Programmed Death Ligand 1 (PD-L1)-PET Imaging in Patients With (Diffuse) Large B-cell Lymphoma Who Are Treated With CD19-directed CAR T-cell Therapy: a Pilot Study
Phase: PHASE2
Conditions: Large B-cell Lymphoma
Sponsor: University Medical Center Groningen

 
 
 
 
 
 
 
 
 
 
Programmed death ligand 1 (PD-L1)-PET imaging in patients with (Diffuse) Large B-
cell Lymphoma who are treated with CD19-directed CAR T-cell therapy: a pilot study.   
12-01-2022 
 
 

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PROTOCOL TITLE:  
Programmed death ligand 1 (PD-L1)-PET/CT imaging  in patients with (Diffuse) Large B-cell 
Lymphoma who are treated with CD19-directed CAR T-cell therapy.  
Protocol ID 
202100663 
Short title 
PD-L1 PET imaging in the setting of CAR T-cell therapy  
EudraCT number  
2021-005192-39 
Version 
2.0 
Date 
12-01-2022 
Principal investigator 
 
T van Meerten, MD, PhD  
Department of Hematology, UMCG 
E-mail: t.van.meerten@umcg.nl 
Coordinating investigator 
 
 J.W. de Boer, MD 
Department of Hematology, UMCG 
E-mail: j.w.de.boer@umcg.nl 
Sponsor  
University Medical Center Groningen 
Hanzeplein 1 
9713 GB Groningen 
The Netherlands 
Subsidizing party 
 
Independent expert  
A. Rutgers, MD, PhD 
Department 
of 
Rheumatology 
and 
Clinical 
Immunology, UMCG 

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E-mail: a.rutgers@umcg.nl 
Laboratory sites 
UMCG 
Co-investigators 
Department of Hematology 
J.A. van Doesum, MD 
M. Nijland, MD, PhD 
Department of Medical Oncology 
E.G.E. de Vries, MD, PhD 
L. Kist de Ruiter, MD 
Danique Giesen, PharmD 
Department of Radiation Oncology 
A.G.H. Niezink, MD 
Nuclear Medicine Department 
W. Noordzij, MD, PhD 
Clinical Pharmacy and Pharmacology 
M. Lub – de Hooge, PharmD 

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PROTOCOL SIGNATURE SHEET 
 
 
 
 

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TABLE OF CONTENTS 
1. 
SUMMARY 
12 
1.1 
Rationale: 
12 
1.2 
Objectives: 
13 
1.3 
Study design: 
15 
1.4 
Study population: 
15 
1.5 
Intervention: 
15 
1.6 
Main study parameters/endpoints: 
16 
1.7 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: 
17 
2. 
INTRODUCTION AND RATIONALE 
19 
2.1 
Disease background: 
19 
2.2 
Anti-CD19 CAR T-cell therapy, principle 
19 
2.3 
CD19-directed CAR T-cell therapy, results 
20 
2.4 
Bridging therapy 
20 
2.5 
Mechanisms of CAR T-cell therapy failure 
21 
2.6 
Rationale for molecular imaging 
23 
2.7 
Anti-PD-L1 PET imaging 
23 
3. 
OBJECTIVES 
25 
4. 
STUDY DESIGN 
27 
4.1 
CAR T-cell therapy standard of care 
27 
4.2 
89Zr-atezolizumab PET/CT Imaging 
28 
4.3 
CAR T-cell expansion in peripheral blood 
29 
4.4 
Tumor Biopsy 
29 
5. 
STUDY POPULATION 
31 
5.1 
Population 
31 
5.2 
Inclusion criteria 
31 
5.3 
Exclusion criteria 
32 
5.4 
Sample size calculation 
33 
6. 
TREATMENT OF SUBJECTS 
34 
6.1 
Investigational treatment 
34 
6.2 
CAR -T-cell therapy 
34 

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6.3 
Use of co-intervention 
35 
6.4 
Escape medication 
35 
7. 
INVESTIGATIONAL PRODUCT 
36 
7.1 
Name and description of investigational product(s) 
36 
7.2 
Summary of findings from non-clinical studies 
36 
7.3 
Summary of findings from clinical studies 
37 
7.4 
Summary of known and potential risks and benefits 
38 
7.5 
Description and justification of route of administration and dosage 
40 
7.6 
Dosages, dosage modifications and method of administration 
40 
7.7 
Preparation and labeling of Investigational Medicinal Product 
40 
7.8 
Drug accountability 
41 
8. 
NON-INVESTIGATIONAL PRODUCT 
42 
9. 
METHODS 
44 
9.1 
Study parameters/endpoints 
44 
9.2 
Main study parameters/endpoints 
44 
9.3 
Secondary study parameters/endpoints 
44 
9.4 
Randomization, blinding and treatment allocation 
45 
9.5 
Study procedures 
46 
9.6 
Screening 
46 
9.7 
Assessment of the medical history and demographics 
46 
9.8 
Physical examination and vital signs 
47 
9.9 
Laboratory assessments 
47 
9.10 
Tumor evaluation 
48 
9.11 
Cardiac and pulmonary function tests 
48 
9.12 
89Zr-atezolizumab injection 
49 
9.13 
89Zr-atezolizumab PET/CT procedure 
49 
9.14 
Venous blood sampling 
50 
9.15 
Biopsy 
50 
9.16 
End of study 
51 
9.17 
Molecular analysis 
51 
9.18 
Tumor tissue analysis 
52 
9.19 
Withdrawal of individual subjects 
53 

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9.20 
Specific criteria for withdrawal 
53 
9.21 
Replacement of individual subjects after withdrawal 
53 
9.22 
Follow-up of subjects withdrawn from the study 
54 
9.23 
Premature termination of the study 
54 
10. 
SAFETY REPORTING 
55 
10.1 
Section 10 WMO event 
55 
10.2 
AEs, SAEs and SUSARs 
55 
10.2.1 
Adverse events (AEs) 
55 
10.2.2 
Serious adverse events (SAEs) 
55 
10.2.3 
Suspected unexpected serious adverse reactions (SUSARs) 
57 
10.2.4 
Adverse events of special interest 
58 
10.3 
Annual safety report 
58 
10.4 
Follow-up of adverse events 
59 
10.5 
Data Safety Monitoring Board (DSMB)/Safety Committee 
59 
11. 
STATISTICAL ANALYSIS 
60 
11.1 
Primary study parameters 
60 
11.1.1 
Measurement of 89Zr-atezolizumab uptake 
61 
11.1.2 
Methods of assessments (Lugano Criteria (20)) 
62 
11.1.3 
General definitions 
62 
11.1.4 
Complete Response 
64 
11.1.5 
Partial Response 
66 
11.1.6 
Stable Disease 
67 
11.1.7 
Progression and relapse 
68 
11.1.8 
Endpoint definitions 
69 
11.2 
Secondary study parameter(s) 
71 
11.2.1 
Correlative expression analysis between 89Zr-atezolizumab-distribution and CAR T-
cell peak expansion and persistence 
71 
11.2.2 
Correlative expression analysis between 89Zr-atezolizumab uptake and CAR T-cell 
therapy related grade 1-5 adverse events (cytokine release syndrome and immune effector 
cell associated neurotoxicity syndrome). 
71 
11.2.3 
Correlation of tumor 89Zr-atezolizumab uptake and tumor cell or tumor infiltrating cell 
PD-L1-expression as assessed by a fresh contemporaneous tumor biopsy 
76 

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11.2.4 
Comparison of 89Zr-atezolizumab distribution in irradiated vs non-irradiated 
lymphoma lesions 
77 
11.2.5 
Determination of the incidence of a treatment-related inflammatory reaction 
(histiocytic/sarcoid-like reaction) after CAR T-cell therapy . 
77 
12. 
ETHICAL CONSIDERATIONS 
78 
12.1 
Regulation statement 
78 
12.2 
Recruitment and consent 
78 
12.3 
Objection by minors or incapacitated subjects 
79 
12.4 
Benefits and risks assessment, group relatedness 
79 
12.5 
Compensation for injury 
82 
12.6 
Incentives 
83 
13. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
84 
13.1 
Handling and storage of data and documents 
84 
13.2 
Monitoring and Quality Assurance 
85 
13.3 
Auditing and inspections 
85 
13.4 
Amendments 
85 
13.5 
Annual progress report 
86 
13.6 
End of study report 
86 
13.7 
Public disclosure and publication policy 
87 
14. 
STRUCTURED RISK ANALYSIS 
88 
14.1 
Potential issues of concern 
88 
14.2 
Synthesis 
90 
15. 
APPENDIX 
91 
15.1 
Lymphoma biopsy: Safety 
91 
15.2 
Adverse Event Severity Grading Scale 
94 
15.3 
Timeline and procedures 
96 
15.4 
CAR T-cell inclusion criteria 
100 
16. 
REFERENCES 
104 
 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
89Zr 
Zirconium-89 
ALK 
Anaplastic Lymphoma Kinase 
ALT 
Alanine aminotransferase 
ANA 
Antinuclear antibodies 
ANC 
Absolute neutrophil count 
ANCA 
Anti-neutrophil cytoplasmic antibody 
anti-dsDNA 
anti-double stranded DNA 
AUC 
Area under the concentration-time curve 
aPTT 
Activated partial thromboplastin time 
AST 
Aspartate aminotransferase 
BA CRO 
Bioanalytical contract research organization. 
BUN 
Blood urea nitrogen 
CBC 
Complete blood count 
CD 
Cluster of differentiation 
Cmax 
Maximum serum concentration 
CNS 
Central nervous system 
CPET 
Serum concentration at the time of the 89Zr-atezolizumab-PET scan 
(LD-)CT 
(Low dose-) Computed Tomography 
CTLA4 
cytotoxic T-lymphocyte antigen-4 
CTCAE 
Common Terminology Criteria for Adverse Events 
DNA 
Deoxyribonucleic acid 

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DSMB 
Data Safety Monitoring Board 
ECG 
Electrocardiogram 
ECOG 
Eastern Cooperative Oncology Group 
EGFR 
Epidermal Growth Factor Receptor 
EudraCT 
European drug regulatory affairs Clinical Trials  
HBcAb 
Hepatitis B core antibody 
HBsAg 
Hepatitis B surface antigen 
HBV 
Hepatitis B virus 
HCV 
Hepatitis C virus 
HER2 
Human epidermal growth factor receptor 2 
HIV 
Human immunodeficiency virus 
IC 
Immune checkpoint inhibitor 
IHC 
Immunohistochemistry  
IMPD 
Investigational Medicinal Product Dossier  
INR 
irAE 
IST 
International normalized ratio 
Immune related adverse event 
Investigator sponsored trial 
LDH 
Lactate dehydrogenase 
METC  
Medical research ethics committee (MREC); in Dutch: medisch ethische 
toetsing commissie (METC) 
MRI 
Magnetic Resonance Imaging 
NCI 
National Cancer Institute 
NSCLC 
Non-small-cell lung cancer 

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ORR 
PBMCs 
Overall response rate 
Peripheral blood mononuclear cells 
PD 
Pharmacodynamics 
PD-1 
Programmed cell death protein 1 
PD-L1 
Programmed death-ligand 1 
PET 
Positron emission tomography 
PK 
Pharmacokinetics 
RBC 
Red blood count 
RNA 
Ribonucleic acid 
(S)AE 
SST 
(Serious) Adverse Event  
Serum separator tube 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
SUV  
Standardized uptake value 
Tmax 
Time to reach maximum serum concentration 
TSH 
Thyroid-stimulating hormone 
ULN 
Upper limit of normal 
WBC 
WFI 
White blood count 
Water for Injection 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 

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1. SUMMARY 
 
1.1 Rationale:  
Anti-CD19 Chimeric Antigen Receptor (CAR)-T cell therapy has changed the treatment 
landscape of patients with (Diffuse) Large B-cell Lymphoma (LBCL) and other types of Non-
Hodgkin Lymphoma (NHL). Patients with LBCL who do not respond to first-line therapy, have 
a relapse within 6 months (primary refractory), or after second-line therapy, including high-
dose chemotherapy and autologous stem cell rescue, have a poor prognosis and only 6% of 
these patients have a long-term survival. The recent results of 3 pivotal studies with 3 
different anti-CD19 CAR T-cell products administered to patients with relapsed/refractory 
(R/R) LBCL resulted in high response rates and long-term remissions in almost half of the 
patients. Unfortunately, some patients do not respond to CAR T-cell therapy and their 
prognosis is extremely poor. 
Possible mechanisms of non-responsiveness include loss of CD19 expression on the tumor 
cells, upregulation of immune checkpoint proteins such as programmed death-ligand 1 (PD-
L1, CD274) or impaired T-cell function due to an immunosuppressive microenvironment. 
However the role each of these mechanisms play in the case of a diminished or non-
response to CAR T-cell therapy is not yet defined.   
Engagement of the PD-1/PD-L1 pathway is known to result in the reduction of T-cell 
activation, proliferation, and survival. Moreover, PD-L1 expression can be altered by 
radiotherapy, used as bridging therapy in CAR T-cell therapy. Hypothetically, patients with 
high or upregulated PD-L1 expression are more likely to be non-responders to CAR T-cell 
therapy and these patients might benefit from additional PD-1/PDL-1 blocking therapy. 
However, the exact role of PD-L1 expression in CAR T-cell therapy is not yet defined.  

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Currently, predicting tumor PD-L1 expression is not optimal, because it requires invasive 
sequential biopsies that are often subject to the errors and limitations of invasive tissue 
collection. Recently, positron-emission tomography computed topography (PET/CT) imaging 
with 89Zr-atezolizumab (formerly known as 89Zr-MPDL3280A), a PET-labeled antibody against 
PD-L1, was found to correlate better with response to immune checkpoint inhibition than 
immunohistochemistry- or RNA-sequencing-based predictive biomarkers in different solid 
tumors. By performing a 89Zr-atezolizumab-PET/CT scan prior to CD19-directed CAR T-cell 
therapy PD-L1 expression could be defined in a non-invasive manner.   
Additionally, repeated 89Zr-atezolizumab-PET/CT scan in the case of a suspected relapse or 
non-response might distinguish between lymphoma activity and a treatment-related 
inflammatory reaction without the need to take invasive biopsies. Fluor-18-deoxyglucose 
(18F-FDG)-PET/CT scans are currently used for the diagnosis, staging, and evaluation of 
response in patients with NHL. However, response assessment with 18F-FDG-PET/CT may 
regularly be false-positive as a result of pseudo-progression or local immune activation 
(histiocytic or sarcoid-like reactions). Most often these reactions are mediated by 
macrophages/histiocytes which are known to express PD-L1 at high levels on their cell 
surface compared to lower levels on malignant B-cells. Therefore, a 89Zr-atezolizumab could 
be used to differentiate between a relapse/non-response or a treatment-related 
inflammatory reaction.  
 
1.2 Objectives:  
Primary objectives: The first primary objective is to study the expression of PD-L1  in normal 
tissue and lymphoma lesions before CAR T-cell therapy in LBCL patients by 89Zr-atezolizumab 
PET/CT imaging and to correlate the pretreatment 89Zr-atezolizumab distribution to the 

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response to CD19-directed CAR T-cell therapy thereby identify clinically relevant PD-L1 
expression.  
The second primary objective is to study whether the amount of 89Zr-atezolizumab uptake, 
measured by the intensity of 89Zr-atezolizumab PET/CT imaging (SUV),  can be used to 
differentiate between lymphoma activity and a treatment-related inflammatory reaction 
(histiocytic/sarcoid-like reaction) in patients with an end-of-treatment 18F-FDG-positive 
PET/CT signal.  
Secondary objectives:  
a) To correlate the pretreatment 89Zr-atezolizumab distribution to CAR T-cell peak expansion 
and persistence.  
b) To correlate the pretreatment 89Zr-atezolizumab uptake to CAR T-cell therapy related 
grade 1-5 adverse events (cytokine release syndrome (CRS) and immune effector cell 
associated neurotoxicity (ICANS)).  
c) To correlate tumor 89Zr-atezolizumab uptake with tumor and immune cell PD-L1-
expression as assessed by immunohistochemistry on a fresh contemporaneous tumor 
biopsy.  
d) To compare the 89Zr-atezolizumab distribution in irradiated versus non-irradiated 
lymphoma lesions in patients who require radiotherapy as a bridging strategy prior to CAR T-
cell infusion. If possible, these results will be compared to tumor and immune cell PD-L1 
expression as assessed by immunohistochemistry on a fresh contemporaneous tumor biopsy 
of an irradiated lymphoma lesion. 

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e) To determine the incidence of a treatment-related inflammatory signal on 18F-FDG-PET/CT 
scan (histiocytic/sarcoid-like reaction) after CAR T-cell therapy.  
 
1.3 Study design: 
This is a single-center, single-arm pilot trial designed to evaluate the expression of PD-L1 in 
patients with LBCL and its role in non-responsiveness to CAR T-cell therapy in a non-
invasive manner. Moreover, we aim to study the possibility of PD-L1 PET/CT imaging to 
distinguish between lymphoma activity and a treatment-related inflammatory signal 
(histiocytic/sarcoid-like reaction) in case of a relapse or non-response after CAR T-cell 
therapy.  
 
1.4 Study population: 
Patients with relapsed/refractory LBCL after 2 prior lines of therapy and who qualify for 
CD19-directed CAR T-cell therapy. Eligibility for CAR T-cell therapy is based on the criteria 
posed by the Dutch Immune Effector Cell working group tumor board. 
 
1.5 Intervention:  
In this imaging trial, the purpose is to explore the feasibility of anti-PD-L1 PET/CT imaging in 
patients to gain insights into clinically relevant PD-L1 expression in the setting of CD19-
directed CAR T-cell therapy. Patients with R/R LBCL after 2 prior lines of therapy will receive 
standard of care CD19-directed CAR T-cell therapy according to the eligibility criteria as 
formed by the Dutch Immune Effector Cell tumor board. 
 

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1.6 Main study parameters/endpoints: 
Primary endpoints: 
- 
to study the expression of PD-L1 in normal tissue and lymphoma lesions before 
CD19-directed CAR T-cell therapy in LBCL patients by 89Zr-atezolizumab PET/CT 
imaging and to correlate pretreatment 89Zr-atezolizumab uptake to response to 
CD19-directed CAR T-cell  therapy and thereby identify clinically relevant PD-L1 
expression. Heterogeneity of 
89Zr-atezolizumab uptake will be evaluated by 
measuring standardized uptake value (SUV) on the 89Zr-atezolizumab PET/CT scan. 
- 
To study whether the amount of 89Zr-atezolizumab uptake,  measured by the 
intensity of 89Zr-atezolizumab PET/CT imaging (SUV), can be used to differentiate 
between 
lymphoma activity 
and 
treatment-related 
inflammatory 
reaction 
(histiocytic/sarcoid-like reaction) in patients with an end-of-treatment 18F-FDG-
positive PET/CT signal.  
Secondary endpoints: 
-  To correlate the pretreatment 89Zr-atezolizumab distribution to CAR T-cell peak 
expansion and persistence. 
- To correlate the pretreatment 89Zr-atezolizumab  uptake to CAR T-cell therapy 
related grade 1-5 adverse events (cytokine release syndrome (CRS) and immune 
effector cell associated neurotoxicity syndrome (ICANS)).   
- To correlate tumor 89Zr-atezolizumab uptake with tumor and immune cell PD-L1 
expression as assessed by immunohistochemistry on a fresh contemporaneous 
tumor biopsy. 
- To compare the 89Zr-atezolizumab distribution in irradiated versus non-irradiated 

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lymphoma lesions in patients who require radiotherapy as a bridging strategy prior 
to CAR T-cell infusion. If possible, these results will be compared to tumor and 
immune cell PD-L1 expression as assessed by immunohistochemistry on a fresh 
contemporaneous tumor biopsy of an irradiated lymphoma lesion. 
- To determine the incidence of a treatment-related inflammatory signal on 18F-FDG-
PET/CT scan (histiocytic/sarcoid-like reaction) after CAR T-cell therapy. 
 
1.7 Nature and extent of the burden and risks associated with participation, 
benefit and group relatedness:  
For this imaging study, patients have to make a maximum of 6 extra visits to the clinic for 
screening, 89Zr-atezolizumab injection, a PET/CT-scan visit and a biopsy taken within 7 
days of the PET-scan visit. In case of an end-of-treatment positive 18F-FDG PET/CT signal 
two visits are needed for the second 89Zr-atezolizumab injection and the PET/CT-scan. The 
biopsy taken afterwards is part of standard procedure of care.  
In case of bridging with radiotherapy and depending on the accessibility of the tumor 
lesions, patients will be asked if they are willing to undergo an extra biopsy. This extra 
biopsy will not be obligatory to participate in the study.  In practice, most procedures will 
be combined with visits to the hospital in the context of clinical care, to minimize patient 
burden.  
The intravenous tracer injection 89Zr-atezolizumab is between day -19 to day -10 of the 
CAR T-cell infusion (depending on time needed for bridging strategy). All patients will be 
observed for at least 30 minutes after 89Zr-atezolizumab injection to monitor for possible 
acute infusion related adverse events. The subsequent 89Zr-atezolizumab PET/CT imaging 
scan is 4 or 7 days (day -15 to day -6). The PET/CT-imaging is followed by a biopsy within 7 

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days, but the biopsy will always take place before day -5. Optimal time point for the 89Zr-
atezolizumab-PET/CT-scan will be determined in the first 3 patients, as they will receive a 
89Zr-atezolizumab-PET/CT-scan on day 4 and 7.  
In case of an end-of-treatment 18F-FDG-positive PET/CT signal another 89Zr-atezolizumab 
injection will be given followed by a 89Zr-atezolizumab PET/CT imaging and a biopsy within 
7 days (standard procedure).  
The radiation burden following administration of 37 MBq of 89Zr-atezolizumab is 18.1 mSv, 
in addition to 1 mSv per low-dose attenuation correction CT-scan. Thus, patients will 
receive 37 or 74 MBq doses of 89Zr-atezolizumab and undergo to 1 or 2 low-dose CT-scans. 
The radiation exposure will be 19.1  mSv per imaging round. Besides PET/CT imaging, 
patients will be asked to provide 12 blood samples (108 mL), which are taken in 
combination with standard clinical and outpatient care. The easiest and safest accessible 
tumor lesion will be biopsied within 7 days of the 89Zr-atezolizumab PET/CT scans. Based 
on a literature review, the risk of tumor biopsies is considered low with a small risk of 
significant or major complications or death. The risk associated with the 89Zr-atezolizumab 
is considered acceptable based on extensive preclinical testing of separate components 
and clinical safety data from the first-in-human 89Zr-atezolizumab-study where only one 
low-grade adverse event (pruritus, grade 1) has been reported. Although patients do not 
directly benefit from this study, results from this study will be valuable for our 
understanding of the tumor immune response and will guide further prospective research 
and hopefully, treatment decisions. After participation within the imaging trial, eligible 
patients will proceed with CD19-directed CAR T-cell treatment, provided they continue to 
meet the eligibility criteria to receive CD19-directed CAR T-cells. 
 
 

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2. INTRODUCTION AND RATIONALE 
 
2.1 Disease background: 
(Diffuse) Large B-cell Lymphoma (LBCL) is an aggressive cancer and the most common B-cell 
Non-Hodgkin Lymphoma (NHL) and if left untreated, fatal within months. Patients with LBCL 
who do not respond to first-line therapy, have a relapse within 6 months (primary 
refractory), or after second-line therapy, including high-dose chemotherapy and autologous 
stem cell rescue, have a poor prognosis with long term survival in only 6% (1). 
2.2 Anti-CD19 CAR T-cell therapy, principle 
CD19-directed Chimeric Antigen Receptor (CAR) T-cell therapy is a novel cellular treatment 
approach that has been studied in patients with relapsed/refractory LBCL. CD19-directed 
CAR T-cells are generated from autologous T-cells collected during apheresis during a multi-
step process. The collected white blood cells will be enriched for T-cells and via viral 
transduction the genomic code for the anti-CD19-CAR and a costimulatory domain, CD28, 
will be introduced into the T-cells. Subsequently, these generated CD19-directed CAR T-cells 
are multiplied, cryopreserved and shipped to the treatment site, the place where the cells 
are infused, in a single infusion, into the patient. The process of collection of cells, the 
genetic modification, expansion, quality check, transports, and finally, the infusion of cells 
may take up to 4 weeks (2). 
Prior to CAR T-cell infusion (day 0), the patients will receive a lympho-depleting 
chemotherapy regimen (fludarabine-cyclophosphamide) to create a more favorable 
environment for the CAR T-cells and to minimize the risk of premature CAR T-cell elimination 
by the endogenous T-cells (day -5 to day -3). Thus, after CAR T-cell infusion, the expansion of 
T-cells is considered to be the expansion of only CAR T-cells (3). 

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2.3 CD19-directed CAR T-cell therapy, results 
Recently, the results of 3 pivotal studies with 3 different CD19-directed CAR T-cell products 
administered to patients with relapsed/refractory DLBCL were presented. Patients received 
a single injection of CAR T-cells, and all studies reported long-term remissions and disease-
free survival for more than 2 years in about half of the patients (2, 4, 5). For example, in the 
ZUMA-1 study, an infusion of 2×106 CD19-directed CAR T-cells per kg of body weight 
resulted in a complete response (CR) rate of 58%. Of those who achieved a CR at month 3, 
72% have a progression-free survival (PFS) of more than 2-years (2, 6). This milestone is of 
high importance, as the 2-year PFS in this disease is a surrogate for the overall survival (7). 
2.4 Bridging therapy  
As the process of CAR T-cell manufacturing and infusion may take up to 4 weeks some 
patients (with highly aggressive disease or bulky masses) might require bridging therapy to 
be able to receive the CAR T-cells. Radiation therapy is an acknowledged approach as a 
bridge to CAR T-cell therapy (8, 9). In general, after apheresis, patients may receive 
radiotherapy (usually 20Gy in 5 fractions) to reduce total tumor burden, on bulky or 
symptomatic sites, or highly proliferative/progressive lesions (near vital organs), while 
preserving lymphoma lesions that are not directly necessary to irradiate. In this way, the 
remaining lymphoma lesions will ascertain the necessary antigen exposure for CAR T-cell 
activation and proliferation.  In case of clinical need, there might be a possibility that a 
patient will be bridged with systemic therapy like chemotherapy or steroids or a 
combination of systemic therapy and radiotherapy.  

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2.5 Mechanisms of CAR T-cell therapy failure 
Unfortunately, half of the patients do not respond to CD19-directed CAR T-cell therapy and 
their prognosis is extremely poor. (10) Several mechanisms of resistance to CAR T-cell 
therapy have been identified, namely loss of CD19 expression, impaired T-cell function due 
to lost death receptor signaling and a T-cell unattractive tumor microenvironment related to 
M2 macrophages. (11,12,13) 
 In addition, the existence of a number of immunosuppressive pathways can restrict the full 
potential of CAR T-cell therapy,as increased expression of these pathways can limit the 
duration and strength of the adaptive T-cell response(14, 15, 16) This includes increased 
expression of inhibitory immune receptors following T-cell activation such as T-cell 
membrane protein-3 (TIM-3), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and/or 
programmed cell death-1 (PD-1) or its ligand programmed cell death ligand 1 (PD-L1) on T-
cells. Moreover, PD-L1 expression can be altered by radiotherapy, nowadays used as 
bridging strategy before CAR T-cell therapy, through the mechanism of enhanced interferon 
signaling of CD8+ T-cells. (17) However, the role of the PD-1/PD-L1 axis in CAR T-cell therapy 
is not yet defined.  
In general, the PD-1/PD-L1 axis is upregulated on many different tumor types. Blocking this 
pathway using antibodies and thereby restoring natural immune response against cancer 
cells  has shown to be a promising therapy in patients with advanced melanoma, non-small 
cell lung, renal cell, as well as hematologic malignancies. (18, 19, 20)  Response to blockade 
therapy and level of PD-L1 expression on tumor cells are significantly correlated. (21) 
Moreover, Zolov et al. recently demonstrated that the upregulation of the PD-1/PD-L1 axis 
leads to diminished cytokine production and proliferative capacity of CD28-costimulated 

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CAR T-cells. Thereby emphasizing the possible contribution of the PD-1/PD-L1 pathway to 
unresponsiveness to CAR T-cell therapy. (22) 
To identify patients who are likely to benefit from PD-L1 blockade therapy accurate 
diagnostics of expression levels are needed. Nowadays immunohistochemistry is used to 
quantify these levels, but unfortunately it is subjected to errors and limitations of invasive 
tissue collection.   
Positron-emission tomography computed tomography (PET/CT) imaging with Zirconium-89- 
labeled anti-PD-L1 antibodies (89Zr-atezolizumab) is a non-invasive approach that might 
identify patients with R/R DLBCL with clinically relevant PD-L1 expression. (23) 
In addition to the identification of patients with relevant PD-L1 expression, PET/CT PD-L1 
imaging might also identify patients with false positive 18F-FDG PET scans after CAR T-cell 
therapy. 18F-FDG-PET/CT scans are the gold standard for staging and response assessment in 
DLBCL, but 18F-FDG-PET/CT scans are inaccurate in differentiating between lymphoma 
activity or a treatment-related inflammatory reaction (histiocytic/sarcoid-like reaction), as 
these nonmalignant events also consume high levels of glucose. Evaluation of biopsies taken 
from 18F-FDG-avid lesions of two patients did not show lymphoma activity, but inflammatory 
changes compatible with a histocytic/sarcoid-like reaction and demonstrate a change to very 
high and intense PD-L1 staining compared to pre-CAR T-cell therapy biopsies. (data not 
published). Therefore, 89Zr-atezolizumab PET/CT imaging could be used to distinguish 
between 
lymphoma 
activity 
or 
a 
treatment-related 
inflammatory 
reaction 
(histiocytic/sarcoid-like reaction).  

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2.6 Rationale for molecular imaging 
As mentioned above, accurate quantification of PD-L1 expression can identify patients who 
will benefit from blockade therapy during CAR T-cell therapy and patients who develop a 
treatment-related inflammatory reaction (histiocytic/sarcoid-like reaction) after CAR T-cell 
therapy.  
However, the approved diagnostics are revealed to be inaccurate for predicting response to 
blockade therapy and require an invasive biopsy to perform quantification. In several cases 
lesions are not applicable for biopsies, causing a gap of essential information in developing 
an effective treatment strategy.  
Recently, pre-treatment PD-L1 PET/CT imaging with 89Zr-atezolizumab was found to better 
correlate with response than immunohistochemistry-or RNA-sequencing-based predictive 
biomarkers in different solid tumors. (23) Moreover, molecular imaging may be able to 
noninvasively monitor whole-body systemic and intratumoral PD-L1 expression serially 
before and during therapy, preventing misinterpretation due to tumor heterogeneity or 
sampling error. This would be an essential step in quantification of PD-L1 expression in a 
noninvasive manner, increasing the potential of identifying patients with a lower to non-
response to CAR T-cell therapy due to PD-L1 expression and being able to provide this 
patient blockade therapy. Moreover, this imaging approach could be able to distinguish 
malignant tissue from inflammatory tissue and reduce the frequency of biopsies.  
2.7 Anti-PD-L1 PET imaging 
89Zr-atezolizumab constitutes of the monoclonal anti-PD-L1 antibody atezolizumab labeled 
with the positron-emitter Zirconium-89, allowing whole-body PET-imaging of PD-L1 
expression. The separate components Zirconium-89 (for tumor PET-imaging) and 

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atezolizumab (in higher dosage for the treatment of several cancers) have already been used 
in various studies and regular health care. 
A first-in-human study showed safe injection of 89Zr-atezolizumab, with only one related 
low-grade adverse event (pruritus, grade 1). The optimal tracer protein dosage was 
determined to be 10 mg (unlabeled atezolizumab in combination with 2,5 mg (37 MBq) of 
89Zr-atezolizumab), corresponding with a serum atezolizumab dosage 100-fold lower than 
reached with the recommend treatment dosage. Optimal imaging time points are 4 and 7 
days after tracer injection.  
Evaluation of 89Zr-atezolizumab biodistribution showed high uptake in intestines, liver and 
kidney, reflecting antibody metabolism and elimination. Moreover, lymphoid tissue, 
including the spleen, uptake was high. All tumor localizations of non-small cell lung cancer, 
triple-negative breast cancer and bladder cancer showed evident uptake of 
89Zr-
atezolizumab. (23) 
 
 

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3. OBJECTIVES 
 
4.1 Primary objective:  
• 
to study the expression of PD-L1 in normal tissue and lymphoma lesions before CD19-
directed CAR T-cell therapy in LBCL patients by 89Zr-atezolizumab PET/CT imaging and 
to correlate pretreatment 89Zr-atezolizumab uptake to response to CD19-directed 
CAR T-cell  therapy and thereby identify clinically relevant PD-L1 expression. 
Heterogeneity of 
89Zr-atezolizumab uptake will be evaluated by measuring 
standardized uptake value (SUV) on the 89Zr-atezolizumab PET/CT scan. 
• to study whether the amount of 89Zr-atezolizumab uptake, measured by the intensity 
of 89Zr-atezolizumab PET/CT imaging (SUV), can be used to differentiate lymphoma 
activity and treatment-related inflammatory signal (histiocytic/sarcoid-like reaction) 
in patients with an end-of-treatment 18F-FDG-positive PET/CT signal. Confirmation of 
the presence of treatment-related inflammatory reaction (histiocytic/sarcoid-like 
reaction) will be determined by an experienced pathologist on a fresh 
contemporaneous biopsy of the positive FDG-PET lesion or in case of non-
accessibility of the lesion by an experienced clinician via a wait-and-see strategy with 
repeated FDG-PET scans.  
  
4.2 Secondary Objectives 
• to correlate the pretreatment 89Zr-atezolizumab distribution, as measured by the 
uptake of 89Zr-atezolizumab on PET/CT imaging in lymphoma, to CAR T-cell peak 
expansion and persistence as measured in peripheral blood. 
• To correlate the pretreatment 89Zr-atezolizumab uptake to CAR T-cell therapy related 

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grade 1-5 adverse events (cytokine release syndrome (CRS) and immune effector cell 
associated neurotoxicity syndrome (ICANS)). Heterogeneity of 89Zr-atezolizumab 
uptake will be evaluated by measuring SUV on the 89Zr-atezolizumab PET/CT scan. 
CAR-T cell therapy related toxicities such as CRS and ICANS will be assessed using 
2018 ASBMT Consensus Grading (24).  
•  to correlate tumor 89Zr-atezolizumab uptake with tumor and tumor infiltrating cell 
PD-L1 expression as assessed by immunohistochemistry on a fresh contemporaneous 
tumor biopsy.  
• To compare the 89Zr-atezolizumab distribution in irradiated versus non-irradiated 
lymphoma lesions in patients who require radiotherapy as a bridging strategy prior to 
CAR T-cell infusion. If possible, these results will be compared to tumor and tumor 
infiltrating cell PD-L1 expression as assessed by immunohistochemistry on a fresh 
contemporaneous tumor biopsy of an irradiated lymphoma lesion. 
• To determine the incidence of a treatment-related inflammatory reactions 
(histiocytic/sarcoid-like reaction) after CAR T-cell therapy.  
 

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4. STUDY DESIGN  
 
4.1 CAR T-cell therapy standard of care 
The treatment of patients with a relapse or refractory DLBCL after 2 prior lines of therapy 
with CAR T-cell therapy (eg axicabtagene ciloleucel; Yescarta) is considered to be standard of 
care. The infusion of CAR T-cells is defined as Day 0. Therefore, apheresis is performed Day -
28, as the manufacturing, quality controls and logistics of the autologous CAR T-cells will 
take up to 28 days. In case patients have bulky, symptomatic and/or high proliferative 
disease, debulking/bridging therapy (radiotherapy, systemic therapy (e.g. chemotherapy or 
steroids) or a combination) is allowed after apheresis (Day -27 to Day -10). Prior to lympho-
depleting chemotherapy with fludarabine and cyclophosphamide (Day -5 to Day -3), a 
standard 18F-FDG PET/CT scan will be performed to determine the active disease status prior 
to the CAR T-cell therapy. Standard 18F-FDG PET/CT response assessment will be performed 
at Months +1, +3, +6, and +12. 
 
Figure 1. Overview of standard CAR T-cell therapy and the study procedures. 

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4.2 
89Zr-atezolizumab PET/CT Imaging 
The study will include ~20 patients that will be imaged with 89Zr-atezolizumab PET/CT 
imaging. Patients will be imaged with 89Zr-atezolizumab PET/CT imaging before CAR T-cell 
therapy (see figure 1). Patients who do have an end-of-treatment 18F-FDG-positive signal will 
undergo a second 89Zr-atezolizumab PET/CT scan.  
In order to determine the PD-L1 expression, we first inject the tracer consisting of 10mg 
total protein tracer dosage (37 MBq (1 mCi)/~2,5mg  89Zr-atezolizumab and unlabeled 
atezolizumab) at day -19 to day -10 (depending on time needed for optional bridging 
therapy) subsequently the whole-body 89Zr-atezolizumab PET scan will be acquired at day -
15 to day -6 ((4 or 7 days after tracer injection). The optimal time point of PET imaging scan 
4 or 7 days after tracer injection will be determined in the first 3 patients. 
SUVmax and SUVpeak (on tumor lesion) and the geometric SUVmax, SUVpeak (sum of all  
tumor samples) will be determined by using spherical VOIs with predefined sizes. 
Within 7 days of the 89Zr-atezolizumab PET/CT scan but no later than day -6, a tumor biopsy 
will be obtained. For revealing tracer distribution at the microscopic level, formalin-fixed 
tumor sections (10 µM) of post-tracer tumor biopsies will be exposed to a phosphorimaging 
screen for 24 hr and will be scanned with a Cyclone phosphorimager. Subsequent sections of 
the same tumor tissue will be stained for hematoxylin and eosin, PD-L1, and T-cell and 
macrophage markers. 
Safety is assessed according to CTCAEv5.0, CRS and ICANS grading will be evaluated 
according to the ASBMT criteria (24). CAR T-cell treatment response is assessed based on the 

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Lugano criteria (with 18F-FDG-PET CT scans) at day 28, month 3, month 6, and at month 12 
(standard procedure).    
4.3 CAR T-cell expansion in peripheral blood  
In addition, in these patients, CAR T-cell expansion in peripheral blood will be measured with 
peak levels at day 5-7, and persistence at day 7, 14, 28 with an anti-CAR T-cell monoclonal 
antibody. T-cell subsets (CD4 / CD8 ratio, different T-cell phenotypes) will be determined on 
day 5. Cytokines (IL-2, IL-6, IL-10, IFN) will be measured in serum at days -28, 0, 5, 7, 14, and 
28 to determine the pro-inflammatory and immune-modulating cytokines in response to 
CAR T-cell therapy. Samples will be collected during venipuncture as part of the standard 
procedure,  thereby minimizing patient burden.  
4.4 Tumor Biopsy  
As mentioned above tumor biopsies will be taken within 7 days of the 89Zr-atezolizumab 
PET/CT scan. Moreover, in case of bridging with radiotherapy and depending on the 
accessibility of the tumor lesion, patients will be asked if they are willing to undergo an extra 
biopsy. This biopsy will be used to compare 89Zr-atezolizumab uptake in a irradiated lesion 
on PET-imaging to PD-L1 expression measured via immunohistochemistry. This extra biopsy 
will not be obligatory to participate in the study.   
PD-L1 expression in post-tracer tumor biopsies will be assessed centrally. We will evaluate 
PD-L1 staining on tumor cells (TC) and on tumor infiltrating immune cells (IC). PD-L1 
expression will be scored as negative (IC0 or TC0: staining on <1% of IC or TC, respectively; 
IHC score 0) or positive (IC1/2/3 or TC1/2/3: staining on ≥1% of IC or TC; IHC score 1/2/3 
depending on the highest staining for either IC or TC). In addition, we will evaluate T-cell 
infiltration as measured by CD8 and CD4 expression and will determine the polarization of 
macrophages and histiocytes. RNA from post-tracer tumor biopsies will be isolated for gene 

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expression analysis by TruSeq RNA Access RNA-seq (Q2Labsolutions). 
Autoradiography of the biopsies will be performed using a phosphorimaging screen for 24 hr 
and will be then scanned with a Cyclone phosphorimager. 
 

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5. STUDY POPULATION 
 
5.1 Population 
Patients with R/R LBCL after two or more lines of therapy who fulfill the eligibility 
criteria for CD19-directed CAR T-cell therapy according the Immune Effector Cell 
Working Group Tumorboard. 
 
5.2 Inclusion criteria 
In order to be eligible to participate in this study, subject must meet the following: 
1. Histologically confirmed DLBCL and associated subtypes, defined by WHO 2016 
classification: DLBCL not otherwise specified (NOS), High-grade B-cell lymphoma 
with MYC and BCL2 and/or BCL6 rearrangements with DLBCL histology (DHL/THL) 
and FL3B, Aggressive B-cell lymphoma, T-cell/histiocyte rich B-cell lymphoma, 
Primary mediastinal B-cell lymphoma, EBV+ DLBCL, transformed lymphoma (e.g. 
transformed follicular or marginal zone lymphoma)  
2. Tumor lesion(s) of which a histological biopsy can safely be obtained according to 
standard clinical care procedures. 
3. Measurable disease, as defined by Lugano criteria (see section 11.1) 
4. If has history of CNS disease, then must have 
a. No signs or symptoms of CNS disease  
b. No active disease on magnetic resonance imaging (MRI)  
c. Absence of large cell lymphoma in cerebral spinal fluid (CSF) on cytospin 
preparation and flow cytometry, regardless of the number of white blood cells. 

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5.  If has history of cerebral vascular accident (CVA) 
a. The CVA event must be >12 months prior to apheresis 
b. Any neurological deficits must be stable 
6. Signed informed consent. 
7. Age ≥18 at the time of signing informed consent. 
8. Life expectancy ≥12 weeks. 
9. Eastern Cooperative Oncology Group (ECOG) performance status 0-1 
10. Ability to comply with the protocol. 
 
5.3 Exclusion criteria 
A potential subject, who meets any of the following criteria, will be excluded from 
participation in this study: 
1. Signs or symptoms of active infection within 2 weeks prior to 89Zr-atezolizumab 
injection, unless treated to resolution. 
2. Prior CD19-directed CAR T-cell therapy or other bi-specific antibodies targeting CD19 
receptor (e.g. blinatumomab). 
3. History of severe allergic, anaphylactic, or other hypersensitivity reactions to 
chimeric or humanized antibodies or fusion proteins. 
4. Any other diseases, metabolic dysfunction, physical examination finding, or clinical 
laboratory finding giving reasonable suspicion of a disease or condition that 
contraindicates the use of 89Zr-atezolizumab, or that may affect the interpretation 
of the results or render the patient at high risk from complications. 
 

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5.4 Sample size calculation 
This exploratory study will be a proof-of-concept, open-label, single-center study to explore 
the expression of PD-L1 in normal tissue and lymphoma lesions before CAR T-cell therapy in 
LBCL patients by 89Zr-atezolizumab PET/CT imaging and to correlate these expression levels 
to response to CAR T-cell therapy. Thereby this study aims to define clinically relevant PD-L1 
expression in CAR T-cell therapy. In addition, we will investigate the ability of 89Zr-
atezolizumab PET-imaging to differentiate between lymphoma activity and a treatment-
related inflammatory reaction (histiocytic/sarcoid-like reaction) in case of a suspected 
relapse or non-response.  
All patients must have measurable disease at the start of apheresis as determined by 18F-
FDG PET/(diagnostic)CT scan. In case, radiation as bridging therapy is required, at least 1 
non-irradiated lesion must remain, according to the general practice of CAR T-cell therapy, 
which will allows for 89Zr-atezolizumab PET/CT imaging in both non-irradiated and irradiated 
lesions. 
Based on previous studies, we estimate that approximately 50% of the patients will have a 
CR upon CAR T-cell therapy, and the vast majority of the patients will reach this CR within 3 
months. Non-response or relapses will in general also occur within 3 months. We, therefore, 
consider inclusion of 20 patients (with ~10 relapses/non-responders) to yield sufficient 
information for this proof-of-concept study.  
With an expected accrual rate of 1 patient a month, the study could be completed within 2 
years. 
 

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6. TREATMENT OF SUBJECTS 
6.1 Investigational treatment 
For extensive information about the investigational product 89Zr-atezolizumab see IMPDs 
“IMPD 89Zr-atezolizumab, version 8”.  
In this study, patients will receive a defined dose of radioactivity of 37 MBq 89Zr-
atezolizumab and defined dose of approximately 10 mg of protein (sum of 89Zr-
atezolizumab and unlabeled atezolizumab). 
89Zr-atezolizumab conjugation and radiolabeling is performed in the radiopharmacy unit of 
the department of Nuclear Medicine and Molecular Imaging, University Medical Center 
Groningen. Previous results showed efficient radiolabeling with preservation of antigen-
binding capacity. No radioactive impurities were detected. For further details, see the IMPD 
of 89Zr-atezolizumab, version 8.  
The 89Zr-atezolizumab in combination with the unlabeled atezolizumab (total protein 
dosage 10mg, 37 MBq) will be injected as a continuous intravenous bolus injection followed 
by physiologic saline to flush the line (infusion rate will be comparable to the infusion rate 
of administration of a treatment of dose of atezolizumab (250ml/hr)). 
The 89Zr-atezolizumab PET/CT scan will be performed before CAR T-cell therapy using 2,5 
mg of 89Zr-atezolizumab (see Figure). In patients with a relapse or non-response after CAR 
T-cell therapy, a second 89Zr-atezolizumab PET/CT series will be performed in the same 
manner. 
 
6.2 CAR -T-cell therapy 
The treatment of patients with a relapse or refractory DLBCL after 2 prior lines of therapy 
with CAR T-cell therapy (e.g., axicabtagene ciloleucel, Yescarta). The infusion of CAR T cells is 

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defined as Day 0. Therefore, apheresis is performed Day -28, as the manufacturing, quality 
controls and logistics of the autologous CAR T-cells will take up to 28 days. In case patients 
have bulky and/or high proliferative disease, debulking/bridging therapy (radiotherapy, 
systemic therapy (eg chemotherapy or steroids) or a combination) is allowed after apheresis 
(Day -27 to Day -10). Prior to lymphodepleting chemotherapy with fludarabine and 
cyclophosphamide (Day -5 to Day -3), a standard 18F-FDG PET/CT scan will be performed to 
determine the active disease status prior to the CAR T-cell therapy. Standard 18F-FDG PET/CT 
response assessment will be performed at months +1, +3, +6, and +12. 
6.3 Use of co-intervention 
Female patients of childbearing potential and male patients with partners of childbearing 
potential must agree (by patient and/or partner) to use a highly effective form of 
contraception (e.g., surgical sterilization, a reliable barrier method, birth control pills, or 
contraceptive hormone implants) 
 
6.4 Escape medication 
Not applicable 
 

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7. INVESTIGATIONAL PRODUCT 
All information on the investigational product 89Zr-atezolizumab (89Zr-MPDL3280A) can 
be found in the investigational medicinal product dossier (IMPD) of 89Zr-atezolizumab : 
“IMPD [89Zr]-Atezolizumab, version 8”.  
7.1 Name and description of investigational product(s) 
In this Clinical Trial Application, 89Zr-atezolizumab will be administered in combination 
with unlabeled atezolizumab as an injection. The 89Zr-atezolizumab injection contains a 
maximum amount of 2.5 mg 
89Zr-atezolizumab, supplemented with unlabeled 
atezolizumab to obtain a total protein dose of 10 mg.  
Atezolizumab is a humanized IgG1 monoclonal antibody which targets human PD-L1 on 
antigen-presenting cells or tumor cells. It is provided as a solution of 60 mg/ml in 20 
mM Histidine Acetate, 120 mM Sucrose, 0.04% PS20, pH 5.8.  
89Zr-atezolizumab is a radiolabeled monoclonal antibody directed against PD-L1. [89Zr] 
is a positron emitter with a physical half-life of 3.27 days. The 89Zr-atezolizumab drug 
product is a clear, colorless liquid solution for iv administration which is free of visible 
particles. The product is sterile and free of endotoxins, with a pH of 6-7. The target 
radioactivity for 89Zr-atezolizumab is 37 MBq at injection time, with a targeted volume 
of 2.5 mg.  
 
7.2 Summary of findings from non-clinical studies 
No toxicology or pharmacokinetics studies are performed on 89Zr-atezolizumab based on 
abundant safety data available on the separate components. Several studies with 89Zr-
labeled antibody tracers have demonstrated safe use of the isotope. Safety studies have 
been performed on atezolizumab and showed safe use in patients.  It is recently 

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registered for the treatment of urothelial carcinoma and non-small cell lung cancer. A 
summary of non-clinical studies is provided below. For more details, refer to the IMPD of 
89Zr-atezolizumab.  
Binding studies of atezolizumab showed similar affinity to human and cynomolgus 
monkey T-cells. Therefore cynomolgus monkey was selected as primary and relevant 
nonclinical model. Furthermore, it was defined that atezolizumab binds to target with 
high affinity and completely blocks binding to receptors B7-1 (PD-L1) and PD-1.  
Toxicology studies of atezolizumab were perfomed in mice and cynomolgus monkeys. 
The 8-week in vivo toxicity and toxicokinetic evaluation in Cynomolgus monkeys did not 
result in any local or systemic adverse effects, including no effects on safety 
pharmacology or clinical, immunologic and anatomic pathology endpoints. 
In vitro internalization rates of 89Zr-atezolizumab were determined in two tumor cell lines 
and healthy volunteers peripheral blood mononuclear cells (PBMCs). Tumor cells showed 
a high internalization rate, whereas lower rates were found in human PBMCs and T-cells. 
(23) 
7.3 Summary of findings from clinical studies 
First-in-human study, performed in the UMCG, to assess the feasibility and biodistribution 
of imaging with 89Zr-atezolizumab included 25 patients. Tracer injection was safe, with 
only one related low-grade adverse event (pruritus, grade 1). The optimal tracer injection 
protocol was the combination of unlabeled atezolizumab administration with the 
radioactive labeled 89Zr-atezolizumab (total protein dosage 10 mg, 2.5 mg 89Zr-
atezolizumab (37 MBq)) , and the optimal moment for PET scanning was 4 or 7 days after 
tracer administration. Pharmacokinetic analysis confirmed  that the circulating tracer 

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dose corresponded with a serum concentration of atezolizumab reached with 0,1-0,3 
mg/kg atezolizumab, which is almost 100-fold lower than that reached with recommend 
treatment dose. Day 4 blood pool, liver and kidney 89Zr-atezolizumab uptake was 
comparable to the results of other 89Zr-labeled antibody tracers.  
The tracer showed high uptake in lymphoid tissue, at sites of inflammation and at all main 
metastatic sites. Low uptake was observed in the brain, subcutaneous tissue, muscle, 
compact bone and lung. No signs of toxicity were observed.  
Moreover, the internalization rates of 89Zr-atezolizumab in PBMCs of patients receiving 
89Zr-atezolizumab tracer injection were determined to be 0.3% and 0.4% on day 4 (n=2) 
and 0.6% on day 7(n=1).  
89Zr-atezolizumab tumor uptake, based on PET/CT imaging, was correlated to patient 
response to atezolizumab therapy (anti-PD-L1 blockade therapy) and to target lesion size 
change during atezolizumab therapy.  Furthermore, the geometric mean SUVmax of 89Zr-
atezolizumab was significantly associated with progression free survival and overall 
survival even after adjustment of tumor type and tumor load, whereas PD-L1 IHC assays 
of the same patients showed moderate to poor discrimination for patient outcome. In 
conclusion, It has been shown that 89Zr-atezolizumab is capable of detecting clinical 
significant PD-L1 expression. (23) 
 
7.4 Summary of known and potential risks and benefits 
Measuring the PD-L1 expression in patient tumors may help to understand the influence 
of PD-L1 expression in the effectiveness of CAR T-cell therapy and evaluate the possible 
diagnostic 
tool 
to 
identify 
a 
treatment-related 
inflammatory 
reaction 
(histiocytic/sarcoid-like reaction) in patients with potential relapse after CAR T-cell 

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therapy. The potential future benefit of 89Zr-atezolizumab is to enable systemic 
assessments of PD-L1 distribution at baseline to identify patients who will potentially 
benefit from PD-1/PD-L1 blockade therapy and after CAR T-cell therapy to identify 
treatment-related inflammatory reactions (histiocytic/sarcoid-like reaction) without the 
need for an invasive biopsy. 
The potential risk associated with 89Zr-atezolizumab administration is considered 
acceptable based on the non-clinical assessments and the clinical experience obtained 
so far. 89Zr-atezolizumab administration has showed safety in the first-in-human study 
in het UMCG (See Section 7.3). Potential risks may include infusion-related reactions  
and injection site reactions. An additional risk associated with this study involves the 
exposure to radiation that is associated with all positron-emitting agents used in PET 
imaging studies. To date, several 89Zr-labeled antibodies have been evaluated in vivo in 
animals and in clinical studies in patients without any sign of toxicity.  
As there is radioactivity involved for tumor detection, the patient is exposed to ionizing 
radiation. The radioactive dose caused by a single administration of 37 MBq of 89Zr-
atezolizumab will be 18.1 millisieverts (mSv) based on the available dosimetric data. As 
a comparison, the radiation dose from an 18fluorodeoxyglucose (18F-FDG) PET/CT scan is 
usually in the range of 5.7-11.4 mSv, corresponding to 300-600 MBq. Generally, if 18F-
FDG PET/CT scans are part of the pharmacological response assessment in a clinical 
study, each patient would undergo at least 3 (or even more) 18F-FDG PET/CT scans, 
corresponding to a similar or even higher cumulative radiation dose than the total 
radiation dose patients will receive in this imaging study. Of note, based on the median 
age of the patients at diagnosis of LBCL, it is expected that most patients who will 

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participate in the study will be aged over 60 years.  The risk of adverse effects from the 
anticipated exposure to ionizing radiation is therefore considered minimal. 
The tracer doses used in this study and the experience of the investigators in the 
execution of PET studies in humans, in addition to the potential benefit of the 89Zr-
atezolizumab PET/CT for diagnostic and pharmacodynamic assessments make the 
relative risk/benefit acceptable for the proposed study. 
 
7.5 Description and justification of route of administration and dosage 
The proposed total protein tracer injection dose of 10 mg is mainly based on imaging 
and pharmacokinetic considerations as described previously. (23) This dosage provides 
sufficient PET-signal without any high-grade adverse events or toxicity. Circulating 
tracer dose corresponds with a serum atezolizumab concentration 100-fold lower than 
reached with the recommend treatment dose.  
 
7.6 Dosages, dosage modifications and method of administration 
Based on the study in the UMCG in patients with solid cancer, the tracer formulation 
with 89Zr-atezolizumab (37 MBq, 10 mg total protein dosage) is optimal. (23) 
 
7.7 Preparation and labeling of Investigational Medicinal Product 
Atezolizumab is provided as a solution of 60 mg/ml in  20 mM Histidine Acetate, 120 
mM Sucrose, 0.04% PS20, pH 5.8. The final 89Zr-MPDL3280A product is a clear light 
opalescent, colourless to light yellow liquid contained in a 10 mL sterile syringe for i.v. 
injection, containing ~2,5mg 89Zr-atezolizumab (37 mBq) in 0,9% NaCl.   
 

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7.8 Drug accountability 
Atezolizumab is shipped at 2-8˚C. Upon receipt at the UMCG, the product is released by 
the QP for administration as injection and for use in the manufacturing of the 89Zr-
atezolizumab drug product. If applicable, the product will be destroyed according to 
local regulations. 
89Zr-atezolizumab drug product is manufactured by the UMCG. The product is released 
by the QP for administration as injection. 89Zr-atezolizumab is stored at 2-8˚C. If 
applicable, product will be destroyed according to local regulations. 
 
 
 

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8. NON-INVESTIGATIONAL PRODUCT 
 
All information about the non-investigational product 18F-FDG PET/CT-scan can be found in 
het UMCG FDC SmPC (Version 5) included as attachment.  
All information about the non-investigational product “Yescarta” (axicabtagene ciloleucel), in 
this protocol referred to as CAR T-cell therapy, can be found in the SmPC of the European 
Medicines Agency.   
8.1 Name and description of non-investigational product(s) 
UMCG-FDG is indicated for imaging in patients as part of oncologic diagnostic procedures, 
using fludeoxyglucose (18F) in combination with positron emission tomography (PET). 
Fludeoxyglucose is a glucose analogue which is accumulate in all cells using glucose as 
primary energy source. Therefore it is accumulate in tumours with a high glucose turnover.  
Yescarta is a CD19-directed genetically modified autologous T-cell immunotherapy.  Patient’s 
own T-cells are harvested and genetically modified ex vivo to express a chimeric antigen 
receptor (CAR), consisting of an anti-CD19 single chain variable fragment linked to a CD28 
co-stimulatory domain and CD3-zeta signalling domain (second generation CAR). The CAR T-
cells are expanded and infused into the patient. 
 
8.2 Dosages, dosage modifications and method of administration 
The recommend activity for an adult weighing 70kg is 100 to 400 MBq (depending on 
body weight, type of camera and acquisition mode) and is administered by direct 
intravenous injection. The activity of fludeoxyglucose has to be measured with 
activimeter immediately prior to injection.  

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Each patient specific single infusion bag of Yescarta contains a dispersion of CAR T-cells in 
approximately 68 mL for a target dose of 2 x106 CAR T-cells/kg body weight (range 1x106-
2 x106), with a maximum of 2 x106 CAR T-cells.  No dose adjustment is required in patients 
≥ 65 year. Yescarta is administered via intravenous infusion after a lymphodepleting 
chemotherapy regimen consisting of cyclophosphamide 500 mg/m2 intravenous and 
fludarabine 30 mg/m2 intravenous on the 5th, 4th, 3rd day before infusion of Yescarta. 
Yescarta infusion should begin within 30 minutes of thaw completion and the total 
Yescarta infusion time should not exceed 30 minutes.  
8.3 Preparation and labelling of Non Investigational Medicinal Product 
UMCG-FDG is manufactured at the department of Nuclear Medicine and Molecular Imaging 
of the UMCG. A part of the quality control is done by the department of Hospital Pharmacy 
of the UMCG. UMCG-FDG is provided in a 25 ml glass vial containing 25 mL of the solution 
which corresponds to 600 MBq per mL at calibration time. The product should be stored a 
temperature below 25 °C for a maximum of 10 hours after production time.  
Yescarta is provided from Kite Pharma EU B.V. in an ethylene-vinyl acetate cryostorage bag 
with sealed addition tube and two available spike ports, containing approximately 68 mL of 
cell dispersion. Yescarta is stable for 1 year when stored frozen in the vapour phase of liquid 
nitrogen (≤-150 °C). Thawed product should not be refrozen.  
 
 
 

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9. METHODS 
9.1 Study parameters/endpoints 
 
9.2 Main study parameters/endpoints 
- 
to study the expression of PD-L1 in normal tissue and lymphoma lesions before 
CD19-directed CAR T-cell therapy in LBCL patients by 89Zr-atezolizumab PET/CT 
imaging and to correlate pretreatment 89Zr-atezolizumab uptake to response to 
CD19-directed CAR T-cell  therapy and thereby identify clinically relevant PD-L1 
expression. Heterogeneity of 
89Zr-atezolizumab uptake will be evaluated by 
measuring standardized uptake value (SUV) on the 89Zr-atezolizumab PET/CT scan. 
- 
to study whether the amount of 89Zr-atezolizumab uptake, measured by the intensity 
of 89Zr-atezolizumab PET/CT imaging (SUV), can be used to differentiate between 
lymphoma activity and treatment-related inflammatory signal (histiocytic/sarcoid-
like reaction) in patients with an end-of-treatment 18F-FDG-positive PET/CT signal. 
Confirmation of the presence of treatment-related inflammatory reaction 
(histiocytic/sarcoid-like reaction) will be determined by an experienced pathologist 
on a fresh contemporaneous biopsy of the positive FDG-PET lesion or in case of non-
accessibility of the lesion by an experienced clinician via a wait-and-see strategy with 
repeated 18F-FDG PET/CT scans. 
 
9.3 Secondary study parameters/endpoints 
- 
to correlate the pretreatment 89Zr-atezolizumab distribution, as measured by the 
uptake of 89Zr-atezolizumab on PET/CT imaging in lymphoma,  to CAR T-cell peak 
expansion and persistence as measured in peripheral blood.  
- 
To correlate the pretreatment 89Zr-atezolizumab  uptake to CAR T-cell related grade 
1-5 adverse events (cytokine release syndrome (CRS) and immune effector cell 

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associated neurotoxicity syndrome (ICANS)). Heterogeneity of 89Zr-atezolizumab 
uptake will be evaluated by measuring SUV on the 89Zr-atezolizumab PET/CT scan. 
CAR-T cell therapy related toxicities such as CRS and ICANS will be assessed using 2018 
ASBMT Consensus Grading (24). 
- 
to correlate tumor 89Zr-atezolizumab uptake with tumor and tumor infiltrating cell 
PD-L1 expression as assessed by immunohistochemistry on a fresh contemporaneous 
tumor biopsy. Heterogeneity of 89Zr-atezolizumab uptake will be evaluated by 
measuring standardized uptake value (SUV) on the 89Zr-atezolizumab PET/CT scan. 
- 
To compare the 89Zr-atezolizumab distribution in irradiated versus non-irradiated 
lymphoma lesions in patients who require radiotherapy as a bridging strategy prior to 
CAR T-cell infusion. If possible, these results will be compared to tumor and tumor 
infiltrating cell PD-L1 expression as assessed by immunohistochemistry on a fresh 
contemporaneous tumor biopsy of an irradiated lymphoma lesion. 
- 
To determine the incidence of a treatment-related inflammatory signal on 18F-FDG 
PET/CT scan (histiocytic/sarcoid-like reaction) after CAR T-cell therapy. Confirmation 
of the presence of treatment-related inflammatory reaction (histiocytic/sarcoid-like 
reaction) will be determined by an experienced pathologist on a fresh 
contemporaneous biopsy of the positive 18F-FDG-PET/CT lesion or in case of non-
accessibility of the lesion by an experienced clinician via a wait-and-see strategy with 
repeated 18F-FDG-PET/CT scans.  
 
9.4 Randomization, blinding and treatment allocation 
This is a single arm, non-blinded study. Patients will therefore not be randomized. 
Treatment with CD19-directed CAR T-cell therapy is standard of care. 

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9.5 Study procedures 
Flow charts of scheduled study assessments are provided in 15.3. 
 
9.6 Screening 
Patients with R/R LBCL after 2 or more lines of treatment who qualify for CD19-directed 
CAR T-cell therapy will be asked to participate in this imaging trial. Written informed 
consent for participation in the imaging trial, must be obtained before performing any 
study specific screening tests or evaluations. All screening evaluations must be 
completed and reviewed to confirm that patients meet all eligibility criteria before 
enrollment. The investigator will maintain a screening log to record details of all 
patients screened and to confirm eligibility or record reasons for screening failure, as 
applicable. 
 
9.7 Assessment of the medical history and demographics 
Medical history includes clinically significant diseases, surgery/operations, cancer 
history (including prior cancer therapies and procedures), reproductive status, and all 
medication (e.g. prescription drugs, over-the-counter drugs, herbal or homeopathic 
remedies, nutritional supplements) used by the patient within 7 days prior to the 
screening visit. A history of pleural or pericardial effusion or of ascites requiring 
intervention should be entered in the medical history. Demographic data will include 
age, sex, and self-reported race/ethnicity. Results of prior analysis of tumor 
characteristics will be recorded in the electronic case record form. If applicable, 
immunohistochemistry on archival tissue will be repeated as formulated in the CD19-

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directed CAR T-cell treatment protocol, and results need to be available before 89Zr-
atezolizumab injection. 
 
9.8 Physical examination and vital signs 
A complete physical examination should include evaluating the head, eyes, ears, nose, 
and throat and the cardiovascular, dermatological, musculoskeletal, respiratory, 
gastrointestinal, and neurological systems. Any abnormality identified at baseline 
should be recorded. The performance score, height and weight should be measured and 
recorded. Vital signs will include measurements of pulse rate, blood oxygen saturation, 
respiratory rate, systolic and diastolic blood pressures while the patient is in a seated 
position, and temperature. 
 
9.9 Laboratory assessments 
Samples for hematology, serum chemistries, coagulation, urinalysis, and the pregnancy 
test will be analyzed at the study site’s local laboratory. 
Local laboratory assessments will include the following: 
● Hematology (CBC, including RBC count, hemoglobin, hematocrit, WBC with 
differential [neutrophils, eosinophils, lymphocytes, monocytes, basophils, 
and other cells], and platelet count) 
● Serum chemistries (glucose, urea, creatinine, sodium, potassium, 
magnesium, chloride, bicarbonate, calcium, phosphorus, total bilirubin, ALT, 
AST, alkaline phosphatase, LDH, CRP, total protein, CRP, and albumin) 
● Coagulation (aPTT, PT, fibrinogen, D-dimeer) 

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● Urinalysis: Urine assessment will include the following: leukocytes, nitrite, 
haem, pH, protein (including total amount of protein), glucose, ketones, 
bilirubin, urobilinogen, erythrocytes and bacteria. 
 
9.10 Tumor evaluation 
Screening assessments must include diagnostic 18F-FDG-PET and contrast-enhanced CT 
scans of the neck, chest, abdomen, and pelvis. A 18F-FDG-PET/CT scan, which has been 
performed >14 days but ≤ 42 days before 89Zr-atezolizumab injection, can be used for 
assessment of the eligibility criteria, if no systemic or local treatment has been applied 
in between. The 18F-FDG-PET/CT scan used for baseline assessment of Lugano criteria 
measurements must be performed ≤14 days before 89Zr-atezolizumab injection, 
otherwise, a new scan will be performed with the 89Zr-atezolizumab PET/CT scan. 
 
9.11 Cardiac and pulmonary function tests 
● Electrocardiogram (ECG): Twelve-lead ECG is required at screening and as 
clinically indicated. ECGs should be obtained on the same machine 
whenever possible. Lead placement should be as consistent as possible. ECG 
recordings should be performed after the patient has been resting in a 
supine position for at least 10 minutes. For safety monitoring purposes, the 
investigator must review, sign, and date all ECG tracings. Paper copies of 
ECG tracings will be kept as  part of the patient's permanent study file at the 
site. Any morphologic waveform changes or other ECG abnormalities must 
be documented. 
● Echocardiograms: Echocardiograms or MUGA scan is considered standard of 

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care and is required as vital CAR T-cell therapy eligibility criterium.  
 
9.12 89Zr-atezolizumab injection 
89Zr-atezolizumab injection will be administered as described in section 6 . 1  (see 
chapter “Investigational treatment”). Sequentially, a defined dose (10 mg) of unlabeled 
atezolizumab will be followed by the radioactive dose of 89Zr-atezolizumab (37 MBq [~2,5 
mg]). After 89Zr-atezolizumab injection, patients will be observed for 30 minutes for 
safety reasons. Patient’s vital signs (pulse rate, respiratory rate, blood pressure, and 
temperature) will be evaluated. Patients will be discharged when clinically stable after 
the observation period (30 minutes) or otherwise on investigators discretion. 
All patients will be informed about the possibility of delayed post-infusion symptoms 
and instructed to contact their study physician if they develop such symptoms. As 
clinically indicated, on the days of PET scanning or otherwise, limited, symptom-
directed physical examinations will be performed. Changes from baseline abnormalities 
should be recorded in patient notes. New or worsened clinically significant 
abnormalities should be recorded as adverse events. 
 
9.13 89Zr-atezolizumab PET/CT procedure 
All scans will be obtained in total body mode (trajectory mid-thigh-skull vertex), using 
low-dose (LD) CT for attenuation correction and localization purposes. For all PET scans, 
acquisition will comprise approximately 14 bed positions. For 89Zr-atezolizumab PET/CT 
imaging, the harmonization procedures, comparable to the EARL PET/CT accreditation 
and European Association of Nuclear Medicine guidelines, as described by Makris et al. 
(26) will be applied. PET/CT imaging of the first 3 patients will be performed on day 4 

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and 7 to determine the optimal time point. Afterwards PET/CT imaging will be 
performed on the determined (4 or 7 days) time point.  
 
9.14 Venous blood sampling 
CAR T-cell expansion in peripheral blood will be measured with expected peak levels at 
days 5-7, and persistence at day 7, 14, 28 with an anti-CAR T-cell monoclonal antibody. T-
cell subsets (CD4 / CD8 ratio, different T-cell phenotypes) will be determined on day 5. 
Cytokines (IL-2, IL-6, IL-10, IFN) will be measured in serum at days -28, 0, 5, 7, 14, and 28 
to determine the pro-inflammatory and immune-modulating cytokines in response to 
CAR T-cell therapy. Samples will be collected during venipuncture as part of the standard 
procedure,  thereby minimizing patient burden. 
 
9.15 Biopsy 
All patients participating in the imaging trial will undergo at least one mandatory tumor 
biopsy. Biopsies will be taken, preferably directly but within 7 days after the 89Zr-
atezolizumab-PET scan. In case there is a suspicion of a relapse or refractory disease 
after CAR T-cell therapy based on 18F-FDG PET/CT-scan a second tumor biopsy will be 
obtained within 7 days of the second 89Zr-atezolizumab.  Moreover, in case of bridging 
with radiotherapy and depending on the accessibility of the tumor lesion, patients will 
be asked if they are willing to undergo an extra biopsy. This biopsy will be used to 
compare 89Zr-atezolizumab uptake in a irradiated lesion on PET/CT imaging to PD-L1 
expression measured via immunohistochemistry. This extra biopsy will not be obligatory 
to participate in the study.   
Determination of the biopsy site will be based on tumor localization on previous 

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conventional imaging and will be performed according to current safety criteria. The 
location of the biopsy site should be determined before 89Zr-atezolizumab PET/CT 
examination to minimize the bias. The biopsy will be performed according to standard 
clinical care. Fine-needle aspiration, brushing, cell pellets from pleural effusion, and 
lavage samples are not acceptable. Acceptable samples include core needle biopsies for 
deep tumor tissue or excisional, incisional, punch, or forceps biopsies for cutaneous, 
subcutaneous, or mucosal lesions. For core needle biopsy specimens, at least 2-3 cores 
should be obtained for molecular evaluation. Biopsies will be taken by the intervention-
radiologist guided by CT or ultrasound.  
 
9.16 End of study 
According standard procedure of care patients will be regularly followed after CAR T-cell 
therapy (day +28, 3/6/12 mth.). Study follow-up will take place during these visits. 
Patients will be eligible for a second 89Zr-atezolizumab PET/CT scan within 1 year after 
CAR T-cell administration.  
Adverse events related to the 89Zr-atezolizumab administration will be followed 30±2 
days after last 89Zr-atezolizumab injection, or until the last (serious) adverse event 
related 
to 
the 
89Zr-atezolizumab administration 
or other study procedure 
(venipuncture, biopsy) has resolved.  
 
9.17 Molecular analysis 
On-site, UMCG laboratories will perform the histology, CD8, PD-L1, and additional 
biomarker assessment on tumor tissues, and assessments of autoradiography on tumor 

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tissues.  
 
9.18 Tumor tissue analysis 
Representative tumor specimens in paraffin blocks (preferred) or at least 15 unstained 
slides from freshly cut, serial sections, should be available at the study site. Tumor 
tissue should be of good quality based on total and viable tumor content. Tissue 
samples will be collected to evaluate the relationship between tumor 89Zr-atezolizumab 
uptake and tumor tissue PD-L1 expression before CAR T-cell treatment. Moreover, in 
case of a suspicion of relapse or refractory disease tissue samples will be obtained to 
evaluate the risk between 
89Zr-atezolizumab uptake and a treatment-related 
inflammatory reaction (histiocytic/sarcoid-like reaction). In addition, in case of bridging 
with radiotherapy and depending on the accessibility of the tumor lesion, patients will 
be asked if they are willing to undergo an extra biopsy. This biopsy will be used to 
compare 89Zr-atezolizumab uptake in a irradiated lesion on PET/CT imaging to PD-L1 
expression measured via immunohistochemistry. This extra biopsy will not be obligatory 
to participate in the study.   
Analyses include but are not restricted to PD-L1 expression on tumor cells and tumor 
infiltrating lymphocytes, as well as the evaluation of T-cell infiltration, measured by CD8 
en CD4 expression, and the determination of histiocytes and macrophages. RNA from 
post-tracer biopsies will be isolated for gene expression analysis by TruSeq RNA Acces 
RNA-seq (Q2Labsolutions). Furthermore, the samples might be used to develop 
biomarker or diagnostic assays and establish the performance characteristics of these 
assays. Autoradiography on obtained tumor tissue will be performed to evaluate the 
relationship between marker expression and local radioactivity by tracer accumulation 

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within a tissue sample. 
 
9.19 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. If the patient wants to leave the trial, it will be enough if he/she informs 
one of the investigators of the decision. The decision will be documented in the 
(electronic) patient dossier. The investigator can decide to withdraw a subject from the 
study for urgent medical reasons. 
 
9.20 Specific criteria for withdrawal 
Reasons for withdrawal from the study are the following: 
● Patient withdrawal of consent at any time. 
● Any medical condition that the principal investigator determines may 
jeopardize the patient’s safety if he or she continues in the study. 
● Investigator or Sponsor determines it is in the best interest of the patient. 
● Patient non-compliance. 
 
9.21 Replacement of individual subjects after withdrawal 
Subjects that withdraw from the study before the 89Zr-atezolizumab PET/CT scan before 
CAR T-cell therapy will be replaced. Subjects with an indication for a second 89Zr-
atezolizumab PET/CT scan that that withdraw from the study before this scan will not be 
substituted.  
 

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9.22 Follow-up of subjects withdrawn from the study 
Every effort should be made to obtain information on patients who withdraw from the 
study. However, patients will not be followed, besides the standard follow-up after CAR 
T-cell treatment, for any reason after consent has been withdrawn. 
 
9.23 Premature termination of the study 
Premature termination will be applied when serious unforeseen adverse events are 
detected, which could be clearly attributed to the investigational product, and which 
prompt a premature termination of the study.  
 
 
 

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10. SAFETY REPORTING 
10.1 Section 10 WMO event 
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the 
subjects and the reviewing accredited METC if anything occurs, on the basis of which it 
appears that the disadvantages of participation may be significantly greater than was 
foreseen in the research proposal. The study will be suspended pending further review by 
the accredited METC, except insofar as suspension would jeopardize the subjects’ health. 
The investigator will take care of all subjects being kept informed. 
 
10.2 AEs, SAEs and SUSARs 
10.2.1 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject during the 
study, whether or not considered related to the 89Zr-atezolizumab injection. All (serious and 
non-serious) adverse events, whether reported by the patient or noted by authorized study 
personnel, will be recorded. For each AE and SAE recorded on the AE CRF, the investigator 
will make an assessment of seriousness, severity and causality. The AE grading (severity) 
scale found in the NCI CTCAE v5.0 will be used for assessing AE severity. 
In the context of AEs related to CAR T-cell treatment as the standard of care parameter, see 
section 4.1. 
 
10.2.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that at any dose:  
o results in death; 
o is life threatening (at the time of the event); 

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o requires hospitalization or prolongation of existing inpatients’ 
hospitalization; 
o results in persistent or significant disability or incapacity; 
o Any other important medical event that may not result in death, be 
life-threatening, or require hospitalization, may be considered a 
serious adverse experience when, based upon appropriate medical 
judgment, the event may jeopardize the subject or may require an 
intervention to prevent one of the outcomes listed above. 
 
SAE will be monitored until 30 (±2) days after the injection of the tracer. 
Medical and scientific judgment should be exercised in deciding whether expedited 
reporting to the sponsor is appropriate in other situations, such as important medical events 
that may not be immediately life-threatening or result in death or hospitalization but may 
jeopardize the patient or may require intervention to prevent one of the outcomes listed in 
the definitions above.  
 
Causality is initially assessed by the investigator. For serious adverse events, causality can be 
one of 2 possibilities: 
● No (unrelated; equals not drug related).  
● Yes (remotely, possibly, probably or definitely drug related). 
 
The sponsor will report the SAEs through the web portal ToetsingOnline to the accredited 
METC that approved the protocol, within 15 days after the sponsor has first knowledge of 
the serious adverse events. SAEs that result in death or are life-threatening should be 

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reported expedited. The expedited reporting will occur not later than 7 days after the 
responsible investigator has first knowledge of the adverse event. This is for a preliminary 
report with another 8 days for completion of the report.  
 
10.2.3 Suspected unexpected serious adverse reactions (SUSARs) 
An unexpected Adverse Event is one of which nature or severity is not consistent with the 
applicable product information. 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1. the event must be serious (see chapter 9.2.2); 
2. there must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the medicinal product under investigation, regardless of 
the administered dose; 
3. the adverse reaction must be unexpected, that is to say, the nature and severity 
of the adverse reaction are not in agreement with the product information as 
recorded in: 
o Summary of Product Characteristics (SPC) for an authorized medicinal 
product; 
o Investigator’s Brochure for an unauthorized medicinal product. 
 
The sponsor will report expedited the following SUSARs through the web portal 
ToetsingOnline to the METC: 
o SUSARs that have arisen in the clinical trial that was assessed by the METC; 

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o SUSARs that have arisen in other clinical trials of the same sponsor and with 
the same medicinal product and that could have consequences for the safety 
of the subjects involved in the clinical trial that was assessed by the METC. 
The remaining SUSARs are recorded in an overview list (line-listing) that will be submitted 
once every half year to the METC. This line-listing provides an overview of all SUSARs from 
the study medicine, accompanied by a brief report highlighting the main points of concern. 
The expedited reporting of SUSARs through the web portal ToetsingOnline is sufficient as 
notification to the competent authority. 
 
The sponsor will report expedited all SUSARs to the competent authorities in other Member 
States, according to the requirements of the Member States. The expedited reporting will 
occur not later than 15 days after the sponsor has first knowledge of the adverse reactions. 
For fatal or life-threatening cases the term will be maximal 7 days for a preliminary report 
with another 8 days for completion of the report.  
 
10.2.4 Adverse events of special interest  
Not applicable.  
 
10.3 Annual safety report 
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a year 
throughout the clinical trial, a safety report to the accredited METC, competent authority, 
and competent authorities of the concerned Member States. 
 
This safety report consists of: 

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● a list of all suspected (unexpected or expected) serious adverse reactions, along with 
an aggregated summary table of all reported serious adverse reactions, ordered by 
organ system, per study; 
● a report concerning the safety of the subjects, consisting of a complete safety 
analysis and an evaluation of the balance between the efficacy and the harmfulness 
of the medicine under investigation. 
 
10.4 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been reached. 
Depending on the event, follow up may require additional tests or medical procedures as 
indicated, and/or referral to the general physician or a medical specialist. SAEs need to be 
reported till end of study, as defined in the protocol.  
 
10.5 Data Safety Monitoring Board (DSMB)/Safety Committee 
An independent DSMB has been established for reviewing all participants in this imaging 
trial. The DSMB will perform safety reviews for this study that includes a review of all AEs 
and SAEs reported for each patient for the period as defined in section 10.2.2. The DSMB will 
receive reports every six months on all AEs and SAEs reported for this trial. The DSMB will 
minimally consist of the following independent members: 1, prof. dr. Andor Glaudemans, 
nuclear medicine specialist 2) prof dr. E Buskens, epidemiologist and 3) dr. R. Fehrmann, 
internist-medical oncologist.  The advice(s) of the DSMB will only be sent to the sponsor of 
the study. Should the sponsor decide not to fully implement the advice of the DSMB, the 
sponsor will send the advice to the reviewing METC, including a note to substantiate why 
(part of) the advice of the DSMB will not be followed.  
 
 

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11. STATISTICAL ANALYSIS 
 
Data of the CD19-directed CAR T-cell trial with 89Zr-atezolizumab PET/CT imaging will be 
analyzed in a pre-planned joined analysis. The statistics will be mainly descriptive due to the 
small sample size. Correlations between imaging variables, response measures and 
pathological assessments will be assessed through data tabulations and graphical 
techniques. Sample size permitting, statistical measures of correlations such as Spearman's 
rho coefficient may also be calculated.  
 
11.1 Primary study parameters 
The first primary endpoint of this study is to study the expression of PD-L1 in normal tissue 
and lymphoma lesions before CD19-directed CAR T-cell therapy in LBCL patients by 89Zr-
atezolizumab PET/CT imaging and to correlate the pretreatment 89Zr-atezolizumab uptake to 
response to CD19-directed CAR T-cell  therapy and thereby identify clinically relevant PD-L1 
expression. Heterogeneity of 89Zr-atezolizumab uptake will be evaluated by measuring 
standardized uptake value (SUV) on the 89Zr-atezolizumab PET/CT scan. 
The second primary endpoint is to study whether the amount of 89Zr-atezolizumab uptake 
differentiates between lymphoma activity and treatment-related inflammatory signal 
(histiocytic/sarcoid-like reaction) in patients with an end-of-treatment 18F-FDG-positive 
PET/CT signal. Confirmation of the presence of treatment-related inflammatory reaction 
(histiocytic/sarcoid-like reaction) will be determined by an experienced pathologist on a 
fresh contemporaneous biopsy of the positive FDG-PET lesion or in case of non-accessibility 
of the lesion by an experienced clinician via a wait-and-see strategy with repeated FDG-PET 

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scans. Heterogeneity of 
89Zr-atezolizumab uptake will be evaluated by measuring 
standardized uptake value (SUV) on the 89Zr-atezolizumab PET/CT scan. 
Response assessment will be performed according to the Lugano criteria. (i.e., revised 
criteria of the International Working Group, rIWG) using local assessments at 4 weeks, 12 
weeks, 6 and 12 months after CD19-directed CAR T-cell infusion. Criteria are summarized in 
12.1.3-12.1.8. 
 
 
11.1.1 Measurement of 89Zr-atezolizumab uptake  
89Zr-atezolizumab tumor uptake will be assessed, evaluated by measuring standardized 
uptake value (SUV) on the 89Zr-atezolizumab PET/CT scans 4 or 7 days after 89Zr-
atezolizumab injection. The optimal time point will be determined in the first 3 patients (4 or 
7 days).  
89Zr-atezolizumab tumor uptake and organ distribution will be scored visually and 
quantitatively. Qualitative assessment of 89Zr-atezolizumab uptake in non-malignant 
lymphoid tissue identified in the PET/CT scan will also be performed. Quantification of 89Zr-
atezolizumab distribution will be performed using AMIDE software (Stanford University, Palo 
Alto, CA) and Accurate software (developed by R. Boellaard). 89Zr-atezolizumab uptake will 
be corrected for body weight and injected dose and be quantitatively assessed as 
standardized uptake value, which is calculated using the formula: [tissue activity 
concentration (MBq/g)]/[(injected dose (MBq)/body weight (g)]. The SUV of all tumor lesions 
and in relevant normal tissues will be calculated on all 89Zr-atezolizumab PET scans. 
 
 

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11.1.2 Methods of assessments (Lugano Criteria (20)) 
 
Efficacy  
Response assessment will be performed at time points as given in the visit schedule. 
Assessment of response includes a careful physical exam, evaluation of the peripheral blood, 
bone marrow histology if clinically indicated and either CT or MRI in cases of CT IV contrast 
contraindication or FDG-PET-CT. Response is assessed based on Lugano criteria. (27) 
The Lugano criteria are summarized in the following. 
 
11.1.3 General definitions 
The following categories of response are defined: CR, PR, SD, relapse and progression (PD). 
In the case of SD, follow-up assessments must have met the SD criteria at least once after 
entry to that step at a minimum interval of eight weeks. 
 
The following guidelines are to be used for establishing tumor measurements at the baseline 
of each treatment step and for subsequent comparison: 
The six largest measurable nodes or extranodal masses must be identified as target lesions 
at baseline. If there are 6 or fewer measurable nodes and extranodal masses, all must be 
listed as Target Lesions. 
If there are more than 6 involved measurable nodes or extranodal masses, the 6 largest 
nodes or extranodal masses should be selected as Target Lesions according to the following 
features: a) they should be clearly measurable in at least two perpendicular measurements; 
b) they should be from as disparate regions of the body as possible; and c) they should 
include mediastinal and retroperitoneal areas of disease whenever these sites are involved. 
When there are more than 6 involved measurable nodes or extranodal masses, any lesions 

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that are not included within these 6 Target Lesions will be considered non- measured 
lesions. 
 
Non-measured lesions: Any disease not selected as measured, dominant disease and truly 
assessable disease should be considered not measured. These sites include any nodes, nodal 
masses, and extranodal sites not selected as dominant or measurable or that do not meet 
the requirements for measurability but are still considered abnormal, as well as any site of 
suspected disease that would be difficult to follow quantitatively with measurement, 
including pleural effusions, ascites, bone lesions, leptomeningeal disease, abdominal masses, 
and other lesions that cannot be confirmed, measured or followed by imaging. 
 
Measurements for all target lesions will be reported at baseline of each treatment step. 
Measurements for non-measured lesions are not required. 
 
The lymph nodes or extranodal masses selected as target lesions for measurement should 
be measured in two perpendicular diameters, one of which is the longest perpendicular 
diameter. The lymph nodes should be measured in centimeters to the nearest one-tenth of a 
centimeter (e.g., 2.0 cm, 2.1 cm, 2.2 cm, etc.). A measurable node must have a longest 
diameter (LDi) >1.5 cm. Measurable extranodal disease (e.g., hepatic nodules) may be 
included in the six representative, measured lesions. A measurable extranodal lesion should 
have an LDi greater than 1.0 cm. 
 
The two measured diameters of each target lesion should be multiplied, giving a product for 
each nodal site or extranodal mass. The product of each site should be added, yielding the 

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sum of products of the diameters (SPD). The SPD will be used in determining the definition 
of response for those who have less than a complete response. 
 
Tumor assessment response with standard FDG-PET-CT-based evaluation should use the 
following 5 point scale: PET 5 point scale: 1, no uptake above background; 2, uptake 
≤mediastinum; 3, uptake >mediastinum but ≤liver; 4, uptake moderately >liver; 5, uptake 
markedly higher than liver and/or new lesions; X, new areas of uptake unlikely to be related 
to lymphoma. 
 
11.1.4 Complete Response 
Complete disappearance of all detectable clinical evidence of disease, and disease-related 
symptoms if present prior to therapy. 
 
18F-FDG PET/CT Based Criteria 
• Complete metabolic response with a 5-point scale score of 1, 2 or 3, with or without a 
residual mass. 
• In patients with bone marrow involvement before treatment there must be no residual 
FDG uptake in the marrow. 
• In patients with a typically FDG-avid lymphoma with no pre-treatment PET scan, or for 
lymphomas for which the FDG-PET scan was positive prior to therapy: a post-treatment 
residual mass of any size is permitted as long as it is FDG-PET-negative. 
 
 
 

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CT Based Criteria 
• For variably FDG-avid lymphomas without a pretreatment FDG-PET scan, or if a 
pretreatment PET scan was negative: all lymph nodes and extranodal masses must have 
regressed on CT to normal size (≤1.5 cm in their greatest transverse diameter for nodes >1.5 
cm prior to therapy). Previously involved nodes that were 1.1-1.5 cm in their long axis and 
>1.0 cm in their short axis prior to treatment must have decreased to ≤1 cm in their short 
axis after treatment. 
• The spleen and/or liver, if considered enlarged prior to therapy on the basis of a physical 
examination or CT scan, should not be palpable on physical examination, and nodules 
related to lymphoma should disappear. However, no normal size can be specified because of 
the difficulties in accurately evaluating splenic and hepatic size and involvement. E.g., a 
spleen considered normal size may contain lymphoma, whereas an enlarged spleen may not 
necessarily reflect the presence of lymphoma, but variations in anatomy, blood volume, the 
use of hematopoietic growth factors, or other causes. 
• If the bone marrow was involved by lymphoma prior to treatment, the infiltrate must 
have cleared on repeat bone marrow biopsy. The biopsy sample on which this determination 
is made must be adequate (with a goal of >20 mm unilateral core). If the sample is 
indeterminate by morphology, it should be negative by immunohistochemistry. A sample 
that is negative by immunohistochemistry but demonstrating a small population of clonal 
lymphocytes by flow cytometry will be considered a CR until data become available 
demonstrating a clear difference in subject outcome. 
Of note, the definitions of CR (as given in this section) and of PR (as given in Section 11.1.5) 
eliminate the category of complete remission/unconfirmed. 
 

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11.1.5 Partial Response 
The designation of PR requires all of the following: 
 
PET-CT Based Criteria 
• Partial metabolic response with reduced uptake compared with baseline AND a 5-point 
scale score of 4 or 5. 
• For a typically FDG-avid lymphoma with no pretreatment FDG-PET scan or one that was 
FDG-PET-positive prior to therapy, the post-treatment FDG-PET should be positive at any 
previously involved sites. 
• In patients with bone marrow involvement before treatment, residual uptake higher than 
uptake in normal marrow but reduced compared with baseline (diffuse uptake compatible 
with reactive changes from chemotherapy allowed). If there are persistent focal changes in 
the marrow in the context of a nodal response, consideration should be given to further 
evaluation with MRI or biopsy or an interval scan. 
 
CT Based Criteria 
• For variably FDG-avid lymphomas/FDG-avidity unknown, without a pretreatment PET 
scan, or if a pretreatment FDG- PET scan was negative, CT scan criteria should be used. 
• A ≥50% decrease in sum of the product of the diameters (SPD) of up to 6 of the largest 
target lesions. These nodes or masses should be selected according to the following: (a) they 
should be clearly measurable in at least 2 perpendicular dimensions; if possible, they should 
be from disparate regions of the body; (b) they should include mediastinal and 
retroperitoneal areas of disease whenever these sites are involved. 
• No increase in the size of other nodes, liver or spleen. 

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• Bone marrow assessment is irrelevant for the determination of a PR if the sample was 
positive prior to treatment. However, if positive, the cell type should be specified, e.g., large-
cell lymphoma or small cleaved cell lymphoma. 
• No new sites of disease 
• Patients who achieve CR by the above criteria, but who have persistent morphologic bone 
marrow involvement will be considered PR 
• When the bone marrow was involved before therapy and a clinical CR was achieved, but 
with no bone marrow assessment after treatment, patients should be considered PR 
 
11.1.6 Stable Disease 
PET/CT Based Criteria: 
• Absence of metabolic response, with a score of 4 or 5 AND no significant change from 
baseline at interim or end of treatment. 
• In patients with bone marrow involvement before treatment, there must be no change 
from pre-treatment FDG-PET scan. 
• No new areas of FDG uptake 
 
CT Based Criteria 
• For variably FDG-avid lymphomas/FDG-avidity unknown: for patients without 
pretreatment FDG-PET scan or if the pre-treatment FDG-PET was negative, there must be no 
change in the size of the previous lesions on the post-treatment CT scan. 
• Less than 50% decrease from baseline in SPD of up to 6 target lesions 
• No increase in organ enlargement and non-measurable lesions compatible with PD 
 

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11.1.7 Progression and relapse 
PET/CT Based Criteria 
Progressive metabolic disease: 
• Individual target nodes and nodal masses must present increase intensity of uptake from 
baseline, with a 5-point score of 4 or 5, or 
• Extranodal lesions with new FDG-avid foci consistent with lymphoma at interim or end of 
treatment assessment, or 
• New FDG-avid foci consistent with lymphoma rather than another etiology (e.g. infection, 
inflammation). If uncertain regarding the etiology of new lesions, a biopsy or repeat imaging 
scan should be considered. 
 
CT Based Criteria 
• For determination of relapsed and progressive disease, lymph nodes should be 
considered abnormal if the long axis is >1.5 cm, regardless of the short axis. If a lymph node 
has a long axis of 1.1 to 1.5 cm, it should only be considered abnormal if the short axis is >1 
cm. Lymph nodes ≤1 cm x ≤1 cm will not be considered as abnormal for relapse or 
progressive disease. 
• At least a 50% increase from the nadir in the SPD of any previously involved target 
lesions, or in a single involved node or extranodal mass, or the size of other lesions (e.g., 
splenic or hepatic nodules). 
 • To be considered PD, a lymph node or extranodal mass with a diameter of the long or 
short axis of ≤2.0 cm must have increased by at least 0.5 cm; lesions >2.0 cm must have 
increased by at least 1.0 cm. 

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• In the setting of splenomegaly, the splenic length must increase by >50% of the extent of 
its prior increase from baseline. If no prior splenomegaly, must increase by at least 2.0 cm 
from baseline. 
• New lesions: Regrowth of previously resolved lesions; or a new lymph node 
>1.5 cm in any axis; or a new extranodal site >1.0 cm in any axis (new extranodal disease 
<1.0 cm in any axis, can be considered PD if its presence is unequivocal and attributable to 
lymphoma). 
• New or recurrent bone marrow involvement 
• Clinical PD can be determined using the following criteria: 
- 
ECOG PS of at least 3 
- 
Patient unable to have follow-up radiologic assessment due to performance status 
decline 
- 
Symptomatic decline deemed related to metastatic disease or disseminated disease 
(not toxicity from therapy or concurrent illness) 
 
11.1.8 Endpoint definitions 
Best Overall Response (BOR) 
This is the best response recorded from the start of the treatment until disease 
progression/recurrence (taking as reference for progressive disease the smallest 
measurements recorded since the treatment started). The patient’s best response 
assignment will depend on the achievement of both measurement and confirmation criteria. 
 
 
 

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Duration of response (DOR) 
Duration of response is measured from the time measurement criteria are met for CR or PR 
(whichever is first recorded) until the time of the last assessment of response before the first 
date that SD, or recurrent or progressive disease is objectively documented (taking as 
reference for PD the smallest measurements recorded since the treatment started). 
 
Overall survival (OS) 
This is defined as the date of enrollment to the date of death. 
 
Progression-free survival (PFS) 
PFS is defined as the time from IMP infusion until PD or death from any cause. PFS reflects 
tumor growth and, therefore, occurs prior to the endpoint of OS. PFS is not confounded by 
the administration of subsequent therapy. Progression is defined as the first date of 
documentation of a new lesion or enlargement of a previous lesion, or the date of the 
scheduled clinic visit immediately after radiologic assessment has been completed. If there is 
missing information, censoring of the data may be defined as the last date at which 
progression status was adequately assessed. 
 
11.1.9 Treatment-related inflammatory reaction 
18F-FDG-PET/CT scans will be performed regularly after CAR T-cell treatment (standard 
procedure follow-up day +28, 3/6/12 mth). In case of a positive 18F-FDG-PET signal a second 
89Zr-atezolizumab PET/CT scan and a biopsy will be performed. Confirmation of the presence 
of treatment-related inflammatory reaction (histiocytic/sarcoid-like reaction) will be 
determined by an experienced pathologist on a fresh contemporaneous biopsy of the 

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positive 18F-FDG-PET lesion or in case of non-accessibility of the lesion by an experienced 
clinician via a wait-and-see strategy with repeated 18F-FDG-PET/CT scans.  
 
11.2 Secondary study parameter(s)  
11.2.1 Correlative expression analysis between 89Zr-atezolizumab-distribution 
and CAR T-cell peak expansion and persistence 
Heterogeneity of 89Zr-atezolizumab tumor uptake will be assessed, evaluated by measuring 
standardized uptake value (SUV) on the 89Zr-atezolizumab PET/CT scans 4 or 7 days after 
89Zr-atezolizumab injection. Optimal time point will be determined in the first 3 patients, 
who will receive a 89Zr-atezolizumab PET/CT scan on day 4 and 7. Distribution will be 
correlated to CAR T-cell peak expansion and persistence as measured in peripheral blood 
with an anti-CAR T-cell monoclonal antibody. Peak levels will be determined at day +5 (±2 d). 
Persistence levels will be determined at day 0, 5,28, 90, 365.  
 
11.2.2 Correlative expression analysis between 89Zr-atezolizumab uptake and 
CAR T-cell therapy related grade 1-5 adverse events (cytokine release syndrome 
and immune effector cell associated neurotoxicity syndrome).  
Heterogeneity of 89Zr-atezolizumab tumor uptake will be assessed, evaluated by measuring 
standardized uptake value (SUV) on the 89Zr-atezolizumab PET/CT scan 4 or 7 days after 
89Zr-atezolizumab injection. CAR-T cell therapy related toxicities such as cytokine release 
syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) will be 
assessed using 2018 ASBMT Consensus Grading. (24) Data will be derived from the 
electronic medical dossier in a prospectively observational way, assessed by investigators 
or treating physician’s initiative and graded. 
 

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Definitions of adverse events 
Adverse events (AEs) 
An AE is defined as any untoward medical occurrence in a patient administered with CAR T-
cell administration and which does not necessarily have to have a causal relationship with 
this treatment. An AE can therefore be any unfavorable and unintended sign (including an 
abnormal clinically significant laboratory finding), symptom, or disease temporarily 
associated with the CAR T-cell administration, whether or not causally related to the (CAR 
T-cell administration. 
 
Treatment-emergent adverse event (TEAE) 
TEAEs are undesirable events not present prior to medical treatment, or an already present 
event that worsens either in intensity or frequency following the introduction of the 
investigational therapy. TEAE will be defined as any AE with onset on or after the first dose 
of conditioning chemotherapy and including the MB-CART2019.1 cell infusion, the 28 post-
infusion follow up period, and up to 90 days post-infusion period. 
 
Adverse Reactions (ARs) 
ARs include all untoward and unintended responses to the CAR T-cell administration or to 
the lymphodepletion regimen related to any dose administered. All AEs judged by either 
the reporting investigator or the sponsor as having a causal relationship of possibly, 
probably, or definitely related to the IMP or the lymphodepletion regimen qualify as ARs. 
All ARs are judged by the reporting investigator and the sponsor as having a reasonable 
causal relationship to a medicinal product. The expression “reasonable causal relationship” 
means to convey in general that there is evidence or argument to suggest a causal 

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relationship. 
Events for that no causality is given or that are classified as “not assessable” are considered 
as AR until enough information is provided to allow a proper assessment of the causal 
relationship. 
 
Serious Adverse Events (SAEs) 
An SAE is any untoward medical occurrence or effect that at any dose results in death, is life-
threatening, requires hospitalization or prolongation of existing hospitalization, results in 
persistent or significant disability or incapacity, or is a congenital anomaly or birth defect. A 
serious adverse drug reaction (SAR) is a serious AR. 
In addition, medical and scientific judgment should be exercised in deciding whether other 
conditions should also be considered serious, such as important medical events that may 
not be immediately life-threatening or result in death or hospitalization but may jeopardize 
the patient’s safety or may require intervention to prevent one of the other outcomes listed 
in the definition above. Examples of such events are intensive treatment in an emergency 
room or at home for allergic bronchospasm, blood dyscrasias, or convulsions that do not 
result in hospitalization, or development of drug dependency or drug abuse. 
 
Definition of ‘life-threatening’: 
An AE is life-threatening if the patient was at immediate risk of death from the event 
as it occurred; i.e., it does not include a reaction that if it had occurred in a more 
serious form, it might have caused death. For example, drug-induced hepatitis that 
resolved without evidence of hepatic failure would not be considered life-

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threatening even though drug-induced hepatitis can be fatal. 
 
Definition of ‘disability/incapacitating’: 
An AE is incapacitating or disabling if the event results in a substantial and/or 
permanent disruption of the patient's ability to carry out normal life functions. 
 
Suspected unexpected serious adverse reaction (SUSAR) 
Suspected unexpected serious adverse reactions are serious adverse reactions in 
patients given a drug, that may or may not be dose-related, but are unexpected, as 
they are not consistent with current information (e.g., Investigator’s Brochure of the 
IMP, product information for approved drugs). 
 
Assessment of Severity 
CAR-T cell therapy related toxicities such as CRS and ICANS will be assessed using 2018 ASBMT 
Consensus Grading (24). 
Safety evaluations will be made based on NCI CTCAE Version 5. CTCAE grades help to 
assess the severity of the AE: 
Grade 1 
mild AE 
Grade 2 
moderate AE 
Grade 3 
severe AE  
Grade 4 
life-threatening AE  
Grade 5 
death related to AE 
 

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In case the CTCAE are not applicable, the maximum intensity should be assigned to 
one of the following categories: 
Mild: For example, an AE which is easily tolerated by the patient, causing minimal 
discomfort and not interfering with everyday activities. 
Moderate: For example, an AE which is sufficiently discomforting to interfere with normal 
everyday activities. 
Severe: For example, an AE which is incapacitating and prevents normal everyday activities. 
 
Causality of Adverse Events 
The causality of AEs refers to the relationship of the AE to trial treatment. When completing 
the eCRF, the investigator will be asked to assess the causality of the event as ‘IMP-related’, 
‘concomitant medication-related or ‘other’. Causality will then be categorized by using a 
simple binary decision for causality according to the following criteria for all three 
parameters as applicable: 
 
Not related: 
An AE for which there is no reasonable possibility of a causal relationship with the IMP, with 
the concomitant medication or other causes specified by the investigator.  
 
Related: 
An AE for which there is a reasonable possibility of a causal relationship with the IMP 
concomitant medication or other causes specified by the investigator. This means that 
there are facts (evidence) or arguments to suggest a causal relationship. 

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The assessment of whether there is a reasonable possibility of a causal relationship is 
usually made by the investigator. The causality assessment given by the investigator should 
not be downgraded by the sponsor. If the sponsor disagrees with the investigator’s causality 
assessment, the opinion of both, the investigator and the sponsor, should be provided with 
the report. The following points should be considered during causality assessment of AEs: 
Timing of the event between administration of the drug and the onset of the AE Drug 
levels and evidence, if any, of overdose. 
The expectedness assessment will be done by the sponsor (or delegate). 
 
11.2.3 Correlation of tumor 89Zr-atezolizumab uptake and tumor cell or tumor 
infiltrating cell PD-L1-expression as assessed by a fresh contemporaneous 
tumor biopsy 
PD-L1 expression will be scored based on immunohistochemistry as negative (IC0 or TC0: 
staining on <1% of IC or TC, respectively; IHC score 0) or positive (IC1/2/3 or TC1/2/3: 
staining on ≥1% of IC or TC; IHC score 1/2/3 depending on the highest staining for either IC 
or TC). These IHC results will be compared with tumor 89Zr-atezolizumab uptake. 
In addition, we will evaluate T-cell infiltration as measured by CD8 and CD4 expression and 
will determine the polarization of macrophages and histiocytes. RNA from post-tracer tumor 
biopsies will be isolated for gene expression analysis by TruSeq RNA Access RNA-seq 
(Q2Labsolutions). Autoradiography of the biopsies will be performed using a 
phosphorimaging screen for 24h and will be then scanned with a Cyclone phosphorimager 
and will be compared with PD-L1 expression in adjacent tissue biopsy samples. 
 

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11.2.4 Comparison of 89Zr-atezolizumab distribution in irradiated vs non-
irradiated lymphoma lesions  
In case a patient requires bridging therapy between leukapheresis and standard lympho-
depleting chemotherapy and the subsequent CAR T-cell infusion, radiation therapy might be 
applied. Only lymphoma lesions that are highly proliferative and are at risk for vital organs or 
causes discomfort will be irradiated, while preserving lymphoma lesions that are not directly 
necessary to irradiate. In this way, the remaining lymphoma lesions will ascertain the 
necessary antigen exposure for CAR T-cell activation and proliferation. 89Zr-atezolizumab 
injection will be after the final irradiation day. Heterogeneity of 89Zr-atezolizumab tumor 
uptake in irradiated and non-irradiated lymphoma lesions will be assessed, evaluated by 
measuring SUV parameters on the 89Zr-atezolizumab PET/CT scans 4 or 7 days after 89Zr-
atezolizumab injection. Depending on the accessibility of the tumor lesion, patients will be 
asked if they are willing to undergo an extra biopsy, these results will be compared to tumor 
and tumor infiltrating cell PD-L1 expression as assessed by immunohistochemistry on a fresh 
contemporaneous tumor biopsy of an irradiated lymphoma lesion. This extra biopsy is not 
mandatory for participation in the study.  
 
11.2.5 Determination of the incidence of a treatment-related inflammatory 
reaction (histiocytic/sarcoid-like reaction) after CAR T-cell therapy . 
Standard procedure for patients with an end-of-treatment positive 18F-FDG PET/CT scan 
consists of a biopsy if suspected lesion is accessible or a wait-and-see strategy with repeated 
18F-FDG PET/CT scans. Hence, confirmation of the presence of treatment-related 
inflammatory reaction (histiocytic/sarcoid-like reaction) will be determined by an 
experienced pathologist on a fresh contemporaneous biopsy of this lesion or in case of non-
accessibility determined by an experienced clinician via a wait-and-see strategy with 

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repeated 18F-FDG PET/CT scans, which in case of a treatment-related inflammatory signal 
will show regression of the lesion over time. Descriptive statistics will be used to determine 
the incidence of treatment-related inflammatory signals (histiocytic reaction/sarcoid-like 
reaction).  
 
12. ETHICAL CONSIDERATIONS 
12.1 Regulation statement 
The study will be conducted according to the standards of Good Clinical Practice, in 
agreement with the principles of the Declaration of Helsinki (64, October 2013, Fortaleza, 
Brazil) and with Dutch law, in accordance with the WMO and according to “Besluit 
Stralingsbescherming (BS 2000), artikel 64 – en Nota van Toelichting (Staatsblad 2001, 397)". 
 
12.2 Recruitment and consent 
All patients referred to the UMCG for CAR T-cell therapy are possible candidates for this 
study. Patients will be recruited by the treating physician and be informed by the PI or CI. 
Before they agree to participation in this trial, all patients will be provided with written 
information in the form of a Patient Information Sheet. This document will be submitted for 
approval to the METC along with the protocol. A statement of approval should be provided 
before commencement of the study. Furthermore, the subjects will be informed about the 
CAR T-cell treatment each patient will be given the opportunity to ask questions and will be 
informed about the right to withdraw from the study at any time without prejudice. The 
formal written consent for this trial of a patient must be obtained before initiation of any 
study-specific procedure. Patients must be given adequate opportunity to read the 
information and enquire about the details of the study before consent is given. The informed 
consent procedure takes place according to the ICH guidelines on Good Clinical Practice. This 

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implies that the written informed consent form will be signed and personally dated by the 
patient. The informed consent statement will be signed and dated by the investigator 
afterward and the patient will receive a copy. The general physician of each patient will be 
informed about the enrolment of the patient to the study. 
Subjects are free to decide whether or not to participate in this trial. Non-participation will 
not have any consequences concerning their treatment. If the patient agrees to participate 
in the trial, it will be documented in the (electronic) patient dossier.  
 
12.3 Objection by minors or incapacitated subjects  
Not applicable. 
 
12.4 Benefits and risks assessment, group relatedness 
 
Benefits 
Molecular imaging with an antigen-targeted PET scan is an upcoming technique for obtaining 
non-invasive whole-body information about specific molecular targets. In this study, we 
want to evaluate the uptake and distribution of 89Zr-atezolizumab in patients with R/R LBCL 
who are eligible for CD19-directed CAR T-cell therapy. We will correlate the results with the 
response to CAR T-cell therapy. The 89Zr-atezolizumab distribution might predict which 
patients may  have a diminished response to CAR T-cell therapy and will possibly benefit 
from CAR T-cell therapy in combination with PD-1/PD-L1 blockade therapy.  
In addition, in case of an end-of-treatment 18F-FDG-positive PET/CT signal after CAR T-cell 
therapy the 89Zr-atezolizumab uptake might differentiate between a treatment-related 
inflammatory reaction (histiocytic/sarcoid-like reaction) and lymphoma activity in a non-

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invasive manner. The possible benefits of this study, however, do not directly apply to the 
participants of this study because no decisions will be made based on 89Zr-atezolizumab 
results in this study. Nevertheless, their participation will help to get more insight into the 
tumor immune response and will guide further prospective research. 
 
Risks of 89Zr-atezolizumab  
Recent first-in-human study showed safe administration of 89Zr-atezolizumab in patients 
with solid malignancies, as only one infusion-relation adverse event (pruritus, grade 1) 
was reported (see IMPD). (23) Pharmacokinetic analysis confirmed that the circulating 
tracer dose corresponded with a serum concentration of atezolizumab reached with 
administration of 0,1-0,3 mg/kg, which is almost 100-fold lower than reached with the 
recommend treatment dose.  
Both atezolizumab as 89Zr-labeled antibodies al already used abundantly in various 
studies and/or standard care. Therefore, it already is established that atezolizumab binds 
to its target with high affinity. Moreover, preclinical safety assessments of atezolizumab 
in Cynolmolgus monkeys showed no local or systemic adverse effects in a 8-week in vivo 
toxicity evaluation.  
All patients will be informed about the possibility and symptoms of adverse events 
related to infusion of 89Zr-atezolizumab, including an allergic reaction and pain at the site 
of infusion. First clinical study showed safe injection of 89Zr-atezolizumab, with only one 
related low-grade adverse event. All patients will be observed for at least 30 minutes 
after 89Zr-atezolizumab injection to monitor for possible acute infusion related adverse 
events.  
 

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Radiation burden 
Since 89Zr-atezolizumab is a radioactive compound, it will cause radiation burden to the 
patient. The projected effective dose after receiving 37 MBq of the 89Zr-atezolizumab PET 
scan is 18.1 mSv and 1 mSv for each low dose attenuation correction CT scan. The total 
effective dose for patients will be 19.1 mSv. In case of the need for a second 89Zr-
atezolizumab PET/CT scan because of a suspicion of relapse of refractory disease after CD19-
directed CAR T-cell therapy, the effective dose for those patients will be in total 38.2mSv.  
 
Risks of biopsy and blood sampling 
Besides PET/CT imaging, patients will be asked to give in total, 12 blood samples (108ml), 
which will be collected during venipuncture as part of the standard procedure. Thereby 
minimizing patient burden. In addition, patients will undergo a fresh tumor biopsy. Patients 
will only be eligible for the study if they have a safely accessible lesion as assessed by the 
treating physician and the physician performing the biopsy. Based on a literature review, the 
risk of tumor biopsies is considered low with a small risk on significant/major complications 
(0 to 1.6%) or death (0 to 0.48%). The results of this literature review are depicted in the 
Appendix (section 15.1).  
 
 
 
Time investment 
Whenever possible, to minimize the burden, intervention and patients’ visits are preferably 
planned on the same day. For this study, patients will make maximal 4 extra visits to the 
hospital: 1) Screening visit, 2) 89Zr-atezolizumab injection 3) 89Zr-atezolizumab PET/CT scan 4 

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or 7 days after 89Zr-atezolizumab injection, 4) Biopsy visit within 7 days after 89Zr-
atezolizumab PET/CT scan. In case of a suspicion of a relapse or refractory disease after 
CD19-directed CAR T-cell therapy, 2 extra visits to the hospital have to be made: 1) 89Zr-
atezolizumab injection 2) 89Zr-atezolizumab PET/CT scan 4 or 7 days after 89Zr-atezolizumab 
injection. Biopsy will be obtained within 7 days after 89Zr-atezolizumab PET/CT scan, but is 
considered to be part of standard care procedure. 
 
In conclusion, the risk associated with 89Zr-atezolizumab appears acceptable and although 
patients do not directly benefit from this study, the results of this study will be valuable for 
our understanding of the tumor immune response and will guide further prospective 
research.  
 
12.5 Compensation for injury 
The UMCG as sponsor/investigator has a liability insurance, which is in accordance with 
article 7 of the WMO. 
  
The sponsor (also) has an insurance, which is in accordance with the legal requirements in 
the Netherlands (Article 7 WMO). This insurance provides cover for damage to research 
subjects through injury or death caused by the study. 
The insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study. 
 

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12.6 Incentives 
For each day of patient-related study procedures, the subjects will receive compensation for 
traveling expenses (€ 0.19/km) and a ticket for free parking. 

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13.  ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
13.1 Handling and storage of data and documents 
As a result of the regulations for radiation exposure and safety, the information on the 
administered radioactivity will be stored (non-anonymously) in the patients’ files. Images of 
experimental PET/CT scans that are obtained at the PET/CT center (Department of Nuclear 
Medicine and Molecular Imaging) will internally be stored non-anonymously. However, 
personal information will only be accessible for medical personnel affiliated to that 
department and/or (delegated) members of the research team and handled confidentially. 
Data analysis will be performed on anonymized scan images. 
For other data management, study subjects will receive a code. The key to the code (number 
linked to the patient) is safeguarded by the investigator. The study code assigned to the 
patients will also be used in the collection of all the study results by the investigator who will 
perform the data-analysis. An overview of all data and data-analysis is made according to 
this code so that the final results cannot be traced back to the patients by another person 
than the investigators involved in the study (in compliance with the EU General Data 
Protection Regulation Data will be stored for a maximum period of 15 years after the study is 
finished. In case a patient give permission to store their personal information and samples 
for a langer period of time and to use it in future research in the field of CAR T-cell therapy, 
data will be stored for a maximum period of 25 years.  
The handling of personal data will comply with the EU General Data Protection Regulation, in 
Dutch often referred to as the Algemene Verordering Gegevensbescherming (AVG). 
 

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13.2 Monitoring and Quality Assurance  
On-site monitoring will take place conform the NFU (Nederlandse Federatie van 
Universitaire Medisch Centra)-guideline “Kwaliteitsborging van mensgebonden onderzoek 
2020” by the appointed monitor. Monitoring will take place to assure the quality and validity 
of the research data. The monitor will perform source data verification on the research data 
by comparing the data entered into the CRF with the available source documentation and 
other available documents. Source documents are defined as the patient’s hospital medical 
records, clinician notes, laboratory print outs, digital and hard copies of imaging, memos, 
electronic data etc.  
The monitor will verify the following items: Patient flow (inclusion speed and dropout rate); 
Informed consent forms (presence, dates, signatures); Trial Master File and Investigator Files 
(presence of all documents), in-/exclusion criteria (using source documents); SAEs/SUSARs 
(number, missed, reporting procedures); study product (administration, accountability). 
After each control the monitor will send a written report to the sponsor (including a 
summary; quality assessment; summary of findings, deviations and shortcomings; possible 
solutions to warrant compliance with the protocol; final conclusion). 
 
13.3 Auditing and inspections 
Participation in this study implies acceptance of potential inspection by national health 
authorities. 
 
13.4 Amendments  
A ‘substantial amendment’ is defined as an amendment to the terms of the METC 
application, or to the protocol or any other supporting documentation, that is likely to affect 
to a significant degree: 

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● the safety or physical or mental integrity of the subjects of the trial; 
● the scientific value of the trial; 
● the conduct or management of the trial; or 
● the quality or safety of any intervention used in the trial. 
All substantial amendments will be notified to the METC and to the competent authority. 
Non-substantial amendments will not be notified to the accredited METC and the competent 
authority, but will be recorded and filed by the sponsor.  
 
13.5 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the accredited 
METC once a year. Information will be provided on the date of inclusion of the first subject, 
numbers of subjects included and numbers of subjects that have completed the trial, serious 
adverse events/ serious adverse reactions, other problems, and amendments.  
 
13.6 End of study report 
The sponsor will notify the accredited METC and the competent authority (CA) of the end of 
the study within a period of 90 days. The end of the study is defined as the last patient’s last 
visit. In case the study is ended prematurely, the sponsor will notify the accredited METC 
and the competent authority within 15 days, including the reasons for the premature 
termination. Within one year after the end of the study, the investigator/sponsor will submit 
a final study report with the results of the study, including any publications/abstracts of the 
study, to the accredited METC and the CA.  
 

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13.7 Public disclosure and publication policy 
The sponsor of this investigator driven study is the University Medical Centre Groningen. 
This study is financially supported by PUSH call 2020 and the Cancer research fund UMCG. 
All publication manuscripts will be reviewed by the investigating group before submission.  

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14. STRUCTURED RISK ANALYSIS  
 
14.1 Potential issues of concern 
a. Level of knowledge about mechanism of action 
Mechanism of action of the investigational products, atezolizumab and 89Zr-atezolizumab 
relies on the binding to the PD-L1 receptor on antigen-presenting cells or tumor cells.  
Further information about mechanism of action can be obtained from the IMPD of89Zr-
atezolizumab (version 8) . 
 
b. Previous exposure of human beings with the test product(s) and/or products with a 
similar biological mechanism 
This study in which 89Zr-atezolizumab will be applied in patients with R/R LBCL who are 
eligible for CD19-directed CAR T-cell therapy will be the first study with 89Zr-atezolizumab 
imaging in this specific patient category. Previous study with 89Zr-atezolizumab imaging in 
patients with active solid malignancies were noted to be safe (see IMPD). In addition, several 
studies with 89Zr-labeled antibodies have demonstrated that the isotope used (89Zr) is safe 
for human use. 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo human 
cell material? 
See the “IMPD 89Zr-atezolizumab, version 8”. In addition, several preclinical studies with 89Zr-
labeled antibodies demonstrate the feasibility and safety of PET imaging in cynomolgus 
monkeys. 
 

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d. Selectivity of the mechanism to target tissue in animals and/or human beings 
89Zr-atezolizumab binds selectively to human programmed death-ligand 1 (PD-L1) on 
antigen-presenting cells or tumor cells. 
 
f. Pharmacokinetic considerations 
Pharmacokinetic analysis in a recent first-in-human study showed good correlation with the 
blood pool on PET with a total tracer protein dose of 10 (unlabeled atezolizumab with 
~2,5mg 89Zr-atezolizumab). 89Zr-atezolizumab uptake was comparable to results of other 
89Zr-labeled antibodies. For further information see the “IMPD 89Zr-atezolizumab, version 8”.  
 
g. Study population 
For information about the study population, see section 1.4.  
 
h. Interaction with other products 
The dose of 89Zr-atezolizumab is unlikely to cause any interactions.  
 
i. Predictability of effect 
In this study no pharmacological effect will be pursued with the investigational product, so 
no biomarkers are used to predict a clinical effect. 
 
j. Can effects be managed? 
Subjects will be carefully monitored for side effects of 89Zr-atezolizumab. A protocol for 
intervention in case of infusion reactions is available and known by clinical care staff. 
 

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14.2 Synthesis 
For a full synthesis of risk associated with the investigational product and burden for the 
patient please refer to section 12.4. 

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15. APPENDIX  
15.1 Lymphoma biopsy: Safety  
 
The mandatory biopsy will be taken within 7 days of the 89Zr-atezolizumab PET/CT scan 
before CAR T-cell treatment start.  
In UMCG, histologic and mutation analysis of tumors is currently standard practice. 
Therefore, biopsies are necessary to guide the best treatment. Patients are involved in this 
decision as part of good clinical practice and the procedure is discussed for safety reasons in 
the weekly multidisciplinary board or together with biopsy performing specialist. 
Histological biopsy of a lymphoma lesion will be performed by an interventional-radiologist 
under CT or ultrasound guidance. Registration is needed on which lymphoma lesion the 
biopsy has been performed.  
All biopsy site(s) will be chosen based on safety aspects. Review of the literature shows that 
in general, tumor biopsies can be performed with only minor complications and acceptable 
risks (Table 1). Although biopsies are generally considered to be safe, the location of the 
tumor is an important determinant of the risk associated with the biopsy. The risk 
assessment of liver biopsies shows that overall mortality of ultrasound-guided large-core 
needle biopsy of liver tumors is very small, i.e., < 0.5 %. Large-core needle biopsies of liver 
lesions are performed with an 18G biopsy needle and the use of a 17G guiding needle, which 
is positioned into the target lesion. In general, 2-3 biopsy samples (specimen length 22 mm) 
per lesion are obtained. Complications are generally limited when precautions are taken into 
account (e.g., adequate coagulation status). Bleeding risk, in general, is considered to be 
lower than 10%. This percentage is much lower when CT of the biopsied lesion is carefully 
imaged and evaluated whether vessels are present in the biopsy track. In contrast, biopsies 

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of lung metastases have a risk of causing pneumothorax, whereas biopsies of subcutaneous 
metastases are considered to be very safe. 
Overall, tumor biopsies provide important information and pose a minor burden to the 
patient’s well-being. Biopsies are conducted according to the local clinical protocol. All 
patients will be observed after biopsy according to the local protocol for a full recovery.  
 

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Author 
Study design 
No. 
of 
patients 
(pts) 
No. of biopsies 
Overall 
mortality 
Mortality 
in 
patients 
with 
malignancy 
Significant or major 
complications 
Pain 
Bleeding 
Complications 
associated 
with malignancy 
Meyers, 200825 
Retrospective review of 
records (Percutaneous 
liver biopsies) 
3627 
4275 
6 pts (0.17% 
of pts; 0.14% 
of bps) 
6/? (total no. of 
pts 
with 
malignancies 
NR) 
32 ptsb (0.75% of all 
biopsies) 
22/3627 
(0.51%) 
requiring 
admission 
15/3627c 
(0.35%) 
15/32 
(47%; 
HCC 
n=5; 
metastases 
n=10) 
8/15 
(53%) 
bleeding 
had 
malignancy 
Appelbaum, 
200826 
Retrospective 
study 
(FNAB)  
208 
208 (408 passes) 
0 
0 
0 
10/208 
(4.8%) 
requiring 
analgesics 
0d 
0 (176 Malignant tumors of 
which 128 metastases) 
Thanos, 200527 
Prospective study (18-
gauge 
automated 
biopsy gun) 
767 
(2351 passes) 
0 
0 
0 
8/767 (1.0%) had 
mild pain during 
biopsy 
1e 
NR 
(736/767 
malignant 
tumors; 
of 
which 
193 
metastases) 
Terjung, 200328 
Retrospective review of 
records 
574 
629 
3/629 (0.48%) 
0 
10/629 (1.6%) 
NR 
72/629f 
(11.4%) 
7/79 (8.9%; HCC n=7/26, 
metastases n=0/53) 
McGill, 
199029 
Prospective 
recorded 
data  
9212 
9212 
10/9212 
(0.11%)  
7/1766 (0.4%) 
22/9212 
(0.24%) 
hemorrhage 
NR 
22/9212 
(0.24%) 
significant 
bleeding 
0.57% 
non-fatal 
hemorrhage in cancer pts 
(2-6 x higher risk of bleeding 
than without cancer) 
Piccinino, 
198630 
Retrospective 
multicenter study 
NR 
68276 
6 pts 
3 pts (total no. 
with malignancy 
NR) 
NR 
NR 
42/68276 
(0.06%) 
5/1755 (0.28%) 
NR = not reported; a five pts died due to massive bleeding and one due to aspiration pneumonia and congestive heart failure; b 15/32 (47%) had malignancy; c 8/15 (53%) had malignancy; d not checked for subclinical 
hemorrhage; e one perihepatic hematoma; f symptomatic and asymptomatic 
Table 1 Safety of tumor biopsies 
(from CPCT – 02 biopsy protocol, Development of a platform for next-generation DNA sequencing-based personalized  for cancer patients: 
Protocol to obtain biopsies from patients with locally advanced (incurable) or metastatic cancer, ToetsingOnline nr.: NL35781.041.11) 

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15.2 Adverse Event Severity Grading Scale 
 
Grade  
Severity 
1  
Mild; asymptomatic or mild symptoms; clinical or diagnostic observations 
only; or intervention not indicated 
2  
Moderate; minimal, local, or non-invasive intervention indicated; or limiting 
age-appropriate instrumental activities of daily livinga 
3  
Severe or medically significant, but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; or 
limiting self-care activities of daily livingb,c 
4 
Life-threatening consequences or urgent intervention indicatedd 
5 
Death related to adverse eventd 
 
NCI CTCAE= National Cancer Institute Common Terminology Criteria for Adverse Events. 
Note: Based on the version of NCI CTCAE v4.0, which can be found at: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm 
 
a  Instrumental activities of daily living refer to preparing meals, shopping for groceries or 
clothes, using the telephone, managing money, etc. 
b  Examples of self-care activities of daily living include bathing, dressing and undressing, 
feeding one's self, using the toilet, and taking medications, as performed by patients who 
are not bedridden. 
c  If an event is assessed as a "significant medical event," it must be reported as a serious 
adverse event, per the definition of serious adverse event in 9.2.2. 

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d  Grade 5 events must be reported as serious adverse events, per the definition of serious 
adverse event in Section 10.2.2. 
 
 
 

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15.3 Timeline and procedures 
Study flowchart 
* Exact moment of 89ZR-PET scan (4 or 7 days after tracer injection) will be determined in the first 3 patients.  
** In case of suspicion of a relapse or pseudoprogression based on a FDG-PET scan. 
 
Study flowchart 
Screening 
 
Apheresis 
Radiation 
therapy  
(if 
applicable) 
Infusion 
of tracer 
89Zr - PET 
scan  
 
Biopsy  
FDG-PET 
scan 1 
 
Infusion of 
CAR T-cells 
FDG-PET 
scan 2 
 
Visit 
Infusion 
of tracer 
** 
89Zr - PET 
scan** 
 
Biopsy** 
Window of assessments 
D-35 to  
D-30 
D-28 
D-27  
to -10 
D-19 
 to - 10 
D-12  
to -6 
D-12 
to -6 
D-6 
D0 
D28 
D30±2 
D … 
D … 
D …. 
 
 
 
D X 
D X 
+ 4-7* 
 
 
 
 
 
D X 
D X+ 4-7* 
DX+ 11-
14 
 
 
 
 
 
Signed Informed Consent Forms1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Review of eligibility criteria 
(both CAR T-cell therapy and anti-PD-L1 imaging) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Medical, surgical, and cancer histories, including 
demographic information2 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Concomitant medication3 
X 
X 
X 
 
X 
 
 
 
X 
X 
 
X 
 
Physical examination4 
X 
X 
X 
 
X 
 
 
 
X 
X 
 
X 
 
Vital signs5 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
ECOG performance status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Weight and height 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
HIV, HBV, and HCV serology6 
X 
 
 
 
 
 
 
 
 
 
 
 
 
CRP and ferritin7 
X 
X 
X 
 
 
 
 
X 
X 
 
 
 
 
Blood laboratory tests (including cytokines)8 
X 
 
 
 
 
 
X 
X 
X 
 
 
X 
 
Tumor assessment (FDG-PET CT)9  
X 
 
 
 
 
 
X 
 
X 
 
 
 
 
12-lead electrocardiogram10 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Echocardiogram/MUGA scan11 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Infusion of 89Zr-atezolizumab 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
89Zr-atezolizumab -PET scan12 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
Fresh tumor biopsy13 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
Archive tumor tissue 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 

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1Written informed consent can be obtained up to 30 days prior to study entry and is required for performing any study-specific tests or 
procedures. Results of standard-of-care tests or examinations performed prior to obtaining informed consent and within 28 days prior to study 
entry may be used for screening assessments rather than repeating such tests. 
2 Cancer history includes stage, date of diagnosis, and prior anti-tumor treatment. Demographic information includes sex, age, and self-
reported race/ethnicity. A history of pleural or pericardial effusion or of ascites requiring intervention should be entered in the medical history 
Reproductive status and smoking history should also be captured.  
3 Concomitant medications include any prescription medications or over-the-counter medications. At screening, any medications the patient 
has used within the 7 days prior to the screening visit should be documented. At subsequent visits, changes to current medications or 
medications used since the last documentation of medications will be recorded. 
4 Complete physical examination is defined in Section 9.8. A limited physical examination will be performed at screening visit and on every 
other visit if clinically indicated.  
5 Vital signs at screening visit include pulse rate, blood oxygen saturation, respiratory rate, systolic and diastolic blood pressures while the 
patient is in a seated position, and temperature. On the day of injection, the patient’s vital signs (pulse rate, respiratory rate, blood pressure, 
and temperature) should be determined within 60 minutes before, directly after (± 10 minutes) and 30 (± 10) minutes after injection of the 

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unlabeled dose, as well as directly before and 60 minutes (± 15 minutes) after the infusion dose (pulse rate, blood pressure, and temperature 
only). Additional vital signs should be determined during the infusion if clinically indicated. 
6 All patients will be tested for HIV prior to the inclusion into the study and HIV-positive patients will be excluded from the clinical trial. 
Hepatitis B surface antigen, anti-HBc antibody, anti-HBs antibody, and anti-HCV antibody should be collected during screening. In patients who 
have positive serology for the anti-HBc antibody, HBV DNA should be collected prior to 89Zr-atezolizumab injection. 
7 CRP and ferritin testing will be performed as part of the screening assessments and if clinically indicated. 
8 Hematology consists of CBC, including RBC count, hemoglobin, hematocrit, WBC count with automated differential (neutrophils, lymphocytes, 
eosinophils, monocytes, basophils, and other cells), and platelet count. A manual differential can be done if clinically indicated. Serum 
chemistry includes urea, creatinine, sodium, potassium, magnesium, chloride, bicarbonate, calcium, phosphorus, glucose, total bilirubin, ALT, 
AST, alkaline phosphatase, lactate dehydrogenase, total protein, albumin, and C-reactive protein. Urine analysis includes specific gravity, pH, 
glucose, protein, ketones, and blood. Furthermore, only at screening, a serum pregnancy test will be performed and TSH, free T3, free T4 will 
be assessed. Serum pregnancy test (for women of childbearing potential, including women who have had a tubal ligation) must be performed 
and documented as negative within 14 days prior to 89Zr-atezolizumab injection. Cytokines include IL-2, IL-6, IL-10, IFN. 
9 Tumor assessments (18F-FDG PET/CT scan of chest, abdomen and pelvis) must be performed ≤14 days before injection, otherwise, a new 
diagnostic CT scan needs to be performed with one of the 89Zr-atezolizumab-PET scans. A CT or MRI, which has been performed >14 days but ≤ 

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42 days before injection, can be used for assessment of the eligibility criteria if no systemic or local treatment has been applied in-between. All 
measurable and evaluable lesions should be assessed and documented at the screening visit.  
10 ECG recordings will be obtained during screening. Patients should be resting and in a supine position for at least 10 minutes prior to ECG 
collection. 
11 Required at screening. MUGA is an acceptable modality for this assessment. In such patients, echocardiograms will be performed at 
screening and clinically indicated. Patients who develop new pericardial effusions while on study must be followed by echocardiography. 
12 In part A every patient will undergo 4 mandatory PET scans. Depending on the findings in part A, in part B study PET scans will be performed 
according to the schedule as assessed in part A. 
13 All patients will undergo a mandatory tumor biopsy sample collection, if clinically feasible as determined by the study investigator, preferably 
after the PET scan procedures. Acceptable samples include core needle biopsies for deep tumor tissue or lymph nodes or excisional, incisional, 
punch, or forceps biopsies for cutaneous, subcutaneous, or mucosal lesions. For core needle biopsy specimens, at least 2-3 cores should be 
submitted for evaluation.  
 
 
 

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15.4 CAR T-cell inclusion criteria 
 
Verwijsformulier CAR T-celbehandeling DLBCL/ALL buiten studieverband
 
  
*Let op: 
- 
Dit formulier biedt geen garantie dat de patiënt daadwerkelijk voor CAR-T cel 
behandeling in aanmerking komt.  
- 
Bij afwijkende waarden overleggen met het CAR T-cel centrum.  
- 
Voor CAR T-cel behandeling in studieverband gelden aanvullende in- & exclusiecriteria.  
- 
Dit formulier wordt beoordeeld door de landelijke CAR T-cel tumorboard en naar 
aanleiding hiervan wordt binnen 3 werkdagen contact met de verwijzer opgenomen. 
Als de patiënt aan de voorwaarden lijkt te voldoen én er een slot beschikbaar is voor 
behandeling zal de patiënt binnen een week worden opgeroepen voor screening.  
 
- 
Dit formulier graag zo compleet mogelijk invullen, en samen met verwijsbrief en 
recente/relevante verslagen (lab/beeldvorming/pathologie/etc.) mailen naar het 
regionale CAR T-celcentrum en naar het landelijke email adres: cart-
nl@amsterdamumc.nl.  
 
- 
Recente/relevante beeldvorming ook graag zsm op CDrom opsturen naar het regionale 
CAR T-celcentrum 
 
- 
Tevens patiënt informed consent laten tekenen voor opname van de geanonimiseerde 
verwijsgegevens in de “Database CAR T-celbehandeling Nederland” en informed 
consent meesturen. 
                                                    
Indicatie: 
  
☐ DLBCL : na 2 eerdere lijnen systemische behandeling (DLBCL, PMBL, transformed FL)  
                  (Yescarta) 
☐ ALL: Pediatrische en jongvolwassen patiënten tot 25 jaar, refractair of met een 
             recidief na transplantatie of met tweede/later recidief (Kymriah) 
 
 
 
Info verwijzer: 
 
Naam arts: Klik of tik om tekst in te voeren.    
 
Naam centrum: Klik of tik om tekst in te voeren.     
 
Ingevuld door: Klik of tik om tekst in te voeren.   
 
Datum: Klik of tik om een datum in te voeren. 
 
 
Info patiënt: 
 
Achternaam & initialen:  Klik of tik om tekst in te 
voeren. 
 
 
 
 
 
Geboortedatum: Klik of tik om een datum in te 
voeren. 
                              

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Patiëntgegevens 
Diagnose: 
Lengte:  
Gewicht:  
Reisafstand ≤ 1 uur tot het CAR T-celcentrum:  
Alleenwonend:  
Klik of tik om tekst in te voeren. 
Klik of tik om tekst in te voeren. 
Klik of tik om tekst in te voeren.kg 
 
☐ JA / ☐ NEE 
☐ JA / ☐ NEE 
 
Diagnose: 
Lengte:  
Gewicht:  
Reisafstand ≤ 1 uur tot het CAR T-celcentrum:  
Alleenwonend:  
Klik of tik om tekst in te voeren. 
Klik of tik om tekst in te voeren. 
Klik of tik om tekst in te voeren.kg 
 
☐ JA / ☐ NEE 
☐ JA / ☐ NEE 
 
Voorgeschiedenis 
Hematologische VG 
 Klik of tik om tekst in te voeren. 
Niet-hematologische VG 
 Klik of tik om tekst in te voeren. 
 
Diagnostiek 
Beeldvorming  
(CT, PET/CT, Cerebrale MRI of CT) 
 Klik of tik om tekst in te voeren. 
Pathologie  
(lymfeklier-, beenmergbiopt, liquor) 
 
Klik of tik om tekst in te voeren. 
 
 
 
 
 
 
 
 
 

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Eerdere behandellijnen 
Start- & stopdatum 
Respons 
 
1e lijn: Klik of tik om tekst in te 
voeren. 
Klik of tik om een datum in te voeren. 
Klik of tik om een datum in te voeren. 
 
 Klik of tik om tekst in te voeren. 
 
 
2de lijn: Klik of tik om tekst in te 
voeren. 
Klik of tik om een datum in te voeren. 
Klik of tik om een datum in te voeren. 
 
 Klik of tik om tekst in te voeren. 
 
 
3de lijn: Klik of tik om tekst in te 
voeren. 
Klik of tik om een datum in te voeren. 
Klik of tik om een datum in te voeren. 
 
 Klik of tik om tekst in te 
voeren. 
 
 
4de lijn: Klik of tik om tekst in te 
voeren. 
Klik of tik om een datum in te voeren. 
Klik of tik om een datum in te voeren. 
 
 Klik of tik om tekst in te 
voeren. 
 
 
≥5de lijn: Klik of tik om tekst in 
te voeren. 
Klik of tik om een datum in te voeren. 
Klik of tik om een datum in te voeren. 
 
 Klik of tik om tekst in te 
voeren. 
 
Toxiciteit na eerdere behandelingen ≤ graad 
1 m.u.v. klinisch niet relevante afwijkingen 
zoals haaruitval. 
☐ JA / ☐ NEE 
 Klik of tik om tekst in te voeren. 
 
Kwalificatie 
Leeftijd ≥ 18 
☐ JA / ☐ NEE 
Klik of tik om een datum in te voeren. 
WHO-PS 0 - 2 
☐ JA / ☐ NEE 
Klik of tik om een datum in te voeren. 
Meetbare ziekte (LNN of extranodale laesie ≥ 15 
mm) 
☐ JA / ☐ NEE 
Klik of tik om een datum in te voeren. 
Hematologische parameters: 
- 
ANC ≥ 1000/µL (1x10e9/L) 
- 
Trombocyten ≥ 75,000/µL (75x10e9/L)  
- 
ALC ≥ 100/µL (0.1x10e9/L) 
 
☐ JA / ☐ NEE 
☐ JA / ☐ NEE 
☐ JA / ☐ NEE 
 
Klik of tik om een datum in te voeren. 
Klik of tik om een datum in te voeren. 
Klik of tik om een datum in te voeren. 

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Adequate nierfunctie: 
- 
Kreatinine ≤ 1.5xULN 
- 
Kreatinineklaring > 40 ml/min (Cockcroft) 
 
☐ JA / ☐ NEE  
☐ JA / ☐ NEE 
 
Klik of tik om een datum in te voeren. 
Klik of tik om een datum in te voeren.  
Adequate leverfunctie: 
- 
ALAT/ASAT ≤ 3xULN 
- 
Totaal Bilirubine < 2 mg/dl (34 µmol/L) 
tenzij syndroom van Gilbert 
 
☐ JA / ☐ NEE 
☐ JA / ☐ NEE 
 
Klik of tik om een datum in te voeren. 
CRP < 100 mg/L  
Alternatieve verklaring indien verhoogd? 
☐ JA / ☐ NEE 
Klik of tik om een datum in te voeren. 
Klik of tik om tekst in te voeren. 
Adequate hartfunctie: 
- 
Ejectiefractie > 40% en klinisch goede LV 
functie 
- 
Significant pericardvocht 
- 
Klinisch significante ECG afwijkingen 
 
☐ JA / ☐ NEE 
☐ JA / ☐ NEE 
☐ JA / ☐ NEE 
 
Klik of tik om tekst in te voeren.  
Klik of tik om tekst in te voeren. 
Klik of tik om tekst in te voeren. 
Adequate longfunctie: 
- 
Significant pleuravocht 
- 
Baseline saturatie ≥ 92% bij kamerlucht 
 
☐ JA / ☐ NEE 
☐ JA / ☐ NEE 
 
Klik of tik om tekst in te voeren.  
Klik of tik om tekst in te voeren. 
HIV/HepB/HepC infectie 
☐ JA / ☒ NEE  
Klik of tik om tekst in te voeren. 
Overige actieve infectie 
☐ JA / ☐ NEE  
Klik of tik om tekst in te voeren. 
Verdenking of bewezen CZS lokalisatie lymfoom 
☐ JA / ☐ NEE 
Klik of tik om tekst in te voeren. 
Neurologische afwijkingen 
☐ JA / ☐ NEE 
Klik of tik om tekst in te voeren. 
Auto-immuunziekte 
☐ JA / ☐ NEE  
Klik of tik om tekst in te voeren.  
Andere significante aandoeningen 
☐ JA / ☐ NEE 
Klik of tik om tekst in te voeren. 
Opmerkingen: 
Klik of tik om tekst in te voeren. 
 
 
 
 
 
 
 
Contactpersonen en email adressen CAR T-celcentra: 
Landelijke email adres CAR T-cel tumorboard: CART-NL@amsterdamumc.nl 
Regio Amsterdam UMC (AMC/VUMC): Marie José Kersten: contact: CAR-T-AMC@amsterdamumc.nl 
Regio UMCG: Tom van Meerten: contact: atmp@onco.umcg.nl 
Regio UMCN: Suzanne van Dorp: contact: coordinatiestamceltransplantatie.hemat@radboudumc.nl 
Regio Erasmus MC: Pim Mutsaers & Elly Lugtenburg: contact: SCT@erasmusmc.nl 
Regio UMC Utrecht : Monique Minnema & Margot Jak: contact: sct-hematologie@umcutrecht.nl 
 
 

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