higher mean (half of the design effect size) 
compared to the other experimental treatment, the probability is 96% that the most efficacious 
regimen will be selected with this decision rule.  Even if the increase is only 12.5% (i.e., a fourth 
of the design effect size), the probability is 82% in making the right choice.  Since under the null 


8 
 
(
/
 
 
ATG
ATG GCSF
Cp
Cp



) we consider selecting either treatment a non-error there is no contribution 
to the type I error, and therefore, no further adjustment is required to the α-level of the initial two 
pair-wise tests. 
Sample Size Re-estimation   
The Residual Mean Square Error (RMSE) and the mean of the control group are two 
parameters used in determining the sample size goal (NSSG=78) which are directly tied to the 
study cohort enrolled.  The former value is directly proportional to the sample size and the latter 
applies its influence in defining the effect size.  Thus the advertised statistical power of this trial 
may differ from the actual power if either differs from the initial values assumed.  Consequently, 
the plan is to estimate both values using the accumulated data (internal interim estimate, IIE) 
when approximately half the subjects (NSSG/2) have had their 1 year C-Peptide assessment.  
Since the initial values used are based on real data from four previous TrialNet studies, the re-
estimation of each parameter will be a two-term weighted average using the number of subjects 
on which the IIE is based as the one weight (NIIE) and the remaining number of subjects 
participants to be enrolled as the other weight (NSSG-NIIE).  Algebraically, the re-estimate will be 
computed: 
 
2
2
2
e
ˆ
ˆ
(
)
ˆ
IIE
IIE
SSG
IIE
Initial
R
estimate
SSG
N
N
N
N







  
Similarly, re-estimation of the mean of the control group will be calculated.  These new 
estimates will be used to calculate a new sample size goal for the trial. 
It is important to note that this adaptive procedure is a non-comparative interim analysis (i.e., 
the observed treatment effect has no influence on the sample size re-estimation).  Therefore, 
this analysis has no effect on the type I error.  Note the re-estimation can be conducted by the 
analyst in a blinded fashion.  The goal is to assure adequate statistical power at the completion 
of the trial by assuring the two design parameters reflect accurately the study population being 
enrolled.   
 
6. Interim Monitoring Plan 
 
Interim analyses will be conducted periodically during the study and will be reviewed by the 
TrialNet Data and Safety Monitoring Board (DSMB) for assessment of effectiveness and safety; 
the TrialNet DSMB meets at least every six months to review study progress and safety. An 
independent medical monitor will closely monitor the events in the trial as described in section 
10.4. If a group sequential stopping boundary is crossed, the DSMB may terminate enrollment 
into the trial early.   The Lan-DeMets3 spending function with an O’Brien-Fleming boundary will 
be used to protect the type I error probability for the primary outcome analyses, and to assess 
the significance of the interim results periodically during the trial.  The spending function that 
approximates the O’Brien-Fleming boundaries is: 
 


9 
 
 
 
 
where 
 is the information fraction (
) and 
 is the fixed-sample type I error (i.e., 
0.025). The monitoring plan will allow for early termination based on the treatment effect on C-
peptide values at 1 year of follow-up using the ANCOVA model described above. 
 
The DSMB will also be informed if there is a serious lack of evidence of a treatment effect 
(i.e. futility analysis). The boundaries are based on the paper by Lachin4. The study arm 
should be “closed” based on the futility of rejecting the null hypothesis at the completion of 
the trial if: 
zATG(t
* ) 0.1when 0.5  t*  0.75 or if 
zATG (t
* ) 1.0 when t*  0.75 (z is the Wald test of the treatment effect coefficient). The same rule 
would be applied to the ATG/GCSF treatment group. These t* 0.5 and 0.75 are equivalent to 
when there are 39 and 59 participants with one-year C-peptide results, respectively. Lachin 
showed that a onetime use of either boundary for the design parameters above    
(Z1Z13.00 ) raises the type II error to approximately 0.15414 and 0.15611, 
respectively.  For larger values of t*the increase to the error probability is even less. 
Furthermore, by the laws of probability a single use of each rule will increase the type re than 
the sum of the increase (i.e., 0.00414+0.00611 = 0.0103). 
 
Using Lachin’s formulas a onetime use of either boundary for the design parameters above (
1
1
2.8
Z
Z








) raises the type II error to approximately 0.204 and 0.202, respectively.  For 
larger values of 
 the increase to the error probability is even less.  Furthermore, by the laws of 
probability a single use of each rule will increase the type II error no more than the sum of the 
increase (i.e., 0.15+0.004+0.002 = 0.156).   
 
Additional analysis will assess potential adverse outcomes of treatment and will assess the 
incidence of all severe adverse events. 
Interim Analysis Study Modification  
Whether Lan-DeMets boundary is crossed or a futility decision rule indicates closure of an 
experimental arm, the course of action for the study is as follows. In case the futility decision 
rule indicates closure of one of the experimental arms (and the governing bodies concur), the 
trial should continue randomizing between the other remaining experimental treatment and 
placebo (1:1 ratio) since the corresponding hypothesis remains unaddressed. However, in the 
circumstance where the Lan-DeMets boundary is crossed the normal course of action would be 
to close the placebo arm. This would incapacitate the second formal hypothesis test thus 
leaving only the issue of which experimental treatment is “best” as the remaining question. 
However, it may or may not be reasonable to make this decision as well. To evaluate the 
evidence at the interim the plan is to use stochastic curtailment and compute the probability that 
the currently ‘best’ treatment (as defined by the decision rule above) is not the ‘best’ when the 
full sample size is reached. In the normal employment of stochastic curtailment in futility 
*t
*
0
1
t



*t


10 
 
analysis the parameters used for the simulation are based on the alternative hypothesis. The 
analogous parameters for this situation is to set the mean of the currently ‘best’ treatment to be 
lower than the other experimental treatment mean and determine the probability of a reversal. 
As with the normal application of stochastic curtailment, we will use the probability of 0.20 or 
less to stop accrual completely and invoke the decision rule for selecting the ‘best’ treatment 
immediately. Alternatively, the study would continue to its planned completion, randomizing 
between the two active treatment arms, until the projected probability of a re-ordering of the 
outcome (change in the selection of the superior treatment arm) is less than 0.2. 
 
Our choice for setting the parameters for simulation is to set the ‘best’ treatment group at 25% 
lower than the other experimental treatment mean; these two parameters being centered at the 
midpoint between the two estimated experimental group means. This is consistent with the 
difference between the two treatment arms specified in the Simon selection at the completion of 
the trial (described above), should both arms be superior to the placebo.  
The graph below provides an estimated probability of a ‘best’ selection reversal conditioned on 
the interim data (simulated under the condition of crossing the Lan-DeMets boundary and t* = 
0.5). The probability is primarily a function of the relative position of the ‘lesser’ mean to the 
positions of the placebo mean and the ‘greater’ mean (all means being estimates from the 
interim data). That is, the closer the ‘lesser’ mean is to the placebo, the less likely there could be 
a reversal in the selection of the ‘best’ treatment.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


11 
 
 
 
 
Probability of Reversal Given the Relative Location of the Lesser   
Experimental Group Mean 
 
  
 
 
 
Mean Placebo Grp  
 
 
 
 
 
 
 
 
Mean (Greater) 
Relative Location of Lesser Mean 
 
 
 
 
Additional analysis will assess potential adverse outcomes of treatment and will assess the 
incidence of all severe adverse events. 
 
7. General Considerations 
 
7.1 Timing of Analyses 
 
The final analysis will come after all subjects have had their 1-year MMTT (or been followed 
long enough to determine they will not) to achieve the planned 80% statistical power for the 
primary analysis. 
 
7.2 Analysis Populations 
 
7.2.1 The Intention to Treat Population (ITT) 
The intention to treat population comprises all randomized (as planned) subjects. 
P ro b a b ilit y 
0 . 0   0 . 1   0 . 2   0 . 3   0 . 4   0 . 5   0 . 6   0 . 7   0 . 8   0 . 9   1 . 0 


12 
 
 
7.2.2 Full Analysis Population 
The Full Analysis Set (FAS) will comprise all subjects who received any study drug and who 
participated in at least one post-baseline assessment. These will be analyzed as 
randomized. FAS will be the primary efficacy population. So, FAS is a subset of ITT 
 
 
 
7.2.3 Complete Case Population 
The complete case analysis will comprise all subjects that have had the endpoint measured.  
This is the population on which the primary hypothesis test will be based. 
 
 
 
 
7.2.3 Per Protocol Population 
The Per Protocol Set (PPS) will comprise all subjects who did not substantially deviate from 
the protocol as to be determined on a per-subject basis before data base lock and 
unblinding. These will be analyzed according to actual treatment received and stratum. 
 
7.2.4 Safety Population 
All subjects who received any study treatment (including control) but excluding subjects who 
drop out prior to receiving any treatment. 
 
During accrual we discovered that a couple of subjects enrolled on the >= 18 year of age 
stratum did not have a second OGTT prior to randomization to confirm abnormal glucose 
tolerance.  Eligibility is technically unknown without the results of the missing test.  Given the 
slow accrual of this trial we pursued every reasonable justification to retain such subjects 
blinded from their end result.   Given that the these subjects had an OGTT on study indicated 
abnormal glucose tolerance, not necessarily consecutive, we considered these conditions 
sufficient to retain these subjects for analysis.  This rule will be applied consistently to any 
subjects >=18 that fail to have the confirmatory test. 
 
7.2.5 Adjustment of Confidence Intervals and p-values 
 
 No adjustments will be necessary because no interim analyses were conducted. 
 
8. Safety Analyses 
 
Safety will be evaluated with summary of adverse events for the safety population. 
The following parameters will be assessed during the study:  
 
8.2 Adverse Events 
The summary statistics will be produced in accordance with Section 8. 
Treatment emergent adverse events (AEs) are those events that occur after the baseline 
assessment. Only incidence of the following AEs will be reported: 
 
A tabular summary of AE will present: Number of subjects with any AE; Number of SAEs with 
outcome death; Number subjects with SAE; Number subjects with AEs leading to 
discontinuation of study drug; Number of subjects with AEs leading to discontinuation of study; 
Total number of AEs; Total number of SAEs [TABLE]. 
 
The Adverse Events summary tables will include number of adverse events, the number of 
subjects in each treatment group in whom the event occurred, and the incidence of occurrence 
and should be grouped by system organ class, preferred terms and/or other interested variables 


13 
 
(e.g., relatedness, intensity and seriousness). [TABLE] 
 
When calculating the incidence of adverse events, or any sub-classification thereof by 
treatment, time period, severity, etc., each subject will only be counted once and any repetitions 
of adverse events will be ignored; the denominator will be the total population size. 
 
8.3  Deaths, Serious Adverse Events and other Significant Adverse Events 
 
All formal testing of adverse effects will be based on the subject as the experimental unit.  Thus 
for comparing incidence of AE within system organ by treatment group, a one-sided Fisher’s 
exact test will be conducted at 0.05 level (higher incidence in experimentally treated group is the 
alternative hypothesis).  Also, highest AE grade will be determined for each subject and 
compared by treatment group using a 2 sample Wilcoxon test (one-sided at 0.05).   No 
correction for multiple testing will be employed in order that the statistical power is maintained.  
 
9. Reporting Conventions 
 
P-values ≥0.01 will be reported to 2 decimal places; p-values ≥0.001 but less than 0.01 will be 
reported to 1 decimal place; p-values less than 0.001 will be reported as “<0.001”. The mean, 
standard deviation, and any other statistics other than quantiles, will be reported to one decimal 
place greater than the original data. Quantiles, such as median, or minimum and maximum will 
use the same number of decimal places as the original data. Estimated parameters, not on the 
same scale as raw observations (e.g. regression coefficients) will be reported to 3 significant 
figures. 
 
10. Per Protocol Analysis 
 
Quantifying the evidence of any dose response relationship is part of a complete analysis of any 
well run and completed clinical trial.  This is especially true when the trial’s primary outcome is 
negative to explore whether there is evidence that deviations from the treatment protocol 
(planned or not) may have played a role in the negative outcome.  Given this we plan to assess 
the treatment effect on the c-peptide mean by the degree of compliance to the ideal scheduled 
dose in a quantitative manner. Using the ANCOVA model we will assess the evidence for an 
effect of treatment compliance including the entire cohort.  The number of courses of treatment 
will be introduced into the model to determine its effect on risk (regardless of treatment group).  
If there is evidence that it is predictive (<=0.05, one-sided) then a treatment-courses of therapy 
interaction term will be introduced to see if there is a different compliance gradient between the 
two treatment groups.  The procedure for including covariates, such as age, will follow the set 
up procedure as described above under Primary and Secondary analyses. 
    
 
11. Technical Details 
 
The analysis will be performed in R, S-Plus or SAS. 
The distributional assumptions as well as other assumptions underpinning the planned analyses 
will be checked. Final decisions regarding analysis methods and choice of explanatory variables 
will be taken then. 


14 
 
 
 
 
 
12. References 
 
1. Gitelman SE, Gottlieb PA, Rigby MR, Willi SM, Fisher LK, Moran A, Gottschalk M, Moore 
WV, Pinckney A, Keyes-Elstein L, Aggarwal S, Phippard D, Sayre PH, Ding L, Bluestone JA, 
Ehlers MR, Felner EI, the START Study Team. Antithymocyte globulin treatment for patients 
with recent-onset type 1 diabetes: 12-month results of a randomised, placebo-controlled, phase 
2 trial. The Lancet Diabetes & Endocrinology, 28 August 2013; doi:10.1016/S2213-
8587(13)70065-2. 
2. Simon, R., Wittes RE, Ellenberg SS. Randomized phase II clinical trials. Cancer Treatment 
Reports 1985; 69: 1375-81. 
3. DeMets DL, Lan G: The alpha spending function approach to interim data analyses. Cancer 
Treat Res 75:1-27, 1995. 
4. Lachin JM. A review of methods for futility stopping based on conditional power. Statistics in 
Medicine, 24, 2747-2764, 2005.   
5. Cox DR: Regression model and Life Tables J R Stat Soc 34B:187-220, 1972 
6. Kalbfleisch JD, Prentice RL: The statistical analysis of failure time data., 1980 
7. Diggle PJ, Heagerty PJ, Liang K-Y, Zeger SL: Analysis of longitudinal data. Oxford, UK, 
Oxford University Press, 2002 
8. Reboussin DM, DeMets DL, Kim KM, Lan KK: Computations for group sequential boundaries 
using the Lan-DeMets spending function method. Control Clin Trials 21:190-207, 2000 
10. Wieand S, Schroeder G, O'Fallon JR: Stopping when the experimental regimen does not 
appear to help. Stat Med 13:1453-1458, 1994 
 
 

ICF_001.pdf: 
 
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Model Intervention Informed Consent 
Type 1 Diabetes TrialNet Protocol TN-19 
 
 
Effects of Low Dose ATG (Thymoglobulin®) Used Alone or in  
Combination with GCSF (Neulasta®) on the Progression of  
Type 1 Diabetes in New Onset Subjects  
 
Project Description  
You (you means you or your child) are being asked to take part in this research study 
because you have developed type 1 diabetes within the last 3 months. Type 1 diabetes is 
an autoimmune disease. This means that the immune system, the part of the body which 
helps fight infections, mistakenly attacks cells that produce insulin in your body. The cells 
that produce insulin are called beta cells and are found in your pancreas. As the immune 
system destroys these cells, the ability to produce insulin decreases and diabetes develops.   
 
The investigators carrying out this study are part of a group called TrialNet, which is 
studying type 1 diabetes (T1D). These investigators are testing a medication, called ATG 
(Thymoglobulin) given alone and in combination with GCSF (Neulasta) as a possible 
treatment for people with newly diagnosed type 1 diabetes. 
    
ATG is given by infusion (through a vein) and is approved by the U.S. Food and Drug 
Administration as a treatment to protect organ rejection after transplantation.  GCSF is 
given by injection and is approved for use in people undergoing chemotherapy for cancer. 
GCSF has also been tested in the treatment of autoimmune diseases. We have recently 
completed a small study giving individuals with T1D low dose ATG in combination with 
GCSF.   Results from this study suggest that combination treatment may preserve insulin 
secretion after diagnosis.  
 
Based on these findings, the goal of this study is to conduct a larger, more conclusive study, 
to learn if low dose Thymoglobulin (ATG) used alone or in combination with Neulasta (ATG/ 
GCSF) could help people with newly diagnosed type 1 diabetes by delaying or stopping the 
further destruction of beta cells.  
 
This consent form tells you about the study and what people in the study will be asked to 
do. The study will be explained to you and you will be given the chance to ask questions.  
You will be given a research volunteer handbook that explains the overall study. Taking part 
in this study is your decision.  


 
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If you agree to take part in the study, you will be asked to sign this consent form. You will be 
given a copy of the consent form to keep for your records. 
 
Procedures 
 
There are two parts to this study, a treatment phase that lasts three months and a 
monitoring phase that lasts 21 months.  
  
Treatment Phase 
The treatment phase is completed in the first three months. The first part of the treatment 
phase (inpatient visit) involves you staying overnight for 2-3 days in a hospital or research 
setting. The second part of the treatment phase requires 5 outpatient visits every two 
weeks.  
 
At the in-patient visit, you will be randomly placed into one of three treatment groups. One 
group will receive ATG infusions given through a vein and GCSF injections. Another group 
will receive ATG infusions and placebo injections. The third group will receive placebo in 
both the infusion and injections. A placebo looks like medicine, but has no medicine in it so 
that people in the study will not know whether they are receiving the drug or placebo. You 
will be placed into one of these groups by chance (similar to drawing straws). You will have 
a 1 out of 3 chance of getting ATG alone, ATG and GCSF, or placebo. Neither you nor your 
doctor will be able to choose the group in which you will be placed. Neither you nor your 
doctor will know who is getting ATG or ATG and GCSF and who is getting placebo. 
 
You will receive two treatment infusions of ATG or placebo given at the research site over a 
period of 2-3 days and one treatment injection of GCSF or placebo after completing the 
second infusion.  If you are assigned to receive ATG, you will receive a total of 2.5 mg/each 
kg of body weight.   
 
The study treatment infusion of ATG or placebo is given by placing a needle and/or plastic 
tube (IV) in your arm.  This IV will be left in place for the duration of the visit which will take 
2-3 days. You will be given two infusions of ATG or placebo. The first study treatment 
infusion will be given continuously and will take between 12 to 20 hours.  There will be 12-
24 hour break between infusions. The second infusion will take from 8 to 16 hours. The 
length of the visit and the time for each infusion depends on whether you are have reactions 
or side effects from the study treatment. 
 
At least 30 minutes before each infusion you will receive acetaminophen, an antihistamine 
to prevent or decrease the immediate reactions to the study treatment as well as another 
medication called methylprednisolone or placebo which is used to suppress inflammation.  
Additional acetaminophen or antihistamine will be given as need to treat any reactions to 
the study medication. You will also receive methylprednisolone or placebo 12 hours after 
the start of each infusion.   
 
During the study treatment infusions you will be checked frequently for any side effects. 


 
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Beginning at the time the infusion starts we will check how you are feeling and measure 
your temperature, blood pressure and pulse every 30 minutes for the first two hours and 
then every hour until the infusion is completed. 
   
Study Treatment Injection Procedure: 
Approximately 6 hours after you have completed the study treatment infusion you will get 
the first of 6 study treatment injections. The injection is given subcutaneously (a shot under 
your skin) just like insulin. After you go home, you will return to the study site for 5 additional 
injections to be given every two weeks, over the following ten weeks.  
 
After the treatment phase is done, you will be monitored closely. We will remain in daily 
contact with you the first two weeks of the study.  
 
Monitoring Phase 
After completing the treatment phase, you will be monitored regularly. You will come to the 
study site every three months for 2-hour Mixed Meal Tolerance Tests (MMTT) at the 3, 6, 
and 9 month visits. You will have a 4-hour MMTT at the 12, 18 and 24 month visits. During 
the second year of the study will come in every six months for MMTT visits and other blood 
tests.  
 
Other study procedures: 
During study visits we will ask questions about your health, diet and activity, and experience 
as a research participant. We will periodically perform a physical examination and take 
blood for testing. Females will also give regular urine samples to be checked for pregnancy.  
 
The blood tests will help us monitor your diabetes, your immune system, and your general 
health.  
The total amount of blood drawn for tests done at each visit will not exceed the amount that 
is safe for your age and weight and will not exceed 8 tablespoons. More information about 
the specific blood tests can be found in the research volunteer handbook. 
 
 Mixed Meal Tolerance Test (MMTT) 
The study requires that you have 7 MMTTs to find out how much insulin your pancreas is 
still making. The MMTT will be done every three months during the first year and every six 
months during the remainder of the study.  
 
Before each MMTT, you will get special instructions about diet and insulin dosing.  To make 
the blood sampling easier for the test, an intravenous needle and plastic tube (IV) will be 
placed in your vein. The IV will be kept in place during the test. Two blood samples taken 
ten minutes apart (one teaspoon of blood for each sample) will be taken through the IV. You 
will then be given a drink called Boost, the “mixed meal”. This drink will raise your blood 
sugar and cause your body to produce insulin. After drinking Boost, one-half teaspoon of 
blood will be taken through the IV at regular intervals for 2 hours.  The total amount of blood 
taken for the MMTT will not be greater than 2 tablespoons for the two hour test and 3 
tablespoons for the 4 hour test.  
 


 
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 Diabetes Care 
If you decide to be in this study, you will receive what is called “intensive management” of 
your diabetes. The goal of this type of treatment is to keep your blood sugar as close to 
normal as possible. This will require you to take enough daily insulin injections to meet this 
goal.  You could also be on an insulin pump instead of injections. During the study you will 
need to check your blood sugar levels frequently, and report them as often as once every 
two weeks to the study team. Your research study team will work with your personal 
diabetes health care team to keep your diabetes under good control.  
 
Blood Samples for Understanding Type 1 Diabetes  
 
An important part of this study is to better understand what causes type 1 diabetes, how 
individuals respond to treatments, and to get ideas about new treatments in the future.  
While TrialNet is ongoing, these samples will be used only by TrialNet approved 
researchers. As such, we may be collecting blood samples including genetic samples for 
these studies at most of your visits. You will not routinely be provided with test results from 
these studies.  
 
 Risks and Discomforts  
The treatment and tests involved in this research project have the known risks listed below.  
There may be other risks that are not possible to predict.  
 
ATG:  
Common Side Effects:  
 
Frequent side effects of ATG include: low blood counts for white blood cells (the cells that 
help fight infections), headache, back pain, and joint pain.  
 
During the inpatient visit, you may experience “cytokine release syndrome (CRS)”. 
Symptoms of CRS include; nausea, vomiting, fever, chills, and increased heart rate.   These 
side effects most often resemble a mild flu.  To help reduce these symptoms you will be 
given medications before each ATG infusion (acetaminophen, antihistamine, and 
methylprednisolone as described above). 
 
You may also have another type of reaction called “serum sickness”. If serum sickness 
occurs, it is expected 1-2 weeks after the ATG infusion. The symptoms of serum sickness 
may include the following:  fever, itching, rashes, muscle or joint pain, swelling and a 
general feeling of unease. The study doctors will provide medicines to treat serum sickness 
if it occurs. 
 
 
Uncommon Side Effects: 
 
Rare but serious side effects include infections, allergic reactions, and bleeding.  


 
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Allergic Reactions: A reaction to ATG may occur in people who are allergic to rabbits 
because the drug contains rabbit proteins. This reaction could be a skin rash or trouble 
breathing within the first hour of the infusion.  The study doctors will start the ATG treatment at 
a low dose, and will give it very slowly.  If symptoms develop and become severe, the study 
doctors will slow the infusion even more or stop it for a period of time. If necessary, they can 
give you more of the same medications and/or other medicines to help reduce allergic like 
symptoms. Such side effects include:  chest pain, diarrhea, shortness of breath, fever, 
shivering, low blood pressure, lack of appetite, dizziness, itching, sweating, and hives and 
could occur within the first hour of the infusion. 
 
In very rare situations, some people have a severe allergic reaction to ATG called 
anaphylaxis.  Anaphylaxis could be life-threatening, with symptoms that include difficulty 
breathing and a fall in blood pressure severe enough to cause shock.  Due to the seriousness 
of this reaction, you may require other medicines to recover.  In the unlikely event that this 
happens, you will not receive any more study medication.  
 
Infections: You may be at a higher risk for getting infections for about 3 months after 
receiving ATG. To prevent you from getting infections, you will be followed closely by the 
study doctors. You should contact your study doctor if you develop any infections, or 
bruising or abnormal bleeding, or you are not feeling well at any time.  
  
 You should avoid direct contact with anyone who you know has an infection during this time.  
Bleeding: Rarely, bleeding may occur.  ATG may cause the number of platelet cells in your 
blood to decrease.  Platelets help your blood clot. If they decrease to very low levels there is a 
higher risk for bleeding.  Your platelet count will be monitored each day before ATG is given.  
If the level falls into a range that is considered high risk for bleeding, then the drug dose will 
either be reduced or held.  If the platelet count recovers to an acceptable range in one to two 
days, then the ATG will be given again. 
GCSF:  
There are also some risks associated with receiving GCSF. While taking GCSF your white 
blood cell count may become elevated. This could cause your spleen to become enlarged.  In 
some instances an enlarged spleen could increase your risk for splenic rupture which could 
be life threatening.  Call your study doctor immediately if you have pain in your upper left 
abdomen or left shoulder. GCSF therapy has also been associated with breathing problems in 
a small number of patients who received the drug as part of cancer therapy. GCSF also 
carries a 10-20% risk of bone pain and a less than 10% risk of fever, pain or redness at the 
site of injection, headache, and dizziness.   
 
Unknown Risks: The experimental study treatment may have side effects that no one 
knows about at this time.  Medications like ATG and GCSF that alter responses by the 
immune system can possibly lead to an increased risk of certain types of cancer. The risk of 
cancer is unknown but is thought to be very small. An increase in the rate of cancer has not 
been seen in previous studies of people given either medication. 


 
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 Birth control and pregnancy 
It is not known whether ATG or GCSF can damage unborn babies. If you could become 
pregnant, you will need to use an effective form of birth control during the first 3 months of 
participation in the study. This is for safety during the treatment part of the study.  Women 
must not become pregnant for two years from starting the study. If you become pregnant, 
you must tell the study doctor right away. We will stop your study medicine.   
 
 Intravenous Needle (IV) and Blood Drawing 
While on the study you may have side effects from having your blood taken or IV placed. 
The risks of side effects from these procedures are very small. There is sometimes 
soreness and/or a bruise at the site where the needle goes through the skin. Once in a 
while, people faint. It is rare, but some people may get an infection, a small blood clot, 
swelling of the vein and the area around it or bleeding where the needle goes through the 
skin.  
 
   
 Genetic Testing 
We will try to understand how genes and gene function may relate to diabetes and ATG 
given alone or in combination with GCSF.  We will not provide the results of your genetic 
testing to you or anyone else.  Although we will try very hard to keep any information about 
your genetic testing private, there is a very small possibility that someone else could learn 
about your testing.  
 
 Vaccines:  
Some types of vaccinations (“live” vaccinations) may not be safe during the treatment 
period.  If you need any “live” vaccinations, you should get them more than six weeks 
before enrolling in the study.   Other types of vaccines (other than live vaccines) might be 
less effective in people whose immune systems are less active. We don’t know if ATG will 
have this effect. Nonetheless, we do recommend that you get an influenza vaccine during 
flu season. Be sure to talk to your study team before getting influenza vaccine during flu 
season as the timing of this is important and will depend on when you completed the ATG 
infusion. 
 
 Except for the “flu shot” you should not have any other vaccines during or for three months 
following the treatment phase of the study.  After that time, be sure you let us know if you 
have received any vaccines as part of your usual medical care.  
 
 Mixed Meal Tolerance Test (MMTT):  
The MMTT requires that you drink a product called BOOST which contains milk and soy 
ingredients. People with severe allergies to these could have a reaction. If you have a 
known allergy to either of these ingredients, please let us know. It is possible we may need 
to advise you not to participate in the trial. 
 


 
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Benefits  
If you decide to take part in this study, there is no guarantee that your health will improve.  It 
is hoped that the ATG given alone or in combination with GCSF will help your body continue 
to make insulin, but there is no guarantee that this will happen.  Even if the study drug can 
protect the insulin producing cells that are left, you will still need to take insulin shots.  
Studies have shown that people who continue to make insulin have less trouble with low 
blood sugars and fewer complications from their diabetes than people who no longer make 
their own insulin.  We will follow your health and diabetes closely. 
 
Study Duration and Number of Participants 
There will be at least 84 participants enrolled in the study. You will be a participant in this 
study for two years. After completing the study, we will invite you to be followed in the 
TrialNet Long-Term Investigative Follow-Up Study (LIFT) so we can continue to provide you 
with further information about your diabetes and learn how long the effect of treatment will 
last as well as any possible long-term side-effects.   
 
Alternatives to Participation 
Before you decide to take part in this study, we will talk with you about the other options 
available to you. You may choose not to participate in this study. There may be other 
research studies that you can choose to be in. 
 
Source of Funding  
The National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK) is providing 
major funding for this study. The study is also sponsored by National Institute for Allergy 
and Infectious Diseases (NIAID), the National Institute for Child Health and Human 
Development (NICHD), the National Center for Research Resources (NCRR), the Juvenile 
Diabetes Research Foundation (JDRF), the American Diabetes Association (ADA), and the 
Helmsley Charitable Trust.  Sanofi will provide the ATG (Thymoglobulin) and Amgen will 
provide the GCSF (Neulasta) as well as the placebo injections for the study. Sanofi will also 
be providing additional support for the study. Roche Diagnostics will be donating blood 
glucose meters and strips.  
 
Cost to Subject  
There is no cost to you for being in this study. There will be no charge for procedures or 
drugs required by the study. You will have to pay the costs for most of your usual diabetes 
care supplies (insulin, needles, etc.). 
 
Subject Payment 
If you decide to be in this study you will receive a small compensation for each study visit 
you attend. In addition to this, we will pay for your minor travel or parking costs. By signing 
this consent form, you understand and agree that, if this research project results in the 
development of any product that can be sold, you will not receive a share of any money that 


 
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is made. 
 
Study Withdrawal 
Your choice to be in this study is completely voluntary. You may choose not to be in this 
study or to stop being in this study at any time, and your doctor will still take care of you.   
Your current or future medical care will not be changed if you decide not to be in this study 
or to stop being in this study at any time. Your study doctor may also choose to discontinue 
your study treatment at any time if it is felt that continuing treatment may hurt you. This may 
happen if the side effects are too great, or if you do not follow the study instructions. You 
will be told of any new findings that affect your being in this study.   
 
Invitation for Questions  
You will receive a copy of this consent form. Please ask questions about this study or 
consent at any time. You are welcome to talk about this study or consent with your family, 
doctor, or anyone else. The staff of the research study will be happy to discuss any 
questions with you. You may ask your questions to ___________________ at phone 
____________________. 
 
Confidentiality: 
 
Your consent to be in this study gives the TrialNet researchers permission to collect 
personal information about you and to use it for research purposes.  Personal information is 
information such as your name that directly identifies you. This information may be shared 
with other TrialNet centers as needed to help with the study. Your consent also includes 
permission for the sponsor of this study (NIDDK) and the Food and Drug Administration 
(FDA) to review your records.  
 
If you participate in this study, you will be given a unique study code number.  It will identify 
the information and samples collected from you from study examinations and procedures. It 
will be sent to the central TrialNet Coordinating Center under the supervision of the NIDDK.  
 
A Certificate of Confidentiality has been obtained from the National Institutes of Health 
(NIH). This is intended to further protect the confidentiality of information that we obtain 
about you. By having a Certificate of Confidentiality, TrialNet researchers are not required to 
give information that can be used to identify you.  For example, we cannot be forced to give 
information about you to insurance companies. Also, we cannot be forced to give 
information about you for any civil, criminal, administrative, or legislative proceedings 
whether at the federal, state or local level. However, the Certificate of Confidentiality does 
not prevent you from giving this information to others. Please understand that we will 
maintain the confidentiality of your research record. We cannot guarantee the confidentiality 
of test results provided to you if you wish to share them. 
 
There are some rare exceptions to the protection offered by the Certificate of 
Confidentiality. TrialNet researchers are not prevented from telling about matters such as 
child abuse, certain infectious diseases, or threatened violence to yourself or others.  


 
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TrialNet researchers will consider your records private.  Rarely, representatives of the 
United States Department of Health and Human Services (DHHS) or TrialNet may review or 
ask for a copy of your study records.  If this happens, we will provide your records. Also, for 
auditing purposes, employees of the (institution’s name) _____________________ or its 
agents could be allowed to see your study records to make sure that the study is being 
done properly. 
 
The results of this study may be published for scientific purposes.  By signing this form, you 
are agreeing to this.    Your records and results will not be identified as belonging to you in 
any publication. 
 
Payment for Injury or Harm 
Taking part in this research study may hurt you (this was explained in the section called 
“Risks and Discomforts”). If you need to get medical care right away, you should go to the 
nearest emergency care center. Be sure to explain that you are participating in a research 
study. If you do not need emergency care, the investigator will take care of you or help you 
get the care you need. You will be sent a bill for whatever medical care you receive. You will 
be responsible for any costs not covered by your health insurance. TrialNet and the clinical 
site you go to will not pay for your care. Likewise, TrialNet and your clinical site will not pay 
you for pain, worry, lost income, or non-medical costs that might occur from being in this 
research study. 
 
Additional Information: 
 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov  
as required by U.S. Law. This website will not include information that can identify you. At 
most, the website will include a summary of the results. You can search this website at any 
time. 
 
CONSENT FORM FOR SUBJECTS (18 years of age or older) OR PARENT/GUARDIAN 
(if subject is less than 18 years of age) 
 
 
Storage of Samples in NIDDK Repository 
When TrialNet is over, we intend to put any remaining samples including genetic samples 
into the National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) repository for 
future studies related to type 1diabetes and its complications. They will be stored there 
indefinitely without your name or any other identifying information on them, as such, once in 
the repository you will not be able to have them removed. Researchers must first get 
permission from the National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) 
to use samples from the repository.   
 
The following checkbox gives you the choice of allowing us to put any remaining blood 
samples in the NIDDK repository.  Even if you decide not to have your remaining blood 


 
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samples stored, you can still participate in this study.  
 
Are you willing to allow us to put any remaining blood samples including genetic samples in 
the NIDDK repository (please initial yes or no)?  
 
________YES 
 
_________NO 
 
 
 
 
Authorization 
 
By signing this consent form, you agree that you have read this informed consent form and 
that the study has been explained to you. You also agree that your questions have been 
answered and that you agree to be in this study. You do not give up any of your legal rights 
by signing this informed consent form. You will receive a copy of this consent form. 
 
I have read this paper about the study or it was read to me. I know what will happen, both 
the possible benefits and the possible risks. I choose to be (or to have my child) in this 
study. I know I can stop being in the study at any time, and I will still get the usual medical 
care. I will get a copy of this consent form.  
 
Participant 
Print Name of participant: ________________________________________ 
 
Signature of participant (age 12 or older): ____________________________ 
 
 
 
 
 
Date of participant’s signature: _____________________________________ 
 
 
Parent or guardian (if subject < age 18) 
Print Name of parent or guardian: __________________________________ 
 
Signature of parent or guardian: ____________________________________ 
 
Date of parent’s or guardian’s signature: ______________________________  
 
 
Consent obtained by: 
Print name of researcher: _________________________________________ 
 
Signature of researcher: __________________________________________ 
 
Date of researcher’s signature:_____________________________________ 
 
 
 

Prot_000.pdf: 
TrialNet Protocol TN19 
Protocol Version: 20160330 
 
 
 
 
1 
 
 
 
 
 
Antithymocyte Globulin (ATG) and pegylated 
granulocyte colony stimulating factor (GCSF) 
in New Onset Type 1 Diabetes 
 
 Protocol TN-19 
Version: 3.0 March 30, 2016 
 
 
IND # 107,185 
         
 
Sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the 
National Institute of Allergy and Infectious Diseases (NIAID), the National Institute of Child Health and 
Human Development (NICHD), the National Center for Research Resources (NCRR), the Juvenile 
Diabetes Research Foundation International (JDRF), and the American Diabetes Association (ADA) 


TrialNet Protocol TN19 
Protocol Version: 20160330 
 
 
 
 
2 
 
PREFACE 
 
The Type 1 Diabetes TrialNet Protocol TN-19, ATG-GCSF in New Onset Type 1 Diabetes, 
describes the background, design, and organization of the study.   
 
The protocol will be maintained by the TrialNet Coordinating Center over the course of the study 
through new releases of the protocol, or issuance of updates either in the form of revisions of 
complete chapters or pages thereof, or in the form of supplemental protocol memoranda. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


TrialNet Protocol TN19 
Protocol Version: 20160330 
 
 
 
 
3 
 
Glossary of Abbreviations 
AE 
Adverse event 
APC 
Antigen presenting cell 
ATG 
Anti-Thymocyte Globulin (Thymoglobulin®) 
AUC 
Area Under Curve 
CBC 
Complete blood count 
CFR 
Code of Federal Regulations 
cGMP 
Current Good Manufacturing Practice 
CHO 
Carbohydrates 
CMV 
Cytomegalovirus  
CRF 
Case report form 
CRS 
Cytokine Release Syndrome 
DC 
Dendritic Cell 
DSMB 
Data and Safety Monitoring Board 
EBV 
Epstein-Barr virus 
FACS 
Fluorescence activated cell sorting 
FDA 
US Food and Drug Administration  
FOXP3 
Forkhead box P3 
FWA 
Federal-wide Assurance 
GAD 
Glutamate decarboxylase 
GCP 
Good Clinical Practice 
GCSF 
 Granulocyte colony-stimulating factor (Neulasta®) 
HbA1c 
Hemoglobin A1c 
HBsAg 
Hepatitis B surface antigen 
HIV 
Human immunodeficiency virus 
ICA 
Islet cytoplasmic antibodies 
IEC 
Independent Ethics Committee 
IGRA 
Interferon-γ release assays 
IND 
Investigational New Drug 


TrialNet Protocol TN19 
Protocol Version: 20160330 
 
 
 
 
4 
 
IRB 
Institutional Review Board 
ITN 
Immune Tolerance Network 
JDRF 
Juvenile Diabetes Research Foundation 
LIFT 
Long Term Investigative Follow-Up 
MMTT 
Mixed-meal tolerance test 
NIDDK 
National Institute for Diabetes and Digestive and Kidney Disease 
NIH 
National Institute of Health 
NCI-CTCAE 
National Cancer Institute Common Terminology Criteria for 
Adverse Events 
NOD 
Nonobese diabetic 
OHRP 
Office for Human Research Protections 
PBMC 
Peripheral Blood Mononuclear Cell 
PCR 
Polymerase chain reaction 
PI 
Principal Investigator 
PO 
Per Oral (by mouth) 
QA 
Quality Assurance  
RMSE 
Residual Mean Square Error 
SAE 
Serious adverse event 
SOE 
Schedule of events 
SOP 
 
Standard operating procedure 
T1DM 
Type 1 diabetes mellitus 
Tregs 
Regulatory T cells 
TSDR 
Treg Specific Demethylation region 
ZnT8 
Zinc Transporter 8 
 
 
 
 
 
 
 


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TABLE OF CONTENTS 
1 
INTRODUCTION .................................................................................................................. 8 
1.1 
Study Overview .......................................................................................................... 8 
2 
BACKGROUND AND SIGNIFICANCE ................................................................................. 9 
2.1 
Introduction ................................................................................................................ 9 
2.2 
ATG Monotherapy ...................................................................................................... 9 
2.2.1 
Rationale for ATG Therapy in T1D .......................................................................... 9 
2.2.2 
Potential Mechanism of Action .............................................................................. 10 
2.3 
Granulocyte colony-stimulating factor (GCSF) monotherapy .................................... 11 
2.3.1 
Rationale for GCSF Therapy in T1D ..................................................................... 11 
2.3.2 
Potential Mechanism of Action .............................................................................. 11 
2.4 
Combination of ATG and GCSF ............................................................................... 12 
2.4.1 
GCSF Enhances Reversal of Diabetes Afforded by murine ATG in the NOD ........ 12 
2.4.2 
ATG + GCSF Combination Therapy Induces Immunomodulation ......................... 13 
2.5 
Monotherapy and Combination Therapy Clinical Trial Updates ................................ 14 
2.5.1 
GCSF Monotherapy .............................................................................................. 14 
2.5.2 
ATG Monotherapy ................................................................................................ 14 
2.5.3 
ATG +GCSF ......................................................................................................... 14 
3 
STUDY DESIGN ................................................................................................................ 15 
3.1 
Overview .................................................................................................................. 15 
3.1.1 
Inclusion Criteria ................................................................................................... 15 
3.1.2 
Exclusion Criteria .................................................................................................. 16 
3.2 
Description of Treatment Groups .............................................................................. 17 
3.3 
Treatment Assignment and Double Masking ............................................................ 17 
3.4 
Study Assessments .................................................................................................. 17 
3.5 
Quality Assurance .................................................................................................... 17 
3.6 
Post-treatment Follow-up ......................................................................................... 18 
4 
PATIENT MANAGEMENT .................................................................................................. 18 
4.1 
Overview .................................................................................................................. 18 
4.2 
Screening ................................................................................................................. 19 
4.3 
Randomization ......................................................................................................... 19 
4.4 
Intensive Diabetes Management .............................................................................. 19 
4.5 
Drug Administration .................................................................................................. 20 
4.5.1 
ATG ...................................................................................................................... 20 
4.5.2 
ATG / Placebo Administration ............................................................................... 20 
4.6 
Premedication: ......................................................................................................... 20 
4.6.1 
Cytokine Release Syndrome: ................................................................................ 21 
4.6.2 
Allergic Reactions ................................................................................................. 22 
4.7 
Modification or Discontinuation of ATG ..................................................................... 23 
4.7.1 
Modification of second ATG dose to 1mg/kg ......................................................... 23 
4.7.2 
Discontinuation of ATG ......................................................................................... 24 
4.8 
GCSF ....................................................................................................................... 25 


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4.8.1 
GCSF / Placebo Administration ............................................................................ 25 
4.9 
Modification or Discontinuation of GCSF .................................................................. 25 
4.10 
Concomitant Mediations ........................................................................................... 26 
4.11 
Infectious Disease Screening ................................................................................... 26 
5 
STUDY ASSESSMENTS ................................................................................................... 27 
5.1 
General Assessments .............................................................................................. 27 
5.2 
Laboratory Assessments .......................................................................................... 27 
5.3 
Mechanistic Outcome Assessments ......................................................................... 27 
5.4 
Metabolic Outcome Assessments ............................................................................ 27 
5.5 
Visit Windows ........................................................................................................... 28 
5.6 
Withdrawal from treatment ....................................................................................... 28 
5.7 
Re-Entry into Study Treatment ................................................................................. 29 
6 
PARTICIPANT SAFETY ..................................................................................................... 29 
6.1 
Risk, Benefits and Inclusion of Children ................................................................... 29 
6.2 
Potential Risks ......................................................................................................... 29 
6.2.1 
ATG ...................................................................................................................... 29 
6.2.2 
GCSF ................................................................................................................... 30 
6.3 
Pregnancy ................................................................................................................ 31 
6.4 
Protecting Against or Minimizing Potential Treatment Risks ..................................... 31 
7 
ADVERSE EVENT REPORTING AND SAFETY MONITORING ........................................ 31 
7.1 
Adverse Event Definition .......................................................................................... 31 
7.1.1 
Adverse Event ...................................................................................................... 31 
7.1.2 
Adverse Reaction ................................................................................................. 32 
7.1.3 
Serious Adverse Event/Reaction .......................................................................... 32 
7.1.4 
Unexpected Adverse Event ................................................................................... 33 
7.1.5 
Grading Event Severity and Causality ................................................................... 33 
7.2 
Adverse Event Reporting and Monitoring ................................................................. 33 
8 
STATISTICAL CONSIDERATIONS AND ANALYSIS PLAN ............................................... 34 
8.1 
Primary Outcome and Analyses ............................................................................... 34 
8.2 
Secondary Outcome and Analyses........................................................................... 35 
8.3 
Additional Outcomes and Analyses .......................................................................... 36 
8.4 
Sample Size and Power Calculations ....................................................................... 36 
8.5         Sample Size Re-estimation ...................................................................................... 37 
8.6 
Interim Monitoring Plan............................................................................................. 38 
8.7 
Interim Analysis Study Modification .......................................................................... 38 
9 
ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL PRACTICE . 40 
9.1 
Statement of Compliance ......................................................................................... 40 
9.2 
Participating Centers ................................................................................................ 40 
9.3 
Informed Consent ..................................................................................................... 41 
9.4 
Study Subject Confidentiality .................................................................................... 41 
9.5 
Risks and Benefits .................................................................................................... 41 
10 STUDY ADMINISTRATION ................................................................................................ 42 
10.1 
Organizational Structure ........................................................................................... 42 
10.2 
Role of Industry ........................................................................................................ 42 
10.3 
Groups and Committees ........................................................................................... 42 
10.3.1 ATG-GCSF Study Chair Committee ...................................................................... 42 


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10.3.2 TrialNet Chairman’s Office and TNCC .................................................................. 42 
10.3.3 Clinical Sites ......................................................................................................... 42 
10.3.4 Safety Monitoring Subcommittee .......................................................................... 42 
10.3.5 Clinical Site Monitoring ......................................................................................... 43 
10.3.6 Medical Monitor and Data Safety and Monitoring Board (DSMB) .......................... 43 
10.4 
Sample and Data Storage ........................................................................................ 43 
10.5 
Preservation of the Integrity of the Study .................................................................. 44 
10.6 
Participant Reimbursement and Compensation ........................................................ 44 
APPENDIX 1 - Schedule of Assessments .................................................................................. 45 
11 REFERENCES ................................................................................................................... 47 


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1 
INTRODUCTION 
1.1 Study Overview 
Title 
ATG-GCSF in New Onset Type 1 Diabetes 
IND Sponsor 
 National Institute of Diabetes and Digestive and Kidney 
Diseases (NIDDK) 
Conducted By 
Type 1 Diabetes Trial Network (TrialNet) 
Protocol Chair 
Michael J. Haller, MD 
Accrual Objective 
84 
Study Design 
Three-arm, 1:1:1 randomized, placebo controlled, double- 
blinded trial in which at least 28 subjects will receive active 
ATG-GCSF, at least 28 subjects will receive ATG alone and at 
least 28 subjects will receive placebo alone within 100 days 
from diagnosis of T1D. An adaptive design will be used to 
determine the variance of C-peptide over time and may require 
additional subjects to be enrolled in each group to preserve 
power. 
Treatment Description 
ATG will be administered at a dose of 2.5mg/kg as two divided 
IV infusions of 0.5mg/kg and 2mg/kg. GCSF will be 
administered at a dose of 6mg SC or if <44.5 kg a dose of 100 
mcg/kg every two weeks for a total of 6 doses. 
Study Duration 
Enrollment is expected to occur over two-three years.  Subjects 
will be followed up to 24 months from randomization in this 
protocol.   
Objective 
To determine the safety and ability of low dose ATG plus GCSF 
and low dose ATG alone to retain/enhance C-peptide 
production in new onset T1D patients demonstrating residual 
beta cell function.  
Primary Outcome 
The primary statistical hypothesis to be assessed in the study 
is whether the 2 hour area under the curve (change in baseline 
to 12 months) in residual beta cell function (C-peptide) will 
differ between those treated with ATG and GCSF or ATG alone 
as compared with placebo.  
Secondary Goals 
The study will also examine the effect of the proposed 
treatments on surrogate markers for immunologic and 
metabolic outcomes 
Major Inclusion 
Criteria 
Type 1 diabetes diagnosed within the past 100 days 
Age  > 12 years < 46 
At least one diabetes associated autoantibody 
 
 
 


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2 
BACKGROUND AND SIGNIFICANCE 
 
2.1 Introduction 
Recent advances in our understanding of the pathogenesis of T1D have been somewhat 
overshadowed by our continued inability to develop a long lasting means to either reverse or 
prevent the disease (i.e., identify a cure), using a variety of interventions (e.g., cyclosporine, anti-
CD3, anti-CD20, oral insulin) (1-3). The majority of attempts to reverse T1D in humans have, to 
date, involved the delivery of agents as monotherapy.  As a result, we and others have recently 
questioned whether the delivery of therapeutics in combination might produce a synergistic 
response that would allow for the successful reversal of T1D (2, 3). The success of combination 
therapy for advancing the treatment of patients with HIV and cancer demonstrates a model for 
multi-agent therapy in diseases involving multiple pathways required for their successful 
treatment and cure.  
As described below, based on strong preliminary data demonstrating efficacy and potential 
mechanisms leading to reversal of diabetes in NOD mice with a combination of ATG and GCSF, 
as well as preliminary safety and efficacy studies in humans with T1D utilizing ATG and GCSF 
as monotherapies and in combination, we will test the hypothesis that a short course of low dose 
ATG or a combination of low dose ATG plus GCSF will lead to preservation of C-peptide in 
patients with new onset T1D.  
 
2.2  ATG Monotherapy 
2.2.1 Rationale for ATG Therapy in T1D 
 
Preclinical studies have shown that ALS treatment of the NOD mouse (18) and BB rat (19) with 
recent onset T1D can induce remission, supporting proof of principle for such an approach in 
humans. The case for combination of ATG with other agents received support from follow-up 
studies in new-onset NOD mice involving the addition of Exendin-4, a long-acting GLP-1 agonist 
shown to augment insulin secretion in rodents (22) as well as T1D (23) and T2D (24) subjects, 
where remissions were seen in 90% of combination treated mice. While our own NOD studies 
(Preliminary Data section) found ATG and GCSF to be a superior combination (in comparison to 
ALS and Exendin-4) both sets of data provide additional support for the concept of combination 
therapy and it’s superiority to relatively ineffective monotherapy in terms of reversing T1D.  
As further proof of the potential for translation from mouse to man, studies in human 
transplantation and various autoimmune conditions suggest that ATG may indeed induce 
tolerance.  ATG has been successfully used as combination therapy in preventing rejection 
following organ transplantation. After induction with ATG, transplant recipients can be managed 
successfully with only limited maintenance monotherapy (25, 26), with weaning of some to as 
little as a single dose of tacrolimus per week. ATG, either alone or in combination with other 
agents, has also been used to treat a variety of autoimmune conditions, including Wegner’s 
granulomatosis, lupus, rheumatoid arthritis, multiple sclerosis, scleroderma, aplastic anemia and 
myelodysplastic syndrome; with greater efficacy observed when used in combination (27-38). 
Extensive studies with ATG in a non-human primate model demonstrates its effectiveness as a 
single agent in T-cell depletion intravascularly and in peripheral lymphoid organs, as well as in 
prolongation of allograft transplantation of skin and heart (39).   
Other studies also support ATG’s possible efficacy in T1D.  Specifically, early limited human 
studies with equine ATG and prednisone with new onset T1D suggested efficacy in prolonging 


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the honeymoon phase (11, 40).  In a small randomized, placebo-controlled, single-blinded trial 
with ATG (ATG-Fresenius, Germany), T1D participants aged 18-35 years received a total dose 
of 18 mg/kg of ATG, administered in four infusions.  Of the 17 study participants treated, 11 
received drug while 6 received placebo.  In terms of efficacy, increased glucagon-stimulated C-
peptide levels, a lower insulin requirement, and lower glycosylated hemoglobin levels were 
reported in the ATG group (but not placebo group) at the 12 month study visit. Perhaps most 
promising, two ATG treated subjects achieved remission (i.e., off exogenous insulin for at least 1 
month with fasting blood glucose below 126 mg/dl). (41).   
Recently, a phase 2 study in humans with new-onset T1D and funded by the Immune Tolerance 
Network (ITN) was completed to determine if ATG (6.5mg/kg) could preserve C-peptide in new 
onset T1D. The study tested the hypothesis that selective deletion of lymphocytes would reset 
the immunologic rheostat, effect dynamic immune regulation and perhaps induce and maintain 
tolerance in T1D.  While this study helped to establish the relative safety of ATG in humans with 
T1D, the study failed to demonstrate benefit (41b). Post-hoc analyses have suggested that an 
initial decline in beta cell function amongst those who were treated with active drug was followed 
by a relative preservation of beta cell function. We hypothesize that the initial decline in beta cell 
function was related to the severity of cytokine release syndrome and serum sickness. 
Furthermore, we hypothesize that the currently proposed protocol (utilizing ~1/3 of the ATG dose 
in combination with GCSF) will result in less severe cytokine release/serum sickness and, as is 
supported by our preliminary data, preservation/improvement in beta cell function 12 months 
after therapy. 
2.2.2 Potential Mechanism of Action 
 
ATG appears to induce both generalized immunosuppression and immunoregulation, with 
effects on APC and immunoregulatory cell function (42). It prevents B-cell proliferation and 
differentiation, as well as mediates T-cell suppressive effects via inhibition of proliferative 
responses to mitogens (43, 44).  A single “typical” or “standard” dose of ATG reduces the total 
lymphocyte count by more than 85%. T-cell depletion may result from complement-dependent 
opsonization and cellular lysis, Fc-dependent opsonization, or Fas-mediated apoptosis; 
particularly at lower ATG concentrations, at which ATG exhibits preferential effects on pre-
activated, as opposed to nonactivated, T-cells.  T-cell depletion in peripheral blood persists for 
several days to several months following cessation of ATG administration.  Recovery from 
treatment-induced lymphocyte depletion is gradual and total lymphocyte counts usually return to 
normal with 2 months after ATG administration. 
Following ATG function, the CD4:CD8 T cell ratio remains significantly and persistently lower 
and the ratio may correlate with the outcome in transplantation studies (45-48).  It has been 
hypothesized that ATG administration may induce a regulatory population of CD8+, CD57+ and 
CD28- cells that is critical for selective down regulation of pathogenic self-reactive CD4+ Th1 
cells and the induction of tolerance.   
Other studies have suggested alternative mechanisms to underlie the beneficial effects of ATG 
in vivo. As with anti-CD3, ATG may induce partial T-cell activation, leading to an anergic state 
(39, 53, 54).  In cynomolgus monkeys, ATG appears to coat T cells, leading to a down regulation 
in surface expression of CD2, CD3, CD4, and CD8 molecules, along with impaired immune 
responses in mixed lymphocyte reactions (39).  In addition, antibodies to adhesion molecules 
may interfere with cellular adhesion and endothelial interactions, as well as T-cell migration to 
sites of inflammation. ATG may also prevent co-stimulation of T cells by binding directly to APC, 
and may induce complement-mediated lysis of these cells; particularly in more mature APC (55).  
The survival of immature DC thus may be more tolerogenic.  Antigens recognized by ATG 


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include CD86, CD32, CD4, CD11b, CD29, and CD51/61; some of which are shared by 
lymphocytes and DC.  In addition, ATG contains antibodies that cross-react with B-cell surface 
antigens, allowing for induction of activated B-cell (another source of APC) apoptosis in vitro (56-
58).   
 
2.3 Granulocyte colony-stimulating factor (GCSF) monotherapy 
2.3.1 Rationale for GCSF Therapy in T1D  
 
In a spontaneous diabetes model, GCSF treatment of NOD mice at 4 weeks of age for 5 
consecutive days, repeated every 4 weeks thereafter until 16 weeks of age, prevented disease 
onset and insulitis when compared to injection of excipient (Figure 2) (59). Protection from 
diabetes onset correlated with the recruitment of both DC and CD4+CD25+ Treg cells. Indeed, 
the GCSF recipients showed peri-pancreatic lymph node accumulation of functional 
CD4+CD25+ Treg cells (60). Importantly, co-transfer of the functional Treg populations along 
with diabetogenic splenocytes into secondary NOD-SCID recipients actively suppressed 
diabetes.  In addition, DC transferred from mice given a single 5-day-long GCSF treatment to 
secondary NOD recipients triggered enhanced accumulation of CD4+CD25+ Treg cells 
compared to recipients given non-GCSF treated DC. Thus, GCSF likely restores a balance 
between immunogenic and tolerogenic DC, resulting in the recruitment of functional CD4+CD25+ 
Treg cells.  
Based on supportive data from animal models, several groups have moved on to initiate clinical 
trials of GCSF based therapies in human autoimmune disease. Perhaps the most extensive 
human data derives from studies of GCSF therapy in patients with Crohn disease. Several 
studies have now demonstrated improvement in clinical disease scores in patients with Crohn 
disease provided varying courses of GCSF (61-62).  Specifically, a 12 week course of 300mcg 
daily GCSF, demonstrated marked reduction in the Crohn Disease Activity Index amongst 15 
subjects with active disease (62). In a smaller study of Crohn disease patients with a history of 
severe endoscopic ileitis, a similar 12-week course of GCSF was associated with clinical 
remission of disease in all 5 subjects (61).   
 
2.3.2 Potential Mechanism of Action 
 
GCSF, an agent well known to help mobilize hematopoietic precursors from the bone marrow, 
has been used clinically for nearly 20 years to help repopulate peripheral cell counts in patients 
undergoing cancer therapy. However, a rapidly growing body of experimental and clinical 
evidence suggests that GCSF has the potential to be used in the treatment of autoimmune 
diseases. GCSF has been shown to favor the differentiation and mobilization of Treg cells, 
induce tolerogenic DC, and alter the balance between proinflammatory and anti-inflammatory 
soluble mediators both animals and in humans (Figure 3) (65-67). Specifically, GCSF can 
mobilize functional bone marrow CD4+CD25+FoxP3+ Treg cells. Preclinical models of GCSF-
induced inhibition of autoimmune and allogeneic T cell responses have demonstrated the 
potential efficacy of GCSF in treating Crohn 
disease, myasthenia gravis, and T1D.  
 
Figure 1. Effects of GCSF on immune 
function. The GCSF induced modulation of 
cytokine production, T cell polarization, and 


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DC functional profile is depicted schematically. GCSF can alter the balance between pro-
inflammatory and anti-inflammatory soluble mediators, favor the differentiation and mobilization 
of Treg cells, and induce tolerogenic DC both in animals and in humans. (Adapted from Rutella 
et al, J. Immunology 2005, 175: 7085–7091, (65). 
 
With this extensive experience of clinical use, GCSF therapy is known to be both safe and well 
tolerated. The side effect profile of short course GCSF therapy is limited; with frequency and 
severity of toxicities being dose dependent (greater with larger doses and longer duration) and 
minimal in comparison to many of the agents being used in current T1D intervention studies. 
Specifically, the most common side effect associated with GCSF therapy is bone pain (occurring 
in 10-20% of patients) which occurs 2-3 days after initiation of therapy. This discomfort is 
generally controllable with acetaminophen.  Infrequent side effects include redness at injection 
site, fever, headache, chest pain and dizziness (occurring in less than 10% of patients). 
 
2.4  Combination of ATG and GCSF 
2.4.1 GCSF Enhances Reversal of Diabetes Afforded by murine ATG in the 
NOD 
 
To determine the efficacy of a combination of ATG and GCSF in NOD mice with `new-onset’ 
diabetes, female NOD mice were monitored 3 times per week for hyperglycemia (defined as a 
blood glucose > 240mg/dL) by tail bleed for up to 150 days as shown (Figure 2).  
Figure 2. Blood glucose values were 
obtained for up to 150 days post-onset 
in NOD mice treated with (a) control 
(two IP injections of 500 ug rIgG), (b) 
GCSF (Neupogen (Amgen, Inc.) 6ug 
daily x 8weeks, (c) murine ATG (two 
IP injections of 500 ug murine ATG, or 
(d) murine ATG + GCSF.  All received 
sustained release insulin for 3 weeks.  
As 
shown 
(Figure 
2), 
the 
administration of murine ATG alone to 
new-onset NOD mice resulted in 
durable (i.e., > 150 days post-onset) 
remissions from overt hyperglycemia 
in 33% (5/15) of treated animals, while 
neither 
control 
(0/16) 
nor GCSF 
monotherapy (0/14 mice) provided 
such 
reversions 
However, 
the 
combination of murine ATG and GCSF 
therapy resulted in a remission rate of 75%, a significantly greater rate of remission than was 
seen with murine ATG monotherapy (12/16; P = 0.0000006 versus control, P = 0.013 versus 
murine ATG).  
Interestingly and very importantly, in terms of dosage in humans, GCSF improved the 
therapeutic capacity for diabetes reversal even when in combination with a suboptimal dose 
(290ug) of murine ATG   Moreover, successful treatment with murine ATG was largely limited to 


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values of <380mg/dL (mean 317.2mg/dL; range 256-398mg/dL), whereas combination therapy of 
murine ATG and GCSF significantly increased the therapeutic ceiling to ~500 mg/dL (mean 
401.8 mg/dL; range 264-500mg/dL).  Additional studies demonstrated an improvement in IP 
glucose tolerance tests (IPGTT) at from the 60 to 120 day time points. This improvement in 
glucose control occurred in spite of the cessation of both murine ATG and GCSF therapies prior 
to the 60-day time point. 
 
2.4.2  ATG + GCSF Combination Therapy Induces Immunomodulation 
To address the question of whether GCSF-mediated enhancement of diabetes reversal was due 
to induction of immunoregulation, murine ATG and GCSF (as both mono- and combination-
therapy) were administered to pre-diabetic 12-week-old female NOD mice for up to 8 weeks. 
Analysis of peripheral blood revealed marked leukocyte depletion in murine ATG-treated mice 
versus all other groups at 2 week, with movement back towards pre-treatment levels at 4 and 8 
weeks post-induction. However, the addition of GCSF to murine ATG afforded a significant 
increase in leukocytes at 2 weeks versus murine ATG alone. In particular, GCSF increased the 
percentage of splenic macrophages and neutrophils.  Both murine ATG as well as GCSF have 
been reported to induce a population of Treg in vivo. Predictably, all treatments utilized in these 
efforts herein demonstrated a reduced percentage of Treg at 2 weeks versus control animals 
due to either short-term depletion by murine ATG or mobilization of macrophages and 
neutrophils by GCSF. GCSF therapy led to an increase in Treg versus control as early as 4 
weeks while combination therapy had the greatest increase in Treg versus all other treatments at 
8 weeks. As indicated by the increase in Treg the immunomodulatory benefit afforded by GCSF 
continued through 8 weeks, despite the lack of mobilization of macrophages and neutrophils 
beyond 2 weeks. Thus the greatest percentage of Treg cells was observed in mice receiving 
combination therapy. The health of the islets at the endpoint of the pre-diabetic study was an 
important consideration. As such, insulitis scoring was performed to determine the degree of 
lymphocytic infiltration over the 8 weeks of therapy in pre-diabetic NOD mice. Combination 
therapy resulted in markedly lower insulitis intensity scores when compared with islets from 
control animals after 8 weeks In addition, insulin staining revealed improved beta cell area in 
animals receiving combination therapy versus murine ATG monotherapy, while control animals 
demonstrated a decline in beta cell area over the 8 week period  
By combining these two monotherapies, however, the health of the islets was maintained relative 
to control as measured by insulitis scoring and beta cell area. These results indicate that 
combined treatment of murine ATG with GCSF offers a highly effective means for reversal of 
T1D in NOD mice.  
 
 
 
 
 
 
 
 
 


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2.5  Monotherapy and Combination Therapy Clinical Trial Updates 
2.5.1 GCSF Monotherapy 
 
In August of 2008, the University of Florida T1D research group embarked on a pilot (JDRF/NIH 
supported) study of GCSF monotherapy, in order to firmly establish the safety of GCSF and 
demonstrate the potential for efficacy in mobilizing Treg in the T1D population.  The study used a 
2:1 drug: placebo randomization scheme and AUC C-peptide as the primary efficacy outcome 
measure. Twenty-one study participants completed a 12 week course of the study drug (GCSF 
6mg sq q 2 weeks versus placebo).  Analysis of the 12 month follow-up data did not demonstrate 
any significant preservation of C-peptide in treated subjects. Drug was well tolerated with the 
most frequent complaints being bone pain or headache treatable with ibuprofen or 
acetaminophen. In addition, there were no issues with splenomegaly or concerning elevation of 
the absolute neutrophil count. 
2.5.2 ATG Monotherapy 
 
In late 2006, the ITN approved ATG monotherapy in new-onset T1D at a limited number of sites; 
the START trial (Study of Thymoglobulin to Arrest Type 1 Diabetes). After safety and tolerability 
of the drug was demonstrated, the age group was lowered to 12 years in the Fall of 2009 and the 
study was fully enrolled. The primary endpoint analysis was completed in 2012 and did not 
reveal any benefit for ATG monotherapy (at 6.5mg/kg) in preserving beta cell function in new 
onset subjects (41b).  
2.5.3 ATG +GCSF 
 
A phase IIa study of ATG (2.5mg/kg) and GCSF (6mg q 2 weeks x 6 doses) utilizing a 2:1 
randomization scheme (Drug: placebo) is currently underway in “established” (diagnosed for 4 
months to 2 years) T1D patients (Figure 3). The study is fully enrolled (n=25) and data from all 
25 subjects at the 3, 6, and 9, and 12 month study visits demonstrate increased AUC C-peptide 
amongst the treated subjects in comparison to those who have received placebo.  
 
Figure 3. AUC C-
peptide in Treated 
(ATG/GCSF) and 
Placebo Subjects with 
Established T1D. 
P=0.05 at 12 months  
 
 
 
 
 
 
 
 
D AUC c-peptide
0
3
6
12
-200
-150
-100
-50
0
50
100
Treated
Placebo
Months post  treatment
D AUC (ng/ml/2 hr)


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The dose of ATG for the pilot study and the current protocol was chosen based on a combination 
of the demonstrated safety and efficacy in treating human autoimmune and alloimmune diseases 
in adults and children, and the suggestion from our animal data indicating that lower dose ATG 
could be used successfully when utilized in combination with GCSF. Several autoimmune 
diseases (e.g., SLE, RA, Wegner’s Granulomatosis, Systemic Sclerosis, and T1D) have been 
treated with some success with ATG based protocols utilizing doses from 2.5mg/kg to in excess 
of 10 mg/kg (68). In addition, induction therapy for kidney transplantation using low doses of 
ATG (2.5-5mg/kg) has demonstrated efficacy in protecting the graft and reducing acute rejection 
in both children and adults (69, 70). 
In the ITN START study, subjects received ATG at a total dose of 6.5 mg/kg (0.5 mg/kg Day 1, 
then 2 mg/kg for next 3 doses). This dose has been effective and well tolerated in bone marrow 
and solid organ transplantation, and is well below the dose of 20 mg/kg that has been used for 
some transplantation protocols. Based on the observed efficacy in the mouse with combined 
low-dose ATG and GCSF therapy, the dose of ATG was reduced, albeit somewhat empirically, 
to 2.5 mg/kg with the hopes of minimizing side effects while still providing for efficacy in 
preserving beta cell function.  
The dose and duration of GCSF was chosen on the basis of its safety as demonstrated to date in 
the T1D study completed at the University of Florida, and on the basis of other 12-week GCSF 
studies that have demonstrated potential for benefit in autoimmune disease (71). 
 
Summary pre-clinical and preliminary clinical data: 
 
Preclinical data demonstrate that combination therapy of ATG and GCSF has multiple beneficial 
mechanistic actions (e.g., increased Treg frequency, reduced islet inflammation, improved beta 
cell area, etc.) and dramatically extends the range of beta cell dysfunction allowable for effective 
and durable disease remission. Pilot clinical trial data have demonstrated efficacy in preserving 
C-peptide and have confirmed an acceptable safety profile. Collectively, these studies provide 
strong support for the performance of phase II human T1D trials with this combination of agents.   
 
3 
STUDY DESIGN 
3.1  Overview 
Summary of Inclusion and Exclusion Criteria 
 
3.1.1 Inclusion Criteria 
Potential participants must meet all of the following inclusion criteria: 
1. Must be > 12 years < 46 
2. Must have a diagnosis of T1D for less than 100 days at randomization 
3. Willing to provide Informed Consent or have a parent or legal guardian provide informed 
consent if the subject is <18 years of age 
4. Positive for at least one islet cell autoantibody; GAD65A, mIAA, if obtained within 10 days of 
the onset of insulin therapy, IA-2A, ICA, or ZnT8A 


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5. Must have stimulated C-peptide levels  0.2 pmol/ml measured during a mixed meal 
tolerance test (MMTT) conducted at least 21 days from diagnosis of diabetes.  
Randomization should occur within one month (37 days) of the MMTT.  However, with prior 
approval by TrialNet, this window may be extended to allow randomization within 8 weeks 
(56 days). 
6. Subjects who are EBV seronegative at screening must be EBV PCR negative within 30 days 
of randomization and may not have had signs or symptoms of an EBV compatible illness 
lasting longer than 7 days within 30 days of randomization 
7. Be at least 6 weeks from last live immunization 
8. Participants are required to receive killed influenza vaccination at least 2 weeks prior to 
randomization when vaccine for the current or upcoming flu season is available 
9. Be willing to forgo vaccines during the treatment period and for 3 months following last dose 
of study drug  
10. Be willing to comply with intensive diabetes management 
 
3.1.2 Exclusion Criteria 
Potential participants must not meet any of the following exclusion criteria: 
 
1. Be immunodeficient or have clinically significant chronic lymphopenia:  (Leukopenia (< 3,000 
leukocytes /μL), neutropenia (<1,500 neutrophils/μL), lymphopenia (<800 lymphocytes/μL), 
or thrombocytopenia (<100,000 platelets/μL). 
 
2. Have active signs or symptoms of acute infection at the time of randomization 
 
3. Have evidence of prior or current tuberculosis infection as assessed by PPD, interferon    
gamma release assay or by history 
 
4. Be currently pregnant or lactating, or anticipate getting pregnant within the two year study 
period  
 
5. Require use of other immunosuppressive agents including chronic use of systemic steroids 
 
6. Have evidence of current or past HIV, Hepatitis B or Hepatitis C infection  
 
 
7. Have any complicating medical issues or abnormal clinical laboratory results that may 
interfere with study conduct, or cause increased risk to include pre-existing cardiac disease, 
COPD, sickle cell disease, neurological, or blood count abnormalities  
 
8. Have a history of malignancies other than skin 
 
9. Evidence of liver dysfunction with AST or ALT greater than 3 times the upper limits of normal 
 
10. Evidence of renal dysfunction with creatinine greater than 1.5 times the upper limit of normal 
 
11. Vaccination with a live virus within the last 6 weeks 
 


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12. Current or ongoing use of non-insulin pharmaceuticals that affect glycemic control within prior 
7 days of screening 
  
13.  Active participation in another T1D treatment study in the previous 30 days  
 
14. Prior treatment with abatacept or anti-cd3 
 
15. Known allergy to GCSF or ATG 
  
16. Prior treatment with ATG or known allergy to rabbit derived products  
 
17. Any condition that in the investigator's opinion may adversely affect study participation or  
may compromise the study results 
 
3.2  Description of Treatment Groups 
This protocol will enroll at least 84 participants who will be randomly assigned to the following 
groups: 
o 28 participants will be assigned to receive ATG + GCSF  
o 28 participants will be assigned to receive ATG + Placebo 
o 28 participants will be assigned to receive Placebo + Placebo 
 
3.3  Treatment Assignment and Double Masking 
After participants sign the consent form they will be randomized to one of the three arms.  The 
randomization method will be stratified by TrialNet study site.  The participants will not be 
informed regarding the intervention assignment until the end of the study. The investigator and 
clinic personnel will also be masked as to study assignment.  Laboratories performing assays for 
this protocol will be masked as to the identity of biological material to be studied. 
 
3.4  Study Assessments 
During the course of the study, participants will frequently undergo assessments of their insulin 
production, immunologic status, overall health and well-being and diabetes care (see schedule of 
assessments in Appendix A).  Information about the subject’s experience as a research 
participant will also be collected. The participants’ insulin production will be measured by a series 
of mixed meal glucose tolerance tests (MMTT) conducted regularly during the study.  The 
participants’ diabetes control will be evaluated by measuring glycosylated hemoglobin (HbA1c) 
and clinical records including insulin types, doses, and timing and SMBG records. During the 
course of the study, samples will be drawn for storage in the National Institute for Diabetes and 
Digestive and Kidney Disease (NIDDK) Repository and at TrialNet Sites for future analysis.   
 
3.5  Quality Assurance 
 
During the study, duplicate collections of blood samples for assays will be obtained in a small 
sample of subjects for the purpose of quality surveillance of the performance of the central 
laboratories.  
 
 


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3.6  Post-treatment Follow-up 
 
At the conclusion of the study or when subjects reach 24 months, subjects will be encouraged to 
participate in further follow-up via participation in the TrialNet Long Term Investigative Follow-Up 
(LIFT).  
 
4 
PATIENT MANAGEMENT 
 
4.1 Overview 
 
Eligible subjects will receive either: (1) ATG (2.5mg/kg) IV followed by GCSF SQ (for those ≥ 
44.5 kg, the dose is 6mg; for those <44.5 kg, the doses is 100 mcg/kg q 2 weeks x 6 doses), (2) 
ATG (2.5mg/kg) IV plus placebo, or (3) double placebo. Subjects will undergo screening visits to 
determine eligibility.  Within 8 weeks of screening but preferably less than 37 days, eligible 
subjects will be admitted to an inpatient hospital or research facility for visit 0 and will be 
randomized to one of three treatment groups (ATG/GCSF, ATG/Placebo, and Placebo/Placebo).  
Individuals will receive oral and IV premedication and the first dose of ATG/placebo will be 
administered over a minimum of 12 hours (maximum of 20 hours) (Figure 4).  CBC will be drawn 
8 hours after the completion of dose 1 and will inform timing for infusion number 2. If labs are 
above threshold and at least 12 hours have passed since the completion of the first dose, the 
second dose of ATG/placebo will be administered over a minimum of 8 hours (maximum of 16 
hours).  Each ATG/placebo dose will be followed by steroid/saline infusion.  The first dose of 
GCSF/Placebo will be given 6 hours after the end of the second ATG/placebo dose.  
Subsequent GCSF/placebo will then be given as outpatient every 2 weeks for a total of 6 doses 
over 5 visits. Subsequent visits will occur every 3-6 months through 24 months.  
 
 


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Figure 4. Minimum and Maximum Time Course for ATG Infusion and Hospitalization.   
The top half of the figure demonstrates the ideal scenario for ATG infusion: First dose of ATG 
(0.5mg/kg) completed over 12 hours, CBC drawn 8 hours post infusion, second dose (2mg/kg) 
completed over 8 hours, first GCSF dose given 6 hours after completion of ATG, and D/C 12 
hours after completion of ATG. The bottom half of the figure demonstrates the maximum 
allowable time for ATG infusion and first GCSF dosing if laboratory abnormalities or patient care 
issues require interrupting, postponing, or reducing the rate of ATG infusion  
 
4.2 Screening 
 
After informed consent, subjects will undergo assessments to determine if they meet eligibility 
criteria.  Documentation of the subjects understanding of the risks and benefits of the study will 
be collected through the Volunteer Understanding Assessment. 
 
4.3 Randomization 
 
Eligible study participants will be randomized by the TrialNet Coordinating Center at the baseline 
visit once eligibility has been confirmed. Subjects will be assigned a study randomization number 
corresponding to the treatment group assignment.   
 
 
 
 
4.4 Intensive Diabetes Management 
 
During the study period, all participants will receive “intensive” management of their diabetes.  
The goal of the treatment will be to keep the HbA1c levels within the currently recommended 
American Diabetes Association age-specific target range in the absence of significant or severe 
hypoglycemia or diabetic ketoacidosis.  The primary responsibility for diabetes management will 
be the treating or referring diabetes care provider, but the research study team will provide close 
additional support through regular interaction.  Subjects will not be permitted to use non-insulin 
pharmaceuticals for glycemic control. 
 
Glucose levels should be checked frequently and records of the glucose levels communicated 
regularly to the study team.  Records of communication with the participant will provide source 
documentation of this interaction. 
 
The Clinic Monitoring Group (or designated TrialNet Committee) will be evaluating the HbA1c 
data and provide additional guidance to the clinical site as needed to bring diabetes control 
within goals.  Any episode of severe hypoglycemia will be promptly reviewed by the Safety 
Monitoring Committee with recommendations for changes in diabetes management, if any, 
conveyed to the clinical site in conjunction with the Clinic Monitoring Group. 
 
 
 
 


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4.5 Drug Administration 
 
4.5.1 ATG  
 
ATG is indicated for the treatment of renal transplant acute rejection in conjunction with 
concomitant immunosuppression. Each 10 mL vial contains 25 mg ATG (rabbit) as well as 50 mg 
glycine, 50 mg mannitol, and 10 mg sodium chloride. 
 
4.5.2 ATG / Placebo Administration 
 
All participants will be admitted to the hospital for the duration of the infusions and will be 
discharged no sooner than 12 hours after ATG infusion has been completed. Body weight at 
baseline (Time 0 – admission for the ATG/placebo infusion) will be used in calculating the doses 
for all infusions.   Allowance in dose administration will be within +/- 5% of calculated dose.  
To minimize the risk for thrombophlebitis associated with ATG infusion, 1000 units of heparin 
and 20 mg of hydrocortisone will be added to the ATG infusion bag for each dose given via 
peripheral intravenous administration.  Since there is only a slight risk for thrombophlebitis for 
placebo infusion, the heparin and hydrocortisone will not be included in the infusion bag.  
The first dose (0.5mg/kg) will be infused over a minimum of 12 hours, and the second dose 
(2mg/kg) over a minimum of 8 hours. The second dose should be given no less than 12 and no 
more than 24 hours after the previous dose.  A maximum of 60 hours from the start of the first IV 
infusion will be allowed to complete all IV study drug (see parameters below). If a subject has not 
completed the two IV study drug infusions at the 60th hour, the infusion will be discontinued and 
they will not receive additional study drug. Vital signs will be checked every 30 minutes for the 
first 2 hours of the study drug infusion and thereafter at 60-minute intervals or as indicated for 
clinical signs or symptoms.  Blood for laboratory testing should be drawn at least 8 hours after 
completion of each infusion in order to obtain reliable and comparable results. 
Information about subject’s clinical signs and symptoms related to ATG infusion will be captured 
on infusion CRFs.  Those that are Grade 2 or more will be considered AEs and reported as such. 
 
4.6  Premedication:  
To reduce the risk of adverse reactions to ATG infusion, methylprednisolone 0.25 mg/kg IV will 
be given no less than 30 minutes before each infusion of active drug and 0.25 mg/kg IV will be 
given 12 hours (±15 min) after the start of each infusion of active drug.  
Placebo group: A placebo (saline) infusion similar in appearance will be given no less than 30 
minutes before and 12 hours (±15 min) after the start of each infusion. 
Both groups:  Participants in both groups will be pre-medicated with an antihistamine and 
acetaminophen PO at least 30 minutes before each infusion and every 4–6 hours as needed 
during the infusion, as follows: 
 
Diphenhydramine 1.25 mg/kg/dose to a maximum of 50 mg. 
 
Acetaminophen 10–15 mg/kg/dose to a maximum of 650 mg. 


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4.6.1 Cytokine Release Syndrome:  
 
With ATG infusion, the subject may experience Cytokine Release Syndrome (CRS). The signs 
and symptoms can span a wide clinical spectrum. 
Mild Reactions: For mild (grade 1) reactions per the NCI-CTCAE for CRS, the study medication 
will be continued. The investigator shall take one or more of the following actions, depending on 
the type of the reaction:  
1. Administer additional doses of antihistamine and acetaminophen.                                                              
2. Reduce the rate of infusion by 50% or more. 
3. For chills and rigors, meperidine may be considered. 
Moderate Reactions: For moderate (grade 2) reactions per the NCI-CTCAE for CRS, the study 
medication may be interrupted. The investigator shall take the following actions, depending on 
the type of the reaction: 
1.  Interrupt infusion if any of the following occurs: 
   a. Oral temperature of > 40.0 °C 
   b. Symptomatic bronchospasm or pulmonary edema 
   c. Allergy-related edema 
   d. Hypotension 
2. When the temperature is < 38.5 °C and signs and symptoms improve, restart ATG. 
3. Closely monitor the subject with pulse oximetry and a blood pressure monitoring; provide 
ongoing nursing evaluation until at least 2 hours after the infusion is completed. 
4. If necessary, glucocorticoids can be given every 6 hours at a dose of 0.5 mg/kg of 
methylprednisolone or equivalent. 
5. If a subsequent dose of ATG further exacerbates the signs and symptoms of CRS despite 
following the above guidelines, the study treatment must be permanent discontinued. 
6. Additional supportive or resuscitative measures (such as the use of epinephrine) may be 
needed if clinically indicated. 
Severe Reactions: For severe (grade 3) reactions or greater per the NCI-CTCAE for CRS, the 
study medication will be permanently discontinued. 
 


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, 
 
Figure 5. Rate Adjustments in Response to Cytokine Release Syndrome 
 
4.6.2 Allergic Reactions 
 
Hypersensitivity: In rare cases, patients may experience hypersensitivity, which refers to 
immediate allergic, IgE mediated reactions to ATG. Such patients primarily develop skin rash 
and respiratory distress early in the course of the infusion (usually within the first hour). For such 
reactions, the investigator shall take one or more of the following actions: 
1. Discontinue the infusion. 
2. Apply appropriate resuscitation measures, including administration of 0.3–0.5 mL 
aqueous epinephrine (1:1000 dilution) subcutaneously. 
3. Use other resuscitative measures, as clinically indicated, including oxygen, intravenous 
fluids, antihistamines, corticosteroids, pressor amines, and airway management. 
Mild to Severe Reactions: For mild to severe (grade 3 or less) reactions per the NCI-CTCAE for 
allergic reactions, the study medication may be restarted at the discretion of the investigator. For 
those with severe (grade 3) reactions, any subsequent doses should be accompanied by pre-
medication with additional corticosteroids. 
Life-threatening Reactions: For life-threatening (grade 4) reactions per the NCI-CTCAE for 
allergic reactions, the study medication will be permanently discontinued. 
 


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Figure 6. Rules for Temporary interruption or discontinuation of ATG due to 
Hypersensitivity Reaction 
 
4.7 Modification or Discontinuation of ATG 
 
4.7.1 Modification of second ATG dose to 1mg/kg 
 
All ATG must be administered by the end of 60 hours.  CBC with differential will be obtained 8 
hours after the completion of infusion 1. If initial labs show acceptable values (WBC count 
>2,000 cells/mm3, neutrophil count >1,200 cells/mm3, and platelet count >75,000 cells/mm3), 
ATG will be continued at the full dose. If labs are outside of these parameters a second CBC can 
be obtained up to 24 hours after completion of the infusion. 
The second ATG/placebo dose will be reduced from 2mg/kg to 1mg/kg  if both the a  8 hour post 
infusion AND 24 hour post infusion platelet counts fall between 50,000 and 75,000 cells/mm3, or 
if the neutrophil count falls to a value >800 but <1,200 cells/mm3. Although the ATG package 
insert recommends reducing the dose by 50% if a patient has a total white blood cell (WBC) 
count of between 2000 and 3000 cells/mm3, it has been decided not to reduce the dose in this 
protocol for the following reasons: 
 
Because the primary focus of this study is initial T-cell depletion, premature or unnecessary 
reduction of the ATG dose may result in reduced efficacy. 
 
If the WBC count falls to a significantly lower level, i.e. <2000 cells/mm3, then ATG will be 
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 
Participants will be closely monitored for infectious disease risk and offered prophylaxis as 
warranted to minimize their risk 
 
GCSF is expected to minimize the time/severity of neutropenia 
4.7.2 Discontinuation of ATG 
 
The ATG/placebo will be discontinued if any one of the following is observed: 
 
A total WBC count <2,000 cells/mm3 that persists up to 24 hours after the time of the 
planned infusion. 
 
A neutrophil count <800 cells/mm3 that persists up to 24 hours after the time of the planned 
infusion. 
 
A platelet count <50,000 cells/mm3 that persists up to 24 hours after the time of the 
planned infusion. 
 
The participant experiences exacerbation of Cytokine Release Syndrome (CRS) twice 
consecutively despite the use of glucocorticoids.  
 
If the investigator believes that the study treatment is no longer in the best interest of the 
participant 
Participants who prematurely discontinue study treatment will remain in the study and undergo 
all efficacy and safety assessments. 
 
Figure 7. Rules for Reducing or Holding Second Dose of ATG 
 
 
 
 


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4.7.3 Serum Sickness 
 
Serum sickness from host immunization against rabbit protein may occur 7–15 days after the 
first dose of ATG. The patient may require glucocorticoid treatment for supportive care. The dose 
will depend on the severity of signs and symptoms. Each subject who develops signs or 
symptoms concerning for serum sickness will undergo a centralized review to ensure consistent 
decision making on the use of steroids.  For subjects greater than 50kg, we recommend 
Prednisone as follows: Days 1-3, 50 mg every 12 hours, Day 4, 40 mg every 12 hours, Day 5, 30 
mg every 12 hours, Day 6, 20 mg every 12 hours, Day 7, 10 mg every 12 hours. For subjects 
less than 50kg we will provide Prednisone as follows: Days 1-3, 30mg every 12 hours, Day 4, 
20mg every 12 hours, Day 5, 10 mg every 12 hours, Day 6, 5 mg every 12 hours, Day 7, 5 mg 
once. Clinical judgment should be used in augmenting additional steroid therapy to provide the 
least amount of steroid required to provide symptomatic relief of serum sickness.  
 
4.8 GCSF 
GCSF is a covalent conjugate of recombinant methionyl human granulocyte colony stimulating 
factor (filgrastim) and monomethoxypolyethylene glycol.  GCSF is a Colony Stimulating Factor 
that acts on hematopoietic cells by binding to specific cell surface receptors, thereby stimulating 
proliferation, differentiation, commitment, and end cell functional activation. GCSF is supplied in 
0.6 mL prefilled syringes for subcutaneous injection. Each syringe contains 6 mg GCSF (based 
on protein weight), in a sterile, clear, colorless, preservative-free solution (pH 4.0) containing 
acetate (0.35 mg), sorbitol (30.0 mg), polysorbate 20 (0.02 mg), and sodium (0.02 mg) in water 
for injection, USP. The standard 6mg dose will be given with the exception of subjects who 
weigh less than 44.5 kg. For subjects who weigh less than 44.5kg, the dose of GCSF will be 
given as 100mcg/kg/dose. Dosing calculations will be made based on the weight at the previous 
visit unless there has been more than a 10% change. Allowance in dose administered up to +/-
5% of calculated dose is permitted. 
 
 
4.8.1 GCSF / Placebo Administration 
 
GCSF/Placebo treatment will begin 6 hours after completion of the ATG / Placebo. GCSF/ 
Placebo will be given subcutaneously every 2 weeks for a total of 6 doses. Drug vehicle provided 
by the manufacturer will be used for the placebo injections. 
  
4.9 Modification or Discontinuation of GCSF 
Subjects will have a directed physical exam at each study visit and a CBC obtained every two 
weeks after the initiation of GCSF /placebo until they have completed study drug therapy. If at 
any time during the GCSF /placebo therapy the subject develops an ANC greater than 35 x 
109/L, subsequent  doses of GCSF /placebo will be given as placebo (regardless of study 
randomization) until the ANC is again lower than 35 x 109/L. A repeat CBC will be allowed once 
between scheduled doses to provide subjects an opportunity to normalize a previously elevated 
ANC. In addition, if splenomegaly is noted on physical exam, placebo will be given for the 
remainder of the drug course regardless of randomization. 
 
 
 
 


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4.10 Concomitant Mediations  
The use of concomitant medications will be assessed at each study visit and recorded on an 
appropriate source document and CRF. Participants are allowed to use preparations of insulin as 
advised by the investigator or the referring physician. 
 
Participants will be requested not to use any of the following medications during the study.  
 
Agents that influence insulin sensitivity or secretion (pramlintide, sulfonylureas, 
metformin, diphenylhydantoin, thiazide, or other potassium-depleting diuretics, beta-
adrenergic blockers, niacin). 
 
 Vaccination with live vaccines from 6 weeks before enrollment to 3 months following last 
dose of study drug is not permitted.  Killed vaccines other than influenza are discouraged 
during this time period. 
 
Any medication that may result in immunosuppression or immunomodulation. 
 
Systemic glucocorticoids (unless required during ATG administration or for the treatment 
of cytokine release syndrome or serum sickness). 
 
If participants receive, or if the investigator believes that participants must receive, any of the 
above medications, the case must be discussed with the medical monitor and/or treating 
physician to determine if alternatives are available. The use of these medications must be 
documented on the source document and CRF.  
 
 
 
 
 
4.11 Infectious Disease Screening  
 
In general, no specific infectious diseases prophylaxis is warranted in this study.  This decision is 
based on the limited duration and dose of exposure to ATG. In lieu of active serologic or virologic 
monitoring strategies, all subjects will be counseled on an ongoing basis on the importance of 
notifying their research centers about the presence of signs or symptoms suggestive of infection 
especially over the 1-2 months after the infusion.  They will also be counseled on the importance 
of notifying the research center about potential exposures to varicella, influenza, or other 
infectious illnesses.  In addition, during the first 2 weeks post ATG infusion, all research subjects 
will contacted on a daily basis and queried about the presence of signs or symptoms (e.g. fever, 
rhinorrhea, cough, sore throat, mouth sores, myalgia, arthralgia, etc) that might represent 
markers of active infection.  Specific algorithm(s) for the evaluation and management of those 
patients identified as having signs and/or symptoms concerning for infection are provided in the 
manual of operations for this study and are directed based on the participant’s specific 
symptomatology. This will provide general and in some case specific recommendations for the 
assessment of patients.   
 
All subjects will have EBV serology determined prior to study enrollment. Subjects who are EBV 
seronegative at screening must be EBV PCR negative within 30 days of randomization and may 
not have had signs or symptoms of an EBV compatible illness lasting longer than 7 days within 
30 days of randomization.   Additional EBV or other infectious disease monitoring will be done 
only if clinically indicated. 
 
 


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5 
STUDY ASSESSMENTS 
 
See Appendix 1 for detailed schedule of assessments 
 
5.1 General Assessments 
 
Study visits for all groups will occur according to the Schedule of Assessments .General 
assessments include:  
 
• Medical history including lifestyle and participant experience assessment 
• Physical exam  
• Concomitant medications  
• Adverse events  
 
 
5.2 Laboratory Assessments 
 
The following general laboratory assessments will be performed: 
 
Chemistry (sodium, potassium, chloride, CO2, glucose, BUN, creatinine) 
 
Liver function tests (ALT, AST, alkaline phosphatase, total protein, albumin, total and direct 
bilirubin) 
 
Hematology (complete blood count with differential and platelets) 
 
CD4/CD8 ratio 
 
Serum IgG levels 
 
 
At screening, these additional laboratory assessments will be performed: 
 
 
Interferon-Gamma Release Assay Test (IGRA) 
 
Diabetes Autoantibodies  
 
Antibodies to HIV, hepatitis B (antiHBcAb, HBsAg), hepatitis C (HCV), Cytomegalovirus 
(CMV IgG, IgM), and Epstein-Barr Virus (EBV IgG, IgM, EBNACMV and EBV PCR 
 
Additional assessments include: 
 
Pregnancy test before each injection for all sexually mature women  
 
5.3 Mechanistic Outcome Assessments 
 
TrialNet will perform immune and genetic assays to further understand mechanisms that may be 
underlying the type 1 diabetes disease process and response to therapy.  For this purpose, 
samples for PMBC, DNA, RNA, plasma, and serum may be obtained.   
 
5.4 Metabolic Outcome Assessments 
 
Metabolic assessments will consist of: 
 
Glucose records and reports of hypoglycemia 


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 
Insulin dose 
 
HbA1c 
 
Mixed meal tolerance test (MMTT): 2 hour MMTT at 3, 6, 9 months, 4 hour MMTT at 
screening, 12, 18, and 24 months 
 
 
 
 
5.5 Visit Windows 
 
Randomization must occur within 100 days from diagnosis of T1D. The initial treatment should 
begin within 100 days from the day of diagnosis and generally within 37 days from the screening 
MMTT.  With prior approval, this window may be extended to 56 days.  All subsequent treatment 
visits and follow up visits in Appendix 1 must occur within the time limits specified below: 
 
 
Visit Windows 
Visit -1: 
At least 2 weeks before visit 0 and at 
least 21 days from diagnosis. 
Visit 0: 
Randomization < 100 days from 
diagnosis of T1DM and within 37 days 
from Visit -1. 
Visits 1 through 6: 
±3 days 
Visits 7 through 9: 
±7 days 
Visits 10 and 11: 
±14 days 
 
 
5.6 Withdrawal from treatment 
 
The study will be conducted according to the intent-to-treat principle.  This means that once 
randomized into the study, a participant will be expected to undergo all scheduled follow-up 
assessments and will remain within the assigned treatment group for purposes of statistical 
analysis regardless of the actual course of treatment administered.  Withdrawal from treatment 
does not automatically entail withdrawal from the study.  Withdrawal from the study will only 
occur if the participant dies or withdraws consent.  Subjects who withdraw consent are classified 
as inactive but may again become active upon re-entry into the study, if they so choose.  
 
Withdrawal from treatment can occur for a number of reasons, some of which are outlined below.   
 


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A participant may elect to discontinue study medications, may be unable to continue them, or 
may be withdrawn (temporarily or permanently) at the discretion of the Principal Investigator if 
s/he determines that it is unsafe to continue or there is a significant change in the risk/benefit.  
 
Non-pregnant individuals who are withdrawn from treatment should remain in the study and 
undergo scheduled assessment visits as appropriate and any additional visits as needed to 
address ongoing AEs.  MMTT tests will not occur while an individual is pregnant. 
 
 
5.7 Re-Entry into Study Treatment 
In some circumstances, a participant may temporarily discontinue the study medication and/or 
not return to the study clinic for follow-up visits. If the participant decides to return for study 
injections and/or follow-up assessments at a later date, he or she will be allowed and 
encouraged to do so.  
 
6 
PARTICIPANT SAFETY 
6.1 Risk, Benefits and Inclusion of Children 
 
The risks of this study are presented in this protocol and in the informed consent form. This study 
will examine whether ATG-GCSF will preserve beta cell function, but there is no guarantee that 
this will occur. 
There is the prospect of direct benefit to the individual subjects for their participation in the study.  
These potential benefits include the recognized benefits of being in a clinical study, including 
close monitoring and additional resources available to maintain tight glycemic control offered to 
all subjects, regardless of group assignment.  Further, the intervention has the prospect of direct 
benefit to a given subject and is likely to yield general knowledge about T1DM that is of 
importance for the understanding and amelioration of T1DM in children. 
The study procedures, while possibly slightly greater than minimal risk, offer the possibility of 
benefit in the close monitoring for all children.  Assent of children along with consent of the 
parents will be obtained prior to any study procedures.  This research proposal in children is 
therefore consistent with United States Department of Health and Human Services, Protection of 
Human Subjects, subpart D, section 46.405 (research involving greater than minimal risk but 
presenting the prospect of direct benefit to individual subjects) and with Subpart D. 50.52 
(Clinical investigations involving greater than minimal risk but presenting the prospect of direct 
benefit to individual subjects). 
6.2 Potential Risks 
6.2.1 ATG 
 
ATG has been widely used in transplantation and autoimmune disorders (42). With the low dose, 
the absence of other immunosuppressive medications, and the lack of severe medical conditions 
other than T1D we expect minimal side effects. In the ITN study, subjects receive ATG at a total 


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dose of 6.5 mg/kg, a dose which has been effective and well tolerated in bone marrow and solid 
organ transplantation, and well below the dose of 20 mg/kg that has been used for some 
transplantation protocols. For this study, the doses will be administered as 0.5 mg/kg during the 
first infusion and 2 mg/kg during the second infusion to reduce reactions.  ATG will be given 
slowly by continuous IV infusion over at least a 12 hour period during the first dose and at least 
an 8 hour period during the second dose.  Subjects will receive pre-medication with 
methylprednisolone, acetaminophen and diphenhydramine to minimize side effects from possible 
cytokine release syndrome (CRS), and will be closely observed in the hospital during the 
treatment period. Symptoms of CRS may include fever, chills, rigors, headache, tremor, nausea, 
vomiting, diarrhea, abdominal pain, muscle and joint pain, and malaise.  Anaphylactic reactions 
have been rarely reported with ATG, but delayed allergic reactions with serum sickness are 
occasionally observed.   
ATG contains a variety of antibodies that may cross-react with cell-surface markers. ATG causes 
lymphocyte depletion with marked depletion of lymphocytes occurring acutely. The circulating 
number of T cells increases with cessation of therapy, usually reaching pre-treatment levels by 2 
months.  Subjects are at low risk for opportunistic infection during this window of recovery and 
will receive close surveillance for primary viral infections, viral reactivations, and bacterial 
infections (76, 77).   
ATG may also lead to leukopenia and thrombocytopenia. Effects are dose-dependent and are 
mainly encountered with over dosage.  In up to 3% severe thrombocytopenia may occur but this 
is invariably seen in doses 4-5 times that which we are proposing. Thrombocytopenia in these 
subjects 
often 
occurred 
in 
a 
postoperative 
transplant 
setting 
and 
with 
other 
immunosuppressants.  There was no concern for prolonged leukopenia or thrombocytopenia in 
our pilot ATG-GCSF study. 
Transient abnormalities in liver function tests have been described in patients with aplastic 
anemia treated with ATG preparations. Such adverse events have not been noted in numerous 
other clinical settings in which ATG has been used, and it is not clear if this is related to the 
underlying disease or to associated medications used.  
Conflicting data exists about the risk of EBV-related lymphoproliferative disease. Furthermore, in 
renal transplant patients, the overall risk in this transplant population is low (0.25%-0.85%), and 
patients 
received 
several 
immunosuppressive 
therapies 
and 
were 
on 
continuous 
immunosupression (42). There were no reported cases of EBV-associated lymphoproliferative 
disease in over 1,675 treated subjects (including children) treated for aplastic anemia. 
6.2.2 GCSF 
 
There are some potential risks associated with GCSF therapy. The side effect profile of GCSF 
injection will be monitored by close clinical observation of the subjects for 1 hour after the first 
GCSF injection as well as with daily contact to the subjects during the first 5 days of 
GCSF/placebo therapy and then weekly until the week 12 visit. 
GCSF carries a risk (10-20% of patients) of bone pain and a risk (less than 10%) of headache, 
fever, pain or redness at the site of injection, and dizziness.  According to the company USPI, 
there is also potential risk of Acute Respiratory Distress Syndrome, serious allergic reactions, 
and Sickle Cell crisis. There is also a risk that the subjects’ WBC could become elevated while 
taking GCSF. A complete list of the potential risks associated with GCSF is further described in 
the package insert at http://pi.amgen.com/united_states/neulasta/neulasta_pi_hcp_english.pdf. 
This will be monitored by the study team at each center and the dose of study drug will be 
dispensed by the pharmacy as placebo regardless of randomization (to ensure blinding is 


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protected) if the ANC is > 35 x 109/L. There are also now a small number of reports in which 
recipients of GCSF have developed splenomegaly and/or splenic rupture. This risk will be 
explicitly discussed in the consent form and subjects will have close physical examination and 
follow-up to screen for any signs or symptoms. In addition, weekly contact for the month 
following therapy will be used to document any adverse reactions. The CBC and complete 
metabolic profile at each blood draw will be carefully reviewed for any clinically relevant 
abnormalities.  There are no other known long-term risks from a 12 week course of GCSF.  
 
 
6.3 Pregnancy 
Female subjects with reproductive potential will be instructed to use effective means of birth 
control (which includes abstinence) from randomization until 3 months post last infusion of study 
drug to assure safety.  They will also be asked to avoid pregnancy until last scheduled study visit 
at 24 months post randomization.  This is to assure accurate endpoint measurements. They will 
undergo urine pregnancy testing at the start of every study visit.  All pregnancies that are 
identified during the study must be followed to conclusion and the outcome of each must be 
reported.  The investigator should be informed immediately of any pregnancy occurring in a 
female participant.  Monitoring of the participant should continue until the conclusion of the 
pregnancy. Subjects that are found to be pregnant while on this study shall have treatment 
withheld, but will still be followed for safety and other study measures as appropriate. Treatment 
may only be resumed when subjects are no longer pregnant or nursing. 
 
6.4 Protecting Against or Minimizing Potential Treatment Risks 
Subjects will not be enrolled who have other active serious medical problems.  Frequent 
monitoring of patients with history, physical examination, and laboratory studies will allow for 
early identification of adverse events.  All participants will be required to have adequate 
hemoglobin to allow safe frequent venipuncture.  Every attempt will be made to minimize the 
number of venipunctures.   
All study drug infusions and injections will take place in a facility that has resuscitation 
capabilities, and subjects will be closely monitored during and after the injection.    
Subjects will be counseled about the potential risk for infections and the need to report any 
change in health status between or at the time of visits.  Directed questioning about concurrent 
illness will occur before each injection.  No injection will occur in those with signs or symptoms 
indicative of active infection.   
 
7 
ADVERSE EVENT REPORTING AND SAFETY MONITORING 
 
7.1 Adverse Event Definition 
 
7.1.1 Adverse Event 
 
In this clinical trial, an adverse event is any occurrence or worsening of an undesirable or 
unintended sign, symptom or disease whether or not associated with the treatment and study 
procedures.   


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Throughout the study, the investigator must record all adverse events on source documents.  
Events not related hypoglycemia, or hyperglycemia which are Grade 2 or greater per the NCI 
CTCAE (see Section 7.1.5. Grading Event Severity below) must be reported to TNCC as AE. 
The investigator should treat participants with adverse events appropriately and observe them at 
suitable intervals until the events resolve or stabilize. 
Adverse events may be discovered through: 
 
observation of the participant; 
 
questioning the participant; 
 
unsolicited complaint by the participant. 
Questioning of the participant should be conducted in an objective manner. 
 
7.1.2 Adverse Reaction 
 
An adverse reaction means any adverse event caused by a drug.  Adverse reactions are a 
subset of all suspected adverse events for which there is reason to conclude that the drug 
caused the event.  Suspected adverse reaction means any adverse event for which there is a 
reasonable possibility that the drug caused the adverse event.  For the purposes of safety 
reporting, “reasonable possibility” means there is evidence to suggest a causal relationship 
between the drug and the adverse event. A suspected adverse reaction implies a lesser degree 
of certainty about causality than an adverse reaction, which means any adverse event caused by 
a drug.  Examples of evidence that suggest a causal relationship (reasonable possibility) 
between the drug and the adverse event include: 
 
A single occurrence of an event that is uncommon and known to be strongly associated 
with drug exposure 
 
One or more occurrences of an event that is not commonly associated with drug 
exposure, but is otherwise uncommon in the populations exposed to the drug 
 
An aggregate analysis of specific events observed in a clinical trial (such as known 
consequences of the underlying disease or condition under investigation or other events 
that commonly occur in the study population independent of drug therapy) that indicates 
those events occur more frequently in the drug treatment group than in a concurrent or 
historical control group. 
7.1.3 Serious Adverse Event/Reaction 
 
A serious adverse event (SAE) or reaction is defined as “any adverse event occurring at any 
dose that suggests a significant hazard, contraindication, side effect, or precaution.”  An adverse 
event or suspected adverse reaction is considered “serious” if, in the view of either the 
investigator or sponsor, it results in any of the following outcomes: 
1. Death.  A death that occurs during the study or that comes to the attention of the 
investigator during the protocol-defined follow-up after the completion of therapy 
must be reported whether it is considered to be treatment related or not. 
2. A life-threatening adverse event.  A life-threatening event is any adverse therapy 
experience that, in the view of the investigator, places the participant at immediate 
risk of death from the reaction as it occurred. 


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3. Inpatient hospitalization or prolongation of existing hospitalization with the exception 
of hospitalization relating to initial diagnosis of type 1 diabetes. 
4. Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
5. Congenital anomaly or birth defect. 
6. Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical 
judgment, they may jeopardize the patient or subject and may require medical or 
surgical intervention to prevent one of the outcomes listed above.  
An adverse event or suspected adverse reaction is considered “life-threatening” if, in the view of 
either the investigator or sponsor, its occurrence places the patient or subject at immediate risk 
of death. It does not include an adverse event or suspected adverse reaction that, had it 
occurred in a more severe form, might have caused death. 
Regardless of the relationship of the adverse event to study drug, the event must be reported as 
a serious adverse event if it meets any of the above definitions. 
7.1.4 Unexpected Adverse Event 
 
An adverse event/reaction is considered unexpected when the nature (specificity) or severity of 
the event is not consistent with the risks described in the Investigator’s Brochure or the informed 
consent document. Unexpected refers to an experience that has not been previously 
observed.  This includes events that occur more frequently than expected. 
7.1.5 Grading Event Severity and Causality 
 
TrialNet has adopted usage of the National Cancer Institute (NCI) Common Technology Criteria 
for Adverse Events (CTCAE) and/or study-specific criteria for classification to describe the 
severity of adverse events with the exception of hypoglycemia and hyperglycemia.  
Hypoglycemia and hyperglycemia will be reported as adverse events only in the case of 
requiring the assistance of others due to loss of consciousness or DKA. TrialNet Investigators 
will also provide an assessment of relationship of AE to study drug as not, unlikely, possibly, 
probably, or definitely related.  
 
7.2 Adverse Event Reporting and Monitoring 
 
Adverse events will be reported to the TrialNet Coordinating Center.  The investigator will grade 
their severity according to common toxicity criteria or study-specific criteria and will make a 
determination of their relation to therapy.  Events will be assessed and reported consistent with 
the ICH Guideline for Good Clinical Practice, 21 CFR 312.32 for expedited safety reporting, and 
per the guidance of the DHHS Office for Human Research Protections (OHRP).   
The adverse event case report form for the protocol must be completed for all adverse events 
(AE).  For reporting serious adverse events (SAE), the MedWatch Form should also be 
completed and faxed to the TNCC within 24 hours of when the site was notified of the event.  
This will be reviewed by the TrialNet Medical Monitor, the TrialNet Safety Monitoring Committee, 
and the DSMB as appropriate.  Deaths must be reported immediately.  Event outcome and other 
follow-up information regarding the treatment and resolution of the event will be obtained and 


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reported when available, if not known at the time the event is initially reported.  The follow-up 
information should contain sufficient detail to allow for a complete medical assessment of the 
case and an independent determination of possible causality.  
Adverse events will be assessed by the TrialNet Medical Monitor.  The DSMB will conduct 
regular safety reviews approximately every three to six months (and otherwise as needed) of 
adverse events by treatment group assignment.  Serious adverse events as well as adverse 
events leading to study discontinuation will be reviewed by the DSMB.  
For SAEs that are unexpected and considered possibly or probably drug related, the Medical 
Monitor will provide information on frequency of similar events, and generate FDA form 3500A 
reports (MedWatch form) for distribution to FDA, NIDDK, DSMB and site investigators.  
Expedited safety reports will be submitted to the IND by the NIDDK.  The TNCC will also be 
primarily responsible for sending required safety information to Amgen. 
 
8 
STATISTICAL CONSIDERATIONS AND ANALYSIS PLAN 
Analyses of study data will be conducted to address all objectives of the trial and other 
interrelationships among data elements of interest to the investigators and of relevance to the 
objectives of the study.  Analyses by gender and race/ethnicity, as appropriate, are also planned. 
Primary analysis of treatment effect will be conducted employing the intention-to-treat principle 
where all participants with measured one-year C-Peptide AUC mean are included regardless of 
treatment compliance (Complete Case Analysis).     
8.1 Primary Outcome and Analyses 
The primary outcome of each participant is the area under the stimulated C-peptide curve (
AUC
Y
) 
over the first 2 hours of a mixed meal glucose tolerance test conducted at the one-year visit.  
The 
AUC
Y
 is computed using the trapezoidal rule which translates into a weighted sum of the 
timed C-peptide values over the 120 minute MMTT.  The more appealing quantity is the AUC 
mean which is 120
AUC
Y
 in nmol/L and will be denoted as 
Cp
Y
.   
Let  
/
,
, and 
ATG GCSF
ATG
Control
Cp
Cp
Cp
Y
Y
Y
 represent the C-peptide AUC mean for study patients receiving 
both ATG and GCSF, receiving ATG alone, and those receiving placebo, respectively. Likewise, 
let 
/
,
, and 
ATG GCSF
ATG
Control
Cp
Cp
Cp



 represent the population mean of C-Peptide for these groups at 
one year, respectively. 
The primary statistical hypotheses to be assessed in the study are:  
/
/
0
H : 
 versus H :  
ATG GCSF
Control
ATG GCSF
Control
Cp
Cp
a
Cp
Cp






 
 
0
H : 
 versus H :  
ATG
Control
ATG
Control
Cp
Cp
a
Cp
Cp






 


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The primary analysis will be conducted on a transformed scale using the function log(
1)
Cp
Y

  .  
This provides better normal distributional behavior by the test statistic.  The comparison between 
any two treatment arms will be based on a Wald test of treatment effect in an ANCOVA model 
adjusting for gender, baseline age, and baseline log(
1)
Cp
Y

.   
 
8.2 Secondary Outcome and Analyses 
Additional analyses of the primary outcome to determine the effect of ATG with and without 
GCSF include:  
 
C-peptide AUC mean at 3, 6, 9, 12, 18, and 24 months using the ANCOVA mentioned 
above. 
 
The hazard rate of C-peptide failure (C-peptide failure defined as the first occurrence at 
which the 2 hour peak C-peptide < 0.2 nmol/L during a MMTT) using the proportional 
hazards model82 while adjusting for baseline level of C-peptide, gender and baseline age. 
 
Longitudinal analyses of C-Peptide AUC means 3, 6, 9, 12, 18, and 24 months using a 
mixed effects model72  with a random intercept and slope by subject, adjusted for the 
baseline level of C-peptide, gender and baseline age. The mean intercept and slope will 
be compared between treatment groups. 
 
Treatment interactions with the covariates baseline C-peptide, gender and baseline age 
will be analyzed with a homogeneity test categorizing the continuous variables into 3 
approximately equal groups; ladder plots will be constructed. Other variables to be tested 
for treatment interactions are HbA1c levels, HLA , other genotype and immune 
phenotypes, and race/ethnicity, as appropriate.  
Additional secondary objectives to determine the effect ATG with and without GCSF has on the 
following: 
 
HbA1c, Insulin dose (units/kg) and Blood glucose over time by treatment group using 
ANCOVA. 
 
Adverse events 
o Number and severity. 
o The rates of severe adverse events will be computed (total number of events 
divided by total subject years of follow-up). 
 
Hypoglycemia 
o Number of major hypoglycemic events (defined as loss of consciousness, seizure, 
or requiring assistance from another person because of altered state of 
consciousness). 
o Reported hypoglycemic events confirmed with capillary blood glucose 
measurement less than 70 mg/dl. 
o The rates of severe hypoglycemic will be computed (total number of events 
divided by total subject years of follow-up). 
 
For individuals with continuous glucose monitoring data available 


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o Area under the curve and number of events less than 70 mg/dl on the continuous 
glucose monitoring record. 
o Hyperglycemia measured as the area under the curve and number of events 
greater than 180 mg/dl on the CGMS record prior to each study visit. 
o Glycemia and glycemic variability prior to each MMTT visit 
o The daily mean level of glucose, as well as the levels before and after meals will 
be computed. 
o Measures of diurnal variability in glucose will be measured by the J-value, 
standard deviation of glucose values, and the mean amplitude of glycemic 
excursion (MAGE). 
8.3 Additional Outcomes and Analyses 
Additional outcomes of interest include the effects of ATG with and without GCSF treatment with 
regard to the Mechanistic Studies assessed from blood draws as outlined in the Schedule of 
Assessments.   These measures include, but are not limited to, exploration of pharmaco-genetic 
signatures that may differentiate response to treatment, and the relationship between hsCRP 
and IL-1β levels and beta cell function and/or other metabolic measures. 
Additional analyses will compare the results in this trial to other trials using ATG and GCSF and 
other TrialNet studies.  Data in this trial will be used in conjunction with other TrialNet data for 
exploratory analysis.  
8.4 Sample Size and Power Calculations 
The primary analysis will compare the difference in C-Peptide between experimental and 
placebo treatment groups at 12 months using log(
1)
Cp
Y

 transformation and ANCOVA model 
adjusting for gender, baseline age and the baseline value of log(
1)
Cp
Y

. Estimates of the mean 
and standard deviation of log(
1)
Cp
Y

 (expressed algebraically as: 
log(
1)
ˆ
Cp
Y

 and 
log(
1)
ˆ
Cp
Y

) in the 
placebo group were derived from the last four TrialNet studies of early onset disease. The 90% 
confidence bound estimates were used to provide good confidence that this trial’s study 
population characteristics fall within these limits to assure the advertised statistical power; the 
estimates are 
log(
1)
ˆ
0.360
Cp
Y


 and 
log(
1)|
ˆ
0.167
Cp
Y
X



 (i.e., the residual mean squared error 
regressing on gender, baseline age and C-peptide). The geometric-like mean of 
Cp
Y
 for the 
placebo group is exp(0.36) 1
0.433

nmol/L. 
Using standard equations for the comparison of two means and a 1:1:1 allocation, a sample size 
of 78 participants (26 per group) with complete data would provide power of 85% to detect a 
50% increase in the geometric-like mean in either experimental treatment group (compared to 
the control) using the Wald test (from the adjusted linear model) at the 0.025 level (one-sided).  
Thus the overall Type I error for both tests is approximately 0.05. 
Assuming that 10% of the participants will have missing data (one-year MMTT was not done or 
subject withdrew prior to the one-year assessment), the sample size goal for this study will be 
set at 84 participants (28 per group).  


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The study will be closed to additional participants when the total number then randomized plus a 
fraction of those in screening (i.e., screening for eligibility) is expected to provide the proper 
number of eligible participants.  Participants who had already conducted the initial screening visit 
at that time will be allowed to complete screening and be randomized if both consenting and 
eligible. 
In the situation where both hypotheses are rejected in favor of the experimental regimen, then it 
is appropriate to decide which experimental regimen should be considered first for additional 
clinical trials.  If ATG and ATG+GCSF truly differ in efficacy, it is not expected that the difference 
would be as large as the effect size used in this design.  Thus requiring the difference to reach 
statistical significance would be too stringent due to the lack of statistical power.  The plan is to 
employ the method of Simon81 to select the treatment with the largest geometric-like mean in C-
Peptide regardless of how small the difference is over the other experimental regimen. Formally, 
the decision rule is to select the experimental regimen with highest predicted mean based on the 
fitted linear regression model of C-Peptide used in the formal hypothesis tests.  Given the one 
experimental treatment is associated with 25% higher mean (half of the design effect size) 
compared to the other experimental treatment, the probability is 96% that the most efficacious 
regimen will be selected with this decision rule.  Even if the increase is only 12.5% (i.e., a fourth 
of the design effect size), the probability is 82% in making the right choice.  Since under the null (
/
 
 
ATG
ATG GCSF
Cp
Cp



) we consider selecting either treatment a non-error there is no contribution to 
the type I error, and therefore, no further adjustment is required to the α-level of the initial two 
pair-wise tests. 
8.5 Sample Size Re-estimation   
The Residual Mean Square Error (RMSE) and the mean of the control group are two parameters 
used in determining the sample size goal (NSSG=78) which are directly tied to the study cohort 
enrolled.  The former value is directly proportional to the sample size and the latter applies its 
influence in defining the effect size.  Thus the advertised statistical power of this trial may differ 
from the actual power if either differs from the initial values assumed.  Consequently, the plan is 
to estimate both values using the accumulated data (internal interim estimate, IIE) when 
approximately half the subjects (NSSG/2) have had their 1 year C-Peptide assessment.  Since the 
initial values used are based on real data from four previous TrialNet studies, the re-estimation of 
each parameter will be a two-term weighted average using the number of subjects on which the 
IIE is based as the one weight (NIIE) and the remaining number of subjects participants to be 
enrolled as the other weight (NSSG-NIIE).  Algebraically, the re-estimate will be computed: 
 
2
2
2
e
ˆ
ˆ
(
)
ˆ
IIE
IIE
SSG
IIE
Initial
R
estimate
SSG
N
N
N
N







  
Similarly, re-estimation of the mean of the control group will be calculated.  These new estimates 
will be used to calculate a new sample size goal for the trial. 
It is important to note that this adaptive procedure is a non-comparative interim analysis (i.e., the 
observed treatment effect has no influence on the sample size re-estimation).  Therefore, this 
analysis has no effect on the type I error.  Note the re-estimation can be conducted by the 
analyst in a blinded fashion.  The goal is to assure adequate statistical power at the completion 


TrialNet Protocol TN19 
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of the trial by assuring the two design parameters reflect accurately the study population being 
enrolled.   
8.6  Interim Monitoring Plan 
Interim analyses will be conducted periodically during the study and will be reviewed by the 
TrialNet DSMB for assessment of effectiveness and safety.  The Lan-DeMets74 spending 
function with an O’Brien-Fleming boundary will be used to protect the type I error probability from 
early and multiple testing and to assess the significance of the interim results that emerge during 
the trial82. The spending function that approximates the O’Brien-Fleming boundaries is:    
*
/2
1
*
( )
2
2
Z
t
t









 
where 
*t is the information fraction (
*
0
1
t

),
1
is the 
-level

 of the interim (one-sided) test 
and  is the type I error for one of the pair-wise tests (i.e., 0.025).  The monitoring plan will 
allow for early termination based on the treatment effect on C-peptide values at 1 year of follow-
up using the ANCOVA model described above.   
The DSMB will also be informed if there is a serious lack of evidence of a treatment effect (i.e. 
futility analysis). The boundaries are based on the paper by Lachin83. The study arm should be 
“closed” based on the futility of rejecting the null hypothesis at the completion of the trial if: 
*
( )
0.1
ATG
z
t

 when 
*
0.5
 
0.75
t


 or if 
*
( )
1.0
ATG
z
t

 when 
*
0.75
t 
 (z is the Wald test of the 
treatment effect coefficient).  The same rule would be applied to the ATG/GCSF treatment group. 
These 
*
0.5 and 0.75
t 
 are equivalent to when there are 39 and 59 participants with one-year 
C-peptide results, respectively. Lachin showed that a onetime use of either boundary for the 
design parameters above   (
1
1
3.00
Z
Z








) raises the type II error to approximately 
0.15414 and 0.15611, respectively.  For larger values of *t  the increase to the error probability is 
even less.  Furthermore, by the laws of probability a single use of each rule will increase the type 
II error no more than the sum of the increase (i.e., 0.00414+0.00611 = 0.0103).   
8.7 Interim Analysis Study Modification 
Whether Lan-DeMets boundary is crossed or a futility decision rule indicates closure of an 
experimental arm, the course of action for the study is as follows.  In case the futility decision 
rule indicates closure of one of the experimental arms (and the governing bodies concur), the 
trial should continue randomizing between the other remaining experimental treatment and 
placebo (1:1 ratio) since the corresponding hypothesis remains unaddressed.  However, in the 
circumstance where the Lan-DeMets boundary is crossed the normal course of action would be 
to close the placebo arm.  This would incapacitate the second formal hypothesis test thus 
leaving only the issue of which experimental treatment is “best” as the remaining question.  
However, it may or may not be reasonable to make this decision as well.  To evaluate the 
evidence at the interim the plan is to use stochastic curtailment and compute the probability that 
the currently ‘best’ treatment (as defined by the decision rule above) is not the ‘best’ when the 
full sample size is reached.  In the normal employment of stochastic curtailment in futility 
analysis the parameters used for the simulation are based on the alternative hypothesis. The 


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analogous parameters for this situation is to set the mean of the currently ‘best’ treatment to be 
lower than the other experimental treatment mean and determine the probability of a reversal.  
As with the normal application of stochastic curtailment, we will use the probability of 0.20 or less 
to stop accrual completely and invoke the decision rule for selecting the ‘best’ treatment 
immediately.  Alternatively, the study would continue to its planned completion, randomizing 
between the two active treatment arms, until the projected probability of a re-ordering of the 
outcome (change in the selection of the superior treatment arm) is less than 0.2. 
Our choice for setting the parameters for simulation is to set the ‘best’ treatment group at 25% 
lower than the other experimental treatment mean; these two parameters being centered at the 
midpoint between the two estimated experimental group means. This is consistent with the 
difference between the two treatment arms specified in the Simon selection at the completion of 
the trial (described above), should both arms be superior to the placebo.       
The graph below provides an estimated probability of a ‘best’ selection reversal conditioned on 
the interim data (simulated under the condition of crossing the Lan-DeMets boundary and t* = 
0.5).  The probability is primarily a function of the relative position of the ‘lesser’ mean to the 
positions of the placebo mean and the ‘greater’ mean (all means being estimates from the 
interim data).  That is, the closer the ‘lesser’ mean is to the placebo, the less likely there could be 
a reversal in the selection of the ‘best’ treatment. 
 
Additional analysis will assess potential adverse outcomes of treatment and will assess the 
incidence of all severe adverse events. 
 
 
Relative Location of Lesser Mean
P ro b a b ility
Probability of Reversal Given the Relative Location
of the Lesser Experimental Group Mean
0 .0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
0 .7
0 .8
0 .9
1 .0
Mean
Placebo Grp
Mean
(Greater)


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9 
ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL 
PRACTICE 
9.1 Statement of Compliance 
 
This study will be conducted in compliance with the protocol and consistent with current Good 
Clinical Practices (GCP), adopting the principles of the Declaration of Helsinki, and all applicable 
regulatory requirements (ICH E6, 45CFR46, and FDA 21CFR sections 11, 50, 56, 312). 
Prior to study initiation, the protocol and the informed consent documents will be reviewed and 
approved by an appropriate Independent Ethics Committee/Research Ethics Board (IEC/REB) or 
Institutional Review Board (IRB).  Any amendments to the protocol or consent materials must 
also be approved before they are implemented. 
 
9.2 Participating Centers 
Participating TrialNet clinical sites must have an appropriate assurance, such as a Federal-wide 
Assurance (FWA) or an Unaffiliated Investigators Agreement (UIA), with the Office for Human 
Research Protections (OHRP), since they are actively engaged in research and provide informed 
consent.  The protocol and consent forms will be approved by Institutional Review Boards or 
Ethics Committees/Research Ethics Boards at each of the participating clinical sites.  HIPAA and 
applicable local regulations will be followed by each participating institution in accordance with 
each institution’s requirements.  The participating international sites will obtain approval from 
their corresponding review boards in accordance with their local procedures and institutional 
requirements. 
The investigator is required to keep accurate records to ensure the conduct of the study is fully 
documented.  The investigator is required to ensure that all case report forms are legibly 
completed for every participant entered in the trial. 
The investigational sites participating in this study will maintain the highest degree of 
confidentiality permitted for the clinical and research information obtained from participants 
participating in this study.  When a subject participates in this study at more than one TrialNet 
site, sharing of this information is required.  Sharing of information obtained during this study 
between TrialNet clinical centers and affiliates will be done to assure subject understanding and 
consent, safety, and adherence to protocol.  Medical and research records will be maintained at 
each site in the strictest confidence.  However, as a part of the quality assurance and legal 
responsibilities of an investigation, the investigational site must permit authorized representatives 
of the sponsor(s) and regulatory agencies to examine (and when required by applicable law, to 
copy) records for the purposes of quality assurance reviews, audits and evaluation of the study 
safety and progress.  Unless required by the laws permitting copying of records, only the coded 
identity associated with documents or other participant data may be copied (obscuring any 
personally identifying information).  Authorized representatives, as noted above, are bound to 
maintain the strict confidentiality of medical and research information that may be linked to 
identify individuals.  The investigational site will normally be notified in advance of auditing visits. 
 
 
 
 


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9.3 Informed Consent 
The process of assuring that individuals (and parent/guardian if less than 18 years of age) are 
making an informed decision about participating in this study includes both verbal and written 
communication.  Written materials include a Volunteer Handbook, Volunteer Understanding 
Assessment, and written consent forms.  There are several consent forms for this study.  One is 
a Screening consent form that describes the procedures, risks, and benefits, and determines 
eligibility for the study.  The second is the Intervention consent form, which describes the 
procedures, risks, and benefits for the remainder of the study.  A third consent form is for use at 
clinical sites that will be performing the post-treatment visits, but not the treatment visits.  The 
consent forms will be reviewed with participants (and their parent/guardian in the case of 
participants under 18 years of age) and the participant will be given time to review the written 
consent form and ask questions.  An assent form has also been developed for participants less 
than 18 years of age (unless local IRB/REB requirements differ in procedure).  
As part of the informed consent process, the participant and/or parent or guardian (if the 
participant is less than 18 years of age) will also be required to complete a short, written 
Volunteer Understanding Assessment that is designed to ensure that the subject understands 
the study, as well as what is being asked of him/her. The participant will be given a copy of their 
consent/assent forms.   
The consent process will be conducted by qualified study personnel (the Trial Investigator or 
Study Coordinator and/or other designee).  All participants (or their legally acceptable 
representative) must read, sign and date a consent form prior to participation in the study, and/or 
undergoing any study-specific procedures. 
The informed consent form must be updated or revised whenever there is new, clinically 
significant  information applicable to the safety of the participants,  when indicated for a protocol 
amendment, and/or whenever any new information becomes available that may affect a 
subject’s participation in the study.   
Subjects will be re-consented if they reach the age of 18 years while enrolled in the study. 
 
9.4 Study Subject Confidentiality 
Study subject data, which is for reporting purposes, will be stored at the TrialNet Coordinating 
Center.  Data sent to the Coordinating Center will identify participants by the unique TrialNet 
Identification Number.  The data entry system at the Coordinating Center is a secured, password 
protected computer system.  At the end of the study, all study databases will be archived at the 
Coordinating Center for long-term storage.   
Stored samples including genetic samples could be utilized to learn more about causes of type 1 
diabetes, its complications (such as eye, nerve, and kidney damage) and other conditions for 
which individuals with diabetes are at increased risk, and how to improve treatment.  The results 
of these future analyses, and any mechanistic studies will not be made known to the participant. 
 
9.5 Risks and Benefits 
The risks of this study are presented in this protocol, and informed consent form.  There is no 
guaranteed benefit to subjects for their participation in the study.   
Special consideration regarding risks and benefits for children is described in section 6.2. 


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10 STUDY ADMINISTRATION 
10.1 Organizational Structure 
 
This study is part of Type 1 Diabetes TrialNet, which is funded by the National Institutes of 
Health. Funding will also be provided by the Helmsley Charitable Trust. Funding will cover the 
costs of administration and laboratory tests associated with this study.  
 
10.2 Role of Industry 
Sanofi will provide the ATG for the study, and will also be providing funding to support the 
conduct of the study. Amgen will be providing GCSF/Placebo. 
 
10.3 Groups and Committees 
10.3.1 ATG-GCSF Study Chair Committee 
 
The Study Chair and TrialNet Executive Committee will receive periodic reports from the TNCC 
on the progress of the study.  These will include accrual rates and baseline demographic 
characteristics. Interim data summaries provided to others (except those that could lead to 
unmasking of study outcome) will first be supplied to the Study Chair for review. Criteria and 
results of ongoing monitoring of the TrialNet labs in terms of reproducibility will also be provided 
on a routine basis and reported on during TN19 ATG-GCSF Study Chair Committee meetings, 
as scheduled. As appropriate, abstracts and manuscripts dealing with the progress of the trial 
shall be directed by the TN19 ATG-GCSF Study Chair Committee.  
10.3.2 TrialNet Chairman’s Office and TNCC 
 
The TrialNet Chairman’s Office and TNCC will work together in providing leadership to the 
TrialNet study group to include protocol and manual preparation, training for clinical sites, 
development of statistical design for each study, and analysis of study results.  The TNCC will 
also coordinate interactions among the participating TrialNet Clinical Centers, test laboratories 
including TrialNet Core Laboratories and other subcontract laboratories, NIDDK, and other 
sponsoring agencies. 
10.3.3 Clinical Sites 
 
Principal Investigators at each participating TrialNet clinical site will oversee all operations at that 
site.  The clinical sites will forward all laboratory and data collection form information to the 
TNCC for analysis.  Direct communication and site visits, as needed, will facilitate evaluation of 
the trial management.   
10.3.4 Safety Monitoring Subcommittee 
 
The Type 1 Diabetes TrialNet Safety Monitoring Subcommittee (SMS) is responsible for 
establishing policies and procedures for assurance of safety monitoring of TrialNet protocols and 
of TrialNet subjects. The Safety committee will review all serious AEs and receive summary 
reports of all AEs.  This committee will be masked as to treatment assignment.  


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10.3.5  Clinical Site Monitoring 
 
In order to conduct this study with established research principles, site visits will be conducted 
during the study to evaluate study conduct and ensure subject safety.  All sites will be monitored 
by the TNCC and appropriate TrialNet committees for patient enrollment, compliance with 
protocol procedures, completeness and accuracy of data entry, the occurrence and reporting of 
adverse 
events 
(AEs) 
and 
serious 
adverse 
events 
(SAEs), 
site 
pharmacy 
accountability/operations and to confirm the presence of appropriate IRB/REB regulatory 
approvals/documents.   
10.3.6 Medical Monitor and Data Safety and Monitoring Board (DSMB) 
 
All adverse events will be recorded on the adverse event forms, which will be sent to the local 
IRBs/REBs, per their reporting requirements, and to the Coordinating Center.  
An independent physician will be designated to serve as the medical monitor for this study who 
will maintain regular contact with the study and the Study Chair.  (S)he will review all adverse 
event reports, masked to treatment assignment, and will file event reports with regulatory 
authorities as appropriate.   
The DSMB will meet approximately every 3 months and as needed to review indicators of safety.  
In addition, they will meet every 6 months to review the interim effectiveness and potential 
toxicity of the study treatments based on interim analyses of indicators of effectiveness and 
safety prepared by the TNCC separately by treatment group.  The DSMB will independently 
evaluate whether there are grounds to modify or discontinue the study. 
 
10.4 Sample and Data Storage 
Samples to be stored for research purposes will be located at the NIDDK Repository and at 
TrialNet Laboratory Sites.  While TrialNet is active, the use of the samples will be restricted to 
TrialNet researchers unless researchers from outside of TrialNet obtain approval from the 
TrialNet Steering Committee and the NIDDK to utilize the samples.  All samples will be coded 
with unique study numbers, but TrialNet researchers will be able to identify samples if it is 
necessary to contact participants for reasons of health or for notification to them about future 
studies. Approval from the TrialNet Steering Committee and the NIDDK would be required before 
such linkage could occur.  Researchers from outside of TrialNet will not be permitted to identify 
samples.  
Data collected for this study will be sent to the TNCC.  De-identified data will be stored at the 
NIDDK Repository, under the supervision of the NIDDK/NIH, for use by researchers including 
those outside of TrialNet. 
With permission of the subject, when TrialNet is completed, samples will continue to be stored at 
the NIDDK Repository.  Since the stored data will be fully de-identified upon the completion of 
TrialNet, it will no longer be possible to identify samples.  Thus, whereas a sample can be 
destroyed upon a participant’s request during the existence of the TrialNet, it can no longer be 
destroyed once TrialNet is completed.  However, there will still be the potential to link data 
derived from the samples with data that had been derived from TrialNet studies.  Once TrialNet 
is completed, researchers will only obtain access to samples through grant proposals approved 
by the NIDDK.  The NIDDK will convene an external panel of experts to review requests for 
access to samples. 


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10.5 Preservation of the Integrity of the Study 
The scientific integrity of the trial dictates that results be reported on a study-wide basis; thus, an 
individual Center will not report the data collected from its site alone.  All presentations and 
publications using TrialNet trial data must protect the main objectives of the study.  Data that 
could be perceived as threatening the study outcome will not be presented prior to release of the 
primary study outcomes.  Approval as to the timing of presentations of data and the meetings at 
which they might be presented will be granted by the TrialNet Steering Committee.  Study results 
should be discussed with the news media only upon authorization of the Steering Committee, 
and never before the results are presented.  Any written statements about this study that are 
shared with national media must be approved by TrialNet before release. 
 
10.6 Participant Reimbursement and Compensation 
Participants may be compensated for each visit attended in the study. In compliance with ICH 
Guidance E6, the amount and method of payments to subjects shall be designed to avoid 
coercion or undue influence on the study subjects. Payments to subjects will be prorated and not 
wholly contingent on completion of the trial by the subject.


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APPENDIX 1 - Schedule of Assessments 
Week of Trial 
  
  
 
 
2 
4 
6 
8 
10 
 
 
 
 
 
 
Month of Trial 
  
  
  
 
 
 
 
 
 
3 
6 
9 
12 
18 
24 
Day of Trial 
 
0 
1 
2 
 
 
 
 
 
 
 
 
 
 
 
Visit number 
-1 
0A 
0B 
0C 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
ATG/Placebo: Infusion 1 and 2 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
GCSF/Placebo q 2 wk6 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
History 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical exam 8 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
TB Test – IGRA 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC with differential2 (local) 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
CD4/CD8 Ratio3 
 
X 
 
 
 
X 
 
  X 
 
X 
X 
X 
X 
 
 
Chemistries 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Pregnancy test (if female of reproductive potential) 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis  
X 
X 
 
 
X 
X 
X 
  X 
 
 
 
 
 
 
 
Flu Vaccination7 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV, Hep B, Hep C serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IgG 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
EBV and CMV serology and PCR4  
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Serum for autoantibodies 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Mechanistic Assessments5 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Adverse Events Assessments6 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Hemoglobin A1c 
X 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
Mixed Meal Tolerance Test (2 hr) 
 
 
 
 
 
 
 
 
 
X 
X 
X 
 
 
 
Mixed Meal Tolerance Test (4 hr) 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
Diabetes  Assessment 
 
X 
 
 
X 
X 
 
 
 
X 
X 
X 
X 
X 
X 
2 CBC obtained 8 hours after completion of infusion 1. CBC can be repeated up to 24 hours after completion of infusion 1 if initial CBC is abnormal. The repeat CBC will inform dosing for infusion 2. 
3 CD4/CD8 ratio will continue to be monitored until the CD4 count is above 500. CD4/CD8 ratio can be collected at screening visit or baseline Visit 0A  and may also be done at local laboratory if 
necessary to obtain results. 
4 Viral PCR/Serology: All subjects will have serology and PCR for EBV and CMV at screening and at 1 year. Subjects who are EBV serology negative at screening must be PCR negative within 
30 days of receiving ATG/Placebo. Additional EBV PCR testing may be obtained locally if necessary to obtain results within study windows. Subsequent EBV PCR/Serology will only be 
performed for symptomatic subjects who were EBV seronegative at screening. Subsequent CMV PCR will be performed for symptomatic subjects regardless of initial CMV serology status.  
5 May include samples for RNA, plasma, serum, DNA, measures of B and T cell number and function to understand the effect of therapy on the immune system and infectious disease. The 
schedule for these assessments may vary as appropriate. At no time will the blood draw volume exceed what is allowable according to the subject’s age and body weight (for subjects <18 years, 
5 mL/kg per visit, 9.5 mL/kg in an 8 week period). 
6 Weekly contact with participant will occur. 
*Visit 0 A-C will occur over a 3 day period. During the Baseline Visit, the participant is admitted to the hospital to undergo ATG/placebo infusions. 
7Participants are required to receive killed influenza vaccination at least 2 weeks prior to randomization when vaccine for the current or upcoming flu season is available. 


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8 Full physical exam including Tanner staging at screening.   Full exam also at randomization, week 12, month 12 and month 24.    Tanner staging required at month 12 and 24 if Tanner stage 1 
or 2 on previous exam.   Directed exam as indicated by symptoms in addition to  HEENT and spleen exam at weeks 2-10, month 6,9, and 18.  


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