Clinical trial NCT01583686
Conditions: Cervical Cancer Pancreatic Cancer Ovarian Cancer Mesothelioma Lung Cancer
Keywords: Metastatic Cancer Immunotherapy Gene Therapy Mesothelioma Pancreatic Cancer
Sponsor: National Cancer Institute (NCI)
Start date: 2012-05-04; Completion date: 2018-12-17
Enrollment: 15 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
Background:

The National Cancer Institute (NCI) Surgery Branch has developed an experimental therapy for treating patients with metastatic cancer that involves taking white blood cells from the patient, growing them in the laboratory in large numbers, genetically modifying these specific cells with a type of virus (retrovirus) to attack only the tumor cells, and then giving the cells back to the patient. This type of therapy is called gene transfer. In this protocol, we are modifying the patients white blood cells with a retrovirus that has the gene for anti-mesothelin incorporated in the retrovirus.

Objective:

The purpose of this study is to determine a safe number of these cells to infuse and to see if these tumor fighting cells (anti-mesothelin cells) cause metastatic cancer tumors to shrink.

Eligibility:

\- Adults age 18-70 with metastatic cancer expressing the mesothelin molecule.

Design:

Work up stage: Patients will be seen as an outpatient at the National Institutes of Health (NIH) clinical Center and undergo a history and physical examination, scans, x-rays, lab tests, and other tests as needed

Leukapheresis: If the patients meet all of the requirements for the study they will undergo leukapheresis to obtain white blood cells to make the anti-mesothelin cells. {Leukapheresis is a common procedure, which removes only the white blood cells from the patient.}

Treatment: Once their cells have grown, the patients will be admitted to the hospital for the conditioning chemotherapy, the anti-mesothelin cells, and aldesleukin. They will stay in the hospital for about 4 weeks for the treatment.

Follow up: Patients will return to the clinic for a physical exam, review of side effects, lab tests, and scans about every 1-3 months for the first year, and then every 6 months to 1 year as long as their tumors are shrinking. Follow up visits will take up to 2 days.

Detailed description:
Background:

* We have constructed a single retroviral vector that contains a chimeric T cell receptor (CAR) that recognizes mesothelin, which can be used to mediate genetic transfer of this CAR with high efficiency (\> 50%) without the need to perform any selection.
* In co-cultures with mesothelin expressing cells, anti-mesothelin transduced T cells secreted significant amounts of interferon (IFN)-gamma with high specificity.

Objectives:

Primary Objectives:

* To evaluate the safety of the administration of anti-mesothelin CAR engineered peripheral blood lymphocytes in patients receiving a non- myeloablative conditioning regimen, and aldesleukin.
* Determine if the administration anti-mesothelin CAR engineered peripheral blood lymphocytes and aldesleukin to patients following a nonmyeloablative but lymphoid depleting preparative regimen will result in clinical tumor regression in patients with metastatic cancer.

Eligibility:

Patients who are 18 years of age or older must have

* Metastatic or unresectable cancer that expresses mesothelin;
* Previously received and have been a non-responder to or recurred after standard care;

Patients may not have:

-Contraindications for low dose aldesleukin administration.

Design:

* Peripheral blood mononuclear cells (PBMC) obtained by leukapheresis will be cultured in order to stimulate T-cell growth.
* Transduction is initiated by exposure of approximately 10\^8 to 5 X 10\^8 cells to retroviral vector supernatant containing the anti-mesothelin CAR.
* Patients will receive a nonmyeloablative but lymphocyte depleting preparative regimen consisting of cyclophosphamide and fludarabine followed by intravenous infusion of ex vivo CAR gene-transduced PBMC plus low dose intravenous (IV) aldesleukin
* Patients will undergo complete evaluation of tumor with physical examination, Computed tomography (CT) of the chest, abdomen and pelvis and clinical laboratory evaluation four to six weeks after treatment. If the patient has stable disease (SD) or tumor shrinkage, repeat complete evaluations will be performed every 1-3 months. After the first year, patients continuing to respond will continue to be followed with this evaluation every 3-4 months until off study criteria are met.
* The study will be conducted using a Phase I/II optimal design. The protocol will proceed in a phase 1 dose escalation design. Once the maximum tolerated dose (MTD) has been determined, the study then would proceed to the phase II portion. Patients will be entered into two cohorts based on histology: cohort 1 will include patients with mesothelioma, and cohort 2 will include patients with other types of cancer that express mesothelin.
* For each of the 2 strata evaluated, the study will be conducted using a phase II optimal design where initially 21 evaluable patients will be enrolled. For each of these two arms of the trial, if 0 or 1 of the 21 patients experiences a clinical response, then no further patients will be enrolled but if 2 or more of the first 21 evaluable patients enrolled have a clinical response, then accrual will continue until a total of 41 evaluable patients have been enrolled in that stratum.

Arms: arm 1: Non-myeloablative but lymphodepleting preparative regimen of cyclophosphamide and fludarabine plus anti-mesothelin chimeric T cell receptor (CAR) transduced peripheral blood lymphocytes (PBL) plus low dose aldesleukin. arm 2: Non-myeloablative lymphocyte depleting preparative regimen of cyclophosphamide and fludarabine + anti-mesothelin chimeric T cell receptor (CAR) transduced peripheral blood lymphocytes (PBL) + low-dose aldesleukin
Interventions: intervention 1: Fludarabine intervention 2: Anti-mesothelin chimeric T cell receptor (CAR) transduced peripheral blood lymphocytes (PBL) intervention 3: Cyclophosphamide intervention 4: Aldesleukin
Intervention descriptions: intervention 1: Days -5 to Day -1: Fludarabine 25 mg/m\^2/day intravenous piggyback (IVPB) daily over 30 minutes for 5 days. intervention 2: Day 0: Cells will be infused intravenously (IV) on the Patient Care Unit over 20 to 30 minutes. intervention 3: Days -7 and -6: Cyclophosphamide 60 mg/kg/day X 2 days intravenous (IV) in 250 ml 5% dextrose in water (D5W) with Mesna 15 mg/kg/day X 2 days over 1 hr. intervention 4: Aldesleukin 72,000 IU/kg intravenous (IV) (based on total body weight) over 15 minutes every eight hours (+/- 1 hour) beginning within 24 hours of cell infusion and continuing for up to 5 days (maximum of 15 doses).

Protocol documents:
Prot_SAP_000.pdf: 
Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
 
1 
Abbreviated Title: Anti-mesothelin CAR PBL 
CC Protocol Number: 12-C-0111 P 
IBC Number: RD-12-I-02 
OSP Number: 1112-1139  
NCT Number: NCT01583686  
Version Date: April 12, 2018 
PROTOCOL TITLE 
A Phase I/II Study of Metastatic Cancer Using Lymphodepleting Conditioning Followed by 
Infusion of Anti-mesothelin Gene Engineered Lymphocytes 
 
NIH Principal Investigator: Steven A. Rosenberg, M.D., Ph.D. 
 
 
 
 
 
Chief of Surgery, Surgery Branch, CCR, NCI 
 
 
 
 
 
Building 10, CRC, Room 3-3940 
 
9000 Rockville Pike, Bethesda, MD 20892 
 
 
 
 
 
Phone: 240-760-6218; Email: Steven.Rosenberg@nih.gov 
 
 
Investigational Agents: 
 
Drug Name: 
Anti-mesothelin CAR (coSS1scFv-CD28Z) 
transduced peripheral blood lymphocytes (PBL) 
IND Number: 
14989 
Sponsor: 
Center for Cancer Research 
Manufacturer: Surgery Branch Cell Production Facility 
 
Commercial Agents:  Cyclophosphamide, Fludarabine, and Aldesleukin 
 
 
 
 
 
 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
 
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PRÉCIS 
Background: 
x We have constructed a single retroviral vector that contains a chimeric T cell receptor 
(CAR) that recognizes mesothelin, which can be used to mediate genetic transfer of this 
CAR with high efficiency (> 50%) without the need to perform any selection.    
x In co-cultures with mesothelin expressing cells, anti-mesothelin transduced T cells secreted 
significant amounts of IFN-Ȗ with high specificity).   
 
Objectives: 
Primary objectives:  
x To evaluate the safety of the administration of anti-mesothelin CAR engineered peripheral 
blood lymphocytes in patients receiving a non- myeloablative conditioning regimen, and 
aldesleukin. 
x Determine if the administration anti-mesothelin CAR engineered peripheral blood 
lymphocytes and aldesleukin to patients following a nonmyeloablative but lymphoid 
depleting preparative regimen will result in clinical tumor regression in patients with 
metastatic cancer. 
 
Eligibility: 
Patients who are 18 years of age or older must have  
x metastatic or unresectable cancer that expresses mesothelin; 
x previously received and have been a non-responder to or recurred after standard care;  
x Patients may not have contraindications for low dose aldesleukin administration. 
 
Design: 
x PBMC obtained by leukapheresis will be cultured in order to stimulate T-cell growth. 
x Transduction is initiated by exposure of approximately 108 to 5 X 108 cells to retroviral 
vector supernatant containing the anti-mesothelin CAR.   
x Patients will receive a nonmyeloablative but lymphocyte depleting preparative regimen 
consisting of cyclophosphamide and fludarabine followed by intravenous infusion of ex 
vivo CAR gene-transduced PBMC plus low dose IV aldesleukin  
x Patients will undergo complete evaluation of tumor with physical examination, CT of the 
chest, abdomen and pelvis and clinical laboratory evaluation four to six weeks after 
treatment.  If the patient has SD or tumor shrinkage, repeat complete evaluations will be 
performed every 1-3 months.  After the first year, patients continuing to respond will 
continue to be followed with this evaluation every 3-4 months until off study criteria are 
met. 
x The study will be conducted using a Phase I/II optimal design.  The protocol will proceed 
in a phase 1 dose escalation design.  Once the MTD has been determined, the study then 
would proceed to the phase II portion. Patients will be entered into two cohorts based on 
histology: cohort 1 will include patients with mesothelioma, and cohort 2 will include 
patients with other types of cancer that express mesothelin.   
 
x For each of the 2 strata evaluated, the study will be conducted using a phase II optimal 
design where initially 21 evaluable patients will be enrolled. For each of these two arms of 


Abbreviated Title: Anti-mesothelin CAR PBL 
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the trial, if 0 or 1 of the 21 patients experiences a clinical response, then no further patients 
will be enrolled but if 2 or more of the first 21 evaluable patients enrolled have a clinical 
response, then accrual will continue until a total of 41 evaluable patients have been enrolled 
in that stratum.  


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TABLE OF CONTENTS 
PRÉCIS ....................................................................................................................................... 2 
TABLE OF CONTENTS .......................................................................................................... 4 
1 
INTRODUCTION.............................................................................................................. 8 
1.1 
Study Objectives: ............................................................................................................. 8 
1.1.1 Primary objectives: ...................................................................................................... 8 
1.1.2 Secondary objective: .................................................................................................... 8 
1.2 
Background and Rationale: .............................................................................................. 8 
1.2.1 Prior Surgery Branch Trials of Cell Transfer Therapy Using Tumor Infiltrating 
Lymphocytes in Patients with Metastatic Melanoma .............................................................. 9 
1.2.2 Surgery Branch Trials of Cell Transfer Therapy Using Transduction of Anti-TAA 
TCR Genes into Non-reactive TIL or PBL ............................................................................ 10 
1.2.3 Mesothelin as a Target for Cell Transfer Studies ...................................................... 12 
1.2.4 Preclinical Studies to Provide the Rationale for this Clinical Protocol ..................... 13 
1.2.5 Construction and testing of retroviral vectors expressing CAR against human 
mesothelin .............................................................................................................................. 14 
1.2.6 Safety Considerations ................................................................................................ 15 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT ............................................... 17 
2.1 
Eligibility Criteria .......................................................................................................... 17 
2.1.1 Inclusion Criteria ....................................................................................................... 17 
2.1.2 Exclusion Criteria ...................................................................................................... 18 
2.2 
Screening Evaluation ..................................................................................................... 19 
2.2.1 Within 3 months of prior enrollment ......................................................................... 19 
2.2.2 Within 8 weeks prior to enrollment ........................................................................... 19 
2.2.3 Within 4 weeks prior to enrollment: .......................................................................... 19 
2.2.4 Within 14 days prior to enrollment: ........................................................................... 20 
2.2.5 Within 7 days prior to enrollment: ............................................................................. 20 
2.3 
Protocol Registration ..................................................................................................... 20 
2.3.1 Prior to registration for this protocol: ........................................................................ 20 
2.3.2 Registration Procedure: .............................................................................................. 20 
3 
STUDY IMPLEMENTATION ....................................................................................... 20 
3.1 
Study Design .................................................................................................................. 20 
3.1.1 Treatment Phase: ........................................................................................................ 20 
3.1.2 Phase 1 - Dose Escalation: ......................................................................................... 21 
3.1.3 Phase 2 Portion: ......................................................................................................... 22 
3.1.4 Definition of Dose Limiting Toxicity (DLT): ........................................................... 23 
3.1.5 Protocol Stopping Rules: ........................................................................................... 23 
3.2 
Drug Administration ...................................................................................................... 24 


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3.2.1 Preparative Regimen with Cyclophosphamide and Fludarabine ............................... 24 
3.2.2 Cell Infusion and Aldesleukin Administration .......................................................... 25 
3.2.3 Treatment Schedule ................................................................................................... 26 
3.3 
On-Study Evaluation ...................................................................................................... 26 
3.3.1 Within 28 days prior to starting the preparative regimen .......................................... 26 
3.3.2 Within 14 days prior to starting the preparative regimen .......................................... 26 
3.3.3 During the preparative regimen: DAILY ................................................................... 27 
3.3.4 After Cell Infusion: .................................................................................................... 27 
3.3.5 During Hospitalization (following aldesleukin administration): ............................... 27 
3.4 
Post Treatment (Follow-up) Evaluation......................................................................... 27 
3.4.1 Time period of evaluations ........................................................................................ 28 
3.4.2 At each evaluation, patients will undergo: ................................................................. 28 
3.5 
Criteria for Removal from Protocol Therapy and Off Study Criteria ............................ 28 
3.5.1 Criteria for removal from protocol therapy: .............................................................. 29 
3.5.2 Off Study Criteria: ..................................................................................................... 29 
3.5.3 Off Protocol Therapy and Off Study Procedure ........................................................ 29 
4 
CONCOMITANT MEDICATIONS/MEASURES ....................................................... 29 
4.1 
Infection Prophylaxis: .................................................................................................... 29 
4.1.1 Pneumocystis Jirovecii Pneumonia............................................................................ 29 
4.1.2 Herpes and Varicella Zoster Virus Prophylaxis......................................................... 30 
4.1.3 Fungal Prophylaxis .................................................................................................... 30 
4.2 
Blood Product Support ................................................................................................... 30 
4.3 
Empiric antibiotics ......................................................................................................... 30 
4.4 
Other Concomitant medications to control side effects ................................................. 30 
5 
BIOSPECIMEN COLLECTION ................................................................................... 31 
5.1 
Samples Sent to Dr. Figg’s lab ...................................................................................... 31 
5.2 
Samples Sent to the Surgery Branch Cell Processing Laboratory ................................. 31 
5.3 
Prior to chemotherapy administration ............................................................................ 31 
5.4 
Prior to cell infusion (1-8ml SST) ................................................................................. 31 
5.5 
Post cell infusion evaluations......................................................................................... 32 
5.6 
Immunological Testing .................................................................................................. 32 
5.7 
Monitoring Gene Therapy Trials:  Persistence and RCR .............................................. 32 
5.8 
sample, storage, tracking and disposition for SB Cell Processing Laboratory .............. 33 
5.9 
Sample Storage, Tracking and Disposition for Dr. Figg’s lab ....................................... 33 
5.9.1 Sample Data Collection ............................................................................................. 33 
5.9.2 Sample Storage and Destruction ................................................................................ 34 
6 
DATA COLLECTION AND EVALUATION .............................................................. 34 
6.1 
Data Collection .............................................................................................................. 34 


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6.1.1 Exclusions to Routine Adverse Event Reporting: ..................................................... 35 
6.2 
Data Sharing Plans ......................................................................................................... 35 
6.2.1 Human Data Sharing Plan .......................................................................................... 35 
6.2.2 Genomic Data Sharing Plan ....................................................................................... 36 
6.3 
Response Criteria ........................................................................................................... 36 
6.3.1 Definitions.................................................................................................................. 36 
6.3.2 Disease Parameters .................................................................................................... 36 
6.3.3 Methods for Evaluation of Measurable Disease ........................................................ 37 
6.3.4 Response Criteria ....................................................................................................... 38 
6.4 
Toxicity Criteria ............................................................................................................. 40 
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN ......................................................................................................................................... 40 
7.1 
Definitions...................................................................................................................... 40 
7.1.1 Adverse Event ............................................................................................................ 40 
7.1.2 Suspected Adverse Reaction ...................................................................................... 40 
7.1.3 Unexpected Adverse Reaction ................................................................................... 40 
7.1.4 Serious........................................................................................................................ 40 
7.1.5 Serious Adverse Event ............................................................................................... 40 
7.1.6 Disability .................................................................................................................... 41 
7.1.7 Life threatening Adverse Drug Experience ............................................................... 41 
7.1.8 Protocol Deviation (NIH Definition) ......................................................................... 41 
7.1.9 Non-compliance (NIH Definition) ............................................................................. 41 
7.1.10 Unanticipated Problems ............................................................................................. 41 
7.2 
NCI-IRB and Clinical Director (CD) Reporting ............................................................ 41 
7.2.1 NCI-IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths
 
41 
7.2.2 NCI-IRB Requirements for PI Reporting at Continuing Review .............................. 42 
7.2.3 NCI-IRB Reporting of IND Safety Reports............................................................... 42 
7.3 
Institutional Biosafety Committee (IBC) Reporting Criteria ......................................... 42 
7.3.1 Serious Adverse Event Reports to IBC ...................................................................... 42 
7.3.2 Annual Reports to IBC............................................................................................... 42 
7.4 
IND Sponsor Reporting Criteria .................................................................................... 43 
7.4.1 Wavier of expedited reporting to CCR ...................................................................... 44 
7.4.2 Reporting Pregnancy .................................................................................................. 44 
7.5 
Data and Safety Monitoring Plan ................................................................................... 45 
7.5.1 Principal Investigator/Research Team ....................................................................... 45 
7.5.2 Sponsor Monitoring Plan ........................................................................................... 45 
7.5.3 Safety Monitoring Committee (SMC) ....................................................................... 46 


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8 
STATISTICAL CONSIDERATIONS ........................................................................... 46 
9 
COLLABORATIVE AGREEMENTS........................................................................... 48 
10 HUMAN SUBJECTS PROTECTIONS ......................................................................... 48 
10.1 
Rationale for Patient Selection ....................................................................................... 48 
10.2 
Participation of Children ................................................................................................ 48 
10.3 
Participation of Subjects Unable to Give Consent ......................................................... 49 
10.4 
Evaluation of Benefits and Risks ................................................................................... 49 
10.5 
Consent Process and Documentation ............................................................................. 49 
10.5.1 Informed consent of non-English speaking subjects ................................................. 50 
11 PHARMACEUTICAL INFORMATION ...................................................................... 50 
11.1 
Investigational Regimen ................................................................................................ 50 
11.1.1 Anti-Mesothelin CAR Transduced PBL .................................................................... 50 
11.1.2 Aldesleukin (Interleukin-2, Proleukin, Recombinant Human Interleukin 2) ............ 51 
11.1.3 Fludarabine ................................................................................................................ 52 
11.1.4 Cyclophosphamide ..................................................................................................... 53 
11.2 
Supportive Medications ................................................................................................. 54 
11.2.1 Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex) .............................. 54 
11.2.2 Filgrastim (Granulocyte Colony-Stimulating Factor, G-CSF, Filgrastim, Neupogen)
 
54 
11.2.3 Trimethoprim and Sulfamethoxazole Double Strength (TMP / SMX DS) ............... 54 
11.2.4 Herpes and Varicella Zoster Virus Prophylaxis......................................................... 56 
11.2.5 Fluconazole ................................................................................................................ 56 
11.2.6 Ondansetron hydrochloride ........................................................................................ 57 
11.2.7 Furosemide ................................................................................................................. 57 
12 REFERENCES ................................................................................................................. 58 
13 TABLES, FIGURES, AND APPENDICES: ................................................................. 60 
 
 
 


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1 
INTRODUCTION 
1.1 
STUDY OBJECTIVES: 
1.1.1 Primary objectives:  
x To evaluate the safety of the administration of anti-mesothelin CAR engineered peripheral 
blood lymphocytes in patients receiving the non- myeloablative conditioning regimen, and 
aldesleukin 
x Determine if the administration of anti-mesothelin CAR engineered peripheral blood 
lymphocytes and aldesleukin to patients following a nonmyeloablative but lymphoid 
depleting preparative regimen will result in clinical tumor regression in patients with 
metastatic or unresectable cancer that expresses mesothelin.  
1.1.2 Secondary objective: 
x Determine the in vivo survival of CAR gene-engineered cells. 
 
1.2 
BACKGROUND AND RATIONALE:   
Studies in experimental animals have demonstrated that the cellular rather than the humoral 
arm of the immune response plays the major role in the elimination of murine tumors.  Much 
of this evidence was derived from studies in which the adoptive transfer of T lymphocytes 
from immune animals could transfer resistance to tumor challenge or in some experiments, the 
elimination of established cancer.  Thus, most strategies for the immunotherapy of patients 
with cancer have been directed at stimulating strong T cell immune reactions against tumor-
associated antigens. 
 
In contrast to antibodies that recognize epitopes on intact proteins, T cells recognize short 
peptide fragments (8-18 amino acids) that are presented on surface class I or II major 
histocompatibility (MHC) molecules and it has been shown that tumor antigens are presented 
and recognized by T cells in this fashion.  The molecule that recognizes these peptide 
fragments is the T-cell receptor (TCR).  The TCR is analogous to the antibody 
immunoglobulin molecule in that, two separate proteins (the TCR alpha and beta chains) are 
brought together to form the functional TCR molecule.  An alternate approach to enable T cells 
to recognize targets is to utilize a chimeric T cell receptor which is constructed by using the 
variable regions of the heavy and light chains of an antibody connected by a linker sequence 
and fused to CD28, CD3 zeta and 41BB signaling chains.  Thus, the T cell can recognize and 
signal based on the non-MHC restricted recognition of the target.  The goal of this protocol is 
to transfer genes encoding a chimeric receptor that recognizes mesothelin into normal 
peripheral blood lymphocytes (PBL) derived from cancer patients and to return these 
engineered cells to patients aimed at mediating regression of their tumors. This trial is similar 
to previous Surgery Branch TCR gene transfer adoptive immunotherapy protocols except that 
we will use a receptor that recognizes targets based on the recognition of an antibody reactive 
with the mesothelin molecule.  This has the potential to treat patients with common epithelial 
malignancies of multiple histologies.   
 


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1.2.1 Prior Surgery Branch Trials of Cell Transfer Therapy Using Tumor Infiltrating 
Lymphocytes in Patients with Metastatic Melanoma 
 
Studies in the Surgery Branch, National Cancer Institute, identified genes that encode 
melanoma tumor associated antigens (TAA) recognized by tumor infiltrating lymphocytes 
(TIL) in the context of multiple MHC class I molecules1.  These TAA appeared to be clinically 
relevant antigens responsible for mediating tumor regression in patients with advanced 
melanoma since the TIL used to identify these antigens were often capable of mediating in 
vivo anti-tumor regression.  Two antigens, which were present in virtually all fresh and 
cultured melanomas, were called MART-1 (Melanoma Antigen Recognized by T Cells - 1), 
and gp100. The genes encoding these two antigens have been cloned and sequenced.  The 
MART-1 gene encodes a 118 amino acid protein of 13 kd. The gp100 gene encodes a protein 
identical to that recognized by monoclonal antibody HMB-45. These antigens were thus the 
original targets of our gene therapy cell transfer protocols in patients with metastatic 
melanoma.   
 
In the great majority of murine models demonstrating the therapeutic effectiveness of the 
adoptive transfer of lymphocytes mediating tumor regression, immunosuppression of the host 
prior to the adoptive transfer of lymphocytes was required.  North and colleagues demonstrated 
that adoptive transfer of lymphocytes was not effective unless the mouse was 
immunosuppressed by total body irradiation or chemotherapy prior to adoptive transfer of 
lymphocytes2.  Very similar observations were made in other rodent cancer models3.  We thus 
incorporated a lymphodepleting chemotherapy into our human cell transfer studies.   
 
In the Surgery Branch, NCI, we developed a protocol to rapidly expand heterogeneous TILs 
for adoptive transfer4. TILs were expanded using the rapid expansion protocol (REP) in the 
presence of OKT3, irradiated allogeneic feeder cells and IL-2. These REPed TILs retained 
highly specific in vitro anti-tumor activity, often contained reactivities against several antigenic 
epitopes and contained both CD8+ and CD4+ lymphocytes.  These autologous bulk TIL were 
re-infused to patients following a nonmyeloablative chemotherapy with cyclophosphamide and 
fludarabine.  These patients subsequently received high-dose IL-2 and some received peptide 
immunization when the TIL reactivity was against known MART-1 and gp100 peptides 
(protocol 99-C-0158).   A total of 43 patients received this treatment5,6.  This regimen resulted 
in objective cancer regressions in 49% of patients (21 of 43) with metastatic melanoma (Table 
1).     
 
Murine models predicted that increasing the extent of lymphodepletion could increase the 
effectiveness of the cell transfer therapy7.  Thus, we performed two additional sequential trials 
of ACT with autologous anti-tumor lymphocytes (TIL) in patients with metastatic melanoma. 
Increasing intensity of host preparative lymphodepletion consisting of cyclophosphamide and 
fludarabine with either 200cGy (25 patients) or 1200 cGy (25 patients) total body irradiation 
(TBI) was administered prior to cell transfer.  While non-myeloablative chemotherapy alone 
showed an objective response rate of 49%, when 200cGy or 1200cGy TBI was added the 
response rates were 52% and 72% respectively (Table 1).  TBI appeared to result in increased 
patient survival.  Responses were seen in all visceral sites including brain. Host 
lymphodepletion was associated with increased serum levels of the lymphocyte homeostatic 


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10 
cytokines IL-7 and IL-157. Objective responses were correlated with the telomere length of the 
transferred cells8. 
 
Patients exhibited the expected hematological toxicities associated with the cyclophosphamide, 
fludarabine and TBI preparative regimens. Other toxicities are shown in Table 2.  Patients 
recovered marrow function rapidly after cell infusion with absolute neutrophil counts greater 
than 500 per mm3 by day 12 and sustained platelet counts above 20,000 per mm3 by day 14 
(except 4 patients on the TBI 1200 protocol with platelet recovery on days 16, 17, 20, and 22). 
 
1.2.2 Surgery Branch Trials of Cell Transfer Therapy Using Transduction of Anti-TAA TCR 
Genes into Non-reactive TIL or PBL 
 
It is often not possible to isolate sufficient tumor samples from melanoma patients and even 
when tumor is available, only about 60 to 70% generate melanoma reactive TIL cultures.  As a 
potential alternative to the requirement to establish TIL cultures from melanoma patients, we 
sought methods that could be used to easily obtain a polyclonal population of T cells with anti-
TAA properties.  Transfer of antigen specific TCR genes to PBL is a potential method 
generating large numbers of reactive anti-cancer T cells9.   In a murine model of this approach, 
TCR gene transfer into murine splenocyte T-cells was performed using a retroviral vector10.  
The engineered T cells were shown to expand in vivo upon viral challenge and efficiently 
homed to effector sites.  In addition, small numbers of TCR-transduced T cells promoted the 
rejection of antigen-expressing tumors in the mice.  Retroviral vector mediated gene transfer 
can be used to engineer human T cells with high efficiency.  In published work, the Surgery 
Branch was among the first to demonstrate that retroviral vector-mediated transfer of TCR 
genes could endow human PBL with anti-tumor reactivity11,12.     
 
To expand on and potentially improve on these early results, we next isolated TCR genes from 
both gp100 and MART-1 reactive T cells.  These studies have been published and are 
presented briefly here11,12.  To test the in vivo efficacy of these MART-1 TCR engineered T 
cells, 31 HLA-A*0201 patients with progressive metastatic melanoma were treated.  Results in 
the first 17 patients were published in 200613.  All patients were refractory to prior therapy with 
IL-2.  T cell cultures from all patients were biologically reactive, with specific secretion of 
interferon-ȖIROORZLQJFR-culture with either MART-1 peptide pulsed T2 cells and or 
melanoma cell lines expressing the MART-1 antigen.  Gene transfer efficiencies measured by 
staining for VE12 expression in these lymphocytes ranged from 17% to 67% (mean value 
42%).  Four of the 31 patients demonstrated a sustained objective regression of their metastatic 
melanoma assessed by standard RECIST criteria.  
 
There were no toxicities in any patient attributed to the gene-marked cells.  We thus 
demonstrated for the first time in humans, that normal autologous T lymphocytes, transduced 
ex vivo with anti-TAA TCR genes and reinfused in cancer patients can persist and express the 
transgene long-term in vivo and mediate the durable regression of large established tumors.  
 
A similar study was conducted using gp100 TCR gene marked cells, however this retroviral 
vector had a low titer when produced under GMP conditions.  Fourteen patients were treated 


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11 
on this study.  No antitumor responses have been seen.  There have been no grade 5 toxicities 
observed on this study, and all grade 3 and 4 toxicities observed were expected toxicities 
associated with the non-myeloablative chemotherapy regimen or IL-2. 
 
The low response rate in our prior MART-1 TCR gene transfer protocol led us to identify 
MART-1 reactive TCR with higher avidity than the MART-1 F4 TCR used in the prior gene 
therapy clinical trial14,15.  We treated 24 patients with metastatic melanoma using autologous 
PBL transduced with an improved MART-1 F5 TCR following a non-myeloablative 
chemotherapy.  Six patients (25%) have achieved an objective partial response.  Toxicities 
were similar to those seen in the prior TCR gene therapy trial except that 15 patients developed 
a transient mild anterior uveitis easily reversed by steroid eye drops and ten patients developed 
decreased hearing reversed by middle ear steroid injections.  Transient rashes have also been 
seen.  There were no treatment related deaths. 
 
We have also conducted a clinical trial with a TCR that recognizes the gp100:154-162 
melanoma peptide.  This TCR was raised in an HLA-A2 transgenic mouse immunized with 
this peptide15.  We have now treated 21 patients with metastatic melanoma using autologous 
PBL transduced with this improved gp100 TCR following a non-myeloablative chemotherapy.  
Four patients (19%), have achieved an objective partial response.  Seven patients developed a 
transient mild anterior uveitis reversed by steroid eye drops and ten patients developed 
decreased hearing reversed by middle ear steroid injections.  There were no treatment related 
deaths.  
 
In addition to the studies listed above in patients with metastatic melanoma, we have recently 
initiated several studies for patients with other metastatic cancers using PBL transduced with 
TCR genes or chimeric antigen receptor (CAR) genes.  We have studied PBL transduced with 
TCR genes targeting p53, CEA, NY-ESO-1, TRAIL bound to the DR4 Receptor, and MAGE 
A3/12 and PBL transduced with CAR targeting Her-2, CD19 and VEGFR2. Patients on these 
studies also received a non-myeloablative chemotherapy regimen consisting of 
cyclophosphamide and fludarabine, and high dose IL-2.  In two studies, 08-C-0121 (anti-ESO-
1 TCR) and 09-C-0082 (anti-CD19 CAR), we have seen impressive clinical responses.  In 08-
C-0121, in patients with melanoma, we have observed two confirmed complete responses and 
three confirmed partial responses in the twelve evaluable patients who did not receive 
vaccination with the ALVAC vaccine, and one confirmed complete response and one 
confirmed partial response were observed in the 6 evaluable patients that received the vaccine.  
In the eight evaluable patients with other cancers, there have been 5 confirmed partial 
responses out of the 7 treated patients with synovial sarcoma who did not receive vaccination 
with the ALVAC vaccine, and there have been 3 confirmed partial responses in the 5 evaluable 
patients who received the vaccine.  Therefore, to date the response rate for this study is 50%. In 
study 09-C-0082, eight patients have been treated and are evaluable on this study including six 
patients with CLL, one patient with follicular lymphoma, and one patient with splenic marginal 
zone lymphoma.   We have observed a clinical response rate of 75%, which includes 5 patients 
with a partial response (PR) and 1 patient with a complete response (CR) to treatment.   
Toxicity information regarding TCR and CAR transduced cell therapy studies conducted in the 
Surgery Branch is included in Section 1.2.6 below. 
 


Abbreviated Title: Anti-mesothelin CAR PBL 
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12 
1.2.3 Mesothelin as a Target for Cell Transfer Studies 
 
As discussed above, adoptive cell transfer (ACT) immunotherapy strategies designed to 
directly target antigens expressed on tumor cells can result in durable objective regression in 
patients with metastatic melanoma. Despite its demonstrated effectiveness in experimental 
mouse tumor models and in the human, the application of ACT is limited because of the need 
to identify antigens with highly selective expression in cancer.  Mesothelin as a target for ACT 
can potentially have wide application for the treatment mesothelin-positive cancer histologies 
including, but not limited to, mesothelioma, ovarian and pancreatic cancer.  Redirecting 
immune cells to target mesothelin offers an alternative to overcome the obstacles confronting 
conventional palliative surgery and chemotherapy as well as current tumor-specific 
immunotherapeutic approaches.  
 
Mesothelin is a 40-kDa cell surface glycoprotein that is highly expressed in mesothelioma, 
ovarian, pancreatic and some other cancers16-18.  Small amounts of mesothelin shed into the 
circulation may be detected by sandwich ELISA.  It has been suggested that elevated soluble 
mesothelin levels may correlate with advanced disease stage and total tumor burden.  The 
biological function of mesothelin is unknown.  A trans-intracellular binding activity with 
CA125, a tumor antigen routinely used in the diagnosis and monitoring of ovarian cancer has 
been noted19.  Among normal human tissues mesothelin is expressed on mesothelial cells of the 
pleura, pericardium, and peritoneum, as well as the basal cells of the trachea, tonsil epithelium, 
and cells in the Fallopian tubes but is absent from vital organs including heart, liver, lung, 
kidney and nervous tissue20,21.  There is limited evidence that mesothelin is also expressed to 
some extent on the corneal epithelium22.   
 
Initial immunotherapy efforts to target mesothelin on epithelial cancer cells involved the use of 
an immunotoxin, SS1P.  SS1P is a high affinity anti-mesothelin disulfide-stabilized murine-
antibody Fv genetically combined with PE3823.  PE38 is a fragment of the potently cytotoxic 
Pseudomonas exotoxin from which the native cell-binding domain and other unnecessary 
sequences have been removed.  The chimeric recombinant immunotoxin, SS1(dsFv)PE38 
(referred to as SS1P), kills mesothelin-expressing cells but not similar cells that do not express 
detectable levels of mesothelin.  As an alternate approach to an immunotoxin, a chimeric 
antibody has been developed.  MORab-009 is a chimeric IgG/N antibody in which the murine 
SS1single chain variable fragment (scFv) is fused in frame with the human IgG1 and kappa 
constant regions.  This monoclonal antibody has been shown in vitro to elicit antibody-
dependent cellular cytotoxicity (ADCC) and inhibit tumor growth in a mouse xenograft 
model24.  
 
In order to extend the current immunotherapy approaches, we have developed a chimeric T cell 
receptor (CAR) utilizing SS1scFv fused in frame to the intracellular signaling chains of a 
conventional T cell receptor.  This SS1 CAR, when transduced into lymphocytes, enables the 
lymphocyte to recognize mesothelin-positive targets based on the antigen recognition of the 
antibody scFv. This CAR combined with the antitumor potency of ACT suggested to us that 
the transduction of a chimeric receptor recognizing mesothelin into lymphocytes could produce 
self-replicating T cells capable of selectively destroying mesothelin-expressing tumors. 
 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
 
13 
Cancer histologies such as lung, pancreatic, ovarian and esophageal are some of the leading 
causes of cancer deaths among both men and women today.  These cancer histologies, in 
addition to pleural mesothelioma, have also been shown to express mesothelin.  The incidence 
of mesothelin-positive tumors has been determined by RT-PCR and/or immunohistochemisty 
(IHC).  In many studies, all tumors from all patients with mesothelioma and pancreatic cancer 
express mesothelin (Table 3).  Thus, in all clinical studies targeting mesothelin at the NCI (R. 
Hassan and I. Pastan) no confirmation of mesothelin expression is necessary for patients with 
mesothelioma or pancreatic cancer.  IHC evidence of mesothelin expression will be required 
for patients with other cancer types.  It has been shown that 10/12 (83%) lung 
adenocarcinomas were positive for mesothelin based on IHC data.  Additionally, in the same 
study, 7/9 (78%) of NCI-60 cell lines evaluated were also positive25.  Similarly, Chang et al. 
(1992) demonstrated by IHC that 15/15 (100%) of pleural mesotheliomas, 0/4 sarcomatous 
mesotheliomas, 10/15 (66%) of nonmucinous ovarian cancers and 0/4 mucinous ovarian 
cancers stained positive for mesothelin20,26.  In addition, it was determined by RT-PCR and 
IHC that 4/4 (100%) and 60/60 (100%), respectively, of primary pancreatic adenocarcinomas 
expressed mesothelin27,28.  While the incidence is much lower, mesothelin has been shown to 
be expressed on a variety of other cancer histologies including esophageal, cervical and head 
and neck27.  Based on the date presented herein, mesothelin can have wide application as an 
immunotherapy target.  
  
1.2.4 Preclinical Studies to Provide the Rationale for this Clinical Protocol 
 
SS1P has been used in two Phase I clinical trials.  In the first trial, the SS1P immunotoxin was 
administered as an I.V. bolus.  The primary objectives of the trial were to determine the safety, 
toxicity, maximum tolerated dose (MTD) and pharmacokinetics of SS1P with secondary 
objectives of evaluating effects on tumor growth and disease progression29.  In terms of the 
clinical response, of the 33 evaluable patients, 4 had minor responses, 19 patients had stable 
disease and 10 patients had progressive disease.  The dose limiting toxicity (DLT) was grade 3 
pleuritis in 3/33 patients29.  In a second Phase I trial, the administration of the drug was 
changed from I.V. bolus to continuous infusion.  Of the 24 patients treated, 1 patient had a 
partial response.  Five patients experienced pleuritic chest pain30.   
 
Alternatively, the chimeric anti-mesothelin monoclonal antibody (MORab-009) was evaluated 
in a Phase I clinical trial31.   MORab-009 was determined to be well tolerated with 11/24 
patients treated exhibiting stable disease.  It was reported that 2 patients experienced grade 4 
transaminitis and grade 3 serum sickness.  No other grade 3/4 toxicities were reported.  
MORab-009 is now being evaluated in a multi-center Phase II clinical trial.   
 
Previously, Carpenito et. al. (2009) cloned the murine SS1 CAR directed against mesothelin 
into a lentiviral vector32.  The authors demonstrated that coupling the SS1 scFv to human 
CD28 and CD3] (CD28Z) or CD28, 4-1BB and CD3] (CD28BBZ) resulted in greater than 
80% CAR+ lymphocytes that specifically recognized mesothelin-positive tumor targets.  When 
tested in a mouse xenograft model, both constructs were able to effectively treat large 
established mesothelin-positive tumors when CAR+ lymphocytes were administered 


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14 
intravenously32.  Subsequently, this same group was able to demonstrate similar tumor 
treatment efficacy using RNA-electroporated lymphocytes in the same xenograft model33.  
 
Based on the previously published data, we generated a MSGV-based recombinant retroviral 
vector encoding a second-generation CAR comprised of the codon-optimized anti-human SS1 
single chain variable regions (ScFv) linked to the intracellular human T cell signaling 
sequences derived from CD28 and the CD39 chain of the T cell receptor via the human CD8D 
hinge and transmenbrane regions (referred to as coSS1scFv-CD28Z). The recombinant 
retroviral construct is described schematically in Figure 1A.  Surface expression of the 
retrovirally encoded transgene product in transduced CD3+ primary human T cells was 
determined by flow cytometry. As shown in Figure 1B, the coSS1scFv CAR-expressing vector 
efficiently transduced OKT3-activated human peripheral blood lymphocytes (PBL, range 63-
82% after correcting for background staining).  When co-cultured with mesothelin-positive 
tumor cell lines, the CAR-transduced PBL showed mesothelin-specific recognition of the 
target cells as measured by gamma interferon release. 
 
1.2.5 Construction and testing of retroviral vectors expressing CAR against human 
mesothelin  
 
The ability of the T cell based anti-mesothelin targeting strategy to treat established 
mesothelin-positive tumors in a mouse xenograft model led us to explore the translation of 
these preclinical findings to the treatment of human tumors32,33.  We constructed a recombinant 
retroviral vector expressing CAR against human mesothelin using the sequences from the SS1 
scFv34.  
 
The anti-human mesothelin CAR vector termed coSS1scFv-CD28Z was comprised of a scFv 
derived from a murine antibody fragment, SS1, linked to the hinge and transmembrane 
sequences from the human CD8D, which was in turn fused to the intracellular sequences 
derived from human CD28 and the CD39 chain of TCR (Figure 1A).   
 
The functional integrity of the SS1 CAR-encoding retroviral vector was tested in vitro in 
several assays.  Supernatants from gibbon ape leukemia virus-pseudotyped high titer virus 
producer cells were used to transduce OKT3 activated human PBLs from 3 different donors.  
The SS1 CAR-encoding retroviral vector transduced human PBLs at a high frequency resulting 
in 63 to 82% of the CD3+ T cells expressing the CAR on the cell surface (Figure 1B).  Next, 
we tested the ability of SS1 CAR-modified T cells to recognize mesothelin positive human 
cancer cells as measured by specific IFN-J secretion.  coSS1scFv-CD28Z CAR-transduced 
PBLs from 3 different donors, 8 days post transduction were cocultured for 24 hours with 
mesothelin-negative cells (A431) or mesothelin-positive (A431-H9, a stable transfectant 
expressing mesothelin; OVCAR3 and Panc10.05, Figure 1C).  The surface expression on 
mesothelin-positive and negative cancer cell lines was determined by flow cytometry using the 
MN antibody for mesothelin detection (Figure 2).  CAR-transduced PBL were subjected to a 
secondary or rapid expansion (REP) by exposure to soluble OKT3 antibody, high-dose 
Interleukin 2 (IL-2) and irradiated feeders.  Following a REP, the average cell expansion from 
the 3 patient PBL tested was approximately 180-fold (Figure 3A).  In addition, the CAR-


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
 
15 
transduced cells maintained both CAR surface expression (Figure 3B) and specific recognition 
of mesothelin-positive tumor targets (Figure 3C). 
 
Thus based on the previously published preclinical studies, which provide the rationale for the 
translation of SS1 CAR-transduced cells for the treatment of human cancer and data from the 
experiments presented herein, we selected the coSS1scFv-CD28Z vector for use in our human 
clinical protocol.  A cGMP master cell bank was manufactured and used for the production of 
cGMP Gammaretroviral vector supernatant.  Each of the vector harvests was titered on human 
PBL (Table 4) and then subsequently tested for specific recognition of mesothelin-positive 
tumor targets as measure by specific IFNJ release (Table 5).  All cGMP reagents were 
subjected to and passed all biosafety testing as stipulated by the FDA guidance documents. 
 
1.2.6 Safety Considerations 
 
Several safety concerns regarding the infusion of large numbers of retrovirally modified tumor 
reactive T-cells have been addressed in our previous clinical studies.  The non-myeloablative 
chemotherapy and the administration of high-dose IL-2 have expected toxicities discussed 
earlier.  The non-myeloablative chemotherapy used in this protocol has been administered to 
over 150 patients and all have reconstituted their hematopoietic systems.   
 
CAR transduced human T cells has been administered to humans in several clinical trials.  We 
treated 14 patients with metastatic ovarian cancer utilizing autologous T cells transduced with a 
retroviral vector encoding a single chain antibody that recognized the ovarian cancer-
associated antigen alpha-folate receptor.  The single chain antibody was linked to the signaling 
domain of the Fc receptor gamma chain.  These 14 patients received up to 1.7 x 1011 cells 
intravenously and except for mild malaise and fevers there were no toxicities attributed to the 
cell administration.  Lamers et al. reported on the treatment of three patients who received 
autologous T cells transduced with a retrovirus encoding an antibody against the carbonic 
anhydrase IX molecule expressed on renal cell cancers35.  Three patients were treated and mild 
transaminase elevations precluded the treatment of additional patients.  This was probably due 
to the expression of carbonic anhydrase IX expression on biliary ductal epithelium. 
 
Pule, et al. engineered human T cells to express a CAR directed to the diasialoganglioside 
GD2, a tumor associated antigen expressed by human neuroblastoma cells36.  These CAR 
transduced cells were administered to eleven children between the ages of 3 and 10, all of 
whom had metastatic neuroblastoma.  There were no adverse effects attributable to the 
genetically modified CAR T cells in the eleven subjects that were followed for up to two years 
following cell infusion.  Two patients exhibited an objective response and several additional 
patients exhibited necrosis in established tumors.  Persistence of the transduced cells was 
greater when the CAR were inserted into EBV specific cytolytic T lymphocytes than when the 
CAR were inserted into T cells that were activated by an anti-CD3 monoclonal antibody.   
 
Till et al. reported the adoptive immunotherapy of patients with indolent  non-Hodgkin’s 
lymphomas and mantle cell lymphoma using specific T cells transfected with CAR recognizing 
the CD20 molecule37.  Seven patients were treated and one achieved a partial response.  These 


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16 
cells were administered in conjunction with low dose subcutaneous IL-2.  There were no 
toxicities associated with the cell transfer.  Brentjens et al. recently reported on the 
administration of autologous T cells transduced with a CAR recognizing the CD19 molecule in  
patients with chronic lymphocytic leukemia38. Three patients were treated with modified T 
cells alone without dose limiting toxicities.  However, the first patient who was enrolled in a 
second cohort of T cell administration following a cyclophosphamide lymphodepleting 
regimen developed a syndrome of fever and hypotension and died four days later.  Following 
extensive analysis of this patient it was concluded that the adverse event may have been due to 
an idiosyncratic reaction to the combination of the cyclophosphamide and cell administration.  
 
Most toxicities observed in these studies were expected toxicities of the chemotherapy and 
aldesleukin administration.  However, in several studies, we have observed serious adverse 
events related to the transduced cells.  In 08-C-0121 (anti-ESO-1 TCR transduced PBL), one 
patient experienced grade 3 dyspnea and grade 4 hypoxia resulting in intubation.  These events 
were attributed to the cells, and aldesleukin as well as the extensive pulmonary disease in this 
patient.  In 09-C-0041 (anti-Her2 CAR transduced PBL), the first patient, with metastatic 
colorectal cancer, was treated with 1010 autologous T cells transduced with the retrovirus 
encoding an anti-Her-2 CAR.  This patient developed respiratory distress and died four days 
later.  This toxicity was apparently due to a previously unrecognized ability of this CAR to 
recognize Her-2 expressed on lung epithelial cells although the exact explanation for the 
toxicity is not clear39.   In 09-C-0047 (anti-CEA TCR transduced PBL), all three patients 
treated experienced a variety of gastrointestinal events which were attributed to the gene/cell 
therapy including diarrhea, and colitis21.  All gastrointestinal events have since resolved in 
these patients, and the patients’ colonic mucosa has returned to normal, and the patients have 
normal bowel function.  Grade 3 diarrhea lasting longer than 72 hours is considered a DLT per 
protocol and this event was observed in two of three patients enrolled in protocol 09-C-0047, 
meeting the criteria for stopping protocol accrual.  One patient treated on this study 
experienced a partial response.  In 09-C-0082 (anti-CD19 CAR transduced PBL) SAEs related 
to the cell therapy include dose-limiting toxicities (DLT) of grade 4 somnolence in two 
patients, grade 3 creatinine in one patient and grade 3 confusion (aphasia) and grade 3 
neuropathy (right facial droop) in another patient.  In 11-C-0062 (anti-MAGE A3/12 TCR 
transduced PBL), out of the nine patients treated, four patients were intubated, and three of 
these intubations were the result of toxicities attributed to the cells.  One of these patients 
experienced hypoxia, seizures, and encephalopathy resulting in confusion, dysarthria, ataxia, 
short-term memory loss, non-fluent aphasia, and action myoclonus in the face and limbs.  This 
patient recovered from these toxicities and was discharged.  Two other patients had 
somnolence lasting 3 and 5 months, respectively, before dying from the effects of the 
treatment.  Both patients have had brain biopsies which indicated significant leukomalacia, 
sparing the cortex.  This IND has been placed on clinical hold by the FDA while we investigate 
these SAEs.  In 11-C-0013 (anti-VEGFR2 CAR, two patients experienced elevated liver 
function tests which were attributed to the cell therapy.  One of these patients also had an 
elevated EBV level, increased triglycerides, and bilirubin, hypoxia and hypotension.  After 
consultation with Medical Oncology, this patient was treated with rituximab and prednisone as 
it was suspected that he had hemophagocytic syndrome or lymphoma. This patient experienced 
worsening mental status and was intubated.  Shortly thereafter, this patient died of respiratory 
failure.  In our most recent study, 12-C-0006 (IL-12/anti-ESO-1 TCR transduced PBL), the 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
 
17 
first patient received 1 X107 transduced cells and did well for the first two weeks following cell 
administration.  However, she then began to experience shortness of breath, elevated liver 
enzymes, hypotension and acute renal failure.  She was transferred to the ICU and was 
intubated and underwent CVVH.  Also at this time, skin breakdown was observed, and serum 
cytokine levels, interferon-gamma and IL-12, were extremely elevated.  The patient received a 
total of 5 doses of alemtuzumab, methylprednisone, and a single dose of ustekinumab to 
decrease levels of interferon gamma and IL-12.  She also experienced a gastric bleed and 
extensive lung infiltration.  The patient’s condition continued to deteriorate and she expired 
approximately one month after receiving the cells. 
 
In other protocols we have administered over 3 X 1011 TIL with widely heterogeneous 
reactivity including CD4, CD8, and NK cells without difficulty.  As discussed above, the 
expansion of tumor reactive cells is a desirable outcome following the infusion of antigen 
reactive T-cells.  Some patients receiving gp100 or MART-1 reactive cells have developed 
vitiligo or uveitis probably due to destruction of normal melanocytes though these toxicities 
have been manageable.  We do not believe the transfer of these gene modified cells has a 
significant risk for malignant transformation in this patient population.  While the risk of 
insertional mutagenesis is a known possibility using retroviral vectors, this has only been 
observed in the setting of infants treated for XSCID using retroviral vector-mediated gene 
transfer into CD34+ bone marrow cells.  In the case of retroviral vector-mediated gene transfer 
into mature T-cells, there has been no evidence of long-term toxicities associated with these 
procedures since the first NCI sponsored gene transfer study in 1989.  Although continued 
follow-up of all gene therapy patients will be required, data suggest that the introduction of 
retroviral vectors transduced into mature T-cells is a safe procedure.  While we believe the risk 
of insertional mutagenesis is extremely low, the proposed protocol follows all current FDA 
guidelines regarding testing and follow up of patients receiving gene transduced cells. 
  
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT 
 
2.1 
ELIGIBILITY CRITERIA 
2.1.1 Inclusion Criteria 
a. 
Metastatic or unresectable measurable cancers that express mesothelin. As in other 
protocols conducted by Dr. Hassan in the NCI, epithelial mesotheliomas and pancreatic 
cancers do not need to be assessed for mesothelin expression since all of these tumors 
have been shown to express mesothelin.  Other metastatic or unresectable cancers must 
be shown to expresses mesothelin as assessed by RT-PCR or immunohistochemistry on 
tumor tissue.  Bi-phasic mesotheliomas must express mesothelin on greater than 50% of 
the cells in the epithelial component.  Diagnosis will be confirmed by the Laboratory of 
Pathology, NCI.   
b. 
Patients must have previously received at least one systemic standard care (or effective 
salvage chemotherapy regimens) for metastatic or unresectable disease, if known to be 
effective for that disease, and have been either non-responders (progressive disease) or 
have recurred.  
c. 
Greater than or equal to 18 years of age and less than or equal to 70 years of age. 
d. 
Willing to sign a durable power of attorney 
e. 
Able to understand and sign the Informed Consent Document 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
 
18 
f. 
Clinical performance status of ECOG 0 or 1. 
g. 
Patients of both genders must be willing to practice birth control from the time of 
enrollment on this study and for up to four months after treatment. 
h. 
Serology: 
o Seronegative for HIV antibody.  (The experimental treatment being evaluated in 
this protocol depends on an intact immune system.  Patients who are HIV 
seropositive can have decreased immune-competence and thus be less responsive to 
the experimental treatment and more susceptible to its toxicities.)  
o Seronegative for hepatitis B antigen, and seronegative for hepatitis C antibody. If 
hepatitis C antibody test is positive, then patient must be tested for the presence of 
antigen by RT-PCR and be HCV RNA negative.  
i. 
Women of child-bearing potential must have a negative pregnancy test because of the 
potentially dangerous effects of the treatment on the fetus. 
j. 
Hematology: 
o Absolute neutrophil count greater than 1000/mm3 without the support of filgrastim. 
o WBC (> 3000/mm3). 
o Platelet count greater than 100,000/mm3. 
o Hemoglobin greater than 8.0 g/dl. 
k. 
Chemistry: 
o Serum ALT/AST less or equal to 2.5 times the upper limit of normal. 
o Serum creatinine less than or equal to 1.6 mg/dl. 
o Total bilirubin less than or equal to 1.5 mg/dl, except in patients with Gilbert’s 
Syndrome who must have a total bilirubin less than 3.0 mg/dl. 
l. 
More than four weeks must have elapsed since any prior systemic therapy at the time 
the patient receives the preparative regimen, and patients’ toxicities must have 
recovered to a grade 1 or less (except for toxicities such as alopecia or vitiligo).  
Note: Patients may have undergone minor surgical procedures within the past 3 weeks, 
as long as all toxicities have recovered to grade 1 or less. 
m. 
Subject’s must be co-enrolled in protocol 03-C-0277. 
  
2.1.2 Exclusion Criteria  
a. 
Patients with sarcomatoid mesothelioma as mesothelin is not expressed in this type of 
mesothelioma. 
b. 
Women of child-bearing potential who are pregnant or breastfeeding because of the 
potentially dangerous effects of the treatment on the fetus or infant. 
c. 
Patients with known brain metastases. 
d. 
Patients receiving full dose anticoagulative therapy. 
e. 
Active systemic infections (e.g.: requiring anti-infective treatment), coagulation 
disorders or any other major medical illnesses. 
f. 
Any form of primary immunodeficiency (such as Severe Combined Immunodeficiency 
Disease). 
g. 
Concurrent opportunistic infections (The experimental treatment being evaluated in this 
protocol depends on an intact immune system.  Patients who have decreased immune 
competence may be less responsive to the experimental treatment and more susceptible 
to its toxicities). 


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19 
h. 
Patients with diabetic retinopathy. 
i. 
Concurrent Systemic steroid therapy. 
j. 
History of severe immediate hypersensitivity reaction to any of the agents used in this 
study. 
k. 
History of coronary revascularization or ischemic symptoms. 
l. 
Documented LVEF of less than or equal to 45% tested in patients with: 
x Clinically significant atrial and/or ventricular arrhythmias including but not limited 
to: atrial fibrillation, ventricular tachycardia, second or third degree heart block, 
chest pain, or ischemic heart disease 
x $JH65 years old 
m. 
Documented FEV1 less than or equal to 60% predicted tested in patients with: 
x A prolonged history of cigarette smoking (20 pk/year of smoking within the past 2 
years). 
x Symptoms of respiratory dysfunction 
n. 
Patients who are receiving any other investigational agents. 
 
2.2 
SCREENING EVALUATION   
Note: Testing for screening evaluation is conducted under our companion protocol, 99-C-0128. 
 
2.2.1 Within 3 months of prior enrollment 
a) HIV antibody titer and HBsAg determination, anti HCV  
b) Anti CMV antibody titer, HSV serology, and EBV panel, (Note: patients who are 
known to be positive do not need to be retested) 
c) Immunohistochemistry or PCR of tumor tissue for expression of mesothelin (testing is 
permitted to be conducted at any time prior to this point). 
d) Confirmation of diagnosis of metastatic cancer by the Laboratory of Pathology of the 
NCI. (Note: Testing is permitted to be conducted at any time prior to enrollment.) 
 
2.2.2 Within 8 weeks prior to enrollment 
a) Pulmonary Function Testing will be conducted in all patients.   
b) Cardiac Evaluation (stress thallium, echocardiogram, MUGA, etc.) for patients who are 
greater than or equal to age 60, or who have a history of ischemic heart disease, chest 
pain, or clinically significant atrial and/or ventricular arrhythmias including but not 
limited to: atrial fibrillation, ventricular tachycardia, heart block.  Patients with a LVEF 
of less than or equal to 45% will not be eligible.  Patients under the age of 60 who have 
cardiac risk factors may also undergo cardiac evaluation as noted above (e.g., diabetes, 
hypertension, and obesity).  
 
2.2.3 Within 4 weeks prior to enrollment:  
a. 
Complete history and physical examination, including weight and vital signs, and noting 
any organ system involvement and any allergies/sensitivities to antibiotics. (Note: 
patient history may be obtained within 8 weeks)  


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20 
b. 
Baseline imaging  to determine the status of disease. This may include CT, MRI, PET, 
or Photography. 
 
2.2.4 Within 14 days prior to enrollment: 
a) Screening blood test 
x Chemistries: Creatinine, ALT/GPT, AST/GOT, and Total Bilirubin 
x CBC with differential and platelet count 
x Coagulation profile including INR, PT/APTT, and fibrinogen. 
b) Urinalysis and culture, if indicated  
 
2.2.5 Within 7 days prior to enrollment:  
a. 
ȕ-HCG pregnancy (serum or urine) test on all women of child-bearing potential 
b. 
ECOG performance status of 0 or 1  
 
2.3 
PROTOCOL REGISTRATION  
2.3.1 Prior to registration for this protocol: 
Patients will initially be registered on protocol 03-C-0277 (Cell Harvest and Preparation for 
Surgery Branch Adoptive Cell Therapy Protocols) for apheresis to obtain PBL prior to 
transduction of PBL cells, by the clinical fellow or research nurse. 
 
Once cells exceed the potency requirement and are projected to exceed the minimum number 
specified in the Certificate of Analysis (CoA), patients will sign the consent document for this 
protocol.  
 
2.3.2 Registration Procedure: 
Authorized staff must register an eligible candidate with NCI Central Registration Office 
(CRO) within 24 hours of signing consent.  A registration Eligibility Checklist from the web 
site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and sent via 
encrypted email to: NCI Central Registration Office ncicentralregistration-l@mail.nih.gov.  
After confirmation of eligibility at Central Registration Office, CRO staff will call pharmacy to 
advise them of the acceptance of the patient on the protocol.  Verification of Registration will 
be forwarded electronically via e-mail to the research team. A recorder is available during non-
working hours. 
 
3 
STUDY IMPLEMENTATION 
3.1 
STUDY DESIGN 
3.1.1 Treatment Phase: 
Patients will receive one course of treatment.  The start date of the course will be the start date 
of the chemotherapy; the end date will be the day of the first post-treatment evaluation. 


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Version Date: April 12, 2018 
 
21 
3.1.1.1 Performed on 03-C-0277 
PBL will be grown, transduced with the anti-mesothelin CAR genes and expanded for this trial 
according to standard operating procedures submitted in the IND.  PBMC will be obtained by 
leukapheresis (approximately 1 X 1010 cells).  PBL will be cultured in the presence of anti-
CD3 (OKT3) and aldesleukin in order to stimulate T-cell growth.  Transduction is initiated by 
exposure of approximately 1 X 107 to 5 X 108 cells to supernatant containing the anti-
mesothelin CAR retroviral vector.  These transduced cells will be expanded and tested for their 
anti-tumor activity.  Successful CAR gene transfer will be determined by FACS analysis for 
the CAR protein and anti-mesothelin reactivity will be tested by cytokine release as measured 
on transfected cells.  Successful CAR gene transfer for each transduced PBL population will be 
defined as >30% CAR positive cells and for biological activity, gamma-interferon secretion 
must be at least 200pg/ml, and greater than twice background. 
 
3.1.2 Phase 1 - Dose Escalation: 
Initially, the protocol will enroll 3 patients in each dose cohort unless a patient experiences a 
dose limiting toxicity (DLT).  If dosing is escalated to cohort 10, 6 patients will be accrued to 
this cohort in order to further characterize the safety of the maximum tolerated dose prior to 
starting the phase II portion.   
 
The total number of anti-mesothelin engineered cells transferred for each cohort will 
be: 
o 
Cohort 1 
 106 cells 
 
De-escalation cohorts: 
x 
Cohort -1:  3 X105 cells 
x 
Cohort -2:  105 cells 
o 
Cohort 2 
3 X 106 cells 
o Cohort 3  
 107 cells 
o Cohort 4 
3 X 107 cells 
o Cohort 5  
 108 cells 
o Cohort 6 
3 X 108 cells 
o Cohort 7  
 109 cells 
o Cohort 8 
3 X 109 cells 
o Cohort 9 
1010 cells 
o Cohort 10 
3 X 1010 cells 
 
Dosing is based on total cell number rather than % transduced cells since our transduction 
efficiency with the anti-mesothelin CAR genes is between 70-90% in PBL. 
 
In each cohort, if one of the first three patients treated experiences a DLT, three more patients 
would be treated at that dose to confirm that no greater than 1/6 patients have a DLT prior to 
proceeding to the next higher level.  If a level with 2 or more DLTs in 3-6 patients has been 
identified, three additional patients will be accrued at the next-lowest dose, for a total of 6, in 
order to further characterize the safety of the maximum tolerated dose prior to starting the 
phase II portion.  If a dose limiting toxicity occurs in the first cohort, that cohort will be 
expanded to n=6 patients.  If there are 1 or fewer DLTs in the first cohort, the study will 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
 
22 
proceed to the second cohort. If two DLTs occur in the first cohort, the dose will be de-
escalated as described above in cohort -1.  If 2 DLTs occur in cohort -1, the dose of cells will 
be de-escalated as described above in cohort -2.  If two DLTs occur in cohort -2, the study will 
be terminated.    
 
Patients will be enrolled sequentially, therefore enrollment will not proceed to a higher dose 
level until all patients have been treated in the prior cohort. If sufficient cells cannot be grown 
to meet the criteria for the assigned cohort, the patient will be enrolled in the appropriate cohort 
for the number of cells infused.   
 
The maximum tolerated cell dose is the highest dose at which < 1 of 6 patients experienced a 
DLT or the highest dose level studied if DLTs are not observed at any of the dose levels. 
 
Prior to receiving the engineered PBL cells, patients will receive a nonmyeloablative but 
lymphocyte depleting preparative regimen consisting of cyclophosphamide and fludarabine 
followed in one to four days by intravenous infusion of in vitro tumor reactive, CAR gene-
transduced PBL plus IV aldesleukin (72,000 IU/kg q8h for a maximum of 15 doses).  
 
3.1.2.1 Safety Assessments: 
A staggering period of 21 days following regimen completion (defined as the last dose of 
aldesleukin) will be conducted for the first 3 patients enrolled in this study.  We will proceed 
with a 2-week inter-cohort staggering period following regimen completion (defined as the last 
dose of aldesleukin) if the cytokine profiles from the first 3 patients support safety.   
 
A two-week safety assessment period following regimen completion (defined as the last dose 
of aldesleukin) will be conducted between each patient in every cohort. 
 
If any unexpected grade 4 or greater toxicity occurs in a patient treated in either the phase 1 or 
phase 2 portions of this study, a thorough evaluation of the available safety information will be 
conducted to justify a decision to continue enrolling new subjects into the study.  The study 
will be halted until this evaluation has occurred and results discussed with FDA. 
 
3.1.3 Phase 2 Portion: 
Similar to the Phase 1 portion, prior to receiving the engineered PBL cells, patients in the 
phase 2 portion will receive a nonmyeloablative but lymphocyte depleting preparative regimen 
consisting of cyclophosphamide and fludarabine followed in one to four days by intravenous 
infusion of in vitro tumor reactive, CAR gene-transduced PBL plus IV aldesleukin (72,000 
IU/kg q8h for a maximum of 15 doses).  
 
The phase 2 portion of the protocol will proceed utilizing the MTD of anti-mesothelin CAR 
engineered cells as determined in the phase 1 portion. Patients will be entered into two cohorts 
based on histology: cohort 1 will include patients with mesothelioma, and cohort 2 will include 
all other cancer types that express mesothelin.   
 


Abbreviated Title: Anti-mesothelin CAR PBL 
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23 
See section 3.1.2.1 for the safety assessment to be conducted for any incidence of unexpected 
grade 4 or greater toxicity. 
 
3.1.4 Definition of Dose Limiting Toxicity (DLT): 
Dose-limiting toxicity is defined as follows:  
x Grade 2 or greater allergic reaction or reaction that involves bronchospasm or 
generalized urticaria  
x All grade 3 or greater toxicities with the exception of: 
o Grade 3 or 4 myelosuppression, defined as lymphopenia, neutropenia, decreased 
hemoglobin and thrombocytopenia,  
o Expected chemotherapy toxicities as defined in the Pharmaceutical Information 
section 11. 
o Aldesleukin expected toxicities as defined in Appendix 1 and Appendix 2. 
o Immediate hypersensitivity reactions occurring within 2 hours of cell infusion 
(related to cell infusion) that are reversible to a grade 2 or less within 24 hours of 
cell administration with standard therapy. 
o Grade 3 Fever 
o Grade 3 Metabolic Laboratory abnormalities without significant clinical sequel that 
resolve to grade 2 within 7 days 
o Grade 3 autoimmunity, that resolves to less than or equal to a grade 2 autoimmune 
toxicity within 10 days 
 
3.1.5 Protocol Stopping Rules: 
The study will be halted pending discussions with the FDA and NCI IRB if the following 
conditions are met: 
 
x If two DLTs occur in the first cohort of the Phase 1 portion of this study. 
x During the phase 1 portion of the study -  if two or more patients develop a grade 3 or 
greater toxicity related to the cell product, with the exception of: 
o Grade 3 metabolic laboratory abnormalities without significant clinical sequela that 
resolve to grade 2 or less within 7 days 
o Grade 3 fever  
x If 1 of the first 3 patients (OR 2 of the first 6 patients, OR 3 of the first 9 patients, OR 4 
of the first 12 patients) develop grade 3 autoimmunity, that cannot be resolved to less 
than or equal to a grade 2 autoimmune toxicity within 10 days, or any grade 4 or greater 
autoimmune toxicity. 
x If EBV lymphoma in another EBV negative subject occurs in this or any other SB 
adoptive cell therapy study, accrual of EBV negative subjects to all such studies must 
halt pending review of the event by the IRB and FDA 
x If one or more treatment related deaths occur due to the cell infusion, we will promptly 
discuss this with the NCI IRB and FDA. 
x During the phase 2 portion of the study - once five or more patients have been enrolled, 
if 20% or more patients cumulatively enrolled develop a DLT, as described in Section 
3.1.4 


Abbreviated Title: Anti-mesothelin CAR PBL 
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24 
 
3.2 
DRUG ADMINISTRATION 
3.2.1 Preparative Regimen with Cyclophosphamide and Fludarabine 
(Starting on day-6, study medication start times for drugs given once daily should be given 
within 2 hours of the scheduled time.    Administration of diuretics, electrolyte replacement and 
hydration and monitoring of electrolytes should all be performed as clinically indicated.  
Chemotherapy infusions maybe slowed or delayed as medically indicated.) 
 
Day -7 and –6  
Approximately 6 hours Prior to Cyclophosphamide 
Hydrate: Begin hydration with 0.9% Sodium Chloride Injection containing 10 meq/L of 
potassium chloride at 1.5 – 2.6 ml/kg/hr (starting approximately 6 hours pre-cyclophosphamide 
and continue hydration until 24 hours after last cyclophosphamide infusion). At any time 
during the preparative regimen, if urine output is <1 ml/kg/hr or if body weight is >2 kg over 
pre-cyclophosphamide value, furosemide 10-20 mg IV maybe administered.  The hydration 
rate will be capped at 250mL/hr. 
 
Approximately 1 hour pre-Cyclophosphamide  
Ondansetron (0.15 mg/kg/dose [rounded to the nearest even mg dose between 8 mg and 16 mg 
based on patient weight] IV every 8 hours X 3 days) will be given for nausea.  
 
 
 
Cyclophosphamide 60 mg/kg/day X 2 days IV in 250 ml D5W with mesna 15 mg/kg/day X 2 
days over 1 hr. If patient is obese (BMI > 35) drug dosage will be calculated using practical 
weight as described in Table 6. 
 
A decreased dose of cyclophosphamide at 30mg/kg/day (x2 days) will be considered for 
patients who have a history of prolonged hematologic recovery from prior chemotherapy 
treatments. 
 
 
Immediately following the end of Cyclophosphamide  
 
Begin mesna infusion at 3 mg/kg/hour intravenously diluted in a suitable diluent (see 
pharmaceutical section 11) over 23 hours after each cyclophosphamide dose. If patient is obese 
(BMI > 35) drug dosage will be calculated using practical weight as described in Table 6.  
 
 
Day -7 to Day -3: 
Fludarabine 25 mg/m2/day IVPB daily over 30 minutes for 5 days. If patient is obese (BMI > 
35) drug dosage will be calculated using practical weight as described in Table 6. (The 
fludarabine will be started approximately 1-2 hours after the cyclophosphamide and mesna on 
Days -7 and -6) 
 


Abbreviated Title: Anti-mesothelin CAR PBL 
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25 
3.2.2 Cell Infusion and Aldesleukin Administration 
The following criteria must be met for the subject to receive the cell infusion:  T<38.3ºC, 
Pulse <100 BPM, O2 Sat >92% and systolic BP either >90mm Hg systolic or within 
20mm Hg of pre-treatment.   
 
The anti-mesothelin CAR PBLs are delivered to the patient care unit by a staff member from 
the Tumor Immunology Cell Processing Laboratory.  Prior to infusion, the cell product identity 
label is double-checked by two authorized staff (MD or RN), an identification of the product 
and documentation of administration are entered in the patient’s chart, as is done for blood 
banking protocols.  The cells are to be infused intravenously over 20-30 minutes or as 
clinically determined by an investigator for patient safety via non-filtered tubing, gently 
agitating the bag during infusion to prevent cell clumping.  Please note, aldesleukin is being 
administered at the lower dose of 72,000 IU/kg/dose. 
 
Aldesleukin (based on total body weight) will be administered at the lower dose of 72,000 
IU/kg as an intravenous bolus over a 15-minute period approximately every 8 hours beginning 
within 24 hours of cell infusion and continuing for up to 5 days (maximum 15 doses). Doses 
will be preferentially administered every eight hours; however, up to 24 hours may elapse 
between doses depending on patient tolerance.  Aldesleukin dosing will be stopped if toxicities 
are not sufficiently recovered by supportive measures within 24 hours of the last dose of 
aldesleukin.  Dosing will be delayed or stopped if patients reach Grade 3 or 4 toxicity due to 
aldesleukin except for the reversible Grade 3 toxicities common to aldesleukin such as 
diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, or 
constitutional symptoms and laboratory changes as detailed in Appendix 1.  Toxicities will be 
managed as outlined in Appendix 2.  Dosing may be held or stopped at the discretion of the 
treating investigator.  (Appendix 3 lists the toxicities seen in patients treated with aldesleukin 
at the NIH Clinical Center). 
 
Because confusion is a possible side effect of aldesleukin administration, a Durable Power of 
Attorney will be signed by the patient to identify a surrogate to make decisions if a patient 
becomes unable to make decisions. 
 
Day 0 (two to four days after the last dose of fludarabine):  
x Cells will be infused intravenously (i.v.) on the Patient Care Unit over 20 to 30 minutes 
or as clinically determined by an investigator for patient safety (between one and four 
days after the last dose of fludarabine). 
x Aldesleukin will be given as described above.  
 
Day 0-4 (Day 0 is the day of cell infusion):  
x Beginning on day 1 or 2, filgrastim will be administered subcutaneously at a dose of 5 
mcg/kg/day (not to exceed 300 mcg/day).  Filgrastim administration will continue daily 
until neutrophil count > 1.0 x109/L X 3 days or > 5.0 x109/L. 
x Aldesleukin will be given as described above. 
 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
 
26 
3.2.3 Treatment Schedule 
                                                      
Day 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
0 
1 
2 
3 
4 
Therapy 
 
 
 
 
 
 
 
 
 
 
 
 
Cyclophosphamide (60 mg/kg) 
X 
X 
 
 
 
 
 
 
 
 
 
 
Fludarabine (25 mg/m2) 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
Anti-mesothelin CAR PBL 
 
 
 
 
 
 
 
X1 
 
 
 
 
Aldesleukin 
 
 
 
 
 
 
 
X2 
X 
X 
X 
X 
Filgrastim3  (5 mcg/kg/day) 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
TMP/SMX4 
160mg/800mg (example) 
 
 
 
 
 
 
 
X 
 
X 
 
X 
Fluconazole5 (400 mg po) 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
Valacyclovir po or Acyclovir IV6 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
1One to four days after the last dose of fludarabine 
2Initiate within 24 hours after cell infusion 
3Continue until neutrophils count > 1X109/L for 3 consecutive days or > 5x109/L. 
4The TMP/SMX schedule should be adjusted to QD three times per week (Monday, Wednesday, Friday) 
and continue for at least six months and until CD4 > 200 X 2, starting day 0 or within one week of 
anticipated lymphopenia 
5Continue until ANC > 1000/mm3 
6In patients positive for HSV or VZV continue until CD4 >200 X 2 
 
3.3 
ON-STUDY EVALUATION 
Note:  Please refer to section 5 for research evaluations. 
 
3.3.1 Within 28 days prior to starting the preparative regimen 
x EKG  
x coagulation profile including INR, APTT, platelets and fibrinogen. 
 
3.3.2 Within 14 days prior to starting the preparative regimen 
x Apheresis as indicated. 
x Baseline blood test 
o Complete Blood Count with differential 
o Acute Care Panel (sodium, potassium, chloride, bicarbonate, creatinine, glucose, 
BUN), Hepatic Panel (alkaline phosphatase, AST, ALT, total bilirubin, direct 
bilirubin), Mineral Panel (albumin, calcium, magnesium, phosphorus), Uric 
Acid, Creatinine Kinase, Lactate Dehydrogenase, Protein, total 
o PT/PTT 
o TBNK 
o Thyroid Panel 
o Urinalysis 
x Anti CMV antibody titer, HSV serology, and EBV panel. (may be performed within 3 
months of chemotherapy) 
x Chest x-ray 


Abbreviated Title: Anti-mesothelin CAR PBL 
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27 
x EKG 
3.3.3 During the preparative regimen: DAILY 
x Complete Blood Count with differential 
x Acute Care Panel (sodium, potassium, chloride, bicarbonate, creatinine, glucose, BUN), 
Hepatic Panel (alkaline phosphatase, AST, ALT, total bilirubin, direct bilirubin), 
Mineral Panel (albumin, calcium, magnesium, phosphorus), Uric Acid, Creatinine 
Kinase, Lactate Dehydrogenase, Protein, total 
x Urinalysis as needed 
x Daily weight as indicated 
x PT/PTT (every 3 days) 
3.3.4 After Cell Infusion: 
x Vital signs will be monitored hourly (+/- 15 minutes) for four hours and then routinely 
(every 4-6 hours) unless otherwise clinically indicated. 
x Blood samples will be collected at approximately 1 hour, 4 hours, 24 hours, and 1 week 
following infusion for analysis of the cytokines IL-6, TNF-Į, IFN-J and GM-CSF, and 
circulating mesothelin. 
x EKGs and a coagulation profile including INR, APTT, platelets and fibrinogen will be 
conducted 7 days (+/- 2 days) and 14 days (+/- 2 days) after cell administration.  If the 
patient is discharged prior to 14 days following cell infusion, an EKG and coagulation 
profile will be conducted prior to discharge. 
x Once total lymphocyte count is greater than 200/mm3, TBNK for peripheral blood CD4 
count will be drawn weekly (while the patient is hospitalized).   
3.3.5 During Hospitalization (following aldesleukin administration):  
Every 1-2 days 
x A review of systems and physical exam as clinically indicated 
x CBC with differential 
x Acute Care Panel (sodium, potassium, chloride, bicarbonate, creatinine, glucose, BUN), 
Hepatic Panel (alkaline phosphatase, AST, ALT, total bilirubin, direct bilirubin), 
Mineral Panel (albumin, calcium, magnesium, phosphorus), Uric Acid, Creatinine 
Kinase, Lactate Dehydrogenase, Protein, total 
x Other tests will be performed as clinically indicated. 
x Vital signs will be monitored as clinically indicated. 
 
3.4 
POST TREATMENT (FOLLOW-UP) EVALUATION  
All patients will return to the NIH Clinical Center for their 1st evaluation for response 6 weeks 
(+/- 2 weeks) following the administration of the cell product.   
 
Please note: Patients who have received multiple transfusions during the treatment phase or 
discharged with grade 3 or greater significant adverse events should be evaluated by referring 
physician and repeat labs as appropriate within 2 weeks of discharge.  Labs drawn should be 
faxed to the study coordinator. 
 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
 
28 
3.4.1 Time period of evaluations 
Patients who experience stable disease, a partial response, or a complete response or have 
unresolved toxicities will be evaluated as noted below:  
x Week 12 (+/- 2 weeks)  
x Every 3 months (+/- 1 month) x3  
x Every 6 months (+/- 1 month) x 2 years 
x As per PI discretion for subsequent years  
Note: Patients may be seen more frequently as clinically indicated 
3.4.2 At each evaluation, patients will undergo: 
x Physical examination, including weight and vital signs  
x Acute Care Panel (sodium, potassium, chloride, bicarbonate, creatinine, glucose, BUN), 
Hepatic Panel (alkaline phosphatase, AST, ALT, total bilirubin, direct bilirubin), 
Mineral Panel (albumin, calcium, magnesium, phosphorus), Uric Acid, Creatinine 
Kinase, Lactate Dehydrogenase, Protein, total 
x Complete blood count with differential 
x Urinalysis as indicated 
x Thyroid panel as clinically indicated 
x TBNK, until CD4 > 200 X 2 
x EKG and a coagulation profile including INR, PTT/APTT, platelets and fibrinogen. 
x Toxicity assessment, including review of systems  
x Imaging (CT, MRI, and/or PET) as performed at baseline. This end of course evaluation 
will be used to determine tumor response.  
x A 5 liter apheresis may be performed.  If patient is unable to undergo apheresis, 
approximately 96 ml of blood may be obtained at the first follow up visit. Subsequently, 
approximately 60 ml of blood will be obtained at follow up visits (approximately 
monthly) for at least 3 months. Peripheral blood mononuclear cells will be 
cryopreserved so that immunologic testing may be performed. This will be performed on 
03-C-0277. 
x Detection of RCR and persistence of CAR gene transduced cells: (see section 5.7).  This 
will be performed on 03-C-0277. 
x Long-term follow up of patients receiving gene transfer:  Physical examinations will be 
performed and documented annually for 5 years following cell infusion to evaluate long-
term safety.  After 5 years, health status data will be obtained from surviving patients via 
telephone contact or mailed questionnaires.  The long term follow up period for 
retroviral vectors is 15 years.  This will be performed on 09-C-0161. 
x Note: Patients who are unable or unwilling to return for follow up evaluations will be 
followed via phone or email contact.  A request will be made to send laboratory, 
imaging and physician exam reports performed by their treating physician; and any 
outstanding toxicities will be reviewed with the patient. 
 
3.5 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA 
Prior to removal from study, effort must be made to have all subjects complete a safety visit 
approximately 30 days following the last dose of study therapy. 


Abbreviated Title: Anti-mesothelin CAR PBL 
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29 
3.5.1 Criteria for removal from protocol therapy: 
Patients will be taken off treatment (and followed until progression of disease) for the 
following: 
x Completion of protocol therapy 
x Participant requests to be withdrawn from active therapy 
x Investigator discretion. 
x Positive Pregnancy Test 
 
3.5.2 Off Study Criteria: 
Patients will be taken off study for the following: 
x Completion of study follow up period. 
x The participant requests to be withdrawn from study 
x Radiographic or clinical disease progression.  
x Lost to follow-up 
x Death 
 
Note: Once a subject is taken off study, no further data can be collected. 
 
Note: Patients who are taken off study for progressive disease or study closure may be 
followed on protocol 09-C-0161 “Follow up Protocol for subjects Previously Enrolled in 
Surgery Branch Studies.”. 
 
 
3.5.3 Off Protocol Therapy and Off Study Procedure 
Authorized staff must notify Central Registration Office (CRO) when a subject is taken off 
protocol therapy and when a subject is taken off-study. A Participant Status Updates Form 
from the web site (http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must 
be completed and sent via encrypted email to:  NCI Central Registration Office 
<ncicentralregistration-l@mail.nih.gov>. 
 
4 
CONCOMITANT MEDICATIONS/MEASURES 
4.1 
INFECTION PROPHYLAXIS: 
Note: Other anti-infective agents may be substituted at the discretion of the treating physician. 
4.1.1 Pneumocystis Jirovecii Pneumonia 
All patients will receive the fixed combination of trimethoprim and sulfamethoxazole 
(TMP/SMX) as double strength (DS) tab (DS tabs = TMP 160 mg/tab, and SMX 800 mg/tab) 
P.O. daily three times a week on non-consecutive days, beginning day 0 or within 1 week of 
anticipated lymphopenia.  
 
Dapsone (in G6PD sufficient patient), Atovaquone or Pentamidine may be substituted for 
TMP/SMX-DS in patients with sulfa allergies.  


Abbreviated Title: Anti-mesothelin CAR PBL 
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30 
 
4.1.2 Herpes and Varicella Zoster Virus Prophylaxis 
Patients with positive HSV or VZV serology will be given valacyclovir orally at a dose of 500 
mg daily starting the day of cell infusion, or acyclovir, 250 mg/m2 IV every 12 hours if the 
patient is not able to take medication by mouth.  Reversible renal insufficiency has been 
reported with IV but not oral acyclovir.  Neurologic toxicity including delirium, tremors, coma, 
acute psychiatric disturbances, and abnormal EEGs have been reported with higher doses of 
acyclovir.  Should this occur, a dosage adjustment will be made or the drug will be 
discontinued.  Acyclovir will not be used concomitantly with other nucleoside analogs which 
interfere with DNA synthesis, e.g. ganciclovir.  In renal disease, the dose is adjusted as per 
product labeling. 
 
Prophylaxis for Pneumocystis Varicella Zoster and Herpes will continue for 6 months post 
chemotherapy.  If the CD4 count is less than 200 at 6 months post chemotherapy, prophylaxis 
will continue until CD4 count is greater than 200 X 2. 
 
4.1.3 Fungal Prophylaxis 
Patients will start Fluconazole 400 mg p.o. the day of cell infusion and continue until the 
absolute neutrophil count is greater than 1000/mm3.  The drug may be given IV at a dose of 
400 mg in 0.9% sodium chloride USP daily in patients unable to take it orally. 
 
4.2 
BLOOD PRODUCT SUPPORT 
Using daily CBC’s as a guide, the patient will receive platelets and packed red blood cells 
(PRBC’s) as needed.  Attempts will be made to keep Hb >8.0 gm/dl, and plts >10,000/mm3.  
All blood products will be irradiated.  Leukocyte filters will be utilized for all blood and 
platelet transfusions to decrease sensitization to transfused WBC’s and decrease the risk of 
CMV infection. 
 
4.3 
EMPIRIC ANTIBIOTICS 
Patients will start on broad-spectrum antibiotics in accordance with current institutional 
guidelines for fever of 38.3°C once or two temperatures of 38.0°C or above at least one hour 
apart, AND an ANC <500/mm3. Infectious disease consultation will be obtained for all patients 
with unexplained fever or any infectious complications. 
 
4.4 
OTHER CONCOMITANT MEDICATIONS TO CONTROL SIDE EFFECTS 
Concomitant medications to control side effects of therapy may be given.  Meperidine (25-50 
mg) will be given intravenously if severe chilling develops.  Other supportive therapy will be 
given as required and may include acetaminophen (650 mg q4h), indomethacin (50-75 mg q8h) 
and ranitidine (150 mg g12h). If patients require steroid therapy, they will be taken off 
treatment.  Patients who require transfusions will receive irradiated blood products.  
Ondansetron 0.15 mg/kg/dose IV every 8 hours will be administered for nausea and vomiting.  
Additional anti-emetics will be administered as needed for nausea and vomiting uncontrolled 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
 
31 
by ondansetron.  Antibiotic coverage for central venous catheters may be provided at the 
discretion of the investigator. 
 
5 
 
BIOSPECIMEN COLLECTION 
Blood and tissue are tracked at the patient level and can be linked to all protocols on which the 
patient has been enrolled.  Samples will be used to support the specific objectives listed in the 
treatment protocol(s), e.g., immunologic monitoring, cytokine levels, persistence, as well as to 
support long term research efforts within the Surgery Branch and with collaborators as 
specified in our companion protocol, 03-C-0277 (Cell Harvest and Preparation for Surgery 
Branch Adoptive Cell Therapy Protocols). 
 
The amount of blood that may be drawn from adult patients for research purposes shall not 
exceed 10.5 mL/kg or 550 mL, whichever is smaller, over any eight-week period. 
 
5.1 
SAMPLES SENT TO DR. FIGG’S LAB 
x Venous blood samples will be collected in either a 4ml or an 8ml SST tube to be 
processed for serum and stored for future research. Record the date and exact time of 
draw on the tube. Blood tubes may be kept in the refrigerator until pickup. 
x For sample pickup, page 102-11964. 
x For immediate help, call 240-760-6180 (main blood processing core number) or, if no 
answer, 240-760-6190 (main clinical pharmacology lab number). 
x For questions regarding sample processing, contact Julie Barnes by e-mail or at 240-
760-6044. 
x The samples will be processed, barcoded, and stored in Dr. Figg’s lab until requested 
by the investigator. 
 
5.2 
SAMPLES SENT TO THE SURGERY BRANCH CELL PROCESSING LABORATORY 
x Venous blood samples will be collected in 8ml CPT tubes to be processed and stored 
for future research. Record the date and exact time of draw on the tube. Blood tubes are 
kept at room temperature until pickup. 
x Samples will be pick-up by the research nurse or designee and transported to the SB 
Cell Processing Laboratory within 24 hours of blood draw. 
x The samples will be processed, barcoded, and stored in SB Cell Processing Laboratory. 
5.3 
PRIOR TO CHEMOTHERAPY ADMINISTRATION 
x 5 CPT tubes (8ml each) – send to SB lab 
x 1 SST tube (8ml) – send to Figg’s lab 
x 1 SST tube (4 ml) daily; starting day of chemotherapy – send to Figg’s lab 
5.4 
PRIOR TO CELL INFUSION (1-8ML SST) 
x Blood samples for analysis of the cytokines IL-6, TNF-Į,)1-J and GM-CSF. – send to 
Figg’s lab 


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32 
5.5 
POST CELL INFUSION EVALUATIONS 
x Once total lymphocyte count is greater than 200/mm3, the following samples will be 
drawn and sent to the TIL lab on Monday, Wednesday and Friday x 5 days, then weekly 
(while the patient is hospitalized): 
o 5 CPT tubes (8ml each) – send to SB lab 
o 1 SST tube (8ml)  - send to Figg’s lab 
 
5.6 
 IMMUNOLOGICAL TESTING 
x Apheresis may be performed, prior to and 4-6 weeks after the treatment.  At other time 
points, patient peripheral blood lymphocytes (PBL) will be obtained from whole blood by 
purification using centrifugation on a Ficoll cushion. Aliquots of these PBMC will be: 
o Cryopreserved for immunological monitoring of cell function, 
o Subjected to DNA and RNA extraction for PCR analysis of TCR and vector copy 
number estimation 
x Lymphocytes will be tested directly and following in vitro culture. Direct immunological 
monitoring will consist of quantifying T cells reactive with mesothelin by FACS analysis 
using tetramer staining.  Ex vivo immunological assays will consist of cytokine release 
by bulk PBL (+/- peptide stimulation) and by other experimental studies such as cytolysis 
if sufficient cells are available. If cell numbers are limiting, preference will be given to 
the direct analysis of immunological activity.  Immunological assays will be standardized 
by the inclusion of 1) pre-infusion PBMC and 2) an aliquot of the engineered PBL 
cryopreserved at the time of infusion. In general, differences of 2 to 3 fold in these assays 
are indicative of true biologic differences.  Foxp3 levels will be analyzed by 
semiquantitative RT-PCR to evaluate for mRNA on PBL samples obtained prior to cell 
infusion and at the follow up time point. 
 
5.7 
MONITORING GENE THERAPY TRIALS:  PERSISTENCE AND RCR 
x Engineered cell survival.  TCR and vector presence will be quantitated in PBMC samples 
using established PCR techniques.   Immunological monitoring using both tetramer 
analysis and staining for the TCR will be used to augment PCR-based analysis.  This will 
provide data to estimate the in vivo survival of lymphocytes derived from the infused 
cells. In addition, measurement of CD4 and CD8 T-cells will be conducted and studies of 
these T-cell subsets in the circulation will be determined by using specific PCR assays 
capable of detecting the unique DNA sequence for each retroviral vector engineered T-
cell. Note: samples will be batched and assayed at the conclusion of the study. 
x All patients will be co-enrolled on protocol 09-C-0161 “Follow up Protocol for Subjects 
Previously Enrolled in NCI Surgery Branch Studies”.  Patients’ blood samples will be 
obtained and undergo analysis for detection of Replication Competent Retroviruses 
(RCR) by PCR prior to cell infusion and RCR PCR will be performed at 3 and 6 months, 
and at one year post cell administration.  Blood samples will be archived annually 
thereafter if all previous testing has been negative with a brief clinical history. If a patient 
dies or develops neoplasms during this trial, efforts will be made to assay a biopsy 
sample for RCR. If any post-treatment samples are positive, further analysis of the RCR 
and more extensive patient follow-up will be undertaken, in consultation with the FDA.   


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RCR PCR assays detect the GaLV envelop gene and are performed under contract.  The 
results of these tests are maintained by the contractor performing the RCR tests and by 
the Surgery Branch research team. 
 
5.8 
SAMPLE, STORAGE, TRACKING AND DISPOSITION FOR SB CELL PROCESSING 
LABORATORY 
Blood and tissue collected during the course of this study will follow the Cell Tracking and 
Labeling System established by the Tumor Immunology Cell Processing Laboratory.  The 
Cell Tracking and Labeling System is designed to unambiguously ensure that patient/data 
verification is consistent.  The patients' cell samples (blood or tissue) are tracked by distinct 
identification labels that include a unique patient identifier and date of specimen collection.  
Cryopreserved blood and tissue samples also bear the date the sample was frozen.  All 
cryopreserved samples are tracked for freezer location and storage criteria.  All samples are 
stored in monitored freezers/refrigerators in 3NW Surgery Branch Laboratories at specified 
temperatures with alarm systems in place.  Serum samples will be sent to the Blood 
Processing Core (BPC) for storage.  Samples will be barcoded and stored on site or offsite at 
NCI Frederick Central Repository Services in Frederick, MD.  All samples (blood or tissue) 
are entered into a central computer database with identification and storage location, and this 
database is backed up every night. 
 
If, at any time, a patient withdraws from the study and does not wish for their existing 
samples to be utilized, the individual must provide a written request. Following receipt of this 
request, the samples will be destroyed (or returned to the patient, if so requested), and 
reported as such to the IRB. Any samples lost (in transit or by a researcher) or destroyed due 
to unknown sample integrity (i.e. broken freezer allows for extensive sample thawing, etc.) 
will be reported as such to the IRB. 
 
Note:  Blood and tissue collected during the course of this study will be stored, tracked and 
disposed of as specified in our companion protocol 03-C-0277, (Cell Harvest and Preparation 
for Surgery Branch Adoptive Cell Therapy Protocols). 
 
5.9 
SAMPLE STORAGE, TRACKING AND DISPOSITION FOR DR. FIGG’S LAB 
5.9.1 Sample Data Collection 
 
All samples sent to the Blood Processing Core (BPC) will be barcoded, with data entered and 
stored in the LABrador (aka LabSamples) utilized by the BPC, and data will be updated to the 
Surgery Branch central computer database weekly. This is a secure program, with access to 
LABrador limited to defined Figg lab personnel, who are issued individual user accounts. 
Installation of LABrador is limited to computers specified by Dr. Figg. These computers all 
have a password restricted login screen. All Figg lab personnel with access to patient 
information annually complete the NIH online Protection of Human Subjects course.  
 
LABrador creates a unique barcode ID for every sample and sample box, which cannot be 
traced back to patients without LABrador access. The data recorded for each sample includes 


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34 
the patient ID, name, trial name/protocol number, time drawn, cycle time point, dose, material 
type, as well as box and freezer location. Patient demographics associated with the clinical 
center patient number are provided in the system. For each sample, there are notes associated 
with the processing method (delay in sample processing, storage conditions on the ward, etc.). 
 
5.9.2 Sample Storage and Destruction 
 
Barcoded samples are stored in barcoded boxes in a locked freezer at either -20 or -80°C 
according to stability requirements.  These freezers are located onsite in the BPC and offsite at 
NCI Frederick Central Repository Services in Frederick, MD. Visitors to the laboratory are 
required to be accompanied by laboratory staff at all times.  
 
Access to stored clinical samples is restricted. Samples will be stored until requested by a 
researcher named on the protocol. All requests are monitored and tracked in LABrador. All 
researchers are required to sign a form stating that the samples are only to be used for research 
purposes associated with this trial (as per the IRB approved protocol) and that any unused 
samples must be returned to the BPC. It is the responsibility of the NCI Principal Investigator 
to ensure that the samples requested are being used in a manner consistent with IRB approval. 
 
Following completion of this study, samples will remain in storage as detailed above. Access 
to these samples will only be granted following IRB approval of an additional protocol, 
granting the rights to use the material.  
 
If, at any time, a patient withdraws from the study and does not wish for their existing samples 
to be utilized, the individual must provide a written request. Following receipt of this request, 
the samples will be destroyed (or returned to the patient, if so requested), and reported as such 
to the IRB. Any samples lost (in transit or by a researcher) or destroyed due to unknown 
sample integrity (i.e. broken freezer allows for extensive sample thawing, etc.) will be reported 
as such to the IRB. 
 
Sample barcodes are linked to patient demographics and limited clinical information. This 
information will only be provided to investigators listed on this protocol, via registered use of 
the LABrador. It is critical that the sample remains linked to patient information such as race, 
age, dates of diagnosis and death, and histological information about the tumor, in order to 
correlate genotype with these variables. 
 
Note: Blood and tissue collected during the course of this study will be stored, tracked, and 
disposed of as specified in our companion protocol, 03-C-0277 (Cell Harvest and Preparation 
for Surgery Branch Adoptive Cell Therapy Protocols). 
 
6 
DATA COLLECTION AND EVALUATION 
6.1 
DATA COLLECTION 
The PI will be responsible for overseeing entry of data into an in-house password protected 
electronic system and ensuring data accuracy, consistency and timeliness. The principal 


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35 
investigator, associate investigators/research nurses and/or a contracted data manager will 
assist with the data management efforts.   
 
All data obtained during the conduct of the protocol will be kept in secure network drives or in 
approved alternative sites that comply with NIH security standards. Primary and final analyzed 
data will have identifiers so that research data can be attributed to an individual human subject 
participant. Data will be entered into the NCI CCR C3D database. 
 
All AEs, including clinically significant abnormal findings on laboratory evaluations, 
regardless of severity, will be followed until return to baseline or stabilization of event.  
Patients will be followed for adverse events until their first week 6 follow-up evaluation or 
until off-study, whichever comes first. 
 
An abnormal laboratory value will be considered an AE if the laboratory abnormality is 
characterized by any of the following: 
x Results in discontinuation from the study 
x Is associated with clinical signs or symptoms  
x Requires treatment or any other therapeutic intervention 
x Is associated with death or another serious adverse event, including hospitalization.  
x Is judged by the Investigator to be of significant clinical impact 
x If any abnormal laboratory result is considered clinically significant, the investigator 
will provide details about the action taken with respect to the test drug and about the 
patient’s outcome. 
 
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH 
Intramural Records Retention Schedule as applicable.  
 
Loss or destruction of data: Should we become aware that a major breach in our plan to 
protect subject confidentiality and trial data has occurred, the IRB will be notified. 
 
6.1.1 Exclusions to Routine Adverse Event Reporting: 
Patients will be receiving multiple agents which include commercially available agents 
(fludarabine, cyclophosphamide, aldesleukin and supportive medications) in combination with 
the investigational agents; therefore, grade 1 events not related to the cell product will not be 
reported/recorded. 
 
6.2 
DATA SHARING PLANS 
6.2.1 Human Data Sharing Plan 
De-identified human data generated for use in future and ongoing research will be shared 
through a NIH-funded or approved repository (ClinicalTrials.gov) and BTRIS.  At the 
completion of data analysis, data will be submitted to ClinicalTrials.gov either before 
publication or at the time of publication or shortly thereafter. Data may also be used to support 
long term research efforts within the Surgery Branch and de-identified data may also be shared 


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36 
with collaborators as specified in our companion protocol, 03-C-0277 (Cell Harvest and 
Preparation for Surgery Branch Adoptive Cell Therapy Protocols). 
 
6.2.2 Genomic Data Sharing Plan 
The NIH Genomic Data Sharing Policy does not apply to this study. 
 
6.3 
RESPONSE CRITERIA 
For the purposes of this study, patients should be re-evaluated for response at 6 and 12 weeks 
(+/- 2 weeks), then every 3 months (+/- 1 month) x3, then every 6 months’ (+/- 1 month) x 2 
years, then as per PI discretion.  In addition to a baseline scan, confirmatory scans may be 
obtained at least 4 weeks (not less than 4) following initial documentation of objective 
response. 
 
Clinical Response will be determined using the Response Evaluation Criteria in Solid Tumors 
(RECIST) guideline (version 1.0).   
 
6.3.1 Definitions 
Evaluable for toxicity:  All patients will be evaluable for toxicity from the time of their first 
treatment with Cyclophosphamide. 
 
Evaluable for objective response: Only those patients who have measurable disease present at 
baseline, have received at least one course of therapy, and have had their disease re-evaluated 
will be considered evaluable for response.  These patients will have their response classified 
according to the definitions stated below.  (Note:  Patients who exhibit objective disease 
progression prior to the end of course 1 will also be considered evaluable.) 
 
Evaluable Non-Target Disease Response:  Patients who have lesions present at baseline that 
are evaluable but do not meet the definitions of measurable disease, have received at least one 
course of therapy, and have had their disease re-evaluated will be considered evaluable for 
non-target disease.  The response assessment is based on the presence, absence, or unequivocal 
progression of the lesions. 
 
6.3.2 Disease Parameters 
Measurable disease:  Measurable lesions are defined as those that can be accurately measured 
in at least one dimension (longest diameter to be recorded) as >20 mm by chest x-ray, as >10 
mm with CT scan, or >10 mm with calipers by clinical exam.  All tumor measurements must 
be recorded in millimeters (or decimal fractions of centimeters). 
 
Malignant lymph nodes:  To be considered pathologically enlarged and measurable, a lymph 
node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness 
recommended to be no greater than 5 mm).  At baseline and in follow-up, only the short axis 
will be measured and followed. 
 


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37 
Non-measurable disease:  All other lesions (or sites of disease), including small lesions 
ORQJHVWGLDPHWHUPPRUSDWKRORJLFDOO\PSKQRGHVZLWKWRPPVKRUWD[LVDUH
considered non-measurable disease.  Bone lesions, leptomeningeal disease, ascites, 
pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, and 
abdominal masses (not followed by CT or MRI), are considered as non-measurable. 
 
Target lesions:  All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in 
total, representative of all involved organs, should be identified as target lesions and recorded 
and measured at baseline.  Target lesions should be selected on the basis of their size (lesions 
with the longest diameter), be representative of all involved organs, but in addition should be 
those that lend themselves to reproducible repeated measurements.  It may be the case that, on 
occasion, the largest lesion does not lend itself to reproducible measurement in which 
circumstance the next largest lesion which can be measured reproducibly should be selected.  
A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all 
target lesions will be calculated and reported as the baseline sum diameters.  If lymph nodes 
are to be included in the sum, then only the short axis is added into the sum.  The baseline sum 
diameters will be used as reference to further characterize any objective tumor regression in the 
measurable dimension of the disease. 
 
Non-target lesions:  All other lesions (or sites of disease) including any measurable lesions 
over and above the 5 target lesions should be identified as non-target lesions and should also be 
recorded at baseline.  Measurements of these lesions are not required, but the presence, 
absence, or in rare cases unequivocal progression of each should be noted throughout follow-
up. 
 
6.3.3 Methods for Evaluation of Measurable Disease 
All measurements should be taken and recorded in metric notation using a ruler or calipers.  
All baseline evaluations should be performed as closely as possible to the beginning of 
treatment and never more than 4 weeks before the beginning of the treatment. 
The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be 
imaged but are assessable by clinical exam. 
 
Clinical lesions:  Clinical lesions will only be considered measurable when they are superficial 
(e.g., skin nodules and palpable lymph nodes) and t10 mm diameter as assessed using calipers 
(e.g., skin nodules).  In the case of skin lesions, documentation by color photography, 
including a ruler to estimate the size of the lesion, is recommended.  
 
Conventional CT and MRI:  This guideline has defined measurability of lesions on CT scan 
based on the assumption that CT slice thickness is 5 mm or less.  If CT scans have slice 
thickness greater than 5 mm, the minimum size for a measurable lesion should be twice the 
slice thickness.  MRI is also acceptable in certain situations (e.g. for body scans).  Ideally, the 
same type of scanner should be used and the image acquisition protocol should be followed as 


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38 
closely as possible to prior scans.  Body scans should be performed with breath-hold scanning 
techniques, if possible. 
 
FDG-PET:  While FDG-PET response assessments need additional study, it is sometimes 
reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in 
assessment of progression (particularly possible 'new' disease).  New lesions on the basis of 
FDG-PET imaging can be identified according to the following algorithm:  
x Negative FDG-PET at baseline, with a positive FDG-PET at follow-up is a sign of PD 
based on a new lesion. 
x No FDG-PET at baseline and a positive FDG-PET at follow-up:  If the positive FDG-
PET at follow-up corresponds to a new site of disease confirmed by CT, this is PD.  If 
the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, 
additional follow-up CT scans are needed to determine if there is truly progression 
occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-
PET scan).  If the positive FDG-PET at follow-up corresponds to a pre-existing site of 
disease on CT that is not progressing on the basis of the anatomic images, this is not 
PD. 
x FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy 
in cases where a residual radiographic abnormality is thought to represent fibrosis or 
scarring.  The use of FDG-PET in this circumstance should be prospectively described 
in the protocol and supported by disease-specific medical literature for the indication.  
However, it must be acknowledged that both approaches may lead to false positive CR 
due to limitations of FDG-PET and biopsy resolution/sensitivity. 
Note:  A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake greater 
than twice that of the surrounding tissue on the attenuation corrected image. 
 
6.3.4 Response Criteria 
6.3.4.1 Evaluation of target lesions1  
x Complete Response (CR):  Disappearance of all target lesions 
x Partial Response (PR):  At least a 30% decrease in the sum of the longest diameter (LD) 
of target lesions taking as reference the baseline sum LD. 
x Progression (PD):  At least a 20% increase in the sum of LD of target lesions taking as 
reference the smallest sum LD recorded since the treatment started or the appearance of 
one or more new lesions. 
x Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase 
to qualify for PD taking as references the smallest sum LD. 
6.3.4.2 Evaluation of non-target lesions2  
x Complete Response (CR): Disappearance of all non-target lesions and normalization of 
tumor marker level. 
x Non-Complete Response:  Persistence of one or more non-target lesions 
x Progression (PD):  Appearance of one or more new lesions.  Unequivocal progression of 
existing non-target lesions 
----------------------------------------------------------------------------------- 


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39 
1 
All measurable lesions up to a maximum of 10 lesions representative of all involved organs should be 
identified as target lesions and recorded and measured at baseline.  Target lesions should be selected on 
the basis of their size (lesions with the longest diameter) and their suitability for accurate repetitive 
measurements (either by imaging techniques or clinically).  A sum of the longest diameter (LD) for all target 
lesions will be calculated and reported as the baseline sum LD.  The baseline sum LD will be used as 
reference to further characterize the objective tumor response of the measurable dimension of the disease. 
2 
All other lesions (or sites of disease) should be identified as non-target lesions and should also be recorded 
at baseline.  Measurements are not required, and these lesions should be followed as “present” or “absent.” 
 
6.3.4.3 Evaluation of best overall response 
The best overall response 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. 
 
Target Lesions 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
CR 
No 
CR 
CR 
Non-CR/Non-PD 
No 
PR 
PR 
Non-PD 
No 
PR 
SD 
Non-PD 
No 
SD 
PD 
Any 
Yes or No 
PD 
Any 
PD 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
 
6.3.4.4 Confirmatory Measurement/Duration of Response 
Confirmation 
To be assigned a status of PR or CR, changes in tumor measurements must be confirmed by 
repeat studies that should be performed at least 4 weeks after the criteria for response are first 
met.  In the case of SD, follow-up measurements must have met the SD criteria at least once 
after study entry at a minimum interval of 6-8 weeks. 
 
Duration of Overall Response 
The duration of overall response is measured from the time measurement criteria are met for 
CR/PR (whichever is first recorded) until the first date that recurrent or progressive disease is 
objectively documented (taking as reference for progressive disease the smallest measurements 
recorded since the treatment started). 
 
The duration of overall complete response is measured from the time measurement criteria are 
first met for CR until the first date that recurrent disease is objectively documented. 
 
Duration of Stable Disease 
Stable disease is measured from the start of the treatment until the criteria for progression are 
met, taking as reference the smallest measurements recorded since the treatment started.  
 


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40 
6.4 
TOXICITY CRITERIA 
The following adverse event management guidelines are intended to ensure the safety of each 
patient while on the study.  The descriptions and grading scales found in the revised NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for 
AE reporting.  All appropriate treatment areas should have access to a copy of the CTCAE 
version 4.0.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_40). 
 
 
 
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN 
7.1 
DEFINITIONS 
7.1.1 Adverse Event 
Any untoward medical occurrence in a human subject, including any abnormal sign (for 
example, abnormal physical exam or laboratory finding), symptom, or disease, temporally 
associated with the subject’s participation in research, whether or not considered related to the 
subject’s participation in the research.  
 
7.1.2 Suspected Adverse Reaction 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event. For the purposes of IND safety reporting, ‘reasonable 
possibility’ means there is evidence to suggest a causal relationship between the drug and the 
adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any adverse event caused by a drug. 
7.1.3 Unexpected Adverse Reaction 
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in 
the investigator brochure or is not listed at the specificity or severity that has been observed; or, 
if an investigator brochure is not required or available, is not consistent with the risk 
information described in the general investigational plan or elsewhere in the current 
application. "Unexpected”, also refers to adverse events or suspected adverse reactions that are 
mentioned in the investigator brochure as occurring with a class of drugs or as anticipated from 
the pharmacological properties of the drug, but are not specifically mentioned as occurring 
with the particular drug under investigation. 
7.1.4 Serious 
An Unanticipated Problem or Protocol Deviation is serious if it meets the definition of a 
Serious Adverse Event or if it compromises the safety, welfare or rights of subjects or others. 
7.1.5 Serious Adverse Event 
An adverse event or suspected adverse reaction is considered serious if in the view of the 
investigator or the sponsor, it results in any of the following: 
x Death, 
x A life-threatening adverse drug experience 


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41 
x Inpatient hospitalization or prolongation of existing hospitalization 
x Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
x A congenital anomaly/birth defect. 
x Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may 
require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. 
7.1.6 Disability 
A substantial disruption of a person’s ability to conduct normal life functions. 
7.1.7 Life threatening Adverse Drug Experience 
Any adverse event or suspected adverse reaction that places the patient or subject, in the view 
of the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., 
it does not include a reaction that had it occurred in a more severe form, might have caused 
death. 
7.1.8 Protocol Deviation (NIH Definition) 
Any change, divergence, or departure from the IRB approved research protocol.  
7.1.9 Non-compliance (NIH Definition) 
The failure to comply with applicable NIH Human Research Protections Program (HRPP) 
policies, IRB requirements, or regulatory requirements for the protection of human research 
subjects. 
7.1.10 Unanticipated Problems 
Any incident, experience, or outcome that: 
x  Is unexpected in terms of nature, severity, or frequency in relation to  
(a) the research risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study documents, and 
(b) the characteristics of the subject population being studied; AND 
x Is related or possibly related to participation in the research; AND 
x Suggest that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized 
7.2 
NCI-IRB AND CLINICAL DIRECTOR (CD) REPORTING 
7.2.1 NCI-IRB and NCI CD Expedited Reporting of Unanticipated Problems and Deaths 
The Protocol PI will report in the NIH Problem Form to the NCI-IRB and NCI Clinical 
Director: 
x All deaths, except deaths due to progressive disease  
x All Protocol Deviations 
x All Unanticipated Problems 
x All non-compliance 


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Reports must be received within 7 days of PI awareness via iRIS. 
7.2.2 NCI-IRB Requirements for PI Reporting at Continuing Review 
 
The protocol PI will report to the NCI-IRB:  
1. A summary of all protocol deviations in a tabular format to include the date the 
deviation occurred, a brief description of the deviation and any corrective action. 
2. A summary of any instances of non-compliance. 
3. A tabular summary of the following adverse events: 
x 
All Grade 2 unexpected events that are possibly, probably or definitely related to 
the research;  
x 
All Grade 3 and 4 events that are possibly, probably or definitely related to the 
research;  
x 
All Grade 5 events regardless of attribution;  
x 
All Serious Events regardless of attribution.  
NOTE: Grade 1 events are not required to be reported.  
7.2.3 NCI-IRB Reporting of IND Safety Reports 
Only IND Safety Reports that meet the definition of an unanticipated problem will need to be 
reported to the NCI IRB. 
 
7.3 
INSTITUTIONAL BIOSAFETY COMMITTEE (IBC) REPORTING CRITERIA 
7.3.1 Serious Adverse Event Reports to IBC 
The Principal Investigator (or delegate) will notify IBC of any unexpected fatal or life-
threatening experience associated with the use of the anti-mesothelin CAR PBL as soon as 
possible but in no event later than 7 calendar days of initial receipt of the information.  Serious 
adverse events that are unexpected and associated with the use of the anti-mesothelin CAR 
PBL, but are not fatal or life-threatening, must be reported to the NIH IBC as soon as possible, 
but not later than 15 calendar days after the investigator’s initial receipt of the information.   
Adverse events may be reported by using the FDA Form 3500a. 
 
7.3.2 Annual Reports to IBC 
Within 60 days after the one-year anniversary of the date on which the IBC approved the initial 
protocol, and after each subsequent anniversary until the trial is completed, the Principal 
Investigator (or delegate) shall submit the information described below.  Alternatively, IRB 
continuing review report can be sent to the IBC in lieu of a separate report.  Please include the 
IBC protocol number on the report. 
7.3.2.1 Clinical Trial Information  
A brief summary of the status of the trial in progress or completed during the previous year. 
The summary is required to include the following information:  
x the title and purpose of the trial  
x clinical site 


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43 
x the Principal Investigator 
x clinical protocol identifiers  
x participant population (such as disease indication and general age group, e.g., adult or 
pediatric);  
x the total number of participants planned for inclusion in the trial; the number entered 
into the trial to date whose participation in the trial was completed; and the number who 
dropped out of the trial with a brief description of the reasons  
x the status of the trial, e.g., open to accrual of subjects, closed but data collection 
ongoing, or fully completed,  
x if the trial has been completed, a brief description of any study results.  
7.3.2.2 Progress Report and Data Analysis  
Information obtained during the previous year's clinical and non-clinical investigations, 
including:  
x a narrative or tabular summary showing the most frequent and most serious adverse 
experiences by body system 
x a summary of all serious adverse events submitted during the past year 
x a summary of serious adverse events that were expected or considered to have causes 
not associated with the use of the gene transfer product such as disease progression or 
concurrent medications 
x if any deaths have occurred, the number of participants who died during participation in 
the investigation and causes of death 
x a brief description of any information obtained that is pertinent to an understanding of 
the gene transfer product’s actions, including, for example, information about dose-
response, information from controlled trials, and information about bioavailability.  
 
7.4 
IND SPONSOR REPORTING CRITERIA 
Up until the first follow-up evaluation (6 weeks (± 2 weeks) following the administration of the 
cell product), the investigator must immediately report to the sponsor, using the mandatory 
MedWatch FDA Form 3500a or equivalent, any serious adverse event, whether or not considered 
drug related, including those listed in the protocol or investigator brochure and must include an 
assessment of whether there is a reasonable possibility that the drug caused the event. For serious 
adverse events that occur after the first follow-up evaluation, only those events that have an 
attribution of at least possibly related to the agent/intervention will be reported. 
Required timing for reporting per the above guidelines: 
x Death (except death due to progressive disease) must be reported via email within 24 
hours. A complete report must be submitted within one business day. 
x Other serious adverse events as well as deaths due to progressive disease must be reported 
within one business day 
Study endpoints that are serious adverse events (e.g. all-cause mortality) must be reported in 
accordance with the protocol unless there is evidence suggesting a causal relationship between 
the drug and the event (e.g. death from anaphylaxis).  In that case, the investigator must 
immediately report the death to the sponsor. 
 


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44 
Events will be submitted to the Center for Cancer Research (CCR) at: 
CCRsafety@mail.nih.gov  and to the CCR PI and study coordinator. 
 
7.4.1 Wavier of expedited reporting to CCR 
The investigators are requesting a waiver from reporting specific events in an expedited 
manner to the CCR.  Patients will be receiving commercially available agents, such as 
fludarabine, cyclophosphamide, and aldesleukin.  The majority of toxicities observed on 
Surgery Branch Adoptive Cell Therapy protocols are expected toxicities of the non-
myeloablative chemotherapy regimen or IL-2 and occur in approximately 95% of the patients 
enrolled, therefore, we are requesting a waiver from reporting the following events in an 
expedited manner to the CCR.   
 
x Grade 3 or greater myelosuppression, defined as lymphopenia, neutropenia, decreased 
hemoglobin, and thrombocytopenia. 
x Grade 3 or greater nausea, vomiting, mucositis - oral, anorexia, diarrhea, fever, chills, 
fatigue, and rash maculo-papular. 
x Grade 3 hematuria, hypotension, sinus tachycardia, urine output decreased, confusion, 
infections, and febrile neutropenia. 
 
The PI will submit a summary table of all grade 3-5 events, whether or not considered related 
to the product, every 6 months.   The report shall include the number of patients treated in the 
timeframe, the number of events per AE term per grade which occurred in the 6-month 
timeframe and in total since the start of the study, attribution, and type/category of serious. 
Reports will be submitted to the Center for Cancer Research (CCR) at: 
CCRsafety@mail.nih.gov 
 
7.4.2 Reporting Pregnancy  
7.4.2.1 Maternal exposure 
If a patient becomes pregnant during the first four months following treatment the pregnancy 
should be reported to the Sponsor. The potential risk of exposure of the fetus to the 
investigational agent(s) or chemotherapy agents (s) should be documented in box B5 of the 
Medwatch form “Describe Event or Problem”. 
 
Pregnancy itself is not regarded as a SAE unless there is a suspicion that the study treatment 
under study may have interfered with the effectiveness of a contraceptive medication. 
However, as patients who become pregnant on study risk intrauterine exposure of the fetus to 
agents which may be teratogenic, the CCR is requesting that pregnancy should be reported in 
an expedited manner as Grade 3 “Pregnancy, puerperium and perinatal conditions - Other 
(pregnancy)” under the Pregnancy, puerperium and perinatal conditions SOC.  
 
Congenital abnormalities or birth defects and spontaneous miscarriages should be reported and 
handled as SAEs. Elective abortions without complications should not be handled as AEs. The 
outcome of all pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, 
normal birth, or congenital abnormality) should be followed up and documented. 


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If any pregnancy occurs in the course of the study, then the investigator should inform the 
Sponsor within 1 day, i.e., immediately, but no later than 24 hours of when he or she becomes 
aware of it. 
 
The designated Sponsor representative will work with the investigator to ensure that all 
relevant information is provided to the Sponsor within 1 to 5 calendar days for SAEs and 
within 30 days for all other pregnancies. 
 
The same timelines apply when outcome information is available. 
7.4.2.2 Paternal exposure 
Male patients should refrain from fathering a child or donating sperm during the study and for 
120 days after the last dose of aldesleukin. 
Pregnancy of the patient’s partner is not considered to be an AE. However, the outcome of all 
pregnancies (spontaneous miscarriage, elective termination, ectopic pregnancy, normal birth, 
or congenital abnormality) occurring from the date of the first dose until 120 days after the last 
dose should, if possible, be followed up and documented. 
 
7.5 
DATA AND SAFETY MONITORING PLAN 
7.5.1 Principal Investigator/Research Team 
 
The clinical research team will meet on a regular basis when patients are being actively treated 
on the trial to discuss each patient. Decisions about enrollment will be made based on the 
toxicity data from prior patients.  
 
All data will be collected in a timely manner and reviewed by the principal investigator. 
Adverse events will be reported as required above. Any safety concerns, new information that 
might affect either the ethical and or scientific conduct of the trial, or protocol deviations will 
be immediately reported to the IRB using iRIS.  
 
The principal investigator will review adverse event and response data on each patient to 
ensure safety and data accuracy. The principal investigator will personally conduct or supervise 
the investigation and provide appropriate delegation of responsibilities to other members of the 
research staff.  
 
7.5.2 Sponsor Monitoring Plan 
As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring 
program. The CCR’s program allows for confirmation of: study data, specifically data that 
could affect the interpretation of primary study endpoints; adherence to the protocol, 
regulations, and SOPs; and human subject’s protection. This is done through independent 
verification of study data with source documentation focusing on: 
x Informed consent process 
x Eligibility confirmation 


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46 
x Drug administration and accountability 
x Adverse events monitoring 
x Response assessment. 
The monitoring program also extends to multi-site research when the CCR is the coordinating 
center. 
 
This trial will be monitored by personnel employed by an NCI contractor. Monitors are 
qualified by training and experience to monitor the progress of clinical trials. Personnel 
monitoring this study will not be affiliated in any way with the trial conduct. 
7.5.3 Safety Monitoring Committee (SMC) 
This protocol will require oversight from the Safety Monitoring Committee (SMC).  Initial 
review will occur as soon as possible after the annual NCI-IRB continuing review date.   
Subsequently, each protocol will be reviewed as close to annually as the quarterly meeting 
schedule permits or more frequently as may be required by the SMC.   For initial and 
subsequent reviews, protocols will not be reviewed if there is no accrual within the review 
period. Written outcome letters will be generated in response to the monitoring activities and 
submitted to the Principal investigator and Clinical Director or Deputy Clinical Director, CCR, 
NCI. 
8 
STATISTICAL CONSIDERATIONS  
The primary objectives of this trial is to determine safety and also to determine whether the 
combination of low-dose aldesleukin, lymphocyte-depleting chemotherapy, and an infusion of 
anti-mesothelin CAR-gene engineered lymphocytes is able to be associated with a modest 
fraction of patients that can experience a clinical response (PR +CR) to therapy.  The duration 
of the clinical response will be followed to assess the efficacy of the regimen.  A secondary 
objective is to have sufficient patients in order to do exploratory evaluations of survival of 
cells.    
 
Initially, the protocol will enroll 3 patients in each cohort unless that patient experiences a dose 
limiting toxicity (DLT).  If dosing is escalated to cohort 10, 6 patients will be accrued to this 
cohort in order to further characterize the safety of the maximum tolerated dose prior to 
starting the phase II portion.  The total number of anti-mesothelin engineered cells transferred 
for each cohort will be: 
o 
Cohort 1 
 106 cells 
 
De-escalation cohorts: 
x 
Cohort -1:  3 X105 cells 
x 
Cohort -2:  105 cells 
o 
Cohort 2 
3 X 106 cells 
o Cohort 3  
 107 cells 
o Cohort 4 
3 X 107 cells 
o Cohort 5  
 108 cells 
o Cohort 6 
3 X 108 cells 
o Cohort 7  
 109 cells 
o Cohort 8 
3 X 109 cells 
o Cohort 9 
1010 cells 


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47 
o Cohort 10 
3 X 1010 cells 
 
In each cohort, if one of the first three patients treated   experiences a DLT, three more patients 
would be treated at that dose to confirm that no greater than 1/6 patients have a DLT prior to 
proceeding to the next higher level.  If a level with 2 or more DLTs in 3-6 patients has been 
identified, three additional patients will be accrued at the next-lowest dose, for a total of 6, in 
order to further characterize the safety of the maximum tolerated dose prior to starting the 
phase II portion.  If a dose limiting toxicity occurs in the first cohort, that cohort will be 
expanded to n=6 patients.  If there are 1 or fewer DLTs in the first cohort, the study will 
proceed to the second cohort.  If two DLTs occur in the first cohort, the dose will be de-
escalated as described above in cohort -1.  If 2 DLTs occur in cohort -1, the dose of cells will 
be de-escalated as described above in cohort -2.  If two DLTs occur in cohort -2, the study will 
be terminated.    
 
Once the MTD has been determined, the study then would proceed to the phase II portion.  
Patients will be enrolled into individual strata depending on their specific histology.  This 
stratification is being used to separate patients who have mesothelioma, and patients with all 
other histologies.  The six patients accrued at the MTD in the phase I portion of this study will 
be included in the strata for the appropriate histology in the phase II portion. 
 
For each of the two strata, the phase II portion of the study will be conducted using a phase II 
optimal design (Simon R, Controlled Clinical Trials 10:1-10, 1989). For the two strata, the 
objective will be to determine if the combination of low dose aldesleukin, lymphocyte 
depleting chemotherapy, and anti-mesothelin CAR-gene engineered lymphocytes is able to be 
associated with a clinical response rate that can rule out 5% (p0=0.05) in favor of a modest 
20% PR + CR rate (p1=0.20).   
 
In patients in each of the two strata, the following design will be used.  For each strata, with 
alpha=0.05 (5% probability of accepting a poor therapy) and beta=0.10 (10% probability of 
rejecting a good therapy,), initially 21 evaluable patients will be enrolled.  If 0 or 1 of the 21 
patients experiences a clinical response, then no further patients will be enrolled.  If 2 or more 
of the first 21 evaluable patients enrolled have a clinical response, then accrual will continue 
until a total of 41 evaluable patients have been enrolled.  As it may take several weeks to 
determine if a patient has experienced a clinical response, a temporary pause of up to 6 months 
in the accrual to the trial may be necessary to ensure that enrollment to the second stage is 
warranted.  If 2 to 4 of the 41 have a clinical response, then this will be considered inadequate 
for further investigation. If 5 or more of 41 patients have a clinical response, then this will 
indicate that this strategy provides a new approach that may be worthy of further consideration.  
Under the null hypothesis (5% response rate), the probability of early termination is 72%. 
 
Further, to help ensure that maldistribution of patients who are either particularly responsive or 
unresponsive in the first stage does not materially interfere with the intended use of the two-
stage design, we will aim to enroll 4-5 patients of each allowed histology among the first 21 
enrolled in the ‘other histology’ arm.  Although this has its own inherent issues due to limited 
sample size, since we believe that these ‘other histologies’ will behave the same clinically, it 
will permit us to evaluate the different response rates in a limited number of subjects and 


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48 
determine if they differ markedly or not.  Since this would merely be an exploratory analysis, 
we will also look at minor response as well to help evaluate for hints of efficacy.  If the 
response rates do seem to potentially differ markedly by histology, despite our hypothesis that 
this will not happen, we may consider amending the protocol when appropriate to try to restrict 
enrollment to those histologies with stronger evidence of responsiveness.  For patients with 
chemotherapy-sensitive tumors (i.e. sarcoma), only responses seen at day 28 and maintained at 
4 months will be considered a positive response for accrual to the second phase of this study. 
 
To complete the phase 1 dose-escalation stage, and both cohorts in the phase 2 portion of the 
study, a total of up to 136 patients may be required (10 possible escalations in the phase 1 
portion with a maximum of 60 patients (but likely 20-30 patients will be accrued depending on 
the toxicity observed), and 2 phase 2 cohorts with a combined total of 76 patients, assuming 41 
patients per cohort, but that 6 of the 82 will be those patients treated at the MTD).  Provided 
that about 1-2 patients per month will be able to be enrolled onto this trial, approximately 6 
years may be needed to accrual the maximum number of required patients.   
 
9 
COLLABORATIVE AGREEMENTS  
We have established a Cooperative Research and Development Agreement (CRADA #02716) 
with Kite Pharma, Inc., and will be sharing data with them. 
 
10 HUMAN SUBJECTS PROTECTIONS 
10.1 RATIONALE FOR PATIENT SELECTION 
The patients to be entered in this protocol have metastatic cancer which is refractory to 
standard therapy, and limited life expectancies. 
 
Subjects from both genders and all racial/ethnic groups are eligible for this study if they meet 
the eligibility criteria.  To date, there is no information that suggests that differences in drug 
metabolism or disease response would be expected in one group compared to another.  Efforts 
will be made to extend accrual to a representative population, but in this preliminary study, a 
balance must be struck between patient safety considerations and limitations on the number of 
individuals exposed to potentially toxic and/or ineffective treatments on the one hand and the 
need to explore gender and ethnic aspects of clinical research on the other hand.  If differences 
in outcome that correlate to gender or to ethnic identity are noted, accrual may be expanded or 
a follow-up study may be written to investigate those differences more fully. 
10.2 PARTICIPATION OF CHILDREN 
The use of the nonmyeloablative regimen in this protocol is a major procedure which entails 
serious discomforts and hazards for the patient, such that fatal complications are possible.  It is 
therefore only appropriate to carry out this experimental procedure in the context of life 
threatening metastatic cancer.  Since the efficacy of this experimental procedure is unknown, it 
does not seem reasonable to expose children to this risk without further evidence of benefit.  
Should results of this study indicate efficacy in treating metastatic cancer, which is not 
responsive to other standard forms of therapy, future research can be conducted in the pediatric 
population to evaluate potential benefit in that patient population.   


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10.3 PARTICIPATION OF SUBJECTS UNABLE TO GIVE CONSENT 
Adults unable to give consent are excluded from enrolling in the protocol.  However re-consent 
may be necessary and there is a possibility, though unlikely, that subjects could become 
decisionally impaired. For this reason and because there is a prospect of direct benefit from 
research participation (section 10.4), all subjects will be offered the opportunity to fill in their 
wishes for research and care, and assign a substitute decision maker on the “NIH Advance 
Directive for Health Care and Medical Research Participation” form so that another person can 
make decisions about their medical care in the event that they become incapacitated or 
cognitively impaired during the course of the study. Note: The PI or AI will contact the NIH 
Ability to Consent Assessment Team for evaluation.  For those subjects that become 
incapacitated and do not have pre-determined substitute decision maker, the procedures 
described in MEC Policy 87-4 for appointing a surrogate decision maker for adult subjects who 
are (a) decisionally impaired, and (b) who do not have a legal guardian or durable power of 
attorney, will be followed. 
10.4 EVALUATION OF BENEFITS AND RISKS 
The experimental treatment has a chance to provide clinical benefit though this is unknown.  
The risks in this treatment are detailed in section 11.  The goal of this study is to improve upon 
the number of patients who may benefit from adoptive cell therapy by using patients own 
transduced T-cells without the need to identify anti-tumor T cells uniquely from each patient as 
was required in several prior protocols.  Aldesleukin is an approved and effective treatment in 
some patients with metastatic melanoma and renal cancer.  In this study, aldesleukin is 
administered after cell infusion to promote growth and survival of the infused cells. Although 
aldesleukin can cause serious adverse events, it’s use in this protocol is essential to keep the 
cells alive. The success of this effort cannot be predicted at this time.  Because all patients in 
this protocol have metastatic cancer and limited life expectancies the potential benefit is 
thought to outweigh the potential risks. 
10.5 CONSENT PROCESS AND DOCUMENTATION 
Patients initially signs a consent when they agree to have PBMC obtained for study and growth 
on 03-C-0277, Cell Harvest and Preparation for Surgery Branch Adoptive Cell Therapy 
Protocols.  If the lymphocytes can be generated for infusion and the patient meets the thorough 
screening for eligibility, the patient, with family members or friends at the request of the 
patient, will be presented with a detailed description of the protocol treatment.  The specific 
requirements, objectives, and potential advantages and disadvantages will be presented.  The 
Informed Consent document is given to the patient, who is requested to review it and to ask 
questions prior to agreeing to participate in the treatment portion of this protocol.  The patient 
is reassured that participation on trial is entirely voluntary and that he/she can withdraw or 
decide against treatment at any time without adverse consequences. The research nurse, 
principal investigator, associate investigator, or clinical associate is responsible for obtaining 
written consent from the patient. 
 


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50 
10.5.1 Informed consent of non-English speaking subjects 
If there is an unexpected enrollment of a research participant for whom there is no translated 
extant IRB approved consent document, the principal investigator and/or those authorized to 
obtain informed consent will use the Short Form Oral Consent Process as described in MAS 
Policy M77-2, OHSRP SOP 12, 45 CFR 46.117 (b) (2), and 21 CFR 50.27 (b) (2).  The 
summary that will be used is the English version of the extant IRB approved consent 
document.  Signed copies of both the English version of the consent and the translated short 
form will be given to the subject or their legally authorized representative and the signed 
original will be filed in the medical record. 
 
Unless the PI is fluent in the prospective subject’s language, an interpreter will be present to 
facilitate the conversation (using either the long translated form or the short form). Preferably 
someone who is independent of the subject (i.e., not a family member) will assist in presenting 
information and obtaining consent. Whenever possible, interpreters will be provided copies of 
the relevant consent documents well before the consent conversation with the subject (24 to 48 
hours if possible).  
 
We request prospective IRB approval of the use of the short form process for non-English 
speaking subjects and will notify the IRB at the time of continuing review of the frequency of 
the use of the Short Form. 
 
11 PHARMACEUTICAL INFORMATION 
Cyclophosphamide, fludarabine, and aldesleukin, the commercial drugs used in this study will 
not alter labelling of the FDA approved drugs. The investigation is not intended to support a 
new indication for use or any other significant changes to labeling or advertising in either 
Cyclophosphamide, Fludarabine, or Aldesleukin. The investigation does not involve a route of 
administration or dosage level in use in a patient population or other factor that significantly 
increases the risks (or decreases the acceptability of the risks) associated with the use of the 
drug products. 
 
11.1 INVESTIGATIONAL REGIMEN 
11.1.1 Anti-Mesothelin CAR Transduced PBL 
The procedure for expanding the human PBL and the Certificate of Analysis (CoA) are similar 
to those approved by the Food and Drug Administration, and used at the NCI in ongoing 
protocols evaluating cell therapy in the Surgery Branch.  The CoA is included in Appendix 4 
for this protocol, and in the IND submission for these cells along with the Standard Operating 
Procedures for the growth of the PBL, and transduction with retroviral supernatant containing 
the chimeric anti-mesothelin CAR genes. Note: Penicillin, Streptomycin, and gentamycin will 
not be used in the manufacture of products for patients with documented allergies to these 
drugs.  
   


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11.1.1.1 Retroviral Vector Containing the Anti-mesothelin CAR Gene  
The retroviral vector supernatant [PG13-coSS1scFv-CD28Z (D6)] encoding a chimeric antigen 
receptor (CAR) directed against mesothelin was prepared and preserved following cGMP in 
the Surgery Branch Vector Production Facility (SBVPF). The retroviral vector utilizes the 
MSGV retroviral vector backbone and consists of 7024 bps including the 5’ LTR from the 
murine stem cell virus (promoter), packaging signal including the splicing donor (SD) and 
splicing acceptor (SA) sites, SS1 anti-mesothelin-based  CAR protein  (coSS1scFv-CD28Z) 
containing a signal peptide (GMCSFR2), codon-optimized SS1 light chain variable region 
(SS1 VL), linker peptide, codon-optimized SS1 heavy chain (SS1 VH), CD8 (hinge and 
transmembrane regions), CD28 (cytoplasmic region) and TCR zeta (cytoplasmic region), 
followed by the murine stem cell virus 3’LTR. The physical titer was determined by retroviral 
transduction of human peripheral blood lymphocytes followed CAR detection by FACS 
according to sponsor certificate. The supernate will be stored at SBVPF upon the completion of 
production at –80° C or shipped on dry ice and stored at Fisher Bioservices, Rockville, MD.  
Both storage facilities are equipped with around-the-clock temperature monitoring. Upon 
request, supernatant will be delivered on dry ice to be used in ex vivo PBL transduction. There 
will be no re-use of the same unit of supernate for different patients. Retroviral titer has been 
shown to be stable after immediate thawing and immediate administration (coating the tissue 
culture wells previously coated with Retronectin). Handling of the vector should follow the 
guidelines of Biosafety Level-2 (BSL-2).   The specific guidelines for Biosafety Level-2 (BSL-
2) can be viewed at http://bmbl.od.nih.gov/sect3bsl2.htm.   
 
11.1.2 Aldesleukin (Interleukin-2, Proleukin, Recombinant Human Interleukin 2) 
How Supplied: Aldesleukin will be provided by the NIH Clinical Pharmacy Department from 
commercial sources. 
 
Formulation/Reconstitution: Aldesleukin, is provided as single-use vials containing 22 million 
IU (1.3 mg) aldesleukin as a sterile, white to off-white lyophilized cake plus 50 mg mannitol 
and 0.18 mg sodium dodecyl sulfate, buffered with approximately 0.17 mg monobasic and 0.89 
mg dibasic sodium phosphate to a pH of 7.5 (range 7.2 to 7.8).  The vial is reconstituted with 
1.2 mL of Sterile Water for Injection, USP, and the resultant concentration is 18 million IU/ml 
or 1.1 mg/mL.  Diluent should be directed against the side of the vial to avoid excess foaming.  
Swirl contents gently until completely dissolved.  Do not shake.  Since vials contain no 
preservative, reconstituted solution should be used with 24 hours. 
 
Storage: Intact vials are stored in the refrigerator (2° - 8°C) protected from light.  Each vial 
bears an expiration date. 
 
Dilution/Stability: Reconstituted aldesleukin should be further diluted with 50 mL of 5% 
Human Serum Albumin (HSA).  The HSA should be added to the diluent prior to the addition 
of aldesleukin.  Dilutions of the reconstituted solution over a 1000-fold range (i.e., 1 mg/mL to 
1 mcg/mL) are acceptable in either glass bottles or polyvinyl chloride bags.  Aldesleukin is 
chemically stable for 48 hours at refrigerated and room temperatures, 2°– 30°C. 
 
Administration:  The final dilution of aldesleukin will be infused over 15 minutes.  Aldesleukin  


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52 
will be administered as an inpatient.   
 
Toxicities:  Expected toxicities of aldesleukin are listed in the product label and in Appendix 1 
and 2.  Grade 3 toxicities common to aldesleukin include diarrhea, nausea, vomiting, 
hypotension, skin changes, anorexia, mucositis, dysphagia, or constitutional symptoms and 
laboratory changes as detailed in Appendix 1.  Additional grade 3 and 4 toxicities seen with 
aldesleukin are detailed in Appendix 2.   
 
11.1.3 Fludarabine 
Description: (Please refer to package insert for complete product Information) Fludarabine 
phosphate is a synthetic purine nucleoside that differs from physiologic nucleosides in that the 
sugar moiety is arabinose instead of ribose or deoxyribose.  Fludarabine is a purine antagonist 
antimetabolite.  
 
How Supplied:  It will be purchased by the NIH Clinical Pharmacy Department from 
commercial sources. Fludarabine is supplied in a 50 mg vial as a fludarabine phosphate powder 
in the form of a white, lyophilized solid cake.   
 
Stability:  Following reconstitution with 2 mL of sterile water for injection to a concentration 
of 25 mg/ml, the solution has a pH of 7.7.  The fludarabine powder is stable for at least 18 
months at 2-8°C; when reconstituted, fludarabine is stable for at least 16 days at room 
temperature.  Because no preservative is present, reconstituted fludarabine will typically be 
administered within 8 hours.  Specialized references should be consulted for specific 
compatibility information.  Fludarabine is dephosphorylated in serum, transported 
intracellularly and converted to the nucleotide fludarabine triphosphate; this 2-fluoro-ara-ATP 
molecule is thought to be required for the drug’s cytotoxic effects.  Fludarabine inhibits DNA 
polymerase, ribnucleotide reductase, DNA primase, and may interfere with chain elongation, 
and RNA and protein synthesis. 
 
Storage:  Intact vials should be stored refrigerated (2-8°C).  
Administration:  Fludarabine is administered as an IV infusion in 100 ml 0.9% sodium 
chloride, USP over 15 to 30 minutes.  The doses will be based on body surface area (BSA).  If 
patient is obese (BMI > 35) drug dosage will be calculated using practical weight as described 
in Table 6.  
 
Toxicities:  At doses of 25 mg/m2/day for 5 days, the primary side effect is myelosuppression; 
however, thrombocytopenia is responsible for most cases of severe and life-threatening 
hematologic toxicity.  Serious opportunistic infections have occurred in CLL patients treated 
with fludarabine.  Hemolytic anemia has been reported after one or more courses of 
fludarabine with or without a prior history of a positive Coomb’s test; fatal hemolytic anemia 
has been reported.  In addition, bone marrow fibrosis has been observed after fludarabine 
therapy.  Other common adverse effects include malaise, fever, chills, fatigue, anorexia, nausea 
and vomiting, and weakness.  Irreversible and potentially fatal central nervous system toxicity 
in the form of progressive encephalopathy, blindness, and coma is only rarely observed at the 
currently administered doses of fludarabine.  More common neurologic side effects at the 


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53 
current doses of fludarabine include weakness, pain, malaise, fatigue, paresthesia, visual or 
hearing disturbances, and sleep disorders.  Adverse respiratory effects of fludarabine include 
cough, dyspnea, allergic or idiopathic interstitial pneumonitis.  Tumor lysis syndrome has been 
rarely observed in fludarabine treatment of CLL.  Treatment on previous adoptive cell therapy 
protocols in the Surgery Branch have caused persistently low (below 200) CD4 counts, and one 
patient developed polyneuropathy manifested by vision blindness, and motor and sensory 
defects. 
 
11.1.4 Cyclophosphamide  
(Refer to FDA-approved package insert for complete product information) 
 
Description:  Cyclophosphamide is a nitrogen mustard-derivative alkylating agent.  Following 
conversion to active metabolites in the liver, cyclophosphamide functions as an alkylating 
agent; the drug also possesses potent immunosuppressive activity.  The serum half-life after IV 
administration ranges from 3-12 hours; the drug and/or its metabolites can be detected in the 
serum for up to 72 hours after administration.   
 
How Supplied:  Cyclophosphamide will be obtained from commercially available sources by 
the Clinical Center Pharmacy Department.     
 
Stability:  Following reconstitution as directed with sterile water for injection, 
cyclophosphamide is stable for 24 hours at room temperature or 6 days when kept at 2-8°C.   
 
Administration:  It will be diluted in 250 ml D5W and infused over one hour.  The dose will be 
based on the patient’s body weight.  If patient is obese (BMI > 35) drug dosage will be 
calculated using practical weight as described in Table 6. 
 
Toxicities:  Hematologic toxicity occurring with cyclophosphamide usually includes 
leukopenia and thrombocytopenia.  Anorexia, nausea and vomiting, rash and alopecia occur, 
especially after high-dose cyclophosphamide; diarrhea, hemorrhagic colitis, infertility, and 
mucosal and oral ulceration have been reported.  Sterile hemorrhagic cystitis occurs in about 
20% of patients; severity can range from microscopic hematuria to extensive cystitis with 
bladder fibrosis.  Although the incidence of hemorrhagic cystitis associated with 
cyclophosphamide appears to be lower than that associated with ifosfamide, mesna (sodium 2-
mercaptoethanesulfonate) has been used prophylactically as a uroprotective agent in patients 
receiving cyclophosphamide.  Prophylactic mesna is not effective in preventing hemorrhagic 
cystitis in all patients.  Patients who receive high dose cyclophosphamide may develop 
interstitial pulmonary fibrosis, which can be fatal.  Hyperuricemia due to rapid cellular 
destruction may occur, particularly in patients with hematologic malignancy.  Hyperuricemia 
may be minimized by adequate hydration, alkalinization of the urine, and/or administration of 
allopurinol.  If allopurinol is administered, patients should be watched closely for 
cyclophosphamide toxicity (due to allopurinol induction of hepatic microsomal enzymes).  At 
high doses, cyclophosphamide can result in a syndrome of inappropriate antidiuretic hormone 
secretion; hyponatremia with progressive weight gain without edema occurs.  At high doses, 
cyclophosphamide can result in cardiotoxicity.  Deaths have occurred from diffuse 


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54 
hemorrhagic myocardial necrosis and from a syndrome of acute myopericarditis; in such cases, 
congestive heart failure may occur within a few days of the first dose.  Other consequences of 
cyclophosphamide cardiotoxicity include arrhythmias, potentially irreversible cardiomyopathy, 
and pericarditis.  Other reported adverse effects of cyclophosphamide include headache, 
dizziness, and myxedema; faintness, facial flushing, and diaphoresis have occurred following 
IV administration.  Mesna (sodium 2-mercaptoethanesulphonate; given by IV injection) is a 
synthetic sulfhydryl compound that can chemically interact with urotoxic metabolites of 
cyclophosphamide (acrolein and 4-hydroxycyclophosphamide) to decrease the incidence and 
severity of hemorrhagic cystitis.  
 
11.2 SUPPORTIVE MEDICATIONS 
11.2.1 Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex)  
(Please refer to the FDA-approved package insert for complete product information) 
 
Description:  Mesna will be obtained commercially by the Clinical Center Pharmacy 
Department and is supplied as a 100 mg/ml solution.   
Storage:  Intact ampoules are stored at room temperature.   
 
Stability:  Diluted solutions (1 to 20 mg/mL) are physically and chemically stable for at least 
24 hours under refrigeration.  Mesna is chemically stable at room temperature for 48-72 hours 
in D5W, 48-72 hour in D5W/0.45% NaCl, or 24 hours in 0.9% NaCl. 
 
Administration:  Dilute to concentrations less than or equal to 20 mg mesna/ml fluid in D5W 
or 0.9% NaCl and to be administered intravenously as a continuous infusion.  If patient is 
obese (BMI > 35) drug dosage will be calculated using practical weight as described in Table 
6.  Toxicities include nausea, vomiting and diarrhea.  
 
11.2.2 Filgrastim (Granulocyte Colony-Stimulating Factor, G-CSF, Filgrastim, Neupogen) 
Filgrastim will be obtained commercially by the Clinical Center Pharmacy Department and is 
supplied in 300 ug/ml and 480 ug/1.6 ml vials.  G-CSF should be refrigerated and not allowed 
to freeze.  The product bears the expiration date.  The product should not be shaken.  It is 
generally stable for at least 10 months when refrigerated.  The appropriate dose is drawn up 
into a syringe.  G-CSF will be given as a daily subcutaneous injection.  The side effects of G-
CSF are skin rash, myalgia and bone pain, an increase of preexisting inflammatory conditions, 
enlarged spleen with occasional associated low platelet counts, alopecia (with prolonged use) 
elevated blood chemistry levels. 
 
11.2.3 Trimethoprim and Sulfamethoxazole Double Strength (TMP / SMX DS) 
TMP/SMX DS will be obtained by the Clinical Center Pharmacy Department from commercial 
sources.  It will be used for the prevention of PCP pneumonia.  The oral dose is 1 tablet PO 
daily three times a week (MUST be on non-consecutive days) beginning day 0 or within one 
week of anticipated lymphopenia and continuing for at least 6 months and until the CD4 count 
is greater than 200 on 2 consecutive lab studies.  Like other sulfa drugs, TMP/SMX DS can 


Abbreviated Title: Anti-mesothelin CAR PBL 
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55 
cause allergies, fever, photosensitivity, nausea, and vomiting. Allergies typically develop as a 
widespread itchy red rash with fever eight to fourteen days after beginning the standard dose. 
Neutropenia, a reduction in the number of neutrophils, can also occur. Should allergies 
develop, the following medications may be used in place of TMP/SMX DS. 
 
11.2.3.1 Dapsone: 
Dapsone will be obtained by the Clinical Center Pharmacy Department from commercial 
sources. It will be used for the prevention of Pneumocystis pneumonia. The dose is 100mg by 
mouth daily, starting on day 0 (± 7 days) and continuing at least 6 months and until the CD4+ 
count is > 200 on two consecutive lab studies. It is supplied as 25mg and 100mg tablets. 
Dapsone contains a sulfa group, although the cross reactivity in patients with sulfa allergies is 
quite low. Dapsone may be considered in patients with mild to moderate sulfa allergies. 
Dapsone should be avoided in patients with severe (i.e., a history of anaphylaxis or other 
equally serious reaction) reactions to sulfa drugs. Additionally, dapsone has been reported to 
cause hemolytic anemia is patients with G6PD deficiency. It is recommended that patients be 
tested for G6PD deficiency prior to the initiation of dapsone therapy. Dapsone is generally well 
tolerated, but may cause a number of hematologic adverse reactions, including increased 
reticulocyte counts, hemolysis, decreased hemoglobin, methemoglobinemia, agranulocytosis, 
anemia, and leukopenia. Other rare but serious adverse reactions include bullous exfoliative 
dermatitis, Stevens-Johnson syndrome, toxic epidermal necrolysis, pancreatitis, interstitial 
pneumonitis, and pulmonary eosinophilia. For more detailed information about adverse 
reactions, consult the package insert. 
 
11.2.3.2 Atovaquone: 
Atovaquone will be obtained by the Clinical Center Pharmacy Department from commercial 
sources. It will be used for the prevention of Pneumocystis pneumonia in patients who cannot 
tolerate or are allergic to sulfamethoxazole/trimethoprim, dapsone, or pentamidine. 
Atovaquone may be given as a single daily dose of 1500mg orally or the dose may be split into 
750mg given orally twice daily. Atovaquone will be started on day 0 (± 7 days), and will 
continue for at least 6 months and until the CD4+ count is > 200 on two consecutive lab 
studies. Atovaquone is supplied as an oral suspension containing 150mg/mL. Common adverse 
reactions to atovaquone include: headache, rash, diarrhea, nausea, vomiting, abdominal pain, 
cough, and fever. Rare but serious adverse reactions include acute renal failure, hepatitis and 
hepatic failure, angioedema, pancreatitis, and Stevens-Johnson syndrome. For more detailed 
information about adverse reactions, consult the package insert. 
11.2.3.3 Aerosolized Pentamidine:   
Patients with sulfa allergies will receive aerosolized Pentamidine 300 mg per nebulizer with 
one week prior to admission and continued monthly until the CD4 count is above 200 on two 
consecutive follow up lab studies and for at least 6 months post chemotherapy.  Pentamidine 
Isethionate will be obtained by the Clinical Center Pharmacy Department from commercial 
sources.  It will be used to prevent the occurrence of PCP infections.  It is supplied in 300 mg 
vials of lyophilized powder and will be administered via nebulizer.  Toxicities reported with 
the use of Pentamidine include metallic taste, coughing, bronchospasm in heavy smokers and 


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56 
asthmatics; increased incidence of spontaneous pneumothorax in patients with previous PCP 
infection or pneumatoceles, or hypoglycemia.  
 
11.2.4 Herpes and Varicella Zoster Virus Prophylaxis 
11.2.4.1 Valacyclovir (Valtrex) 
Valacyclovir will be obtained by the Clinical Center Pharmacy Department from commercial 
sources.  It will be used orally to prevent the occurrence of herpes virus infections in patients 
with positive HSV serology.   It is supplied in 500 mg tablets.  Valcyclovir will be started at a 
dose of 500 mg orally daily if the patient is able to tolerate oral intake.  See package insert for 
dosing adjustments in patients with renal impairment.  Common side effects include headache, 
upset stomach, nausea, vomiting, diarrhea or constipation.  Rare serious side effects include 
hemolytic uremic syndrome and thrombotic thrombocytopenic purpura.  
   
11.2.4.2 Acyclovir 
Acyclovir will be obtained by the Clinical Center Pharmacy Department from commercial 
sources.  It will be used to prevent the occurrence of herpes virus infections in patients who 
cannot take oral medications.  It is supplied as powder for injection in 500 mg/vials.  
Reconstitute in 10 mL of sterile water for injection to a concentration of 50 mg/mL.  
Reconstituted solutions should be used within 12 hours.  IV solutions should be diluted to a 
concentration of 7mg/mL or less and infused over 1 hour to avoid renal damage.  Reversible 
renal insufficiency has been reported with IV but not oral acyclovir.  Neurologic toxicity 
including delirium, tremors, coma, acute psychiatric disturbances, and abnormal EEGs have 
been reported with higher doses of acyclovir.  Should this occur, a dosage adjustment will be 
made or the drug will be discontinued. Stomach upset, headache or nausea, rash or hives; 
peripheral edema; pain, elevated liver function tests; and leukopenia, diarrhea, 
lymphadenopathy, myalgias, visual abnormalities and elevated creatinine have been reported. 
Hair loss from prolonged use has been reported. Acyclovir will not be used concomitantly with 
other nucleoside analogs which interfere with DNA synthesis, e.g. ganciclovir. In renal disease, 
the dose is adjusted as per product labeling. 
  
11.2.5 Fluconazole 
Fluconazole will be obtained by the Clinical Center Pharmacy Department from commercial 
sources.  It will be used to prophylax against fungal infections.  It is available in 200 mg 
tablets.  It can cause headache, nausea, vomiting, diarrhea or abdominal pain, and liver damage 
which may be irreversible.  It can cause rashes and itching, which in rare cases has caused 
Stevens Johnson Syndrome.  It has several significant drug interactions.  The package insert 
should be consulted prior to prescribing.  For IV administration in patients who cannot tolerate 
the oral preparation, Fluconazole comes in 2 MG/ML solution for injection, and prepared 
according to Clinical Center Pharmacy standard procedures.  It should be administered at a 
maximum IV rate of 200 mg/hr. 
 


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57 
11.2.6 Ondansetron hydrochloride 
Ondansetron hydrochloride will be obtained by the Clinical Center Pharmacy Department from 
commercial sources.  It will be used to control nausea and vomiting during the chemotherapy 
preparative regimen.  It can cause headache, dizziness, myalgias, drowsiness, malaise, and 
weakness.  Less common side effects include chest pain, hypotension, pruritus, constipation 
and urinary retention.  Consult the package insert for specific dosing instructions. 
 
11.2.7 Furosemide 
Furosemide will be obtained by the Clinical Center Pharmacy Department from commercial 
sources.  It will be used to enhance urine output during the chemotherapy preparative regimen 
with cyclophosphamide.  Adverse effects include dizziness, vertigo, paresthesias, weakness, 
orthostatic hypotension, photosensitivity, rash and pruritus.  Consult the package insert for a 
complete list of all side effects. 
 


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58 
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Hassan R, Laszik ZG, Lerner M, Raffeld M, Postier R, Brackett D. Mesothelin is 
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tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma. 
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Brentjens RJ, Rivière I, Park JH, et al. Safety and persistence of adoptively transferred 
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13 TABLES, FIGURES, AND APPENDICES: 


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61 
 
Table 1   
Frequency and duration of objective responses in melanoma patients treated with standard Tumor 
Infiltrating Lymphocytes (TIL) in the Surgery Branch, NCI  (9/1/11) 
 
             
Treatment* 
 
No TBI#  
 
Total 
 
43 
           
PR  
 
16 
  
Į (84, 36, 29, 28, 14,  
12, 11, 7, 7, 7, 7, 4,  
4, 2, 2, 2) 
 
 
 
CR 
 
5 
 
(94+,92+,  
91+ 82+,  
77+) 
  
  
 
OR (%) 
 
21 
(49%)  
 
200cG TBI 
 
 
 
  
 
 
25 
 
 
 
 
  
8 
  
(14, 9, 6, 6, 5, 4, 3, 3)  
 
 
5 
 
(81+, 77+, 73+, 
70+, 67+) 
 
  
 
 
13  
(52.0%)  
1200cG TBI 
 
 
 
  
25 
 
 
 
  
 
8 
  
(21, 13, 7, 6, 6, 5, 
 3, 2)  
 
 
10 
 
(61+, 58+, 57+, 
52+, 51+, 51+, 
51+, 50+, 19) 
 
 
18 
(72% ) 
 
*All patients received cyclophosphamide (60mg/kg x 2d) and fludarabine (25mg/m2 x 5d). 
 
#Patients who received Rapidly Expanded TIL plus the full preparative regimen as a first TIL treatment 
 
Durations of response in months are in parenthesis under the number of responders.  
 
 “+” indicates on ongoing response. 
 
52 responding patients: 42 had prior IL-2, 22 had prior IL-2 + chemotherapy 
20 complete responses: 19 ongoing at 50 to 94 months 


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62 
Table 2 
Time in Hospital and Non-hematological Grade 3 and 4 Toxicities Related 
to Lymphodepleting Chemotherapy and Cell Transfer 
Attribute measured 
Duration, Number or 
Type 
Number of Patients 
(%) 
 
 
 
Days in Hospital1 
6-10 
6 (17%) 
 
11-15 
18 (51%) 
 
16-20 
4 (11%) 
 
21-25 
7 (20%) 
 
 
 
pRBC Transfusions 
0 
2 (6%) 
 
1-5 
18 (51%) 
 
6-10 
13 (37%) 
 
11-15 
2(6%) 
 
 
 
Platelet 
Transfusions 
0 
6 (17%) 
 
1-5 
21 (60%) 
 
6-10 
5 (14%) 
 
11-15 
2 (6%) 
 
16-20 
1 (3%) 
 
 
 
Autoimmunity 
Uveitis 
5 (14%) 
 
Vitiligo 
13 (37%) 
 
 
 
Opportunistic 
Infections 
Herpes zoster 
3 (9%) 
 
Pneumocystis pneumonia 
2 (6%) 
 
EBV-B cell lymphoma 
1 (3%) 
 
RSV pneumonia 
1 (3%) 
 
 
 
Other 
Febrile neutropenia 
13 (37%) 
 
Intubated for dyspnea  
3 (9%) 
 
Cortical blindness 
1 (3%) 
1Measured from the day of cell administration to discharge 
 
 
 
 
 


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63 
Table 3 
 
Mesothelin expression in human cancers by immunohistochemistry 
Cancer histology 
Mesothelin 
Expression (%) 
Comments 
References 
Mesothelioma 
100 
Present in all pleural 
mesotheliomas; absent 
in sarcomatous type 
(Chang et al., 1992a: 
Or Ordonez, 2003) 
Pancreatic 
100 
Absent in normal 
pancreas and chronic 
pancreatitis 
(Argani et al., 2001; 
Hassan et al., 2005b; 
Ordóñez, 2003) 
Ovarian 
67-100 
Present in non-
mucinous ovarian 
cancers; absent from 
mucinous type 
(Chang et al., 1992b; 
Frierson et al., 2003; 
Hassan et al., 2005a; 
Ordóñez, 2003) 
Lung 
adenocarcinoma 
41-53 
Present in lung 
adenocarcinomas and 
some expression in 
squamous and large 
cell lung cancer; absent 
in small cell lung 
cancer 
(Ho et al., 2007; 
Miettinen and 
Sarlomo-Rikala, 2003; 
Ordóñez, 2003) 
Esophageal 
25, adenocarcinoma 
75, squamous carcinoma  
(Ordóñez, 2003) 
Colorectal 
adenocarcinoma 
28 
 
(Ordóñez, 2003) 
Cervical 
25 
Uterine, squamous 
carcinoma 
(Ordóñez, 2003) 
 
 
 


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64 
Table 4 
 
Table 4.  Determination of coSS1scFv-CD28Z
retroviral vector titers.
Harvest 
Titer (TU/mL, x106) 
 
 
1 
1.2 + 0.2 
 
 
2 
1.8 + 0.1 
 
 
3 
2.1 + 0.2 
 
 
4 
2.2 + 0.2 
 
 
5 
1.8 + 0.2 
 
 
6 
1.8 + 0.1 
 
 
 
 
 


A b brevi ate d Title:  A nti- mes ot heli n C A R P B L 
Versi o n D ate: A pril 1 2, 2 0 1 8 
1 2- C- 0 1 1 1
 6 5
T a ble 5 
T a bl e 5.  S p e cifi c c yt o ki n e r el e a s e b y C A R-tr a n s d u c e d P B L.
T u m or C ell Li n e a
M e s o-
M e s o +
P ati e nt
S a m pl e
N o n e
A 4 3 1
A 4 3 1- H 9
H A Y
O V C A R 3
Pt. K
H ar v e st 1
6 3 4 
1 7 4
1 6 2 6 9
1 3 4 7 6
2 1 0 2 0
H ar v e st 2
5 8 3 
1 6 2
3 0 2 2 5
2 6 8 0 8
3 7 3 9 8
H ar v e st 3
7 0 5 
1 9 4
3 2 7 7 3
3 0 1 1 1
4 0 2 4 9
H ar v e st 4
6 4 2 
1 8 8
3 2 5 8 0
2 5 3 4 5
3 2 1 3 0
H ar v e st 5
6 5 7 
1 9 3
2 3 6 2 0
1 9 9 6 5
2 7 0 8 8
H ar v e st 6 
4 5 1 
1 3 2
2 0 3 0 8
1 6 2 3 1
2 1 8 0 0
Pt. L
H ar v e st 1
1 1 2 
1 1 8
3 2 7 7 5
3 9 5 8
2 7 2 1
H ar v e st 2
1 1 3 
1 0 8
2 8 4 8 1
8 2 5 6
4 4 8 6
H ar v e st 3
9 8 
1 0 4
3 6 6 6 1
9 2 0 0
4 6 5 6
H ar v e st 4
1 0 1 
1 0 0
3 6 8 9 3
9 9 0 3
4 7 0 0
H ar v e st 5
8 7 
1 3 5
3 6 6 3 5
5 1 0 0
2 7 1 8
H ar v e st 6 
1 0 2 
1 2 8
2 9 2 8 8
4 3 1 4
2 5 9 7
Pt. M
H ar v e st 1
1 0 2 
8 7
5 6 8 6
2 0 6 8 0
2 6 2 1 0
H ar v e st 2
9 4 
9 4
1 0 0 4 9
2 3 2 6 0
1 6 3 3 7
H ar v e st 3
9 1 
8 0
1 5 0 3 5
2 6 6 7 2
2 0 7 4 0
H ar v e st 4
8 6 
8 4
1 4 1 0 3
2 4 5 3 6
2 2 7 8 4
H ar v e st 5
1 1 6 
7 5
9 3 5 7
2 5 1 6 8
2 1 6 6 9
H ar v e st 6 
1 0 8 
8 4
9 8 3 1
1 8 1 3 4
1 6 4 1 0
a C yt o ki n e r el e a s e w a s m e a s ur e d f oll o wi n g t h e i n c u b ati o n of 1 0
5 tr a n s d u c e d T 
l y m p h o c yt e s wit h 1 05 t u m or t ar g et c ell s i n 2 0 0 m L f or 1 8 h at 3 7o C.  N e g ati v e t u m or 
t ar g et, A 4 3 1 a n d m e s ot h eli n- p o siti v e t u m or t ar g et s i n cl u d e A 4 3 1- H 9 (tr a n sf e ct a nt), H A Y 
(m e s ot h eli o m a c a n c er c ell li n e) a n d O V C A R 3 ( o v ari a n c a n c er c ell li n e).  All c ell li n e s 
w er e c ult ur e d i n R 1 0 m e di u m c o n si sti n g of R P MI 1 6 4 0 c o nt ai ni n g 1 0 % f et al b o vi n e 
s er u m.  Dil uti o n s of c ult ur e s u p er n at a nt w er e t h e n t e st e d f or I F N g b y e n z y m e-li n k e d 
i m m u n o s or b e nt a s s a y ( E LI S A).  D at a r e pr e s e nt e d fr o m 3 s e p ar at e p ati e nt’ s P B L.
 
 
 


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66 
Table 6 
 
Modification of Dose Calculations* in patients whose BMI is greater than 35 
 
Unless otherwise specified in this protocol, actual body weight is used for dose 
calculations of treatment agents.   In patients who are determined to be obese (BMI > 
35), the practical weight (see 3 below) will be used. 
 
 
1. BMI Determination: 
 
BMI = weight (kg) / [height (m)]2 
 
 
2. Calculation of ideal body weight 
 
Male = 50 kg + 2.3 (number of inches over 60 inches) 
Example:  ideal body weight of 5’10’’ male 
50 + 2.3 (10) = 73 kg 
 
Female = 45.5 kg + 2.3 (number of inches over 60 inches) 
Example:  ideal body weight of 5’3’’ female 
45.5 + 2.3 (3) = 57 kg 
 
3. Calculation of “practical weight” 
 
Calculate the average of the actual and the 
ideal body weights.  This is the practical 
weight to be used in calculating the doses of 
chemotherapy and associated agents 
designated in the protocol. 
 
 
*Practical weight will NOT be used in the calculation of dose for aldesleukin. 
 


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67 
Figure 1 
CD28
CD3 zeta
Mesothelin-scFv
LTR
LTR
\
sd
sa
CD8
Anti-mesothelin scFv Clone
(n = 3)
IFNJ (pg/mL)
D6
UT
0
50000
100000
150000
A431
A431-H9
OVACAR3
Panc10.05
C.
ombinant retroviral vector encoding a
in. A) Schematic representation of 
eric antigen receptors (CAR) used in this 
G4S)3 linker to the human CD8D, hinge and 
ellular T cell signaling domains.  B) Cell 
were transduced with the SS1 CAR-encoding 
PBL were stained for CAR+ cells using a 
ptavidin-PE.  Transduction efficiency in 
CAR+ cells.  Data are from three separate 
y secreted IFNJ in response to mesothelin-
1 x 105) and mesothelin-positive target cell 
) at 37ºC and 5% CO2. Supernatants were 
6$WRGHWHFW,)1Ȗ'DWDUHSUHVHQWDWLYHRI
s controls in all experiments (data not shown). 
 
 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
12-C-0111 
68
Figure 2 
Transfectant
Pancreatic
Mesothelioma
Ovarian
Melanoma
A431
A431/H9
Panc 3.014
HTB-80
Panc 6.03
Panc10.05
M30
HAY
You
OVCAR3
OVCAR5
OVCAR8
526mel
888mel
938mel
624mel
anti-Mesothelin (MN)
re 2.  Expression of mesothelin on human tumor cell lines.  Indicated cell lines were incubated
recombinant biotinylated MN monoclonal antibody (a mouse anti-human IgG specific for human 
othelin) or isotype control antibody (mouse IgG) followed by streptavidin-PE.  Samples were analyzed
ACS.  Black histograms indicated staining with the MN antibody and grey histograms , staining wth
sotype control.


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
12-C-0111 
69
Figure 3
A.
B.
C.
Transduced PBL
(n = 3)
Fold Expansion
D6
CTRL
0
100
200
300
Tumor Target
IFNJ (pg/mL)
A431
A431-H9
Panc10.05
OVCAR5
A431
A431-H9
Panc10.05
OVCAR5
A431
A431-H9
Panc10.05
OVCAR5
0
10000
20000
30000
40000
50000
D6
CTRL
Pt. 1
Pt. 2
Pt. 3
69%
55%
70%
Pt. 1
Pt. 3
Pt. 2
Anti-F(ab’)2
ace expression and functional analysis of CAR-transduced PBL following a
A) Expansion of CAR-transduced PBL. CAR-transduced PBL (see Figure 1B) were 
which consists of a second stimulation with OKT3 antibody and cultured at a 
/mL in the presence of IL2 and irradiated feeders.  On day 5 post-REP, fold 
ulated.  B) Cell surface expression of SS1 CAR-transduced PBL following a REP.
, PBL were stained for CAR+ cells using a biotinylated mouse anti-F(ab’)2 antibody 
vidin-PE.  Transduction efficiency in transduced PBL was represented as the 
+ cells.  Data are from the same three separate patient’s PBL as in Figure 1B.  C) 
ed PBL specifically secreted IFNJ in response to mesothelin-positive tumor cell 
ced effector cells (1 x 105) and mesothelin-positive target cell lines (1 x 105) were 
co-FXOWXUHȝODW&DQG&22. Supernatants were harvested for enzyme-
HQWDVVD\(/,6$WRGHWHFW,)1Ȗ'DWDUHSUHVHQWDWLYHRISDWLHQW¶V3%/&'
BL were used as controls in all experiments (data not shown). 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
12-C-0111 
70 
Appendix 1 
 
$'9(56((9(1762&&855,1*,12)3$7,(17675($7(':,7+$/'(6/(8.,1
(n=525)1 
 
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12-C-0111                                                                                                                        71 
Appendix 2 
Expected IL-2 Toxicities and their Management 
 
Expected 
toxicity 
Expected 
grade 
Supportive 
Measures 
Stop Cycle* 
 
Stop Treatment ** 
Chills 
3 
IV Meperidine 25-50 
mg, IV q1h, prn, 
No 
No 
Fever 
3 
Acetaminophen 650 
mg, po, q4h; 
Indomethicin 50-75 
mg, po, q8h 
No 
No 
Pruritis 
3 
Hydroxyzine HCL 
10-20 mg po q6h, 
prn; 
Diphenhydramine 
HCL25-50 mg, po, 
q4h, prn 
No 
No 
Nausea/ Vomiting/ 
Anorexia 
3 
Ondansetron 10 mg, 
IV, q8h, prn; 
Granisetron 0.01 
mg/kg IV daily prn; 
Droperidol 1 mg, IV 
q4-6h, prn; 
Prochlorperazine 25 
mg pr, prn or 10 mg 
IV q6h prn 
No 
No 
Diarrhea 
3 
Loperamide 2mg, po, 
q3h, prn; 
Diphenoxylate HCl 
2.5 mg and atropine 
sulfate 25 mcg, po, 
q3h, prn; codeine 
sulfate 30-60 mg, po, 
q4h, prn 
If uncontrolled after 
24 hours despite all 
supportive measures 
No 
Malaise 
3 or 4 
Bedrest 
If other toxicities 
occur 
simultaneously 
No 
Hyperbilirubinemia 
3 or 4 
Observation 
If other toxicities 
occur 
simultaneously 
No 
Anemia  
3 or 4 
Transfusion with 
PRBCs 
If uncontrolled 
despite all 
supportive measures 
No 
Thrombocytopenia 
3 or 4 
Transfusion with 
platelets 
If uncontrolled 
despite all 
supportive measures 
No 
Edema/Weight gain 
3  
Diuretics prn 
No 
No 
Hypotension 
3 
Fluid resuscitation 
Vasopressor support 
If uncontrolled 
despite all 
supportive measures 
No 


Abbreviated Title: Anti-mesothelin CAR PBL 
Version Date: April 12, 2018 
12-C-0111 
72 
Dyspnea 
3 or 4 
Oxygen or ventilatory 
support 
If requires 
ventilatory support 
No 
Oliguria 
3 or 4 
Fluid boluses or 
dopamine at renal 
doses 
If uncontrolled 
despite all 
supportive measures 
No 
Increased creatinine 
3 or 4 
Observation 
Yes (grade 4) 
No 
Renal failure 
3 or 4 
Dialysis 
Yes 
Yes 
Pleural effusion  
3 
Thoracentesis 
If uncontrolled 
despite all 
supportive measures 
No 
Bowel perforation 
3 
Surgical intervention 
Yes 
Yes 
Confusion 
3 
Observation 
Yes 
No 
Somnolence 
3 or 4 
Intubation for airway 
protection 
Yes 
Yes 
Arrhythmia 
3 
Correction of fluid 
and electrolyte 
imbalances; chemical 
conversion or 
electrical conversion 
therapy 
If uncontrolled 
despite all 
supportive measures 
No 
Elevated Troponin 
levels 
3 or 4 
Observation 
Yes 
If changes in LV function 
have not improved to 
baseline by next dose 
Myocardial 
Infarction 
4 
Supportive care 
Yes 
Yes 
Elevated 
transaminases 
3 or 4 
Observation 
For grade 4 without 
liver metastases 
If changes have not 
improved to baseline by 
next dose 
Hyperbilirubinemia 
3 or 4 
Observation 
For grade 4 without 
liver metastases 
If changes have not 
improved to baseline by 
next dose 
Electrolyte 
imbalances 
3 or 4 
Electrolyte 
replacement 
If uncontrolled 
despite all 
supportive measures 
No 
Neutropenia 
4 
Observation 
No 
No 
*Unless the toxicity is not reversed within 12 hours 
** Unless the toxicity is not reversed to grade 2 or less by next treatment. 
 


 
12-C-0111                                                                                                                        73 
Appendix 3 
Interleukin-2 toxicities observed in patients treated at the NIH Clinical Center 
 


 
12-C-0111 
 
74 
 
Appendix 4  
 
Certificate of Analysis: 
Infused T cells transduced with anti-mesothelin CAR  
 
Date of preparation of final product: 
Patient: 
 
 
Tests performed on final product: 
 
Test 
 
Method 
Limits 
Result 
Tests 
Performed 
by 
Initials
/Date 
Cell viability1 
trypan blue exclusion 
>70% 
 
 
 
Total viable cell 
number1 
visual microscopic count Between 106 and  
5 x 1010 cells 
 
 
 
Identity 
FACs analysis on final 
expanded cells7  
> 80 % CD3+ after 
transduction  
 
 
 
Tumor reactivity2 
J-IFN release vs. cell 
line expressing 
mesothelin 
>200 pg/ml and > 2 
times background 
 
 
 
CAR expression2 
FACS analysis of the 
transduced cells 
PBL, >30% 
 
 
 
Microbiological 
studies  
gram stain1,3,  
no micro-organisms 
seen 
 
 
 
 
aerobic culture3,4 
no growth 
 
 
 
 
fungal culture3,4 
no growth 
 
 
 
 
anaerobic culture3,4 
no growth 
 
 
 
 
mycoplasma test5 
negative 
 
 
 
Endotoxin  
limulus assay1 
(8NJ 
 
 
 
RCR 
S+L- Assay4 
RCR-PCR6 
 
negative 
 
 
 
1 Performed on sample of the final product immediately prior to infusion.  Results are available at the time of infusion. 
2 Performed 2-10 post transduction. Results are available at the time of infusion.  
3 Performed 2-4 days prior to infusion.  Results are available at the time of infusion but may not be definitive. 
4 Sample collected from the final product prior to infusion.  Results will not be available before cells are infused into the patient. 
5 Performed 2-10 days prior to infusion.  Results are available at the time of infusion. 
6 Performed on sample approximately 1-4 days prior to infusion.  Results are available at the time of infusion. 
7 Performed 2-10 days prior to infusion.  Results are available at the time of infusion. REP cells well be tested if intended for 
infusion. 
 
 
Prepared by: 
_________________________________________ 
Date:  ____________________ 
 
 
 
QC sign-off: 
__________________________________________ 
Date:  ____________________ 
Qualified Laboratory or Clinical Supervisor 
 
 
 

ICF_001.pdf: 
MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
INSTITUTE: 
National Cancer Institute 
STUDY NUMBER: 
12-C-0111 
PRINCIPAL INVESTIGATOR: 
Steven A. Rosenberg, MD, PhD 
STUDY TITLE:  
 
A Phase I/II Study of Metastatic Cancer Using Lymphodepleting Conditioning Followed 
by Infusion of Anti-mesothelin Gene Engineered Lymphocytes 
 
Continuing Review Approved by the IRB on 10/25/18 
Amendment Approved by the IRB on 05/01/18 (P) 
           Date posted to web:  11/02/18 
Standard 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent (1) 
 
 
INTRODUCTION 
 
We invite you to take part in a research study at the National Institutes of Health (NIH). 
 
First, we want you to know that: 
 
 
Taking part in NIH research is entirely voluntary. 
 
 
You may choose not to take part, or you may withdraw from the study at any time.  In 
either case, you will not lose any benefits to which you are otherwise entitled.  
However, to receive care at the NIH, you must be taking part in a study or be under 
evaluation for study participation. 
 
 
You may receive no benefit from taking part.  The research may give us knowledge that 
may help people in the future. 
 
Second, some people have personal, religious or ethical beliefs that may limit the kinds of 
medical or research treatments they would want to receive (such as blood transfusions).  If you 
have such beliefs, please discuss them with your NIH doctors or research team before you 
agree to the study. 
 
Now we will describe this research study.  Before you decide to take part, please take as much 
time as you need to ask any questions and discuss this study with anyone at NIH, or with 
family, friends or your personal physician or other health professional. 
 
Why is this study being done? 
You have been diagnosed with metastatic cancer and the standard treatments available have not 
been effective. We have developed an experimental procedure for treating patients with cancer 
that uses blood cells found in their peripheral blood cells.  We genetically modify these cells and 
grow them in the laboratory.  We will be using the anti-mesothelin gene and a type of virus 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 2 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
(retrovirus) in making these special cells (anti-mesothelin cells). The anti-mesothelin cells target 
a protein called mesothelin.  This receptor is located on some types of cancer, including the type 
of cancer you have been diagnosed with.  By blocking mesothelin, we hope that these cells when 
infused will decrease the size of your tumors.  However, it is possible that these cells will not 
have this effect. 
 
The anti-mesothelin cells will be given to you as an intravenous (IV) infusion.  This type of 
experimental regimen is called “gene transfer” and is very closely monitored by the Food and 
Drug Administration (FDA) and other regulatory agencies.  The risks of gene transfer will be 
described later in this document. 
Why are you being asked to take part in this study? 
You are being asked to participate in this study because you have been diagnosed with cancer 
that has the mesothelin molecule on the surface of the tumors. 
How many people will take part in this study? 
Up to 136 patients may participate in this study. 
Description of Research Study  
This study has the following stages outlined below: 
Stage 1 is performed under the screening protocol, 99-C-0128 
Stage 2 is performed under the cell harvest protocol, 03-C-0277 
Stages 3-6 are performed under this protocol 
 
What 
Timeframe 
Location 
Comments & Instructions 
1. Work up 
1-2 weeks 
Inpatient and 
out patient   
Scans, x-rays, labs and other tests 
as needed 
2. Cell 
Manufacturing 
4-6 weeks 
Inpatient or 
Outpatient 
Leukapheresis 
3. Chemotherapy 
 
1 week 
Inpatient 
Receive IV chemotherapy to 
prepare your immune system for 
the cells 
4. Cells and IL-2 
 
1-5 days 
Inpatient and 
possibly ICU 
Receive anti-mesothelin cells, and 
then IL-2 every 8 hours for up to 
15 doses 
5. Recovery 
1-3 weeks 
Inpatient unit 
Recover from the effects of 
chemotherapy and IL-2. 
6. Follow -up 
Ongoing 
until disease 
progression 
Outpatient 
Return to clinic for physical exam, 
review of side effects, labs, scans 
every 1-6 months 
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 3 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
What will happen if you take part in this research study? 
The first few patients enrolled will participate in the Phase I portion of the study, called the “dose 
escalation” phase.  The purpose of dose escalation is to determine the most effective yet safe 
dose of anti-mesothelin cells.  There will be 10 dose levels of anti-mesothelin cells. The first 
patients enrolled get the smallest dose and the dose is increased when a level has been 
determined to be safe.  If the first dose has too many side effects, patients enrolled will receive 
smaller doses.  Discuss with your doctor which dose of anti-mesothelin cells you will be 
receiving.   
 
The major side effects of this experimental regimen (described below) that are most severe 
include:  
 
• Infection and low blood counts caused by the chemotherapy  
• Confusion and changes in mental status caused by the IL-2 
• Fluid retention, low blood pressure, and high heart rate caused by the IL-2 
 
We will discuss the side effects of this experimental regimen with you. You will be given 
medicines, transfusions, and treatments to prevent or treat the side effects including drugs to 
prevent and/or treat different types of infections.  We will try to make you as comfortable as 
possible.   
Before you begin the study 
The following procedures are conducted under 99-C-0128 or 03-C-0277, to which you have 
already enrolled. 
 
Cell harvest and growth 
You underwent a process called apheresis, while enrolled on our companion protocol 03-C-0277.  
This process obtained blood cells from you.  Some of these cells will be grown in the lab and 
genetically modified to recognize a protein on your tumor cells. If your cells do not grow, 
unfortunately, you will not be able to receive the cell infusion.  If that happens, we will look at 
alternative experimental treatments at the NIH Clinical Center or refer you to the care of your 
referring physician.  We usually know after about 4 weeks whether the cells will grow well 
enough to be used as an experimental treatment on this protocol.  At the time we determine that 
your cells are not growing, we will inform you and discuss your options with you. Several 
medications are used during the preparation of your cell product, be sure to tell your doctor if 
you are allergic to any antibiotics. 
 
Work up 
Prior to receiving the experimental regimen, you will undergo many tests. These include imaging 
procedures, heart and lung function tests, eye exams, and laboratory tests.  If you are a woman, 
you will undergo a pregnancy test.    You may be admitted to the hospital for these tests. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 4 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
However, you will be allowed to leave on pass on the days that you are not having tests 
performed. 
 
Catheter insertion 
Prior to beginning the experimental regimen, you will have an intravenous (IV) catheter placed 
in your upper chest. The area will be numbed with an anesthetic before the catheter is put in.  
Although rare, putting these catheters in can sometimes cause collapse of a lung or cause 
bleeding.  Lung collapse is treated by putting a tube into your chest for a few days to allow your 
lung to expand.  Pressure is placed on any area that might bleed.  Other IVs may be needed in 
one or both of your arms if we to give you extra fluids, medicines, or nutrition. 
 
Leukapheresis 
Leukapheresis is a procedure that allows us to remove certain types of blood cells from you and 
return the rest of your blood.  It is a very common procedure that is done routinely here at the 
NIH with very few risks.  During leukapheresis, blood is removed from you through a needle in 
your arm, circulated through a machine that divides whole blood into red cells, plasma (the 
serum part), and lymphocytes (or white cells), and then the plasma and red cells are returned to 
you through a second needle in your other arm.  The white blood cells may be used to help grow 
the cells.  
During the Study 
The following procedures will be conducted under this protocol.   
 
Chemotherapy Regimen (Day -7 through Day -3) 
After we have grown the anti-mesothelin cells to large numbers in the laboratory, you will be 
admitted to the hospital to begin your experimental regimen. You will be given two 
chemotherapy medicines, cyclophosphamide and fludarabine, to suppress your immune system 
so the anti-mesothelin cells can work without any interference from the cells in your immune 
system. (These medicines will not treat your cancer.)  Animal experiments have indicated that 
this can make the cells more effective in fighting cancer tumors, but it is not known whether this 
is true in humans.  The cyclophosphamide will be given into your catheter over 1 hour for two 
days (Day -7 and Day -6) and the fludarabine will be given into your catheter for 30 minutes 
every day for the next five days (Day -7 through Day -3).  The side effects of these medicines are 
described on the following pages. 
 
Cell Infusion and IL-2 Regimen (Day 0 through Day 4) 
One to four days after the last dose of chemotherapy, you will be given the anti-mesothelin cells.  
The anti- mesothelin cells will be given in your catheter over 20-30 minutes.  Within 24 hours 
after the anti- mesothelin cell infusion, you will be given low dose IL-2 through your catheter. 
High dose IL-2 is approved by the FDA for treatment of metastatic melanoma and metastatic 
renal cell cancer.  The purpose of giving the IL-2 with this therapy is to keep the cells we give 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 5 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
you active for as long as possible so they will fight your tumor.  The IL-2 will be given as a 15-
minute infusion every 8 hours for up to five days after the cell infusion (maximum number of 
doses is 15).  Doses may be skipped or delayed depending on how well you tolerate the doses.   
 
The risks of the cells and IL-2 are described on the following pages. The day after you receive 
the anti-mesothelin cells, we will give you more G-CSF (filgrastim) as a shot or injection under 
the skin. This will continue until your white blood cell counts begin to return to normal.  
When you are finished taking the drugs (treatment) 
Recovery 
After your last dose of IL-2, you will recover in the hospital until you are well enough to go 
home.  This usually takes 7 to 21 days; however, you may need to stay in the hospital for longer 
than this before you are well enough to go home. We will continue to give you support 
medications, do laboratory tests, and watch you closely for any side effects until we feel your 
condition is stable.  
 
In addition to the laboratory tests to monitor your condition, we will remove approximately 9 
teaspoons of blood three times per week to study the effects of this regimen on your immune 
system.  The maximum amount of blood for research is approximately 2.3 cups in 8 weeks. 
 
Follow up and Evaluation of Experimental Regimen  
You will need to continue to take Bactrim, an antibiotic, for at least 6 months following your 
regimen. We will ask you to return to NIH approximately 6 and 12 weeks following treatment 
and then if you are responding to the treatment, every 3 months x 3, every 6 months x 2 years 
and then as determined by your physician.  This visit will probably take 2 days.   If your tumor 
shows evidence of shrinking, we will ask you to return for evaluation every month for several 
more months.  If your tumor appears to be growing, we will look for other investigational 
therapies you may be eligible for, or refer you back to the care of your local physician.  At some 
of your follow up visits, you may undergo leukapheresis so that we can see the effect this therapy 
has had on your immune system and if the cells we gave you are still alive.  
 
Birth Control 
If you are a woman who is breast feeding or pregnant, you may not participate in the study 
because we don’t know how this medicine would affect your unborn child or your baby.   If you 
are a woman who can become pregnant, or are the partner of a woman who can become 
pregnant, you will need to practice an effective form of birth control before starting the study 
regimen and for four months after you finish study the regimen.  Males should also refrain from 
sperm donation during this time period. If you think that you or your partner is pregnant, you 
should tell your study doctor or nurse at once.   
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 6 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
Effective forms of birth control include:  
• abstinence 
• intrauterine device (IUD) 
• hormonal [birth control pills, injections, or implants] 
• tubal ligation 
• vasectomy 
 
Gene Therapy Long Term Follow up (Retroviral Vectors)  
Because we do not know the long term side effects of gene therapy, we will collect your blood 
over the next several years, frequently at first and then less frequently.  If you return to your 
referring physician after treatment here we will ask you to have your physician send your blood 
specimens here for this testing.   This testing will determine if the cells have grown or changed in 
your body.  We will test your blood immediately after you receive the cells, and then at 3, 6 and 
12 months (2 teaspoons each time).  If all of the tests are normal and show no change, we will 
collect blood from you every year after that to store in case you develop symptoms later. 
According to FDA requirements, we need you to return annually to the NIH for a physical 
examination for five years after you receive the cells.  After that time, we will be sending you a 
questionnaire to get information regarding your health for the next ten years, for a total follow up 
time period of 15 years.  For this reason, we ask that you continue to provide us with a current 
address and telephone number, even after you complete this research study.   At the time of your 
death, no matter the cause, we may request permission for an autopsy in order to obtain vital 
information concerning the safety of this experimental treatment approach.  Please discuss this 
with your family to inform them of this request. 
 
Risks or Discomforts of Participation 
What side effects or risks can I expect from being in this study?   
The risks and discomforts of this research study can be significant.   This experimental regimen 
can lead to long-term decrease in your immune function.  It is also possible that you may lose 
your fertility following this experimental regimen. It is possible, although unlikely, that this 
experimental regimen may cause your death.  
 
We will discuss the side effects of this experimental treatment with you. You will be given 
medicines, transfusions, and treatments to prevent or treat the side effects including drugs to 
prevent and/or treat different types of infections.  We will try to make you as comfortable as 
possible.  You should talk to your study doctor about any symptoms that you experience while 
taking part in the study. 
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 7 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
Aldesleukin (IL-2) 
When IL-2 is given through a catheter, it can make you feel like you have the flu.  It can also 
cause confusion and mental status changes making you unable to make sound decisions.  Prior to 
beginning the regimen, we will ask you to complete a Durable Power of Attorney so that a 
person of your choosing can make health care decisions for you in case you develop these side 
effects.  In our experience giving IL-2 to over 2,000 patients we have found that these side 
effects go away within a few days of stopping the IL-2. 
 
Anti- mesothelin Cell Infusion (gene transfer)  
The cells we will be giving you have a type of virus (retrovirus) put into them along with the 
anti- mesothelin protein.  Although this retrovirus is not active, there is the rare possibility that it 
may cause infection.  The cells could also cause you to develop another type of cancer, such as 
leukemia or lymphoma.  We do not have much information about the specific side effects of the 
anti- mesothelin cells since this is our first study using these cells.  We have studied similar types 
of cells in patients with cancer in other studies, and have seen the following side effects: 
 
• Fever, chills and shortness of breath, which may last for a few hours (common) 
• Lung congestion  
• Some patients had severe effects on their brain which caused confusion and seizures.  
These patients also had difficulty moving and speaking.  Two of these patients 
remained in the intensive care unit (ICU) for several months with these symptoms, 
and both of these patients died. 
• Immune-type reaction  
o 
 In similar clinical trials with cells targeting a melanoma protein, we have 
observed the following immune-mediated toxicities: loss of skin pigment (known 
as vitiligo), inflammation of the eye (uveitis), hearing loss, and dizziness.  The 
skin, the eye, and the ear are all sites where that targeted melanoma protein is 
known to exist. 
o 
In other clinical trials with a similar type of cell therapy, two patients died due to 
an immune type reaction from the cells.   
• Mesothelin is present on the membranes which surround the lungs, heart, and 
abdomen as well as in cells in the trachea (wind pipe), eye, tonsils and Fallopian 
tubes (tubes in the uterus).  Immunotoxins (antibodies with a toxic agent attached) 
which target mesothelin have been used to treat patients in other studies.  Some of the 
side effects seen in these studies include pleuritis (inflammation of the membrane 
which surrounds the lung) resulting in pain, and pleural effusion (excess fluid in 
between the membranes which surround the lung).  Other side effects include 
shortness of breath, fatigue, nausea and vomiting, weight gain, edema (increase in 
fluid in the body), infection, and capillary leak syndrome. 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 8 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
• You will be treated on this gene transfer protocol with a viral vector that was 
manufactured at the NCI Surgery Branch Vector Production Facility before May 
2016.   An internal review of the facility that made the vector for this protocol 
determined that the facility needed to be closed due to manufacturing issues.  We 
know of no additional risks related to the previously produced vector for patients who 
have received cells with vectors made in this facility as the vectors were extensively 
tested by outside experts.  Therefore, the IRB has determined that the potential 
benefit to you outweighs the potential risks. 
  
There is no data available at this time to guide us in how humans might respond to this type of 
cell infusion.  As this is a new experimental therapy, side effects that we do not anticipate that 
may cause your condition to deteriorate may be encountered.  Any new information that 
becomes available during the course of this study will be shared with you. 
Medications 
The side effects of cyclophosphamide, fludarabine, IL-2 and some of the other medications you 
will receive are listed in below.  
 
Cyclophosphamide and Fludarabine side effects 
Common 
Less Common 
Rare 
 
Changes in blood counts 
including: low red cell count 
(causing fatigue and shortness of 
breath), low platelet count 
(increasing the risk of bleeding 
and bruising), decrease in white 
blood cells (increasing the risk of 
infection and the need for 
treatment with antibiotics or 
other treatment)  
 
Loss of appetite, nausea, 
vomiting,  
 
Diarrhea, stomach pain 
 
Mouth sores 
 
Hair loss 
 
Fatigue 
 
Muscle or joint aches  
 
Bleeding 
 
Infection 
 
Bladder irritation 
with bloody urine 
 
Severe allergic 
reaction (difficulty 
breathing/swelling) 
 
Headache or 
dizziness 
 
Sweating 
 
Swelling of arms or 
legs 
 
Skin changes, rash, 
blisters  
 
Weakness 
 
Hearing loss 
 
Heart damage 
 
Lung damage 
 
Kidney damage 
 
Inflammation of the eye 
resulting in blindness 
 
Inflammation of nervous 
system resulting in death 
 
Epstein Barr Virus 
Lymphoma.  This can be 
fatal (Three patients on 
other studies in the 
Surgery Branch 
developed EBV 
lymphoma, and two died 
as a result of this 
disease.) 
 
Loss of fertility 
 
Support Medications – side effects 
Common 
Less common 
Rare 
Filgrastim (To increase production of white blood cells) 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 9 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Bone Pain 
 
Severe headache 
 
Severe breathing 
problems 
 
Rupture of your spleen 
Bactrim (To prevent a specific type of pneumonia) 
 
 
Fever 
 
Nausea, vomiting,  
 
Skin rash with itching 
 
reduced number of 
white blood cells  
 
Allergic reaction 
 
 
Common 
Less common 
Rare 
Fluconazole: (To prevent fungal infections)  
 
Headache 
 
Nausea, vomiting, 
diarrhea, abdominal 
pain 
 
Itching  
 
 
A skin disorder called Stevens 
Johnson Syndrome, which can be 
fatal 
 
Liver damage which may be 
permanent 
Acyclovir and Valacyclovir 
 
 
Temporary decrease in 
kidney function which 
may not cause any 
symptoms 
 
Nausea, vomiting, 
diarrhea, constipation  
 
Pain and irritation at place 
of injection 
 
Skin rash, hives, itching  
 
Tremors, dizziness, Confusion, 
seizures 
 
Fatigue  
 
Blood in the urine 
 
 
Low dose Aldesleukin Risks and Discomforts 
Common 
Unlikely 
 
Local swelling at the site of 
injection 
 
Low blood pressure 
 
Tiredness 
 
Swelling in hands and feet 
 
Rash 
 
Nausea 
 
Vomiting 
 
Diarrhea 
 
Infection 
 
Laboratory changes including kidney blood tests 
 
Decreased urine 
 
Changes in consciousness 
 
Infections 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 10 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
Prior to and throughout this study you will undergo many tests to determine the size and extent 
of your tumor, as well as the impact of the regimen.  Multiple blood tests will be performed and 
some of your serum and lymphocytes will be stored for future testing.  Blood and tissue samples 
collected from you may be stored and used in the future to study scientific questions related to 
this protocol.  If there are any risks to you or your family associated with these future scientific 
studies which are not covered in this consent form, your consent will be obtained before such 
studies are performed. 
 
If your disease progresses, or recurs after this experimental regimen and you are not eligible for 
retreatment, then you will no longer receive the regimen in this protocol, though you may be 
eligible to be considered for other protocols at the National Cancer Institute, NIH or referred 
elsewhere for treatment.  
 
Gene Therapy Risk of Cancer and Other Diseases  
When retroviral vectors enter a normal cell in the body, the DNA of the vector inserts itself into 
the normal DNA in that cell; this process is called integration.  Most integration is expected to 
cause no harm to the cell or to the patient. However, there is a chance that there may be some 
regions of the normal human DNA where integration of the viral vector’s DNA may result in 
activation of neighboring genes.  For example, if one of these genes were a growth factor, this 
may cause uncontrolled division of the cell, resulting in a cancer.  This type of event has 
occurred in one animal study in mice where the vector integration site correlates with the 
occurrence of cancer in these mice.  Five instances of a similar event have been reported in five 
children out of 22 who received a retroviral vector in two experimental gene transfer studies for 
X-linked Severe Combined Immunodeficiency (SCID) conducted in Europe, not under the 
jurisdiction of the U.S. Food and Drug Administration (FDA).  While most of the children who 
participated in this clinical trial appear to have been cured of their disease, five children 
developed leukemia (a form of cancer of the blood) approximately 2-6 years after receiving the 
gene transfer regimen.  The first patient had extensive testing done to determine the cause of the 
leukemia.  A group of experts in this field have looked at all the test results, and concluded that 
the gene transfer caused the leukemia in the first child.  One of the children died as a result of 
their leukemia.  The risk of another cancer developing in you, including leukemia, is unknown, 
but you need to be aware of this possible risk.  To monitor you for this risk we will be testing 
your blood 3 months after cell infusion, then at 6 and 12 months, and then annually thereafter.  If 
we find that the cells we have given you grow out of control, chemotherapy will be given to you 
to kill the cells, given their risk of causing leukemia or a second cancer. This testing will be 
conducted on our protocol 09-C-0161, “Follow up Protocol for Subjects Previously Enrolled in 
NCI Surgery Branch Studies” to which you will be enrolled following treatment. 
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 11 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
Potential Benefits of Participation 
Are there benefits to taking part in this study?  
The aim of this study is to see if this experimental treatment will cause your tumors to shrink.  
We do not know if you will receive personal, medical benefit from taking part in this study.  
These potential benefits could include shrinking of your tumor or lessening of your symptoms, 
such as pain, that are caused by the cancer.  Because there is not much information about the 
effect of this treatment on cancer, we do not know if you will benefit from taking part in this 
study, although the knowledge gained from this study may help others in the future who have 
cancer. 
Alternative Approaches or Treatments 
What other choices do I have if I do not take part in this study?  
Other options for treatment of your cancer include: 
• Taking part in another study  
• Getting treatment or care for your cancer without being in a study 
• Getting comfort care which is also called palliative care. This type of care helps reduce 
pain, tiredness, appetite problems, and other problems caused by cancer. It does not treat 
the cancer directly, but instead tries to improve how you feel. Comfort care tries to keep 
you as active and comfortable as possible. 
 
Please talk to your doctor about these and other options. 
Research Subject's Rights 
What are the costs of taking part in this study?  
If you choose to take part in the study, the following will apply, in keeping with the NIH policy: 
• You will receive study treatment at no charge to you. This may include surgery, 
medicines, laboratory testing, x-rays or scans done at the Clinical Center, National 
Institutes of Health (NIH), or arranged for you by the research team to be done outside 
the Clinical Center, NIH if the study related treatment is not available at the NIH.  
• There are limited funds available to cover the cost of some tests and procedures 
performed outside the Clinical Center, NIH.  You may have to pay for these costs if they 
are not covered by your insurance company. 
• Medicines that are not part of the study treatment will not be provided or paid for by the 
Clinical Center, NIH. 
• Once you have completed taking part in the study, medical care will no longer be 
provided by the Clinical Center, NIH. 
 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 12 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
Will your medical information be kept private?  
We will do our best to make sure that the personal information in your medical record will be 
kept private.  However, we cannot guarantee total privacy.  Organizations that may look at 
and/or copy your medical records for research, quality assurance, and data analysis include: 
 
• The National Cancer Institute (NCI) and other government agencies, like the Food and 
Drug Administration (FDA), which are involved in keeping research safe for people. 
• National Cancer Institute Institutional Review Board 
• The Study Sponsor, Center for Cancer Research or their agents 
• Kite Pharma 
 
A description of this clinical trial will be available on http://www.Clinicaltrials.gov, as required 
by U.S. Law.  This Web site will not include information that can identify you.  At most the Web 
site will include a summary of the results.  You can search this Web site at any time.  
 
Stopping Therapy  
Your doctor may decide to stop your therapy for the following reasons: 
• if he/she believes that it is in your best interest 
• if your disease comes back during treatment 
• if you become pregnant 
• if you have side effects from the treatment that your doctor thinks are too severe 
• if new information shows that another treatment would be better for you 
 
In this case, you will be informed of the reason therapy is being stopped. 
 
You can stop taking part in the study at any time. However, if you decide to stop taking part in 
the study, we would like you to talk to the study doctor and your regular doctor first. 
 
If you decide at any time to withdraw your consent to participate in the trial, we will not 
collect any additional medical information about you. However, according to FDA 
guidelines, information collected on you up to that point may still be provided to the 
Sponsor. If you withdraw your consent and leave the trial, any samples of yours that have 
been obtained for the study and stored at the NCI can be destroyed upon request. 
However, any samples and data generated from the samples that have already been 
distributed to other researchers or placed in the research databases cannot be recalled and 
destroyed. 
Certificate of Confidentiality  
To help us protect your privacy, we have obtained a Certificate of Confidentiality. The 
researchers can use this Certificate to legally refuse to disclose information that may identify you 
in any federal, state, or local civil, criminal, administrative, legislative, or other proceedings, for 


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 13 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
example, if there is a court subpoena. The researchers will use the Certificate to resist any 
demands for information that would identify you, except as explained below. 
You should also know that there are several circumstances in which the Certificate does not 
provide coverage. These include when information:  
• will be used for auditing or program evaluation internally by the NIH; or  
• must be disclosed to meet the legal requirements of the federal Food and Drug 
Administration (FDA).  
• is necessary for your medical treatment and you have consented to this disclosure;  
• is for other research.  
In addition, identifiable, sensitive information protected by this Certificate cannot be admissible 
as evidence or used for any purpose in any action, suit, or proceeding without your consent.  
You should understand that a Certificate of Confidentiality does not prevent you or a member of 
your family from voluntarily releasing information about yourself or your involvement in this 
research. If an insurer, employer, or other person obtains your written consent to receive research 
information, then the researchers will not use the Certificate to withhold that information. 
The Certificate of Confidentiality will not protect against the required reporting by 
hospital staff of information on suspected child abuse, reportable communicable diseases, 
and/or possible threat of harm to self or others. 
Conflict of Interest 
The National Institutes of Health (NIH) reviews NIH staff researchers at least yearly for conflicts 
of interest.  This process is detailed in a Protocol Review Guide.  You may ask your research 
team for a copy of the Protocol Review Guide or for more information.  Members of the research 
team who do not work for NIH are expected to follow these guidelines but they do not need to 
report their personal finances to the NIH. 
 
Members of the research team working on this study may have up to $15,000 of stock in the 
companies that make products used in this study.  This is allowed under federal rules and is not a 
conflict of interest. 
 
The National Institutes of Health and the research team for this study are using the Anti-
mesothelin CAR developed by the NCI Surgery Branch through a joint study with your 
researchers and the company (Kite Pharma).  Kite Pharma also provides financial support for this 
study. 
Use of Specimens and Data for Future Research 
Blood and tissue collected during the course of this study will be used for future research and 
will be stored, tracked and disposed of under our companion protocol 03-C-0277, (Cell Harvest 
and Preparation for Surgery Branch Adoptive Cell Therapy Protocols) on which you have 
already been enrolled.   


MEDICAL RECORD 
CONTINUATION SHEET for either: 
NIH 2514-1, Consent to Participate in A Clinical Research Study 
NIH 2514-2, Minor Patient’s Assent to Participate In A Clinical Research Study 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 14 of 16 pages 
 
PATIENT IDENTIFICATION 
CONTINUATION SHEET for either: 
NIH-2514-1 (07-09) 
NIH-2514-2 (10-84) 
P.A.:  09-25-0099 
File in Section 4:  Protocol Consent 
 
 
In addition, to advance science, it is helpful for researchers to share information they get from 
studying human samples.  They do this by putting it into one or more scientific databases, where 
it is stored along with information from other studies. A researcher who wants to study the 
information must apply to the database and be approved.  Researchers use specimens and data 
stored in scientific databases to advance science and learn about health and disease.   
 
We plan to keep some of your specimens and data that we collect and use them for future 
research and share them with other researchers. We will not contact you to ask about each of 
these future uses.  These specimens and data will be stripped of identifiers such as name, address 
or account number, so that they may be used for future research on any topic and shared broadly 
for research purposes. Your specimens and data will be used for research purposes only and will 
not benefit you.  It is also possible that the stored specimens and data may never be used.  
Results of research done on your specimens and data will not be available to you or your doctor.  
It might help people who have cancer and other diseases in the future.   
 
If you do not want your stored specimens and data used for future research, please contact us in 
writing and let us know that you do not want us to use your specimens and/or data.  Then any 
specimens that have not already been used or shared will be destroyed and your data will not be 
used for future research. However, it may not be possible to withdraw or delete materials or data 
once they have been shared with other researchers. 
 
 
 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 15 of 16 pages 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH 
STUDY (Continuation Sheet) 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
 
File in Section 4:  Protocol Consent 
 
 
 
OTHER PERTINENT INFORMATION 
1. 
Confidentiality.  When results of an NIH research study are reported in medical journals 
or at scientific meetings, the people who take part are not named and identified.  In most cases, 
the NIH will not release any information about your research involvement without your written 
permission.  However, if you sign a release of information form, for example, for an insurance 
company, the NIH will give the insurance company information from your medical record.  This 
information might affect (either favorably or unfavorably) the willingness of the insurance 
company to sell you insurance. 
The Federal Privacy Act protects the confidentiality of your NIH medical records.  However, you 
should know that the Act allows release of some information from your medical record without 
your permission, for example, if it is required by the Food and Drug Administration (FDA), 
members of Congress, law enforcement officials, or authorized hospital accreditation 
organizations. 
2. 
Policy Regarding Research-Related Injuries.  The Clinical Center will provide short-
term medical care for any injury resulting from your participation in research here.  In general, 
no long-term medical care or financial compensation for research-related injuries will be 
provided by the National Institutes of Health, the Clinical Center, or the Federal Government.  
However, you have the right to pursue legal remedy if you believe that your injury justifies such 
action. 
3. 
Payments.  The amount of payment to research volunteers is guided by the National 
Institutes of Health policies.  In general, patients are not paid for taking part in research studies at 
the National Institutes of Health.  Reimbursement of travel and subsistence will be offered 
consistent with NIH guidelines. 
4. 
Problems or Questions.  If you have any problems or questions about this study, or 
about your rights as a research participant, or about any research-related injury, contact the 
Principal Investigator, Steven A. Rosenberg, M.D., Ph.D., Building 10 CRC, Room 3-3940, 
Telephone:  240-760-6218. You may also call the Clinical Center Patient Representative at 301-
496-2626. If you have any questions about the use of your specimens or data for future research 
studies, you may also contact the Office of the Clinical Director, Telephone:  240-760-6070. 
5. 
Consent Document.  Please keep a copy of this document in case you want to read it 
again. 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 12-C-0111 
CONTINUATION: page 16 of 16 pages 
 
PATIENT IDENTIFICATION 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH 
STUDY (Continuation Sheet) 
• 
Adult Patient or 
• Parent, for Minor Patient 
NIH-2514-1 (07-09) 
P.A.:  09-25-0099 
 
File in Section 4:  Protocol Consent 
 
 
 
 
 
COMPLETE APPROPRIATE ITEM(S) BELOW: 
A. Adult Patient’s Consent 
B. Parent’s Permission for Minor Patient. 
I have read the explanation about this study 
and have been given the opportunity to discuss 
it and to ask questions.  I hereby consent to 
take part in this study. 
I have read the explanation about this study 
and have been given the opportunity to discuss 
it and to ask questions.  I hereby give 
permission for my child to take part in this 
study.   
(Attach NIH 2514-2, Minor’s Assent, if 
applicable.) 
 
  
  
 
 
 
Signature of Adult Patient/ 
Legal Representative 
Date 
Signature of Parent(s)/ Guardian Date 
 
 
Print Name 
 
 
Print Name 
C. Child’s Verbal Assent (If Applicable) 
The information in the above consent was described to my child and my child agrees to 
participate in the study. 
 
  
  
 
Signature of Parent(s)/Guardian 
Date 
Print Name 
THIS CONSENT DOCUMENT HAS BEEN APPROVED FOR USE  
FROM OCTOBER 25, 2018 THROUGH MAY 06, 2019. 
 
 
 
 
 
 
 
 
Signature of Investigator 
Date 
Signature of Witness 
Date 
 
 
Print Name 
 
 
Print Name