Detection of Graft Versus Host Disease With [18F]F-AraG, a Positron Emission Tomography Tracer for Activated T Cells
Phase: PHASE1
Conditions: Graft Versus Host Disease
Sponsor: CellSight Technologies, Inc.

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Detection of Graft Versus Host Disease with [18F]F-AraG, a positron 
emission tomography tracer for activated T cells 
Principal Investigator: 
Sanjiv Sam Gambhir, MD, PhD 
Chairman, Department of Radiology 
Professor of Radiology and Bioengineering 
Director, Molecular Imaging Program at Stanford (MIPS) 
Director, Canary Center at Stanford for Cancer Early Detection 
The James H. Clark Center, 318 Campus Drive 
Stanford, CA 94305 
s.gambhir@stanford.edu
Co-Investigators: 
Robert S. Negrin, MD 
Professor of Medicine (Blood and Marrow 
Transplantation) 
CCSR Room 2205, 
269 West Campus Drive 
Stanford, CA 94305 
negrs@stanford.edu 
Sally Arai, MD 
Associate Professor of Medicine (Blood and 
Marrow Transplantation) 
875 Blake Wilbur Drive, Clinic E, 1st floor 
Palo Alto, CA, 94304 
sarai1@stanford.edu 
Andrei Iagaru, MD 
Associate Professor of Radiology (Nuclear 
Medicine) 
300 Pasteur Drive, Room H-2200 
Stanford, CA 94305 
(650) 725-4711
aiagaru@stanford.edu 
Israt S. Alam, PhD 
Rad/Molecular Imaging Scientist 
The James H. Clark Center,  
318 Campus Drive 
Stanford, CA 94305 
israt@stanford.edu 
Anika Mahavni or Designate 
Clinical Research Coordinator 
300 Pasteur Dr., S095 
Stanford, CA 94305 
mahavni@stanford.edu 
Biostatistician: 
Jarrett Rosenberg, PhD 
Department of Radiology 
Lucas MRS Imaging Center 
1201 Welch Road, Room P-280 
Stanford, CA 94305 
Jarrett.rosenberg@stanford.edu 
IRB number: 
IRB-38850 
OnCore number 
BMT323 
Version date: 
29 February 2020, Amendment 7 
IND number: 
IND 123591 
NCT03367962

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TABLE OF CONTENTS 
SCHEMA 1: Two categories for acute Graft versus Host Disease (aGVHD) group .................... 4 
SCHEMA 2: Healthy subjects ..................................................................................................... 4 
List of Abbreviations and definitions of terms ............................................................................. 5 
1. OBJECTIVES ......................................................................................................................... 6 
1.1. 
Primary objective ............................................................................................................ 6 
1.2. 
Secondary Objectives ..................................................................................................... 6 
2. 
BACKGROUND .............................................................................................................. 6 
2.1. 
Acute Graft versus Host Disease .................................................................................... 6 
2.2 
Study Agent/Device/Imaging procedure .......................................................................... 8 
2.3 
Clinicaltrials.gov ............................................................................................................22 
2.4 
Rationale .......................................................................................................................22 
2.5 
Preliminary results .........................................................................................................22 
2.6 
Study Design .................................................................................................................22 
3 
PARTICIPANT SELECTION AND ENROLLMENT PROCEDURES ..............................23 
3.1 
Inclusion Criteria ............................................................................................................23 
3.2 
Exclusion Criteria ..........................................................................................................24 
3.3 
Informed Consent Process ............................................................................................24 
3.4 
Randomization Procedures ...........................................................................................24 
3.5 
Study Timeline ...............................................................................................................24 
3.5.1 Primary Completion: ......................................................................................................24 
3.6 
Study Completion: .........................................................................................................24 
4 
IMAGING AGENT/DEVICE/PROCEDURE INFORMATION ..........................................24 
4.1 
[18F]F-AraG: 2’-deoxy-2’-[18F]fluoro-9-β-D-arabinofuranosyl guanine ...........................24 
5 
STUDY PROCEDURES ................................................................................................25 
5.1 
Imaging Protocol for Patient Groups ..............................................................................25 
5.2 
Criteria for Removal from Study.....................................................................................28 
6 
STUDY CALENDAR ......................................................................................................29 
6.1 
Healthy Volunteers ........................................................................................................29 
6.2 
Patients highly suspected to have acute Graft versus Host Disease ..............................30 
6.3 
Patients at high risk of developing acute Graft versus Host Disease .... Error! Bookmark 
not defined. 
7 
ADVERSE EVENTS AND REPORTING PROCEDURES ..............................................33 
7.1 
Potential Adverse Events...............................................................................................33 
7.2 
Adverse Event Collection...............................................................................................34 
7.3 
Adverse Event Reporting ...............................................................................................35 
8 
REGULATORY CONSIDERATIONS .............................................................................35 
8.1 
Institutional Review of Protocol ......................................................................................35 
8.2 
Data Management Plan .................................................................................................35 
8.3 
Data and Safety Monitoring Plan ...................................................................................36 

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9 
MEASUREMENTS ........................................................................................................36 
9.1 
Primary and Secondary Outcome measures .................................................................36 
9.1.1 Primary Outcome Measurement Methods .....................................................................36 
9.1.2 Primary Outcome Measurement Time Points ................................................................37 
9.1.3 Response Review .........................................................................................................37 
9.2 
Secondary Outcome ......................................................................................................37 
10 
STATISTICAL CONSIDERATIONS ...............................................................................37 
10.1 
Statistical Design ...........................................................................................................37 
10.2 
Randomization ..............................................................................................................38 
10.3 
Interim analyses ............................................................................................................38 
10.4 
Descriptive Statistics and Exploratory Data Analysis .....................................................38 
10.5 
Analysis .........................................................................................................................38 
10.5.1 Analysis Population .......................................................................................................38 
10.5.2 Analysis Plan .................................................................................................................38 
10.6 
Secondary Analysis .......................................................................................................38 
10.6.1 Analysis Population .......................................................................................................38 
10.6.2 Analysis Plan .................................................................................................................38 
10.7 
Sample Size ..................................................................................................................38 
10.8 
Accrual estimates ..........................................................................................................38 
10.9 
Criteria for future studies ...............................................................................................39 
11 
REFERENCES ..............................................................................................................40 
Appendix A: Eligibility Checklist ................................................................................................43 
 
 
 

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SCHEMA 1: Two categories for acute Graft versus Host Disease (aGVHD) group 
 
Arm 1) Patients highly suspected to have acute Graft versus Host Disease (blue arrow applies) 
Arm 2) Patients at high risk of developing acute Graft versus Host Disease (green arrows apply)  
   
 
 
 
 
SCHEMA 2: Healthy subjects 
 
 
  
 

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List of Abbreviations and definitions of terms 
aGVHD 
Acute Graft versus host disease 
BMT 
Bone marrow transplant 
CD4 
Cluster of differentiation 4 
CD8 
Cluster of differentiation 8 
CDA 
Cytidine deaminase 
[18F]-CFA 
[18F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-
arabinofuranosyl-adenine 
CMC 
Chemistry, Manufacturing and Controls 
dCK 
Deoxycytidine kinase 
dGK 
Deoxyguanosine kinase 
[2]-F-D-AraC 
1-(2′-deoxy-2′-[18F]fluoro-β-D-arabinofuranosyl)cytosine 
[18F]F-AraG 
2’-deoxy-2’-[18F]fluoro-9- β -D-arabinofuranosyl guanine 
F-18, 18F 
Fluorine 18 
GI 
Gastrointestinal 
HCT 
Hematopoietic cell transplant 
HLA 
Human Leukocyte Antigen 
F-18, 18F 
Fluorine 18 
FDA 
Food and Drug Administration 
FDG 
2-deoxy-2-(18F)fluoro-D-glucose 
IND 
Investigational new drug 
IRB 
Institutional Review Board 
IV 
Intravenous 
MRI 
Magnetic resonance imaging 
PET/CT 
Positron emission tomography – computed tomography 
SUV 
Standardized Uptake Value 
ROI 
Region of interest 
IHC 
Immunohistochemistry 
SUV 
Standard uptake value 

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1. OBJECTIVES 
We have developed a Fluorine-18 PET tracer 2’-deoxy-2’-[18F]fluoro-9-β-D-arabinofuranosyl 
guanine ([18F]F-AraG), based on a drug called AraG that is known to be selective towards T 
cells. Our initial findings with this tracer have shown similar mechanism(s) of uptake as AraG 
and preferential accumulation in activated T cells. The selected positron emission tomography 
(PET) tracer ([18F]F-AraG has shown promise in preclinical studies in a mouse model of acute Graft 
versus Host Disease (aGVHD). We have shown the ability to use [18F]F-AraG PET imaging to 
quantitatively visualize locations of T cell accumulation during and early late-stage GVHD in this 
model. Moreover the tracer has already undergone the first in human studies in healthy human 
volunteers where it showed a favorable biodistribution, clearance profile and in vivo stability. With 
promising preclinical and clinical data, the tracer warrants further characterization in aGVHD patients 
where there is a pressing and unmet need for early detection of the disease. We hypothesize that in 
vivo molecular imaging can be safely utilized to identify sites of GVHD and therefore propose the 
following specific aims: 
 
1.1. Primary objective 
Evaluate the  feasibility of [18F]F-AraG PET/CT scanning in patients with graft versus host 
disease 
We hope to determine whether [18F]F-AraG, demonstrates good uptake in regions of GVHD as 
confirmed by GI biopsy. The performance of [18F]F-AraG PET/CT will be assessed on a per-lesion 
based analysis in a cohort of up to 25 GVHD patients.  The tracer’s biodistribution, and dosimetry will 
be studied in all aGVHD patients. All critical data shall be reported to the FDA. 
The ultimate goal of this study is to investigate the predictive power of [18F]F-AraG and whether it 
can be used to diagnose GVHD at its earliest stages to allow for more timely treatments to be 
applied in the future, to monitor treatment efficacy, and to hopefully improve the outcome of 
patient's with this devastating and frequent complication of allogeneic hematopoietic cell 
transplantation. 
1.2. Secondary Objectives 
 
 
The tracer’s pharmacokinetics, biodistribution, dosimetry and toxicity will also be studied in GVHD 
patients having been previously studied in healthy volunteers. Further, extended characterization of 
tracer biodistribution will be carried out in a small number of healthy subjects. 
 
2. BACKGROUND 
2.1. Acute Graft versus Host Disease 
Allogeneic hematopoietic cell transplantation (HCT) is an increasingly used curative option for 
many hematological malignancies, and other life-threatening genetic and hematological 
diseases [3, 4]. HCT is capable of destroying residual tumor cells that persist after radiation 
treatment or chemotherapy, an effect called the graft-versus-tumor reaction. However, the main 
complications of HCT are infections and acute graft-versus-host disease (aGVHD). Whilst 
infections can be treated successfully, patients with aGVHD by the time of diagnosis are less 
responsive to treatments. Thus a principle challenge for more successful implementation of 
HCT has been avoidance or better management of aGVHD. Whilst considerable medical 
advancements have been made in the last two decades, the diagnosis and treatment of this 
devastating disease has only marginally improved during that time. 

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This immunological disorder arises when donor T cells react to “foreign” host human leukocyte 
antigens (HLA), causing a wide spectrum of host tissue injuries in the gastrointestinal (GI) tract, 
liver, skin, and lungs [3, 5]. Two phases of the disease have been described; first the migration 
of CD4+ and CD8+ donor T cells to secondary lymphoid organs where they are activated by 
innate immune cells that become reactive by the conditioning therapy for HCT[6]. The second 
phase is characterized by massive proliferation of these alloreactive T cells which then home to 
various organs (GI tract, liver and skin are tissues that are commonly affected) and start to 
attack host cells via direct cytolysis or by cytokine production causing a wide range of symptoms 
[3]. 
Incidence rates of GVHD are high and depend on the degree of HLA histocompatibility, the 
prophylactic regimen used, patient age, and the number of T cells in the graft [5]. GVHD can 
affect 20-50% of patients receiving related donor transplants [7-10] and upwards of 70% of 
patients receiving HLA-matched unrelated donor transplants [7, 10]. The overall grade of GVHD 
is known to have a major effect on patient outcome, with grade I or II cases associated with little 
morbidity and no mortality, and grade IV cases associated with close to 100% mortality [5]. 
Hence, GVHD is a leading cause of morbidity and mortality of HCT therapy and improved ways 
of managing this complication could lead to more widespread use of this effective therapeutic 
paradigm for hematological malignancies, autoimmune diseases, or organ transplant rejection 
[11, 12]. Since long term survival of grade III-IV aGVHD is a dismal 10-20% [13], it is important 
to detect patients who will progress to high-grade aGVHD earlier in their course. 
Unfortunately, early definitive GVHD diagnosis is often difficult due to its non-specific symptoms 
and numerous differential diagnoses (e.g., drug toxicity, infections, and side effects from 
conditioning regimen) [14, 15]. GVHD is typically diagnosed at late stages when overt 
symptoms such as skin, GI, or liver problems become apparent, and is often one of exclusion, 
delaying the onset and effectiveness of potentially life-saving prophylactic interventions [16, 17]. 
Moreover, invasive endoscopic guided biopsy (often from liver or GI tract) and subsequent 
histology are still the state-of-the-art way to confirm a GVHD diagnosis [7]. These can be 
challenging to perform due to bleeding and critical illness associated with HCT. Dramatic 
differences in estimated response rate at a particular disease stage have been reported 
including 63% to 95% for grade II, 17% to 30% for grade III, and 0% to 6% for grade IV patients 
[18]. The severity of GVHD is also difficult to quantify, with staging relying on the assessment of 
the clinical changes seen in the skin, GI tract, liver, and performance status [5, 16, 17]. 
Therefore, non-invasive methods that can effectively track alloreactive donor T cell 
dynamics, stratify the risk of aGVHD prior to overt symptoms and thus diagnose GVHD at 
earlier stages are urgently needed but entirely lacking. 
 
Present day clinical management  
Several imaging techniques have shown promise for definitive diagnosis of GVHD, particularly 
GI manifestations of the disease, including magnetic resonance imaging (MRI), computed 
tomography (CT), high-resolution transabdominal ultrasound and color Doppler ultrasound [19, 
20]. These techniques primarily focus on structural manifestations such as detection of edema 
and thickening of the intestinal wall that become apparent during advanced stages of GVHD, 
and are therefore likely limited for early disease detection. Molecular imaging of aGVHD, which 
could yield a more sensitive detection, has been very limited focusing mostly on PET imaging 
using [18F]FDG [21]. 
[18F]FDG PET has shown promise for imaging intestinal inflammation associated with GVHD in 
both mouse models and patients with early clinical symptoms, even allowing prediction and 

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monitoring of therapy response in these patients [21].  However, the utility of [18F]FDG PET 
imaging for early detection prior to clinical symptoms is not known and the non-specific nature of 
[18F]FDG imaging for a variety of activated immune cell types should enable a selective 
advantage of [18F]F-AraG PET imaging for this purpose. 
Numerous candidate blood biomarkers are also under active investigation including soluble 
CD30 [22], regenerating islet-derived 3-alpha (REG3a) [23], microRNAs [24], amongst others 
[25, 26]. de Bock et al have also recently identified variations in a panel of proteomic blood 
biomarkers approximately 15 days before definitive GVHD diagnosis [27]. These candidates 
however require further validation prior to widespread clinical use and thus to date not a single 
biomarker has been approved  for early aGVHD diagnosis [27-29]. Cellular blood biomarkers of 
GVHD have also been explored. Bauerlein et al. have shown that, during their migration to 
effector organs, peripheral blood detection of alloreactive T cells expressing homing receptors 
(a4b7 integrin and P-selectin ligand) can be used to define a diagnostic window for the 
improved treatment of GVHD [28]. Notably, if immunosuppressive (i.e., Rapamycin) treatment is 
administered at the time of earliest alloreactive T cell detection then progression to lethal GVHD 
was prevented [28]. We believe that whole body [18F]F-AraG PET imaging that can quantify 
activated T cell accumulation during the initiation phase may provide a highly complimentary 
technique to these in vitro diagnostics to provide more definitive GVHD diagnosis and with 
spatial information. Hypothetically, it may even enable earlier diagnosis than a blood-based 
strategy by detection of T cells in priming sites prior to migration of cells or shedding of cellular 
proteins into the peripheral blood. Overall the continued evaluation of [18F]F-AraG PET imaging 
for GVHD diagnosis and treatment monitoring could enable this technology to fulfill a critical 
need in the management of this terrible disease to allow more widespread success of HCT 
therapy. 
 
2.2 Study Agent/Device/Imaging procedure 
Positron emission tomography (PET) imaging has several key strengths including high 
sensitivity (10-9 to 10-12 M), quantitative capability, and limitless depth of penetration [30]. To 
date, few PET radiotracers have been developed for imaging the immune system and even 
fewer for specific imaging of distinct immune cell types such as activated T cells [2, 31, 32].  
Of the PET tracers that have been developed for the purpose of imaging T cells, to date none 
have been specific for activated T cells [1, 2, 31, 33]. Of these only one is a small molecule 
enzyme substrate, the preferred type of molecular imaging probe due to inherent signal 
amplification from the catalytic trapping of many probes by a single enzyme. This probe is the 
radiotracer 
1-(2’-deoxy-2’-[18F]fluoroarabinofuranosyl 
cytosine 
([18F]F-AraC, 
also 
called 
[18F]FAC). It was identified based on its higher uptake in proliferating versus resting CD8+ T 
cells and was proven useful for visualizing regions of immune activation in vivo through its 
specificity for deoxycytidine kinase (dCK) [31]. However, [18F]F-AraC was found to rapidly 
undergo deamination in the blood by cytidine deaminase (CDA), confounding its utility for 
imaging dCK in species with high CDA levels such as humans [34]. Like drugs with a similar 
structure such as gemcitibine and cytarabine it solely targets dCK activity and is shown to be 
taken up into many immune cell types in vivo, with higher per cell uptake in myeloid (CD11b+) 
and B (B220+) cells compared to T cells [31] and also showed high uptake in dCK+ tumors. In 
fact the final applications of significantly improved [18F]FAC derivatives and similar molecules 
such 
as 
[(18F]Clofarabine; 
2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine 
([18F]CFA), in the clinic has been the imaging of dCK+ tumors. 
At Stanford, we have developed the radiotracer 2’-deoxy-2’-[18F]fluoro-9-β-D-arabinofuranosyl 
guanine ([18F]F-AraG) [35] (Fig. 1b). [18F]F-AraG is an analog of arabinosyl guanine (AraG), a 

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compound identified to have specific cytotoxicity towards T-lymphocytes and T-lymphoblastoid 
cells versus other immune cell types (Fig 1a.) [36-38]. Nelarabine, the water-soluble AraG 
prodrug, is indicated for the treatment of patients with refractory/relapsed T-cell acute 
lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) [39]. AraG enters 
cells via one of two nucleoside transporters followed by the initial and rate-limiting 
phosphorylation of AraG to AraG-monophosphate (AraGMP) by either cytosolic deoxycytidine 
kinase (dCK) or mitochondrial deoxyguanosine kinase (dGK) (Fig. 1c) [39, 40]. Continued 
phosphorylation to AraG-triphosphate (AraGTP) can inhibit DNA synthesis and induce cytotoxic 
effects specifically in T lymphocytes and T-lymphoblastoid cells [37, 39]. Thus [18F]F-AraG PET 
imaging may provide a novel method for imaging of activated T cell dynamics in living subjects 
without toxicity since the tracer is given in very low (ng-µg) mass levels where no therapeutic 
effects are observed.  
 
Fig 1. a) Chemical structure of arabinofuranosyl guanine (AraG) and b) 2’-deoxy-2’-
[18F]fluoro-9-β-D-arabinofuranosyl guanine ([18F]F-AraG). c) Mechanism for cell entry and 
trapping. AraG and putatively the radiotracer [18F]F-AraG, are transported into cells via two 
nucleoside transporters, followed by the rate-limiting conversion to AraGMP or [18F]F-AraGMP 
via phosphorylation by either cytosolic deoxycytidine kinase (dCK) or mitochondrial 
deoxyguanosine kinase (dGK). Further phosphorylation leads to AraGTP or [18F]F-AraGTP 
formation. Bottom left: AraG given at drug doses, once converted to AraGTP, can outcompete 
deoxyGTP to be incorporated into DNA during cellular mitosis, leading to chain termination and 
triggering of the apoptotic cascade in T cells. Bottom right: [18F]F-AraG given at trace doses 
should not cause any pharmacological effects but its accumulation in activated T cells can be 
utilized to visualize these cells in PET imaging studies. 
 
This imaging strategy could allow clinicians to utilize existing or novel interventions in a timely 
fashion and to more effectively monitor the outcome of such treatments. The ultimate utility of 
such efforts would be to prevent progression to higher-grade disease and improve patient 
outcome. 

C O N FI D E N TI A L  
 
I R B-3 8 8 5 0  
Pa ge 1 0  of 4 4  
versi o n 2 0 2 0 -0 2 2 9  
 
Tr a c er S y nt h e si s  
W e h a v e pr e vi o u sl y d e s cri b e d a n d p u bli s h e d t h e s y nt h e si s of [
1 8 F] F -Ar a G vi a a dir e ct 
fl u ori n ati o n m et h o d [ 3 5] al s o  o utli n e d i n S c h e m e 1 a n d 2. S c h e m e 1 s h o w s t h e s y nt h e si s of 
[1 8 F] F -Ar a G pr e c ur s or ; 2- N- A c et yl- 6- O- (( 4-nitr o p h e n yl) et h yl) - 9-( 3, 5-di - O-trit yl- 2-trif yl- β- D-
ri bi of ur a n o s yl) g u a ni n e ( 2). Tr e at m e nt of 2′, 5′- di - O-trit yl g u a n o si n e d eri v ati v e 1 wit h 
C F 3 S O 2 Cl/ D M A P  yi el d s t h e [ 1 8 F] F -Ar a G pr e c ur s or ( 2) wit h a 6 5 % yi el d.   
 
 
N
N
N
A c H N
N
O
O Tr
O
N O 2
1
O H
Tr O
Tr: trit yl,       Tf: trif yl,       A c: a c et yl
N
N
N
A c H N
N
O
O Tr
O
N O 2
2
Tr O
O Tf
C F 3 S O 2 Cl
 
S c h e m e 1. S y nt h e si s of 2 - N-A c et yl - 6- O-(( 4-nitr o p h e n yl) et h yl) - 9-( 3, 5-di - O-trit yl- 2-trif yl-- D-
ri b of ur a n o s yl) g u a ni n e( 2), t h e [ 1 8 F] F -Ar a G pr e c ur s or.  
[1 8 F] -l a b el e d g u a n o si n e d eri v ati v e ( 6) ( S c h e m e 2) i s t h e n pr e p ar e d b y n u cl e o p hili c di s pl a c e m e nt 
of trifl at e i n 2 b y  [1 8 F]fl u ori d e i o n i n D M S O at 8 5° C f or 4 5 mi n. P urifi c ati o n of [ 1 8 F] 6 vi a H P L C i s 
r e q uir e d t o a v oi d c o nt a mi n ati o n of t h e fi n al pr o d u ct [1 8 F] F -Ar a G ( 7) wit h t h e d e -pr ot e ct e d 
st arti n g m at eri al ( 2) ( Ar a G). [1 8 F] 6 i s s m o ot hl y h y dr ol y z e d  fir st b y b a s e ( 0. 5 M N a O C H 3) a n d 
t h e n b y a ci d ( 1 N H Cl) t o yi el d [ 1 8 F] F -Ar a G 7.    
T h e c h e mi c al a n d r a di o c h e mi c al p urit y of t h e fi n al pr o d u ct i s c o nfir m e d b y a r e v er s e-p h a s e 
a n al y ti c al H P L C m et h o d, b y c o -i nj e cti o n of  a c ol d F -Ar a G st a n d ar d  ( Fi g 2.). Purit y  l e v el s 
a c hi e v e d ar e oft e n 9 5 % or m or e.  

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N
N
N
AcHN
N
O
OTr
O
NO2
2
OTf
HN
N
N
H2N
N
O
HO
O
TrO
7
Tr: trityl,       Tf: trifyl,       Ac: acetyl
OH
18F
 [18F]KF/K222
N
N
N
AcHN
N
O
OTr
O
NO2
6
TrO
1) 0.5 N NaOCH3
2) 1N HCl
18F
 
Scheme 2. Synthesis of 2’-deoxy-2’-[18F]fluoro-9-β-D-arabinofuranosylguanine 7 ([18F]F-
AraG). 
 
Fig 2. Confirmation of the purity of 
([18F]F-AraG synthesis by analytical 
HPLC profile of co-injection of [18F]F-
AraG with cold F-AraG standard ( using 
5% acetonitrile: 95% water; 1 mL/min, 254 
nm, Phenomenex Gemini C18 column, 
5μm, 4.6Å~ 250 mm). 
 
 
 
 
To date, [18F]F-AraG has been prepared for human imaging  at UCSF according to the 
approved IND Chemistry Manufacturing and Control (CMC) procedures using the Neptis® 
perform PET synthesizer, a modification our published procedure [35]. The human images are 
shown further below (Fig 9). At Stanford we plan to use an automated preparation for [18F]F-
AraG for human imaging using the GE TRCERLAB FXFN module. Clinical validation runs have 
been completed and approved by the FDA. Briefly, no carrier-added [18F]fluoride ion was 
prepared on the Stanford University GE PET trace cyclotron, transferred to the GE TRCERLAB 
FXFN module  and captured on a QMA cartridge.  The [18F]fluoride ion was eluted with a solution 
of K2CO3 (3.5 mg) and kryptofix 2.2.2 (15 mg) in 1 mL CH3CN/H2O (9:1). The fluoride complex 
was azeotropically dried under vacuum at 65 °C.  The residue is taken up in a solution of 
DMSO(1 mL) containing 5-7 mg of the precursor, 2-N-Acetyl-6-O-((4-nitrophenyl)ethyl)-9-(3,5-
di-O-trityl-2-trifyl- β-D-ribofuranosyl)guanine.  The solution was heated to 115°C for 45 minutes 
followed by the first de-protection for 10 minutes at 100 °C with 0.5 mL of 0.5 M NaOMe in 
methanol.  Excess acid (0.5 mL of 1 M HCl) was added and heated to 95°C for 10 minutes to 
afford the second de-protection. The reaction mixture was cooled to 30 oC and diluted with 0.5 
mL of 1N NaHCO3 in 9.5 mL of water.  The solution in the reactor was transferred through 4 C-
18 plus cartridges. The Cartridges were backwashed to the reactor with 2 mL of methanol. 

C O N FI D E N TI A L  
 
I R B-3 8 8 5 0  
Pa ge 1 2  of 4 4  
versi o n 2 0 2 0 -0 2 2 9  
M et h a n ol w a s r e m o v e d u n d er v a c u u m wit h t h e h eli u m fl o w at 9 0 
o C f or 1 3 mi n. Aft er c o oli n g t h e 
r e a ct or t o 3 0 o C, w at er ( 2 m L) w a s a d d e d a n d t h e s ol uti o n p a s s e d t hr o u g h 0. 4 5 filter i nt o a 
pr e  H P L C  c o nt ai n er.  T h e  r e s ulti n g  mi xt ur e  w a s  p urifi e d  vi a  H P L C  ( P h e n o m e n e x  L u n a  1 0 µ m 
C 1 8, 1 0 x 2 5 0 m m, 9 7 % 5 0 m M a m m o ni u m a c et at e/ 3 % C H 3 C N at 5 m L/ mi n, U V w a s m o nit or e d at 
2 5 4 n m a n d r a di o a cti vit y d et e ct e d wit h a b uilt -i n s y nt h e si z er d et e ct or). T h e pr o d u ct w a s c oll e ct e d 
b et w e e n  1 2  a n d  1 5 mi n ut e s  a n d  dil ut e d  wit h  3 0  m L  of  w at er  a n d  p a s s e d  t hr o u g h  4  C -1 8  pl u s 
c artri d g e s. 1 8 F - D-Ar a G pr o d u ct w a s r e m o v e d fr o m t h e c artri d g e s b y 1 m L et h a n ol f oll o w e d b y 
1 0 m L of s ali n e a n d  p a s s e d t hr o u g h a 0. 2 2 µ m st erili zi n g filt er i nt o a m ulti d o s e vi al.   A s a m pl e 
w a s  c oll e ct e d  f or  q u alit y  t e sti n g.    [1 8 F] F -Ar a G  m et  all  q u alit y  s p e cifi c ati o n s  r e q uir e d  f or  h u m a n 
i ntr a v e n o u s  i nj e cti o n.  T h e  r a di o c h e mi c al  yi el d  w a s  2. 5 -4 %  ( d e c a y  c orr e ct e d, n  =  1 2).   T h e 
c h e mi c al a n d r a di o c h e mi c al p uriti e s of [ 1 8 F] F -Ar a G w er e gr e at er t h a n 9 5 %.   T h e r a di o s y nt h e si s 
ti m e w a s 1 2 0 mi n ut e s a n d s p e cifi c a cti vit y w a s 3 -8 Ci/ µ m ol. Pri or t o i nj e cti o n i n h u m a n s u bj e ct s 
t h e pr e p ar e d [1 8 F] F -Ar a G p a s s e d all q u alit y s p e cifi c ati o n s.      
 
Tr a c e r v ali d ati o n i n vitr o 
 
W e h a v e c arri e d o ut e xt e n si v e i n vitr o a n d i n vi v o v ali d ati o n  of [1 8 F] F -Ar a G t o d at e.  T o e v al u at e 
t h e  p ot e nti al  utilit y  of  [1 8 F] F -Ar a G,  w e  fir st  a s s e s s e d  u pt a k e  a n d  i n  pri m ar y  a cti v at e d  v er s u s 
r e sti n g T c ell s ( b ot h m uri n e; Fi g. 3  a n d h u m a n; Fi g  5 c ).  
 
 
 
Fi g. 3: U pt a k e of [ 1 8 F] F -Ar a G i n 
A cti v at e d V er s u s R e sti n g 
M uri n e T C ell s .  
a)  
A cti v at e d  ( b ott o m)  
m uri n e 
pri m ar y  T  c ell s  di s pl a y e d di s ti n ct 
m or p h ol o gi c al  
c h ar a ct eri sti c s 
c o m p ar e d t o r e sti n g T c ell s (t o p).  
b)   [1 8 F] F -Ar a G  w a s  t a k e n  u p  at 
si g nifi c a ntl y  
hi g h er  
l e v el s  
i n 
a cti v at e d  v er s u s  r e sti n g  c ell s 
(*** p < 0. 0 0 1; n = 3 p er gr o u p).  
 
 
A cti v at e d m uri n e  T  c ell s  ( 2  d a y s  aft er  a cti v ati o n)  a p p e ar e d  m or p h ol o gi c all y  di sti n ct  ( el o n g at e d 
v er s u s  r o u n d)  c o m p ar e d  t o  r e sti n g  c ell s  ( Fi g.   3 a)  a n d  a c c u m ul at e d  si g nifi c a ntl y  hi g h er  ( ~ 7. 8-
f ol d) [1 8 F] F -Ar a G ( p < 0. 0 0 1; Fi g. 3 b). W e h a v e al s o c o m p ar e d t h e u pt a k e of t h e triti at e d v er si o n 
of t h e tr a c er [ 3 H] Ar a G, t o t h e triti at e d v er si o n of t h e tr a c er Ar a C  ([ 3 H] Ar a C ) i n a cti v at e d v er s u s 
r e sti n g pri m ar y h u m a n T c ell s ( Fi g. 4).   
 

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Fig. 4: Uptake and Retention of [3H]AraG and [3H]F-AraC in Resting and Activated Human 
Primary T Cells. Human primary T cells were isolated from PBMC buffy coats, activated in 
culture for 2 days, and incubated with either [3H]AraG or [3H]F-AraC to measure uptake (a) and 
retention (b); n=3 per treatment per tracer). Both tracers were taken up at significantly higher 
levels (~6-7 fold) in activated versus resting T cells (***p<0.001). In contrast, [3H]AraG was 
retained at significantly higher levels (~30% more) within both resting and activated T cells 
versus [3H]F-AraC (*p<0.05). Data in all graphs are expressed as mean ± SD. 
 
A 2-day activation protocol of sorted (~94% CD3+ T cells; data not shown) human peripheral 
blood mononuclear cells (PBMCs) resulted in similar morphological changes as seen in murine 
T cells. Significantly higher uptake in activated versus resting cells was seen with both [3H]AraG 
and [3H]F-AraC, but retention in both cell states was significantly higher with [3H]AraG (p<0.05; 
Fig. 4). Finally, we found significantly higher uptake (~19-fold) of [18F]F-AraG in activated versus 
resting human T cells (p<0.001; Fig. 5c). Although [18F]F-AraG effluxes significantly out of 
activated but not resting cells (p<0.05), absolute levels in activated versus resting cells at all 
time points evaluated were significantly higher (p<0.01). Overall these results support the use of 
[18F]F-AraG  in selective PET imaging of activated versus resting T cells. 
 
We have also evaluated whether [18F]F-AraG accumulates in cells via dCK and/or dGK activity 
using established cell lines [40]. Loss of dCK in mutant CCRF-CEM T lymphoblast cells (dCK-), 
as confirmed with Western blot analysis, (significantly impaired [18F]F-AraG uptake (p<0.001; 
Fig. 5a). Overexpression of dCK in mutant cells (dCK+) showed a trend toward increased tracer 
uptake compared to dCK- cells (p=0.17), whereas dGK overexpression (dGK+) in dCK- cells 
restored tracer uptake to levels seen in wild-type cells. No effects on tracer retention were seen 
across any of these cell lines (Fig. 5b).  
 
Our evidence supports that in contrast to [18F]F-AraC that accumulates via dCK, but in line with 
AraG metabolism [33, 40], [18F]F-AraG accumulates in cells via both dCK and dGK activity. 
These findings support recently published observations in a study on a novel PET tracer 
metabolized by dCK, which also noted a role of dGK in the metabolism of  [18F]F-AraG[33]; 
however, we extend upon these previous findings to highlight the additional role of dCK activity 
in the metabolism of [18F]F-AraG. 
 

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Figure 5: [18F]F-AraG Accumulates in Cells via dCK and dGK Activity and at Increased 
Levels in Activated Versus Resting Primary Human T Cells. 
a) Uptake and b) retention of [18F]F-AraG across wild-type CCRF-CEM T lymphoblasts, mutant 
CCRF-CEM dCK- cells (dCK-), and dCK- cells overexpressing either dCK (dCK+) or dGK 
(dGK+) (n=4 per cell type per time point). Significantly less uptake was seen due to the loss of 
dCK in wild-type cells (CCRF-CEM vs. dCK-), (p<0.001). There was a trend towards higher 
uptake in dCK+ versus dCK- cells, whereas dGK+ cells had significantly higher uptake compared 
to dCK- cells and equivalent uptake compared to wild-type cells. No differences in retention 
were seen across cell types. c) Activated primary human T cells had significantly higher [18F]F-
AraG uptake compared to resting T cells at all time points examined (***p<0.001; n=4 per cell 
state per time point). Data in all graphs are expressed as mean ± SD.  
 
Having confirmed the specificity [18F]F-AraG has for activated T cells in vitro, we assessed its 
suitability for in vivo studies. Serum stability studies showed that the tracer was largely stable at 
37 °C in phosphate buffer saline (PBS), murine serum and in human serum with more than 85% 
of the tracer intact after a two hour incubation in the latter. This indicates that the tracer will stay 
largely intact during in vivo preclinical and human imaging. 
 
 
 

C O N FI D E N TI A L  
 
I R B-3 8 8 5 0  
Pa ge 1 5  of 4 4  
versi o n 2 0 2 0 -0 2 2 9  
 
Fi g. 6: St a bilit y of [1 8 F] F -Ar a G i n 
P B S, m o u s e a n d h u m a n s er u m 
at 3 7  °C a s a s s e s s e d b y H P L C.  
 
 
 
 
 
 
 
 
 
 
 
I n vi v o st u di e s i n a G V H D m o u s e m o d el  
 
T h e  a bilit y  t o  vi s u ali z e  sit e s  of  a cti v at e d  T  c ell  a c c u m ul ati o n  i n  a G V H D  wit h  [ 1 8 F] F -Ar a G  P E T 
i m a gi n g w a s a s s e s s e d i n a w ell-e st a bli s h e d M H C mi s m at c h a G V H D m o u s e m o d el [ 2 8, 4 1-4 3] . 
I n  t hi s  m o d el  d o n or  T  c ell s  c o n stit uti v el y  e x pr e s s  t h e  bi ol u mi n e s c e n c e  r e p ort er  g e n e  fir efl y 
l u cif er a s e ( L u c) e n a bli n g i n vi v o bi ol u mi n e s c e n c e i m a gi n g ( B LI) t o m o nit or sit e s of d o n or T c ell 
a c c u m ul ati o n a n d pr olif er ati o n  o v er ti m e ( Fi g s. 2 a a n d 3 a). B LI e n a bl e s t h e vi s u al iz ati o n of t h e 
i niti ati o n t h e eff e ct or p h a s e of t h e di s e a s e .  T hi s i niti ati o n p h a s e o c c ur s pri or t o o v ert s y m pt o m s 
a n d c o n si st s of T c ell mi gr ati o n t o s e c o n d ar y l y m p h oi d or g a n s w h er e t h e y b e c o m e a cti v at e d b y 
h o st a nti g e n pr e s e nti n g c ell s. T hi s i s f oll o w e d b y t h e eff e ct or p h a s e t h at i n v ol v e s di s s e mi n ati o n 
of h o mi n g r e c e pt or -e x pr e s si n g T c ell s o ut of t h e s e i niti al pri mi n g sit e s t o eff e ct or sit e s s u c h a s 
t h e GI tr a ct, li v er a n d s ki n. 
 
St ati c P E T i m a gi n g  pr o c e d ur e : 
 
W e  p erf or m e d  cli ni c al  s c ori n g, 
i n  vi v o a n d e x  vi v o  B LI,  [ 1 8 F] F -Ar a G  P E T/ C T  i m a gi n g,  tr a c er 
bi o di stri b uti o n  st u di e s,  a n d e x  vi v o  i m m u n ofl u or e s c e n c e  st u di e s  at  b ot h  at  a n  e arl y i niti ati o n 
p h a s e  ( d a y  3 ) a n d l at e eff e ct or  p h a s e  ( d a y  1 0) aft er m o d el  i niti ati o n  i n b ot h  c o ntr ol  (irr a di at e d 
a n d  b o n e  m arr o w  tr a n s pl a nt e d  mi c e  wit h o ut  d o n or  T  c ell s)  a n d  a G V H D  (irr a di at e d  a n d  b o n e 
m arr o w  tr a n s pl a nt e d mi c e wit h  L u c +  d o n or  T  c ell s)  mi c e. C o n si st e nt  wit h pr e vi o u s  r e s ult s  a n d 
b a s e d  o n  a  st a n d ar d  s c ori n g  s y st e m [ 4 4],  b ot h  d a y  3  a n d  d a y  1 0  mi c e  i n  t hi s  m o d el  ar e 
c o n si d er e d wit hi n t h e T c ell a cti v ati o n p h a s e of t h e di s e a s e.  
 
F or P E T/ C T i m a gi n g, mi c e w er e a n e st h eti z e d a n d i nj e ct e d wit h ~ 2 0 0 Ci o f [1 8 F] F -Ar a G vi a t h e 
l at er al  t ail-v ei n.  Si xt y  mi n ut e s  aft er  i nj e cti o n,  st ati c  P E T  i m a g e s  ( 1 0  mi n ut e  a c q ui siti o n  ti m e) 
w er e  c oll e ct e d  u si n g  a  mi cr o P E T/ C T  h y bri d  I n v e o n  s c a n n er  ( Si e m e n s).  A  C T  i m a g e  w a s 
a c q uir e d j u st b ef or e e a c h P E T s c a n t o pr o vi d e a n a n at o mi c r ef er e n c e fr a m e f or t h e r e s p e cti v e 
P E T  d at a.  All  P E T  i m a g e s  w er e  r e c o n str u ct e d  wit h  a  3 -di m e n si o n al  or d er e d -s u b s et s 
e x p e ct ati o n  m a xi mi z ati o n  al g orit h m  a n d  c o -r e gi st er e d  wit h  C T  i m a g e s  u si n g  t h e  I n v e o n 
R e s e ar c h W or k pl a c e (I R W) i m a g e a n al y si s s oft w ar e ( v er si o n 4. 0; Si e m e n s).  T o q u a ntif y tr a c er 
u pt a k e  i n  P E T  i m a g e s,  3 -di m e n si o n al  r e gi o n s  of  i nt er e st  w er e  dr a w n  o v er  t h e  C L N,  a  5 0 % 
t hr e s h ol d w a s a p pli e d t o e a c h s el e ct e d r e gi o n, a n d p arti al v ol u m e c orr e ct e d u pt a k e v al u e s w er e 
e x pr e s s e d a s p er c e nt a g e i nj e ct e d d o s e p er gr a m of ti s s u e ( %I D/ g).  
 

C O N FI D E N TI A L  
 
I R B-3 8 8 5 0  
Pa ge 1 6  of 4 4  
versi o n 2 0 2 0 -0 2 2 9  
Ni n et y mi n ut e s aft er tr a c er a d mi ni str ati o n mi c e w er e e ut h a ni z e d, v ari o u s ti s s u e s ( bl o o d, h e art, 
s m all i nt e sti n e, ki d n e y, li v er, l u n g, m u s cl e, s pl e e n, M L N, a n d C L N) w er e c oll e ct e d, w ei g h e d a n d 
r a di o a cti vit y  w a s  m e a s ur e d  u si n g  a n  a ut o m at e d  c o u nt e r  ( C o br a  II;  P a c k ar d).    R a di o a cti vit y 
w a s  d e c a y -c orr e ct e d  t o  t h e  ti m e  of  r a di otr a c er  i nj e cti o n  u si n g  dil ut e d  ali q u ot s  of  t h e  i niti al 
a d mi ni st er e d  d o s e  a s  st a n d ar d s.   D at a  i s  e x pr e s s e d  a s  p er c e nt a g e  i nj e ct e d  d o s e  p er  gr a m  of 
ti s s u e ( %I D/ g) v al u e s 
 
D a y 3 a G V H D mi c e l a c k e d n oti c e a bl e s y m pt o m s a n d ar e at a ti m e p oi nt w h e n T c ell a cti v ati o n 
i s  b ei n g  i niti at e d,  w h er e a s  d a y  1 0  mi c e  di s pl a y  cli ni c al  s y m pt o m s  a n d  ar e  c o n si d er e d  at  t h e 
p e a k of T c ell a cti v ati o n ( d at a n ot s h o w n). Di s e a s e pr o gr e s si o n i s al s o r efl e ct e d vi a si g nifi c a ntl y 
hi g h er t ot al d o n or T c ell n u m b er s ( hi g h er t ot al b o d y B LI si g n al) at d a y 1 0 ( Fi g. 8 a) v er s u s d a y 3 
( Fi g.  7 a)  ( p < 0. 0 1;  q u a ntit ati v e  d at a  n ot  s h o w n). A s  pr e vi o u sl y  s h o w n  [ 2 8,  4 1,  4 2], i n vi v o ( Fi g. 
7 a)  a n d e x vi v o  B LI  r e v e al e d  t h at  d o n or  T  c ell s  i n  a G V H D  mi c e  at  d a y  3  h a d  a c c u m ul at e d 
pr ef er e nti all y  i n  l y m p h oi d  or g a n s  s u c h  a s  t h e  s pl e e n,  m e s e nt eri c  l y m p h  n o d e s  ( M L N)  a n d 
c er vi c al  l y m p h  n o d e s  ( C L N).  O n  a  s e p ar at e  c o h ort  of  mi c e  r e c ei vi n g  G F P +  d o n or  T  c ell s  w e 
q u alit ati v el y  c o nfir m e d  pr o gr e s si v el y  hi g h er  d o n or  T  c ell  a c c u m ul ati o n  wit hi n  t h e  s pl e e n  a n d 
C L N  fr o m  d a y  3  t o  d a y  1 0.  I m p ort a ntl y,  [ 1 8 F] F -Ar a G  P E T/ C T  i m a g e s  t a k e n  1  h o ur  aft er  tr a c er 
( ~ 2 0 0 Ci )  a d mi ni str ati o n  s h o w e d  q u alit ati v el y  hi g h er  u pt a k e  i n  t h e  C L N  i n  aG V H D  v er s u s 
c o ntr ol  mi c e  ( Fi g.  7 b).  Q u a ntit ati v e  i m a g e  a n al y si s  c orr o b or at e d  o ur  fi n di n g s  r e v e ali n g 
si g nifi c a ntl y hi g h er ( ~ 1. 4 -f ol d) [1 8 F] F -Ar a G u pt a k e i n t h e C L N s of a G V H D mi c e ( p < 0. 0 5, Fi g. 7 c ). 
At  1  h o ur,  tr a c er  u pt a k e  wit hi n  a b d o mi n al  or g a n s  s u c h  a s  t h e  li v er  a n d  ki d n e y s  o b s c ur e d  t h e 
a bilit y t o vi s u ali z e i n vi v o diff er e n c e s i n tr a c er a c c u m ul ati o n wit hi n t h e M L N a n d s pl e e n ( Fi g. 7 b). 
H o w e v er, tr a c er bi o di stri b uti o n st u di e s a s s e s s e d at 9 0 mi n ut e s aft er i ntr a v e n o u s tr a c er i nj e cti o n 
r e v e al e d si g nifi c a ntl y hi g h er [1 8 F] F -Ar a G a c c u m ul ati o n i n b ot h t h e C L N ( p < 0. 0 5; ~ 2. 3 -f ol d) a n d 
t h e  M L N  ( p < 0. 0 5;  ~ 2. 0-f ol d,  ( Fi g.  7 d)),  a n d  a  tr e n d  t o w ar d s  hi g h er  u pt a k e  i n  t h e s pl e e n 
( p = 0. 1 2).  N o  si g nifi c a nt  diff er e n c e s  w er e  n ot e d  a cr o s s  all  ot h er  or g a n s  b et w e e n  c o ntr ol  a n d 
a G V H D mi c e. T h u s, at a n e arl y st a g e of di s e a s e d e v el o p m e nt pri or t o cli ni c al s y m pt o m s i n t hi s 
m o d el,  [
1 8 F] F -Ar a G  a c c u m ul at e d  i n  l y m p h oi d  or g a n s  ( M L N,  C L N,  a n d  s pl e e n)  h ar b ori n g 
a cti v at e d T c ell s a n d [ 1 8 F] F -Ar a G P E T i m a gi n g e n a bl e d vi s u ali z ati o n of e arl y T c ell a c c u m ul ati o n 
wit hi n t h e C L N.  
 

C O N FI D E N TI A L  
 
I R B-3 8 8 5 0  
Pa ge 1 7  of 4 4  
versi o n 2 0 2 0 -0 2 2 9  
 
Fi g ur e  7:  Bi ol u mi n e s c e n c e  a n d  [ 1 8 F] F -Ar a G  P E T  I m a gi n g  of  D o n or  T  C ell  D y n a mi c s  
D uri n g t h e I niti ati o n of T C ell A cti v ati o n i n a M o u s e M o d el of a c ut e G V H D .  
a)  Bi ol u mi n e s c e n c e  i m a gi n g  of  c o ntr ol  a n d  G V H D  mi c e  3  d a y s  aft er  H C T  r e v e al e d  h o mi n g  of 
l u cif er a s e-p o siti v e  d o n or  T  c ell s  t o  s e c o n d ar y  l y m p h oi d  or g a n s  s u c h  a s  t h e  s pl e e n,  c er vi c al 
l y m p h  n o d e s  ( C L N),  a n d  m e s e nt eri c  l y m p h  n o d e s  ( M L N)  i n  G V H D  mi c e. b) R e pr e s e nt ati v e  
[1 8 F] F -Ar a G  P E T/ C T  i m a g e s  ( 1 0  mi n ut e  st ati c  s c a n;  6 0 -7 0  mi n ut e s  p o st -tr a c er  a d mi ni str ati o n; 
~ 2 0 0 Ci )  at  t hi s  ti m e  p oi nt  s h o w e d  vi si bl y  hi g h er  tr a c er  u pt a k e  i n  t h e  C L N  of  G V H D  mi c e 
v er s u s c o ntr ol mi c e ( n = 3 p er gr o u p). c) Q u a ntit ati v e r e gi o n of i nt er e st P E T i m a g e a n al y si s of t h e 
C L N  c orr o b or at e d  o ur  q u alit ati v e  a s s e s s m e nt s,  d e m o n str ati n g  si g nifi c a ntl y  (* p < 0. 0 5)  hi g h er 
tr a c er u pt a k e i n t h e C L N of G V H D mi c e v er s u s c o ntr ol mi c e ( %I D/ g; p er c e nt a g e i nj e ct e d d o s e 
p er  gr a m  of  ti s s u e). d)  Bi o di stri b uti o n  st u di e s  ( 9 0  mi n ut e s  aft er  tr a c er  a d mi ni str ati o n)  al s o 
s u p p ort e d o ur i m a gi n g fi n di n g s s h o wi n g si g nifi c a ntl y (* p < 0. 0 5) hi g h er tr a c er u pt a k e i n C L N s i n 
G V H D v er s u s c o ntr ol mi c e. Si g nifi c a ntl y (* p < 0. 0 5) hi g h er tr a c er u pt a k e w a s al s o a p p ar e nt i n t h e 
T- c ell s h ar b ori n g M L N a n d a tr e n d ( p = 0. 1 2) t o w ar d s hi g h er u pt a k e w a s s e e n i n t h e s pl e e n. D at a 
i n all gr a p h s ar e e x pr e s s e d a s m e a n ± S D. 
 
At d a y 1 0, at t h e p e a k of T c ell a cti v ati o n, [ 1 8 F] F -Ar a G P E T/ C T i m a g e s s h o w e d a n e v e n m or e 
a p p ar e nt i n cr e a s e i n tr a c er a c c u m ul ati o n i n t h e C L N of a G V H D c o m p ar e d t o c o ntr ol mi c e ( Fi g. 
8 b), w hi c h w a s q u a ntit ati v el y c o nfir m e d vi a P E T i m a g e a n al y si s ( Fi g. 8 c). Tr a c er bi o di stri b uti o n 
a n al y si s r e v e al e d si g nifi c a ntl y l o w er tr a c er u pt a k e i n t h e s pl e e n at t hi s ti m e p oi nt ( p < 0. 0 5, Fi g. 
8 d ),  b ut  hi g h er  u pt a k e  i n  b ot h  t h e  C L N  a n d  t h e  M L N  ( p < 0. 0 5  a n d  p < 0. 0 1,  r e s p e cti v el y). 
A m o n g st a G V H D mi c e, w e al s o s h o w e d si g nifi c a ntl y hi g h er B LI a n d P E T si g n al wit hi n t h e C L N s 
at d a y  1 0 v er s u s d a y 3 ( p < 0. 0 5; Fi g. 8 e), i n di c ati n g pr o mi s e f or m o nit ori n g di s e a s e b ur d e n vi a 
t hi s s urr o g at e i m a gi n g bi o m ar k er.  T h e s e fi n di n g s w er e r e c e ntl y p u bli s h e d i n C a n c er R e s e ar c h 
(7 7( 1 1); 2 8 9 3 – 9 0 2 , 2 0 1 7). 

C O N FI D E N TI A L  
 
I R B-3 8 8 5 0  
Pa ge 1 8  of 4 4  
versi o n 2 0 2 0 -0 2 2 9  
 
 
Fi g ur e  8:  Bi ol u mi n e s c e n c e  a n d  [ 1 8 F] F -Ar a G  P E T  I m a gi n g  of  D o n or  T  C ell  D y n a mi c s  
D uri n g t h e P e a k of T C ell A cti v ati o n i n a M o u s e M o d el of a c ut e G V H D  
a)  Bi ol u mi n e s c e n c e  i m a gi n g  of  c o ntr ol  a n d  G V H D  mi c e  1 0  d a y s  aft er  H C T  r e v e al e d  m or e 
wi d e s pr e a d  di stri b uti o n  of  l u cif er a s e -p o siti v e  d o n or  T  c ell s  i n  G V H D  mi c e  b ut  still  l o c ali z e d 
a c c u m ul ati o n wit hi n t h e c er vi c al l y m p h n o d e s ( C L N). N ot e t h e or d er of m a g nit u d e diff er e n c e i n 
s c al e b et w e e n t h e i m a g e s s h o w n h er e a n d t h o s e i n Fi g. 2 a ( D a y 3 aft er c ell tr a n s pl a nt ati o n). b) 
R e pr e s e nt ati v e  [ 1 8 F] F -Ar a G  P E T/ C T  i m a g e s  ( 1 0  mi n ut e  st ati c  s c a n s,  6 0 -7 0  p o st -tr a c er 
a d mi ni str ati o n;  ~ 2 0 0 Ci )  at  t hi s  ti m e  p oi nt  s h o w e d  vi si bl y  hi g h er  tr a c er  u pt a k e  i n  t h e  C L N  of 
G V H D ( n = 4) v er s u s c o ntr ol mi c e ( n = 3). c) Q u a ntit ati v e r e gi o n of i nt er e st P E T i m a g e a n al y si s of 
t h e  C L N  c orr o b or at e d  o ur  q u alit ati v e  a s s e s s m e nt s,  d e m o n str ati n g  si g nifi c a ntl y  hi g h er  tr a c er 
u pt a k e i n t h e C L N of G V H D v er s u s c o ntr ol mi c e (* p < 0. 0 5) ( %I D/ g; p er c e nt a g e i nj e ct e d d o s e p er 
gr a m of ti s s u e). d)  Bi o di stri b uti o n st u di e s ( 9 0 mi n ut e s aft er tr a c er a d mi ni str ati o n) al s o s u p p ort e d 
o ur i m a gi n g fi n di n g s s h o wi n g si g nifi c a ntl y hi g h er tr a c er u pt a k e i n C L N s i n G V H D v er s u s c o ntr ol 
mi c e.  Si g nifi c a ntl y  hi g h er  tr a c er  u pt a k e  w a s  al s o  a p p ar e nt  i n  t h e  M L N  (* p < 0. 0 5).  At  t hi s  ti m e 
p oi nt si g nifi c a ntl y l o w er tr a c er u pt a k e w a s n ot e d wit hi n t h e s pl e e n (* p < 0. 0 5). e) C o m p ari s o n of 
D a y 3 a n d D a y 1 0 bi ol u mi n e s c e n c e a n d P E T i m a g e a n al y si s of C L N s s h o w s b ot h si g nifi c a ntl y 
hi g h er B LI a n d P E T si g n al s o n D a y 1 0 c o m p ar e d t o D a y 3. D at a i n all gr a p h s ar e e x pr e s s e d a s 
m e a n ± S D.  
 
I n o ur pr e cli ni c al m o d el of a G V H D, [1 8 F] F -Ar a G  t h er ef or e e n a bl e d t h e vi s u ali z ati o n of a cti v at e d 
d o n or  T  c ell s,  pri or  t o  o v ert  cli ni c al  s y m pt o m s.  b ef or e  t h e  W hil e  i n  m o u s e  st u di e s  w e  w er e 
u n a bl e t o vi s u ali z e i n vi v o diff er e n c e s of tr a c er u pt a k e i n b ot h t h e M L N a n d s pl e e n, t w o or g a n s 

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we know harbor activated donor T cells. Tracer ex vivo biodistribution results supported higher 
uptake in the MLN of aGVHD versus control mice at both early- and late-stages of disease we 
were unable to identify the MLN in our PET (or CT) images. This was mostly due to high tracer 
uptake in adjacent organs such as the kidneys and liver at 1 hour after tracer administration. 
While this is problematic in mice due to their small size and closely positioned organs, this will 
be less of a concern in humans as supported by the low tracer uptake in the GI tract in human 
PET images of normal volunteers. 
 
Toxicology Study on F-AraG (the non-radioactive version of the tracer) 
A toxicity study of 9-(2-deoxy-2-fluoroarabinofuranosyl) guanine (F-AraG) in Sprague Dawley 
rats was conducted by Sobran, Inc, Bioscience Division, Sobran Rangos Animal Facility 
(Baltimore, MD). A complete report by Sobran entitled “14-Day Single Intravenous Dose Toxicity 
Study of 9-(2-deoxy-2-fluoroarabinofuranosyl) guanine (F-AraG)  in Sprague Dawley Rats” 
dated April 10, 2014, is summarized below. 
Twenty rats of each sex (10/sex/group, 5/sex/sacrifice time point) were assigned to a dose 
group and a vehicle control group and administered 9-(2-deoxy-2-fluoroarabinofuranosyl) 
guanine (F-AraG)  intravenously via the tail vein at 5ml/Kg as a single dose on Study Day 1 with 
either vehicle (3%ethanol and 50mM Ammonium acetate solution in sterile water) or F-AraG at 
44.6ug/kg. Groups of 5 animals/sex and from each sex were sacrificed on Study Days 3 (48 
hours) and 15 for evaluation of clinical pathology and organ toxicity.  
The animals were monitored prior to the administration of the test article, 9-(2-deoxy-2-
fluoroarabinofuranosyl) guanine, and up to 14 days following the administration of the test 
article. Parameters evaluated for test article effect included survival, clinical observations, body 
weight, body weight gain, clinical pathology, gross pathology, organ weights, and microscopic 
pathology. No morbidity or mortality was observed in the study. All rats survived to the 
scheduled termination date and remained bright, alert and responsive during the study. No 
abnormal findings were indicated during mortality checks (cagesides) or hands-on observations. 
There were no treatment-related findings from clinical chemistry, hematology, and coagulation 
samples collected on Study Days 3 and 15. 
Animals remained bright, alert, and responsive at all times and did not exhibit signs of toxicity 
during the conduct of the study. No treatment-related differences were noted in mean body 
weights and body weight changes, clinical chemistry, hematology, or coagulation parameters. In 
addition, no treatment-related effects were observed organ weights or in gross and microscopic 
pathology. Under the conditions of this study, there were no treatment related findings in 
Sprague Dawley rats three or fifteen days after a single intravenous dose of F-AraGat 
44.6μg⁄mg/kg. 
Clinical characterization and dosimetry studies in healthy volunteers  
The suitability of [18F]F-AraG for human studies has been validated in humans at the University 
of California San Francisco (UCSF). [18F]F-AraG PET/CT scans were performed on six healthy 
human volunteers and dosimetry was also calculated. [18F]F-AraG exhibited hepatobiliary and 
renal clearance with highest uptake in associated organs with SUV mean values (normalized to 
body weight) of 13.47 ±1.46, 20.72±5.26 and 17.27±4.19 in liver, right and left kidneys 
respectively at 47-77 minutes post tracer injection (Table 1). High uptake was also observed in 
the myocardium as seen in mice, while relatively low background was observed in the thorax 
and lower abdomen. No significant adverse events or blood lab changes were observed due to 

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tracer administration in any of the volunteers other than slightly increased urine esterase levels. 
Radiation dosimetry results are shown in Table 2.  These findings were recently published in 
Cancer Research (77(11); 2893–902, 2017). 
Fig. 9: Whole body PET 
images of [18F]F-AraG in 
a 
healthy 
human 
volunteer. 
Frontal 
(top 
panel) and dorsal (bottom 
panel) coronal PET images 
taken 
immediately 
after 
intra-venous injection of the 
tracer and at serial time 
points in one of the 6 
humans imaged. Highest 
activity is observed in the 
liver, kidneys and bladder, 
associated with [18F]F-AraG 
clearance from the body. 
Uptake is also observed in 
the heart and spleen while 
relatively low background 
activity is observed in all 
other 
tissues. 
Similar 
images were obtained from 
the other 5 subjects. 
 
 
Site 
SUV-BW mean 
SUV-BW max 
Brain Tissue 
0.02 ±0.01 
0.08±0.02 
Cervical Lymph Node 
5.2 ±1.15 
7.13±1.76 
Heart  
2.12 ±0.72 
4.72 ±1.37 
Myocardium 
3.38 ±1.19 
4.72±1.37 
Liver 
13.47 ±1.46 
16.18±2.00 
Kidney (right) 
20.72±5.26 
44.47±12.44 
Kidney (left) 
17.27±4.19 
41.35±10.65 
Bladder 
8.28 ±6.15 
10.28± 7.66 
Muscle 
0.98 ±0.44 
1.42±0.66 
Table 1: [18F]F-AraG standard uptake values for key tissues- normalized to body weight 
(SUV-BW) at 47-77 minutes post tracer injection in six healthy human volunteers. 
SUV-BW mean and max values shown. Data shown represent averages ± standard deviation; 
n = 6 for each site. Average of both right and left sides of subject shown for brain tissue and 
cervical lymph node. 

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These preliminary studies in healthy human volunteers have been promising with low 
background in the thorax and GI tract, making it favorable for thoracic and pelvic imaging. 
Favorable kinetics of [18F]F-AraG in humans along with the preclinical data support its potential 
as a promising tracer for early detection of GVHD and warrants its further clinical evaluation in 
aGVHD patients. Given the paucity of PET tracers for activated T cells in the clinic this work 
lays the important foundation for [18F]F-AraG PET imaging in aGVHD patients and the potential 
to provide crucial information to aid in the detection of activated T cell dynamics during the 
activation phase of aGVHD. 
Organs 
Male 
Female 
Adrenals  
0.046567 
0.067133 
Brain     
0.006380 
0.007407 
Breasts    
0.005757 
0.007547 
Gallbladder Wall    
0.051400 
0.063033 
LLI Wall  
0.085107 
0.012150 
Small Intestine   
0.047033 
0.073000 
Stomach Wall   
0.018667 
0.025367 
ULI Wall   
0.022600 
0.033067 
Heart Wall    
0.052533 
0.096933 
Kidneys                    
0.877000 
1.133333 
Liver    
0.211000 
0.266667 
Lungs    
0.027167 
0.033133 
Muscle 
0.009190 
0.012167 
Ovaries  
0.016967 
Pancreas   
0.037300 
0.050200 
Red Marrow   
0.013600 
0.016833 
Osteogenic Cells  
0.007390 
0.010593 
Skin   
0.004543 
0.005790 
Spleen   
0.093600 
0.163667 
Testes   
0.002780 
Thymus  
0.005710 
0.007800 
Thyroid  
0.001357 
0.001633 
Urinary Bladder Wall      
0.085800 
0.088167 
Uterus   
0.014133 
0.017500 
Total Body  
0.018767 
0.025200 
  Effective Dose  
 
 
0.0138 mSv/MBq 
Table 2: [18F]F-AraG human radiation dosimetry data. Mean radiation dosimetry data in 3 
males and 3 females in REM/mCi. The kidneys receive the highest radiation dose followed by 
the liver. 

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For our studies at Stanford, the tracer will be administered as a single bolus injection of 
approximately 5 mCi into a hand or arm vein. The rationale for the starting dose is based on 
dosimetry studies already performed in healthy human volunteers where the dose limiting organ 
was found to be the kidney and the annual dose characterized as 5mCi. Please refer to the 
imaging protocol for recommended limits for male and female subjects. The major route of 
clearance based on initial clinical studies is the liver and kidney. 
 
2.3 Clinicaltrials.gov  
We are currently in communication with the company CellSight Technologies, Inc. (Dr. Gambhir 
is a founder) that licensed the [18F]F-AraG patent (US Patent #US20110059014 A1) from 
Stanford. This company has been clinically evaluating this tracer in parallel to our proposed 
studies. Toxicity testing and dosimetry has already been performed by the company under FDA 
IND #123591, and the company has evaluated this tracer in normal subjects prior to testing in 
cancer patients receiving immunotherapy.  
CellSight Technologies has given permission for this study to be submitted to their existing 
IND 123591. 
2.4 Rationale 
Benefits: The results may allow for improved, early and sensitive diagnosis of acute GVHD in 
patients that have undergone a bone marrow transplant.  Please refer to pages 6-9 for 
additional rationale. 
 
2.5 Preliminary results 
Please refer to pages 15-22. 
 
2.6 Study Design  
Study participants will consist of healthy volunteers for extended studies on radiotracer behavior 
and patients belonging to a GVHD group. Two patient categories exist within the GVHD group- 
those highly suspected to have acute GVHD and those that are at high risk for developing acute 
GVHD. We will recruit healthy volunteers through local advertising. We will recruit high risk and 
confirmed GvHD patients from the Bone Marrow and Transplantation Clinic at the Stanford 
Cancer Institute.  
 
Normal subjects will be recruited by publicly disseminating the information about the clinical trial 
through printed media such as a flyer, or by sending out an email to the Stanford community. 
We will ask for volunteers. A consent form for healthy volunteers is also included. Patients will 
undergo preliminary evaluation to ensure eligibility, receive and sign an informed consent, be 
enrolled in the trial, and then have [18F]F-AraG PET/CT.  
 
Highly suspected acute GVHD patients will be scanned following biopsy (taken to confirm acute 
GVHD). The staging and grading of the disease using the Glucksberg grade and International 
bone marrow Transplant Registry Severity Index (IBMTR) at time of enrollment will be noted. 
Their consent for a PET-CT scan will be obtained prior to biopsy. Biopsy tissues of consented 
patients will be analyzed further for T cell involvement.  
High-risk patients (recipients of myeloablative or reduced intensity allogeneic transplants using 
either bone marrow or peripheral blood stem cells from HLA-matched or HLA-mismatched 
related or unrelated donors-protocols 9917, 9142, 9022, 9924) will have to sign consent forms 

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before they undergo a transplant. All those that consent will undergo a PET-CT scan day 4 + 2 
days post transplant. Additionally these patients will be scanned again between day 14-21 post 
transplant. Follow up on these patients will note those that go on to develop aGVHD and the 
clinical end point will be correlated to the scans to verify any predictive potential of the 
radiotracer.  
For both GVHD groups-results of the scan will be compared to biopsy results and 
immunohistochemical staining of the biopsy samples for T cells.  
Investigational agent: [18F]F-AraG will be formulated in a sterile and pyrogen-free isotonic 
solution will be administered in a single slow IV injection. 
This clinical study will be funded by the Parker Institute of Cancer Immunotherapy at Stanford 
Medicine Bench to Bedside Program 2017.  
Additional resources and costs will be supplemented with the NIH RO1 grant  ‘A Novel Positron 
Emission Tomography Strategy for Early Detection and Treatment Monitoring of Graft-Versus-
Host Disease’ (Grant number 5RO1CA201719-02). 
This is a single-arm prospective study of imaging of aGVHD where no randomization will be 
done (please see section 10 for more information on statistical analysis). 
 
3 PARTICIPANT SELECTION AND ENROLLMENT PROCEDURES 
3.1 Inclusion Criteria 
This study will enroll three cohorts of participants: healthy volunteers, patients highly suspected 
to have acute GVHD and requiring systemic therapy, and patients at high risk for developing 
acute GVHD.  For patients highly suspected to have acute GVHD and requiring systemic 
therapy, biopsy of involved tissue is encouraged to support the clinical diagnosis- this will 
normally be taken from the gastrointestinal tract. T cell staining will be incorporated in the 
immunohistochemical analysis of the tissue. 
Participants must meet all of the following relevant criteria to participate: 
1. Must be 21 years of age or older.  
2. Must understand and voluntarily have signed an Informed Consent after its contents 
have been fully explained.   
a. For patients highly suspected to have acute GVHD and requiring systemic 
therapy, informed consent should be signed prior to biopsy taken to support 
clinical diagnosis. 
b. For patients at high risk for developing acute GVHD, informed consent should be 
signed prior to transplant. 
3. For healthy volunteers only: Must have no known medical problems that would make 
undergoing the scan hazardous to the health of the patient or interfere with the results. 
In particular subjects should not have any cardiac or immunological disorders as these 
would likely affect the scan results. Subjects should have had a full physical exam within 
6 months of the study. If healthy volunteers have not had a full medical exam within 6 
months of the study, one of the nuclear medicine physicians will conduct the medical 
exam prior to any study procedures. 
4. For patients highly suspected to have acute GVHD and requiring systemic therapy only: 

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Taking steroid treatment for suspected acute GVHD for 7 days or less. 
5. For patients at high risk for developing acute GVHD only: Recipients of myeloablative or 
reduced intensity allogeneic transplants using either bone marrow or peripheral blood 
stem cells from HLA-matched or HLA-mismatched related or unrelated donors (protocols 
9917, 9142, 9022, 9924) who have not yet been placed on any therapy for acute GVHD. 
 
3.2 Exclusion Criteria 
To participate in this study, subjects must have none of the relevant criteria: 
1. Pregnant or nursing  
2. Individuals with known or suspected substance abuse, obtained by self-reporting. 
3. Uncontrolled infection 
4. Relapsed/persistent malignancy 
5. Currently receiving immunotherapy 
 
3.3 Informed Consent Process 
All participants will be provided a consent form describing the study with sufficient information 
for participants to make an informed decision regarding their participation.  Participants will sign 
the IRB approved informed consent prior to participation in any study specific procedure. The 
participant will receive, as is mandatory, a copy of the signed and dated consent document. The 
original signed copy of the consent document will be retained in the medical record or research 
file.  
3.4 Randomization Procedures 
Patients will not be randomized.  
 
3.5 Study Timeline 
3.5.1 Primary Completion: 
The study will reach primary completion 24 months from the time the study opens to 
accrual. 
 
3.6 Study Completion: 
The study will reach study completion 36 months from the time the study opens to accrual. 
 
4 IMAGING AGENT/DEVICE/PROCEDURE INFORMATION 
4.1 [18F]F-AraG: 2’-deoxy-2’-[18F]fluoro-9-β-D-arabinofuranosyl guanine 
 
The imaging agent to be used in this study is 2’-deoxy-2’-[18F]fluoro-9-β-D-arabinofuranosyl 
guanine. The radionuclide on this imaging agent being Fluorine-18 (18F).  The imaging agent will 
be administered intravenously.  
 
Clinical grade imaging agent will be synthesized under GMP/GLP conditions at the Lucas 
Center Cyclotron and Radiochemistry facility, part of the Molecular Imaging Program at 
Stanford. The imaging agent will be requested as and when subjects become available. The 
radiotracer will be received at the Nuclear Medicine department at Stanford Hospital in a lead 
shielded container by an authorized person and with the appropriate transfer sheet.  
Dosimetry of this imaging agent has previously been characterized in male and female healthy 
subjects and the information is included on page 21. Dosimetric calculations were performed 

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using the OLINDA/EXM 1.1 software using methods previously described using other tracers 
[Yaghoubi, 2001].  
PET/CT is a well established, FDA approved clinical imaging procedure. Each subject will be 
scanned a maximum of 2 times. For each imaging session the amount of radioactivity that will 
be administered will be less than 5.7 and 4.4 mCi for males and females respectively. 
 
5 STUDY PROCEDURES  
5.1 
Imaging Protocol for Patient Groups 
Each subject may have up to two imaging sessions. The procedures for each imaging session is 
detailed below: 
 
Day 1 (Imaging Day) 
Overview of procedures prior and post injection of [18F] F-AraG: 
1. Subject will participate in an informed consent session (approximately 30 minutes) and if 
she/he would like to continue after being fully informed she/he will sign consent form. 
2. Subject will be asked to drink several glasses of water before arriving at the nuclear 
medicine clinic and during the study. 
3. Subject will be interviewed to obtain medical history. 
4. Volunteer height and weight will be measured. 
5. Baseline Heart Rate (HR), Blood Pressure (BP), Blood Oxygen %,Temperature, and 
Respiratory Rate will be collected at baseline (5-120 minutes prior to the injection of the 
tracer). 
6. IV line may be placed in both arms of healthy subjects for PET tracer injection and another 
may be used for blood collection. The GVHD patients will have double lumen catheters 
which can these be used for these purposes. 
7. For baseline CBC analysis- 3 ml of blood will be collected and 5 ml of blood will be collected 
for baseline metabolic panel analysis. 
8. Subject may either be positioned on the PET/CT scanner bed or another bed in the clinic 
(where possible the same PET-CT system should be used especially for the healthy 
subjects). 
9. Injection of [18F] F-AraG and post-injection procedures will follow. 
10. If possible, subjects should be asked to urinate prior to the start of the PET/CT scans. 
11. A bolus < 5.7 mCi of clinical grade [18F] F-AraG for male volunteers or < 4.4 mCi of clinical 
grade [18F]F-AraG for female volunteers will be injected through IV. 
12. A CT image will be acquired with the PET images. 
13. The PET scanning procedure may start at the time of tracer injection or at a later time point 
after tracer injection. The imaging field includes the vertex to thigh for healthy subjects and 
vertex to feet for GVHD patients. Up to two whole-body PET scans may be performed at 
various time points after injection of the tracer, not to exceed 2 hours after tracer injection. 
14. Blood pressure, Temperature, Blood Oxygen saturation, Heart Rate, Respiratory Rate will 
be measured immediately after [18F]F-AraG PET-CT procedure.  
15. A safety follow up will happen at one time point within the time interval (day 2 – day 7) post-
scan. 
16. All observed adverse events and subject complaints will be recorded. 
 

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Healthy subject protocol 
  
1. Volunteer will be asked to drink 1-2 glasses of water before his/her arrival at the clinic. 
2. Volunteer will be consented by a member of the study team  
3. Female subjects will have a urine or serum pregnancy test to rule out pregnancy. 
4. Subject will be weighed.  
5. Subject will have an IV line placed in a hand or arm vein for tracer administration.  
6. Subject will have an IV line placed in a hand or arm vein for blood sampling.  
• 
Subject will have a second IV line placed in the vein of the other hand/arm for 
venous/hand warmed arterialized-venous blood or in a radial artery for arterial blood 
sampling for kinetic and radio-metabolite analysis, over the first 90 minutes post 
tracer injection. The arterial/venous or arterialized-venous  blood will be sampled in 
heparinized coated tubes pre-tracer injection and at  1, 3, 5, 10, 15, 30, 45, 60 and 90 
minutes post tracer injection (10 sampling points). 
• 
During this time, care should be taken to prevent movement in the subject from his or 
her original position in the scanner. The blood sampling volume will be 3 mL, ( 2mL 
for whole blood and plasma radioactivity analysis + 1mL for radiometabolite 
analysis). The exact time point of the collection of each blood sample will be 
recorded using the same clock device.If an automated blood-sampling device is 
available (e.g., Twilite system from Swisstrace GmbH) it should be used for the blood 
sampling procedure, but if this is not available, then the sampling will be done 
manually. 
• 
Radioactivity analysis of whole blood and plasma:  The blood tubes should be 
weighed before and after collecting the blood. Whole blood radioactivity will be 
measured. To measure plasma radioactivity, the whole blood samples will be 
centrifuged. The radiolabeled tracer concentration in whole blood, plasma and pellet 
will be measured by a gamma well counter (1-min per sample). The counting rates 
measured by the well counter will be converted to the PET equivalent counts using a 
conversion factor from a weekly phantom cylinder study. The counts should be 
decay corrected back to the injection time. 
7. Blood pressure, temperature, respiratory rate, heart rate and pulse oximetry measurements 
will be taken at baseline.  
8. A blood sample will be taken prior to the injection of the tracer, to be used for baseline 
chemistry and hematology laboratory testing (hematocrit should be an included measure). 
9. Participant will have EKG leads placed for cardiac gating and then be placed in the scanner. 
10. A low dose attenuation correction CT scan (ACCT) of the heart will be performed for 
attenuation correction.  
11. A small low activity calibration source of known activity, volume, and concentration of F-18 
activity will be taped to the furthest corner of the scanner field of view prior to 
radiopharmaceutical injection.  The 30 minute dynamic PET acquisition of the heart will then 
begin, making sure that the entire heart region is covered in the field of view.  All PET/CT 
scans should be done on the Discovery MI PET/CT scanner or the PET/CT 690 scanner.  
12. Right after the start of the PET acquisition, [18F]F-AraG formulated in a sterile and pyrogen-
free isotonic solution in less than 10 ml will be administered in a single slow IV injection. The 
line will be slowly flushed at a constant rate with at least 10 ml normal saline after injection.  
13. The initial 5 minutes of the dynamic PET scan will be finely acquired at 5 seconds/frame 
(total number of frames in initial 5 minutes =60 frames) so as to capture the peak of the 
cardiac input function and then 1 minute/frame for the next 25 min over the same field of 
view (FOV). The dynamic PET data should be collected in list-mode and saved 

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appropriately. 
14. Participants will have skull base-to-thigh static PET-CT scans (which includes the brain) 
obtained again at 60 and 90 minutes (scans #2 and #3). CT will use 120 kV and dose 
modulation based on body habitus, ranging 10-105 mA for both scans.  
15. Blood pressure, temperature, respiratory rate, heart rate and pulse oximetry measurements 
will be performed post scan 3. 
16. All recordings of important time measurements (e.g. time of injection and acquisitions) will 
be done using the same clock e..g a phone.  
17. Participants will be asked to void frequently to reduce radiation exposure.  
18. Adverse drug events that have developed during the scans will be recorded in source 
documents. Any that develop after 18F]F-AraG injection will also be recorded in the source 
documents when patients seen at the safety follow-up visit at one time point within the time 
interval (day 2 – day 7) post-scan. 
19. After [18F]F-AraG injection, participants will be asked to return to the clinic for a safety follow-
up visit for blood sample collection for blood chemistry and hematology and also for 
measurements of vital signs. Results will be recorded in the case report form.  
 
GVHD patient protocol 
1. Subject will be asked to drink 1-2 glasses of water before his/her arrival at the clinic. 
2. Subject will be consented by a member of the study team  
3. Subject will be weighed.  
4. The subject will likely have an existing port (double lumen catheters) for administration of 
the radiotracer. 
5. A blood sample will be taken 5-120 minutes prior to the injection of the tracer, to be used for 
baseline chemistry and hematology laboratory testing.  Blood pressure, temperature, heart 
rate and pulse oximetry measurements will also be taken up between 5-120 minutes prior to 
injection (baseline). 
6. [18F]F-AraG formulated in a sterile and pyrogen-free isotonic solution will be administered in 
a  single slow IV injection. The line will be flushed with at least 10 ml normal saline after 
injection. The bed strap should be open until scanning starts.  
7. A small low activity calibration source of known activity of F-18 will be placed in the field of 
view in each image collected  
8. Participants will have a static PET scan at 60 minutes after injection of [18F]F-AraG covering 
all positions from vertex to toe. Attenuation correction will be done with a CT scan (120 kv, 
10 mA) usually acquired before the PET scan. There will be 8 bed positions covering head 
to toe (each position will taking approximately 3 minutes). The data should be collected in 
list mode and saved. Patients that are able to tolerate longer times in the scanner will also 
under go an additional scans following the same format described above at 30 minutes post 
tracer injection. 
9. All recordings of important time measurements (e.g. time of injection and acquisitions) will 
be done using the same clock e.g. a phone. 
10. Blood pressure, temperature, respiratory rate, heart rate and pulse oximetry measurements 
will be taken immediately after the [18F]F-AraG PET-CTscan. 
11. Participants will be asked to void as frequently as possible to reduce radiation exposure.  
12. Participants will be given a copy of the consent form they signed and will be dismissed.  
13. Participants will undergo a safety follow-up at one time point within the time interval (day 2 – 
day 7) post-scan for blood sample collection for blood chemistry and hematology and also 
for observation for vital signs. For in-patients we will use the blood samples and vital signs 

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obtained as part of the standard of care by the Bone Marrow and Transplant group. Out-
patients will have to return to the clinic for the safety follow up. 
14. Adverse drug events will be recorded at one time oint with the time interval of 2-7 days after 
[18F]F-AraG injection/scan in the case report form. 
15. Patients in the high risk of developing GVHD arm will undergo the above protocol once more 
10-17 days after the first scan. 
16. A post safety follow up will be conducted for up for 6 months via chart review to assess 
progression or development of aGVHD. 
Safety Follow-up [range: One time point within the interval (Day 2 to Day 7) post-scan 
1. BP, HR, Temperature, Blood Oxygen %, and Respiratory Rate will be recorded.  
2. Blood will be collected for CBC, and Metabolite Analysis. 
3. Adverse events will be collected at one time point within the time interval (day 2 – day 7) 
post scan. 
 
Healthy subjects and out-patients will need to come in to the clinic for the safety follow up.  For 
in-patients (GVHD group)-we will obtain the blood samples and vital signs obtained as part of 
the standard of care by the Bone Marrow and Transplant group. 
Subjects with suspected acute GVHD OR at high-risk of aGVHD will be followed for 6 months 
post their last scan to evaluate disease (progression/development) by chart-review.   
 
5.2 
Criteria for Removal from Study 
Withdrawal of Participants 
The Protocol Director may withdraw participants from the study for one or more of the following 
reasons: failure to follow the instructions of the Protocol Director and/or the study staff;  failure 
to provide informed consent; determination that continuing the participation could be harmful to 
the subject; the study is cancelled or other administrative reasons. 
Discontinuation: Patients can discontinue in case of unacceptable adverse events or patient 
decision to withdrawn from study. 
 

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6 STUDY CALENDAR 
6.1 Healthy Volunteers 
Table 6-1: Study Calendar for Healthy Volunteers 
 
Screening 
Day 1 
Safety 
Follow-Up 
 
Day -30 to 
Day 1 pre-
injection 
Pre-injection of 
[18F]F-AraG 
(Baseline) 
Injection of 
[18F]F-AraGa 
Post-injection of 
[18F]F-AraG 
Day 2-7  
post-scan 
Inclusion/Exclusion 
Criteria 
X 
 
 
 
 
Informed Consent 
Xd 
Xd 
 
 
 
hCGc 
Xd 
Xd 
 
 
 
Height 
 
X 
 
 
 
Weight 
 
X 
 
 
 
Vital signs 
 
X 
 
X 
X 
EKG 
 
X (leads placed 
prior to scan) 
X 
X 
 
CBC w/diff, plts 
 
X 
 
 
X 
Serum chemistryb 
 
X 
 
 
X 
IV administration 
of [18F]F-AraG 
 
 
X 
 
 
PET/CT scan with 
investigational 
Agent  
[18F]F-AraG 
 
 
 
Xe 
 
Arterial/venous 
blood sampling for 
input function 
 
 
 
X  
(0, 1, 3, 5 10, 
15, 30, 45, 60 
and 90 min after 
tracer injection) 
 
Radio-Metabolite 
Analysis 
 
 
 
X 
(0, 1, 3, 5 10, 
15, 30, 45, 60 
and 90 min after 
tracer injection) 
 
Adverse event 
evaluation 
 
 
 
X 
Xf 
a: 
Investigational Agent: Dose as assigned (5 mCi); route/schedule. 
b: 
Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, 
phosphorus, potassium, total protein, SGOT[21], SGPT[45], sodium. 
c: 
Urine or serum pregnancy test (women of childbearing potential). 
d: 
Procedure may be done during screening period or on Day 1 prior to injection of study drug. 
e: 
Scan 1: 30-minute dynamic PET of heart (60x5s+25x1min; Scan 2: vertex to mid-thigh static PET 60-minute post 
injection (includes brain); Scan 3: vertex to thigh static PET 90 minutes post-injection 
f: 
Collected at one time point within the time interval (day 2 – day 7) post scan. 

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6.2 Patients highly suspected to have acute Graft versus Host Disease 
Table 6-2: Study Calendar for Patients highly suspected to have acute GHVD 
 
Screening 
Day 1 
Safety 
Follow-
Up 
Disease 
assessment  
 
Day -30 to 
Day 1 pre-
injection 
Pre-injection 
of [18F]F-
AraG 
(Baseline) 
Injection of 
[18F]F-
AraGa 
Post-
injection of 
[18F]F-AraG 
Day 2-7  
post-scan 
 
Upto 6 month  
post-scan 
Inclusion/Exclusion 
Criteria 
X 
 
 
 
 
 
Informed Consent 
Xd 
Xd 
 
 
 
 
Height 
 
X 
 
 
 
 
Weight 
 
X 
 
 
Xc 
 
Vital signs 
 
X 
 
X 
Xc 
 
CBC w/diff, plts 
 
X 
 
 
Xc 
 
Serum chemistryb 
 
X 
 
 
Xc 
 
IV administration 
of [18F]F-AraG 
 
 
X 
 
 
 
PET/CT scan with 
investigational 
Agent  
[18F]F-AraG 
 
 
 
Xe 
 
 
Adverse event 
evaluation 
 
 
 
X 
Xg 
 
Follow-up for 
development of 
aGVHD 
symptoms/ 
disease 
 
 
 
 
 
 
Xf 
a: 
Investigational Agent: Dose as assigned (5 mCi); route/schedule. 
b: 
Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, 
phosphorus, potassium, total protein, SGOT[21], SGPT[45], sodium. 
c: 
Obtained as part of the standard of care by the Bone Marrow and Transplant group for in-patients (for in-
patients) 
d: 
Procedure may be done during screening period or on Day 1 prior to injection of study drug. 
e: 
Scan 1: head to toe PET/CT scan 60 minutes post injection); OPTIONAL head to toe PET/CT scan 30-minutes 
post-injection 
f: 
Chart review for up to 6 months post scan to monitor disease progression. 
g: 
Collected at one time point within the time interval (day 2 – day 7) post-scan 
 
 
 
 

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6. 3 Patients at high risk of developing acute Graft versus Host Disease 
Table 6-3: Study Calendar for Patients at high risk of developing acute GHVD 
a: 
Investigational Agent: Dose as assigned (5 mCi); route/schedule. 
b: 
Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, potassium, total protein, SGOT[21], 
SGPT[45], sodium. 
c: 
Obtained as part of the standard of care by the Bone Marrow and Transplant group for in-patients (for in-patients) 
d: 
Procedure may be done during screening period or on Day 1 prior to injection of study drug. 
 
Screening 
Day 1 
Safety 
Follow-
Up 1 
Day 11-18:  2nd scan 
Safety 
Follow-
Up 2 
Disease 
assessment 
 
Day -30 to 
Day 1 pre-
injection 
Pre-injection 
of [18F]F-
AraG 
(Baseline) 
Injection of 
[18F]F-
AraGa 
Post-
injection of 
[18F]F-
AraG 
Day 2-7  
post 1st 
scan 
Pre-injection 
of [18F]F-
AraG 
(Baseline) 
Injection of 
[18F]F-
AraGa 
Post-
injection of 
[18F]F-AraG 
2-7 days  
post 2nd 
scan 
 
Upto 6 month  
post-scan 
Inclusion/Exclusion 
Criteria 
X 
 
 
 
 
 
 
 
 
 
Informed Consent 
Xd 
Xd 
 
 
 
 
 
 
 
 
Height 
 
X 
 
 
 
 
 
 
 
 
Weight 
 
X 
 
 
Xc 
X 
 
 
Xc 
 
Vital signs 
 
X 
 
X 
Xc 
X 
 
X 
Xc 
 
CBC w/diff, plts 
 
X 
 
 
Xc 
X 
 
 
Xc 
 
Serum chemistryb 
 
X 
 
 
Xc 
X 
 
 
Xc 
 
IV administration of 
[18F]F-AraG 
 
 
X 
 
 
 
X 
 
 
 
PET/CT scan with 
investigational Agent  
[18F]F-AraG 
 
 
 
Xe 
 
 
 
Xe 
 
 
Adverse event 
evaluation 
 
 
 
X 
Xg 
X 
 
X 
Xg 
 
Follow-up for 
development of aGVHD 
symptoms/ disease 
 
 
 
 
 
 
 
 
 
 
Xf 

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e: 
On Day 1:Scan 1: head to toe PET/CT scan 60 minutes post injection); OPTIONAL head to toe PET/CT scan 30-minutes post-injection; 
On Day 11-18: Scan 2: head to toe PET/CT scan 60 minutes post injection); OPTIONAL head to toe PET/CT scan 30-minutes post-injection 
f: 
Chart review for up to 6 months post scan to monitor disease progression 
g: 
Collected at one time point within the time interval (day  2 – day 7) post-scan #1 and at one time point within the time interval (day 2 – day 7) post-scan #2 

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6 ADVERSE EVENTS AND REPORTING PROCEDURES 
7.1 Potential Adverse Events 
Risks associated with this Investigational Agent 
[18F]F-AraG has been tested in 6 healthy human volunteers, 3 advanced bladder cancer 
patients (2 of whom received 2 doses each), 2 breast cancer patients, and 3 squamous cell 
carcinoma of the head and neck cancer patients (2 of whom received 2 doses each).  Any 
observed side effects were mild.  The only side effect that occurred in more than one 
volunteer/patient was positive WBC esterase in the urinalysis without accompanying urinary 
symptoms. 
Risks associated with Investigational PET/CT Imaging 
The administration of the radioactive substance will feel like a slight pinprick if given by 
intravenous injection.  Patients who are claustrophobic may feel some anxiety while positioned 
in the scanner.  Also, some patients find it uncomfortable to hold one position for more than a 
few minutes.  The subjects will not feel anything related to the radioactivity of the substance in 
their body.  The substance amount is so small that it does not affect the normal processes of the 
body.  Procedures such as isotope injection are standard procedures currently used in the 
clinical PET and Nuclear Medicine clinics and the usual sterile methods will be used. 
Risks from PET imaging include exposure to ionizing radiation and the placement of an 
intravenous line.  Total time required for each PET study will be approximately 90-120 minutes.  
The radiation the participants will receive as a result from participating in this study includes 
radiation from administration of [18F]F-AraG and the accompanying CT scans for attenuation 
correction.  This radiation exposure is for research purposes only.  
• 
The amount of radiation dose the healthy group will receive from one of [18F]F-AraG 
PET/CT is 11.7-12.3 mSv (2.7mSv from 163MBq for females and 3.3 mSv from 211MBq 
of [18F]F-AraG for males plus a further 9mSv from the three CTs (one cardiac and two 
whole body CT scans) for attenuation correction acquired usually before the PET 
acquisitions. 
• 
The amount of radiation dose the GVHD group will receive from one  [18F]F-AraG 
PET/CT session is 5.7-6.3 mSv (2.7mSv from 163MBq for females and 3.3 mSv from 
211MBq of [18F]F-AraG for males plus a further 3mSv from the whole body CT scans for 
attenuation correction. If a patient is able to withstand longer times in the scanner they 
will undergo a further PET acquisition at 30 minutes for which 1 more CT scans will be 
performed usually before the PET acquisition (and an additional 3mSv dose will be 
received). 
This amount of radiation involves minimal risk and is necessary to obtain the research 
information desired.  Procedures will be in place for verification of correct radiopharmaceutical 
dose and route of administration (i.e., each dose will be double checked for dosimetry and 
quality by a researcher and technologist). 
Risks associated with aGVHD 
Patients highly suspected to have aGVHD or who are at high risk of developing aGVHD may 
experience side effects from underlying disease while participating in this study.  Acute GVHD is 
characterized by a wide spectrum of host tissue injuries in the gastrointestinal (GI) tract, liver, 
skin, and lungs; causing a wide range of symptoms [3, 5].  In this setting, a very large number of 

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Grade 1 and 2 adverse events (AEs) are expected to occur, regardless of whether or not a 
patient is participating in a research study.  Expected AEs include a number of specific AEs that 
are associated with acute GVHD. 
The Protocol Director (PD) or designee will assess each AE to determine whether it is 
unexpected according to the Informed Consent, Protocol Document, or Investigator’s Brochure, 
and related to the investigation.  Unless specifically determined otherwise by the investigator, 
the following events are considered expected in the early post-transplant setting.  
• Alopecia  
• Anemia  
• Anorexia  
• Bleeding, including requiring transfusions  
• Cardiac arrhythmias   
• Central venous catheter infections  
• Constipation  
• Diarrhea  
• Edema  
• Fatigue  
• Febrile episodes  
• Gastritis  
• Graft failure  
• Graft versus host disease  
• Hematuria   
• Hypertension  
• Hypotension  
• Hypoxia  
• Incontinence  
• Infections, including sepsis 
• Insomnia  
• Laboratory abnormalities  
• Mental status changes and mood 
alterations  
• Mucositis  
• Nausea  
• Neutropenia  
• Pain  
• Pleural effusion  
• Pneumonitis  
• Rash  
• Seizures  
• Sinusoidal obstructive syndrome  
• Tachycardia  
• Thrombocytopenia  
• Thrombotic microangiopathy  
• Tremor  
• Vomiting 
 
7.2 Adverse Event Collection 
Adverse event collection will be focused on adverse events useful for assessment of the safety 
of [18F]F-AraG.  The following AE monitoring schema is intended to capture all AEs that are 
clinically significant and/or impactful, but minimize “not informative” AE collection.  Adverse 
events will be graded according to CTCAE v4.0.   
• 
All AEs, both non-serious and serious, attributed to investigational agent or study 
procedure will be collected and documented with causality attribution (see Section 7.2 
Adverse Event Attribution) on an adverse event log (AE log).   
o An adverse event is considered serious if it fulfills one of the following criteria per 
21CFR§312.32(a): 
 
Results in death 
 
Life-threatening (patient at risk of death at the time of the event) 
 
Requires inpatient hospitalization or prolongation of existing hospitalization 
 
Results in persistent or significant disability 
 
Other medical events that may not be immediately life-threatening or result in 
death or hospitalization by may jeopardize the patient or require intervention to 
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• 
In addition, all AEs meeting the criteria of “serious” as defined at 21CFR§312.32(a), and 
are Grade 3 or above, will be collected regardless of attribution; and documented on an 
AE log, including causality attribution.   
• 
AEs not meeting these criteria will not be collected, except as follows.  AEs resulting in 
subject withdrawal from the study will be collected. 
All Serious Adverse Events (SAEs) will be tracked until resolution or at least 30 days after the 
last dose of the study treatment.  
The development of acute GVHD, its date of onset and overall grade, is a key component of the 
primary outcome measure for patients on this trial and will be monitored by chart review.  Acute 
GVHD will be recorded on an AE log.  
7.3 Adverse Event Reporting 
Both serious and non-serious AEs will be clearly noted in source documentation and listed on 
study-specific Case Report Forms (CRFs).  Non-serious adverse events will be reported 
annually to FDA via an Annual Report (IND: 123591) and to IRB via Continuing Review.  SAEs 
Grade 3 and above, and all subsequent follow-up reports will be reported to the Stanford 
Cancer Institute Data and Safety Monitoring Committee (DSMC) using the study specific CRF 
regardless of the event’s relatedness to the investigation.  Following review by the DSMC, 
events meeting the IRB definition of ‘Unanticipated Problem’ will be reported to the IRB using 
eProtocol within 10 working days of DSMC review, or within 5 working days for deaths or 
life-threatening experiences. 
Adverse events deemed serious, unexpected (i.e. not described in the protocol, Investigator’s 
Brochure or informed consent documents) and related to study drug must be reported to the 
IND Holder (CellSight Technologies) using the FDA MedWatch form 3500a within 24 hours of 
knowledge of event.  The IND Holder is responsible for deciding whether the event meets IND 
Safety Reporting criteria. 
If the Principal Investigator determines an unanticipated adverse effect presents an 
unreasonable risk to subjects, the study will be terminated as soon as possible, but no later than 
5 working days after the PI makes the determination and no later than 15 working days after first 
receiving notification of the effect. 
 
8 REGULATORY CONSIDERATIONS 
8.1 Institutional Review of Protocol 
The protocol, the proposed informed consent and all forms of participant information related to 
the study (e.g. advertisements used to recruit participants) will be reviewed and approved by the 
Stanford IRB and Scientific Review Committee (SRC).  Any changes made to the protocol will 
be submitted as a modification to FDA and will be approved by the IRB and SRC prior to 
implementation.  The Protocol Director will disseminate the protocol amendment information to 
all participating investigators. 
 
8.2 Data Management Plan 
The study will be registered on Oncore, a centralized database used by CCTO to manage 
clinical trials. All associated approvals and subject information will also be stored on this 
platform.  Any case report forms (CRFs) will be stored in a locked office in the Nuclear Medicine 
clinic. Records will be kept using OnCore.  

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Data will be managed using Excel software where the following important parameters will be 
documented; subject ID (number assigned based on order of scan), the type of subject 
(healthy/highly suspected GVHD or high risk GVHD group), sex of the subject and their weight, 
height and age. The date and time of radiotracer injection, the dose injected, time and duration 
of PET scan will also be noted. 
For data analysis the following parameters will be documented; region of interest (Ie.g. liver, 
heart, cervical lymph nodes), volume of the region of interest and the associated mean, 
maximum and minimum values for the Standard Uptake Values (SUV). 
 
8.3 Data and Safety Monitoring Plan 
During the clinical investigation, the Protocol Director will evaluate the progress of the trial, 
including periodic assessments of data quality and timeliness, participant recruitment, accrual 
and retention, participant risk versus benefit, performance of trial sites, and other factors that 
can affect study outcome.  Monitoring of the trial will occur every 8 weeks and a record of 
monitoring activities will be maintained by the study team.  
The Stanford Cancer Institute Data and Safety Monitoring Committee (DSMC) will audit study 
related activities at least annually in accordance with the DSMC SOP to determine whether the 
study has been conducted in accordance with the protocol, local standard operating procedures, 
FDA regulations, and Good Clinical Practice (GCP).  This may include review of regulatory 
binders, case report forms, eligibility checklists, and source documents.  In addition, the DSMC 
will regularly review serious adverse events and protocol deviations associated with the 
research to ensure the protection of human subjects.  Results of DSMC audits will be 
communicated to the IRB and the appropriate regulatory authorities at the time of continuing 
review, or in an expedited fashion, as needed. 
 
9 MEASUREMENTS 
9.1 Primary and Secondary Outcome measures 
Primary outcomes will be the severity/score of aGVHD or the development of aGVHD in the 
highly suspected and high risk groups respectively. Diagnosis and staging of the disease are 
ultimately based on skin, liver and gut involvement and will be further supported by the 
secondary outcome of IHC analysis of the gastrointestinal tract. The latter mostly focuses on the 
detection of dead regions as determined by haematoxylin and eosin staining. For this study, an 
additional secondary outcome will be to further stain tissues for T cell markers to determine T 
cell infiltration (CD3, CD4 amd CD8) and T cell activation markers (e.g. OX40) to determine T 
cell status since activated T cells underlie the development of aGVHD in the early stages. 
 
9.1.1 Primary Outcome Measurement Methods 
Static images acquired will be used to determine the bio-distribution of the imaging agent in the 
GVHD groups. Past images as well as newly acquired data from healthy subjects will be used 
as comparisons. Regions of interest will be drawn on various organs such as the liver, cervical 
lymph nodes and gut and quantified using imaging software (e.g. Inveon Research Workplace 
or Amide). In addition any areas that show up differently in the GVHD groups, to the normal bio-
distribution seen in healthy subjects will be identified and ROIs will also be drawn over these 
regions. 
 

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Bio-distribution data will be expressed as % injected dose/gram (%ID/g) and/or as standard 
uptake values (SUV) with respect to the organ being studied.  In the high suspected aGVHD 
group, [18F]F-AraG PET results will be compared to any grades and scores of aGVHD severity 
attributed to subjects.  In the case of the high-risk group, after the second scan, patients will be 
monitored for development of aGVHD and the clinical endpoint (whether or not the high risk 
patient develop aGVHD) will represent the primary outcome variable.  In addition, IHC analysis 
of biopsies taken in either GVHD group will help in the interpretation of the primary results and 
thus potentially support interpretation of the PET data. The IHC analysis will involve determining 
the amount of cell death or T cell infiltration and activation. 
 
Vital signs and blood laboratory data collected before and after the i.v. injection of [18F]F-AraG 
will allow evaluation of any unexpected side effects. 
 
To measure plasma radioactivity, whole blood sample will be centrifuged. The radiolabeled 
tracer concentration in whole blood, pellet post centrifugation and plasma will be measured by a 
gamma well counter (1-min per sample). The counting rates measured by the well counter will 
be converted to the PET equivalent counts using a conversion factor from a weekly phantom 
cylinder study. The counts should be decay corrected back to the injection time.  
 
Plasma parent tracer and metabolite concentrations will be determined using solid phase 
extraction combined with HPLC technology. 
 
Clinical assessment of disease severity will be made by the aGVHD grade. Staging and grading 
of the disease will be performed by Clinicians at the Bone Marrow and Transplant Clinic using 
the Glucksberg grade and International bone marrow Transplant Registry Severity Index 
(IBMTR) at time of enrollment and imaging. 
 
9.1.2 Primary Outcome Measurement Time Points 
Duration of the follow up will extend to 6 months post final scan for all aGVHD subjects by chart 
review.  
 
9.1.3 Response Review 
The SUVmax values will be analyzed by Nuclear Medicine and Radiology physicians blinded to 
the diagnosis and results of the other scan, in a randomized order to avoid bias.  
IHC values will be assessed by trained Pathologists.  
 
9.2 Secondary Outcome 
 
Healthy subjects group: Biodistribution information and kinetic behavior of the radiotracer. 
The secondary outcome is the IHC confirmation of abnormal T cell status in those patients who 
have had a biopsy. 
 
10 STATISTICAL CONSIDERATIONS 
10.1 
Statistical Design 
Multi-arm prospective study of imaging of aGVHD (one group of highly suspected aGVHD and 
one group at high risk of developing aGVHD) and healthy volunteers. 

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10.2 
Randomization 
No randomization will be done. 
10.3 
Interim analyses 
No interim analyses are planned. 
10.4 
Descriptive Statistics and Exploratory Data Analysis  
(see below) 
10.5 
Analysis  
10.5.1 Analysis Population 
Analysis population: all ROIs drawn on various organs, as well as areas with elevated SUV 
relative to healthy volunteers. 
 
10.5.2 Analysis Plan 
Although the study will have low power due to the constrained sample size, in addition to 
descriptive statistics we will perform the following statistical analyses: 
 
o In the highly suspected group: 
Spearman rank correlation between median or maximum SUV values across ROIs and 
disease severity 
o In the high-risk group: 
Spearman rank correlation between SUV values in the first and second scans 
prediction of disease status at end of follow-up (aGVHD or no aGVHD) from SUV 
values of the first and second scans (using logistic regression) 
 
10.6 
Secondary Analysis 
10.6.1 Analysis Population 
Analysis population: biopsy samples from the high suspected aGVHD group, as well as any 
later performed on the high-risk aGVHD group. 
 
10.6.2 Analysis Plan 
Although the study will have low power due to the constrained sample size, in addition to 
descriptive statistics we will perform Spearman rank correlation between the SUV values and 
IHC measurements in biopsy locations. 
 
10.7 
Sample Size 
This is a pilot study, for the first time in GVHD patients including up to 25 GVHD patients 
(roughly half each in the highly suspected and high-risk groups).  An additional 5 healthy 
volunteers are also being included to add to existing bio-distribution data crucial to understand 
tracer characteristics.  While the small sample size provides insufficient power for formal 
statistical tests, we will examine the data and expect a high level of agreement between uptake 
of [18F]F-AraG with severity and progression of disease, and IHC results from biopsies 
 
10.8 
Accrual estimates 
We expect the accrual of 5 healthy volunteers and 25 patients over 24-36 months.  
Approximately 1-2 high risk patients for GVHD are seen monthly in the Bone Marrow and 

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Transplant clinic at Stanford Cancer Institute. Established GVHD patients are frequently seen in 
the clinic (approximately 5 high intensity and 10 low intensity patients at a given time).  We 
consider this planned accrual achievable given our experience with other protocols and the 
expressed support from the referring physicians, Professor Negrin and Dr Sally Arai. 
We plan to prioritize patients from Arm 1; imaging up to 10 patients highly suspected to have 
aGVHD and analyze scans to ascertain if the tracer is able to image the disease, before 
recruiting patients from Arm 2 (those that are at high risk of developing the disease post 
transplant). 
 
10.9 
Criteria for future studies 
If the results of this pilot study are encouraging, a larger phase II study will be designed.  

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11 REFERENCES 
 
1. 
Kim, W., et al., [18F]CFA as a clinically translatable probe for PET imaging of 
deoxycytidine kinase activity. Proc Natl Acad Sci U S A, 2016. 
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Tavaré, R., et al., Engineered antibody fragments for immuno-PET imaging of 
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Appendix A:  Inclusion/Exclusion Criteria Checklist 
*All subject files must include supporting documentation to confirm subject eligibility.  The 
method of confirmation can include, but is not limited to, laboratory test results, radiology test 
results, subject self-report, and medical record review.   
Protocol Title: 
Detection of Graft Versus Host Disease with [18F]F-AraG, a positron 
emission tomography tracer for activated T cells 
Protocol Number: 
IRB-38850 / BMT323 
Principal 
Investigator: 
Sanjiv Gambhir, MD, PhD 
 
II.  Subject Information: 
Subject Name/ID: 
 
Gender:    
 Male     
 Female 
III.  Inclusion/Exclusion Criteria 
Inclusion Criteria 
(From IRB approved protocol) 
Yes 
No 
Supporting Documentation* 
1. Must be 21 years of age or older.  
 
 
      
2. Must understand and voluntarily 
have signed an Informed Consent 
after its contents have been fully 
explained.   
a) For patients highly suspected to 
have acute GVHD and requiring 
systemic therapy, informed 
consent should be signed after 
biopsy supporting clinical 
diagnosis. 
b) For patients at high risk for 
developing acute GVHD, 
informed consent should be 
signed prior to transplant. 
 
 
      
3. For healthy volunteers only: Must 
have no known medical problems 
that would make undergoing the 
scan hazardous to the health of the 
patient or interfere with the results. 
In particular subjects should not 
have any cardiac or immunological 
disorders as these would likely 
affect the scan results. Subjects 
should have had a full physical 
 
 
      

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exam within 6 months of the study. If 
healthy volunteers have not had a 
full medical exam within 6 months of 
the study, one of the nuclear 
medicine physicians will conduct the 
medical exam prior to any study 
procedures. 
4. For patients highly suspected to 
have acute GVHD and requiring 
systemic therapy only: Taking 
steroid treatment for suspected 
acute GVHD for 7 days or less. 
 
 
      
5. For patients at high risk for 
developing acute GVHD only: 
Recipients of myeloablative or 
reduced intensity allogeneic 
transplants using either bone 
marrow or peripheral blood stem 
cells from HLA-matched or HLA-
mismatched related or unrelated 
donors (protocols 9917, 9142, 9022, 
9924) who have not yet been placed 
on any therapy for acute GVHD. 
 
 
      
Exclusion Criteria 
(From IRB approved protocol) 
 
1. Pregnant or nursing  
 
 
      
2. Individuals with known or suspected 
substance abuse, obtained by self-
reporting. 
 
 
      
3. Uncontrolled infection 
 
 
      
4. Relapsed/persistent malignancy 
 
 
      
5. Currently receiving immunotherapy 
 
 
      
 
IV.  Statement of Eligibility 
This subject is [ 
 eligible / 
 ineligible ] for participation in the study. 
Signature: 
Date: 
Printed Name: