Conditions: Metastatic Cancer Metastatic Melanoma Renal Cancer
Keywords: Clinical Response Metastatic Cancer Immunotherapy Adoptive Cell Therapy Metastatic Melanoma
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 24
Healthy volunteers: no

Brief summary:
Background:

The National Cancer Institute (NCI) Surgery Branch has developed an experimental therapy for treating patients 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 patient s white blood cells with a retrovirus that has the gene for anti-vascular endothelial growth factor receptor (VEGFR2) incorporated in the retrovirus.

Objectives:

\- To determine a safe number of these cells to infuse and to see the safety and effectiveness of cell therapy using anti-VEGFR2 gene modified tumor white blood cells to treat recurrent or relapsed cancer.

Eligibility:

\- Individuals greater than or equal to 18 years of age and less than or equal to 70 years of age who have been diagnosed with metastatic cancer that has not responded to or has relapsed after standard treatment.

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-VEGFR2 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-VEGFR2 cells and aldesleukin. They will stay in the hospital for about4 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 the Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), which can be used to mediate genetic transfer of this CAR with high efficiency (\> 50%) without the need to perform any selection. Administration of VEGFR2 CAR transduced cells inhibited tumor growth in several different models in different mouse strains.
* In co-cultures with VEGFR2 expressing cells, anti-VEGFR2 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-VEGFR2 CAR engineered cluster of differentiation 8 (CD8)+ peripheral blood lymphocytes in patients receiving a non- myeloablative conditioning regimen, and aldesleukin.
* Determine if the administration of anti-VEGFR2 CAR engineered CD8+ 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.

Secondary objective:

-Determine the in vivo survival of CAR gene-engineered cells.

Eligibility:

Patients who are 18 years of age or older must have:

* metastatic cancer;
* previously received and have been a non-responder to or recurred after standard care for metastatic disease;

Patients may not have:

-contraindications for high dose aldesleukin administration.

Design:

* Peripheral blood mononuclear cells (PBMC) obtained by leukapheresis (approximately 5 times 10(9) cells) 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 10(8) to 5 times 10(8) cells to retroviral vector supernatant containing the VEGFR2 genes.
* 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 CD8+ PBMC plus intravenous (IV) aldesleukin. With approval of amendment C, aldesleukin (based on total body weight) will be administered at a dose of 72,000 IU/kg as an intravenous bolus over a 15 minute period approximately every eight hours (+/- 1 hour) beginning within 24 hours of the cell infusion and continuing for up to 5 days (maximum 15 doses
* 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. Initially, the protocol will enroll 1 patient in each dose cohort unless that patient experiences a dose limiting toxicity (DLT). Should a single patient experience a dose limiting toxicity due to the cell transfer at a particular dose level, additional 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 6 patients. If 2 DLTs are encountered in this cohort, the study will be terminated. If IFN-gamma levels increase substantially (as defined in the protocol) in the patient in a cohort compared to the prior patient, the cohort would be expanded to an n=3 to obtain more data on this phenomenon. If one of these 3 patients experience a DLT, the cohort will be expanded to six patients. Following amendment C, patients will be enrolled in cohorts 8-11, with the non-myeloablative chemotherapy regimen, cells and low dose aldesleukin following a conventional 3+3 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 metastatic melanoma and renal cancer, and cohort 2 will include patients with other types of metastatic cancer.
* 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.
* The objective will be to determine if the combination of aldesleukin, lymphocyte depleting chemotherapy, and anti-VEGFR2 CAR-gene engineered CD8+ 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% partial response (PR) + complete response (CR) rate (p1=0.20).

Arms:
arm 1: Patients will receive (1x10(6) cells plus high dose aldesleukin arm 2: Patients will receive (3x10(6) cells plus high dose aldesleukin arm 3: Patients will receive (1x10(7) cells plus high dose aldesleukin arm 4: Patients will receive (3x10(7) cells plus high dose aldesleukin arm 5: Patients will receive (1x10(8) cells plus high dose aldesleukin arm 6: Patients will receive (3x10(8) cells plus high dose aldesleukin arm 7: Patients will receive (1x10(9) cells plus high dose aldesleukin arm 8: Patients will receive (1x10(9) cells plus low dose aldesleukin arm 9: Patients will receive (3x10(9) cells plus low dose aldesleukin arm 10: Patients will receive (1x10(10) cells plus low dose aldesleukin arm 11: Patients will receive (3x10(10) cells plus low dose aldesleukin

Interventions: intervention 1: Anti-VEGFR2 CAR CD8 plus PBL intervention 2: Cyclophosphamide intervention 3: Aldesleukin intervention 4: Fludarabine
Intervention descriptions: intervention 1: Patients will receive non-myeloablative lymphodepleting preparative regimen consisting of cyclophosphamide and fludarabine followed by the administration of anti-VEGFR CAR CD8+ PBL and high dose (Arms 1-7) or low dose (Arms 8-11) aldesleukin. On day 0, cells will be infused in the Patient Care Unit over 20-30 minutes (Phase 1: 10e6- 3x10e10 cells and Phase 2: maximum tolerated dose of cells from Phase 1) intervention 2: Cyclophosphamide 60 mg/kg/day X 2 days intravenous (IV) in 250 ml dextrose in 5% water (D5W) with mesna 15 mg/kg/day X 2 days over 1 hr intervention 3: Aldesleukin (based on total body weight) will be administered at a dose of 720,000 IU/kg (Arms 1-7) or 72,000 IU/kg (Arms 8-11) as an intravenous bolus over a 15 minute period approximately every eight hours (+/- 1 hour) beginning within 24 hours of the cell infusion and continuing for up to 5 days (maximum 15 doses) intervention 4: Fludarabine 25 mg/m(2)/day intravenous piggyback (IVPB) daily over 30 minutes for 5 days

Protocol document:
Prot_SAP_000.pdf: 
11-C-0013
1
Abbreviated Title: Anti-VEGFR2 CAR CD8+ PBL
OBA: 1004-1036
IBC: RD-10-IV-02
CC Protocol Number: 11-C-0013 L
NCI Protocol Number: P10648
IRB Submission Date: March 8, 2010
Version Date: July 1, 2015
PROTOCOL TITLE
Phase I/II Study of Metastatic Cancer Using Lymphodepleting Conditioning 
Followed by Infusion of Anti-VEGFR2 Gene Engineered CD8+ Lymphocytes
Principal Investigator:
Steven A. Rosenberg, M.D., Ph.D.
Chief of Surgery, NCI
Building 10, CRC, Room 3-3940
Phone: 301-496-4164
Associate Investigators:  
Robert Somerville, Ph.D., Surgery Branch, NCI
Steven A. Feldman, Ph.D., Surgery Branch, NCI 
Nicholas P. Restifo, M.D., Surgery Branch, NCI**
James C. Yang, M.D., Surgery Branch, NCI
Richard Sherry, M.D., Surgery Branch, NCI
Udai S. Kammula, M.D., Surgery Branch, NCI
Stephanie Goff, M.D., Surgery Branch, NCI
Christopher Klebanoff, M.D., Surgery Branch, NCI
Lori McIntyre, R.N., OCD^, NCI
Marie Statler, R.N., OCD^, NCI
Debbie Ann Nathan, R.N., Surgery Branch, NCI**
Monica Epstein, R.N., OCD^, NCI
Abigail Johnson, R.N., OCD^, NCI
Mary Ann Toomey, R.N., Surgery Branch, NCI**
Arati Kamath, Ph.D, Leidos Biomedical Research, Inc.
Daniel Zlott, Pharm. D., Pharmacy Department, NIH Clinical Center
Donald E. White, M.S., Surgery Branch, NCI
Seth M. Steinberg, Ph.D., BDMS, NCI+
Isaac Kriley, M.D., Surgery Branch, NCI
Parisa Malekzadeh, M.D., Surgery Branch, NCI
Kasia Trebska-McGowan, M.D., Surgery Branch, NCI
Referral Contact:
Linda Williams, R.N., Surgery Branch, NCI
Jessica Yingling, R.N., OCD^, NCI
Ellen Bodurian, R.N., OCD^, NCI
+Biostatistics Data Management Section Center for Cancer Research
National Cancer Institute, National Institutes of Health, Bethesda, Maryland
**Not responsible for direct patient care
^Office of the Clinical Director
Drug Name:
anti-VEGFR2 CAR CD8+ peripheral blood lymphocytes (PBL)
IND Number:
IND 14440
Sponsor:
Steven A. Rosenberg, M.D., Ph.D.


1 1- C- 0 0 1 3
2
Précis:
UB ac k gr o u n d:
We ha ve c o nstr uct e d a si n gle r etr o viral v ect or t hat c o ntai ns a c hi meri c T cell rece pt or ( C A R)
t hat rec o g nizes t he Vas c ular E n d ot helial Gr o wt h Fact or R ece pt or 2 ( V E G F R 2), w hic h ca n be 
use d t o me diate ge netic tra nsfer of t his C A R  wit h  hi g h efficie n c y ( > 5 0 %)  wit h o ut t he nee d t o 
perf or m a n y s electi o n.  A d mi nistrati o n of V E G F R 2 C A R tra ns d uce d cells i n hi bite d t u m or 
gr o wt h i n se ver al differ e nt m o de ls i n differe nt m o use strai ns.
I n c o-c ult ures wit h V E G F R 2 e x pressi n g cells, a nti- V E G F R 2 tra ns d u ce d T cells secrete d 
si g nifica nt a m o u nts of I F N-
wit h hi g h s pecificit y.
UO bjecti ves :
Pri mar y o bjecti ves: 
T o e val uate t he s afet y of t he a d mi nistrati o n of a nti- V E G F R 2 – C A R e n gi neere d C D 8 + p eri p heral 
bl o o d l y m p h o c ytes i n patie nts recei vi n g a n o n-  m yel oa blati ve c o n diti o ni n g re gi me n, a n d 
al desle u ki n.
Deter mi ne if t he a d mi nistra ti o n of a nti- V E G F R 2 – C A R e n gi neere d C D 8 + peri p heral bl o o d 
l y m p h oc ytes a n d al desle u ki n t o patie nts f oll owi n g a n o n m yel o a blati ve b ut l y m p h oi d de pleti n g 
pre par ati ve re gi me n will res ult i n cli nical t u m or re gressi o n i n patie nts wit h metastatic ca n cer.
Sec o n dar y o bjecti ve: 
Deter mi ne t he i n vi v o s ur vi val of C A R ge n e-e n gi n eere d cells.
UEli gi bilit y:
Patie nts w h o are 1 8 years of a ge or ol d er m ust ha v e 
metastatic ca n cer;
pre vi o usl y recei ve d a n d ha ve bee n a n o n-res p o n d er t o or rec urre d after sta n dar d care f or 
metastatic disease;
Patie nts ma y n ot ha ve:
c o ntrai n dicati o ns f or hi g h d os e al desle u ki n a d mi nistrati o n.
UDesi g n:
P B M C o btai ne d b y l e u ka p here sis (a p pr o xi matel y 5 X 1 0 9 cells) will be c ult ure d i n t he pres e nce 
of a nti- C D 3 ( O K T 3) a n d al desle u ki n i n  or der t o sti m ulate T-cell gr o wt h.
Tra ns d ucti o n is i nitiate d b y e x p os ure of a p pr o xi matel y 1 0 8 t o 5 X 1 08 cells t o retr o viral vect or 
s u per nata nt c o nt ai ni n g t h e V E G F R 2 ge nes.
Patie nts will recei ve a n o n m yel oa bl ati ve b ut l y m p h oc yt e de pleti n g pre p arati ve re gi me n 
c o nsisti n g of c ycl o p h os p ha mi de a n d fl u dara bi ne f oll o we d b y i ntra v e n o us i nf usi o n of e x vi v o 
C A R ge ne-tra ns d u ce d C D 8 + P B M C pl us I V al desle u ki n.  Wit h a p pr o val of a me n d me nt C, 
al desle u ki n ( base d o n t ot al b o d y w ei g ht) will be  a d mi nistere d at a d ose of 7 2, 0 0 0 I U/ k g as a n 
i ntra ve n o us b ol us o ver a 1 5 mi n ute peri o d a p pr o xi matel y e ver y ei g ht h o urs ( +/- 1 h o ur) 
be gi n ni n g wit hi n 2 4 h o urs of t he cell i nf usi o n a n d c o nti n ui n g f or u p t o 5 d a ys ( ma xi m u m 1 5 
d oses
Patie nts will u n der g o c o m plete e val uati o n of t u m or wit h p h ysical e x a mi nati o n, C T of t he c hest, 
a b d o me n a n d pel vis a n d cli nical la b orat or y e val uati o n f o ur t o si x wee ks after treat me nt.  If t he 
patie nt has S D or t u m or s hri n ka ge, re peat c o m plete e val uati o ns will be perf or me d e ver y 1- 3


11-C-0013
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. Initially, the protocol will enroll 1 patient in each dose cohort 
unless that patient experiences a dose limiting toxicity (DLT).  Should a single 
patient experience a dose limiting toxicity due to the cell transfer at a particular dose level, 
additional 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 6 patients.  If 2 DLTs are encountered in this cohort, the study will be terminated.
If IFN-gamma levels increase substantially (as defined in the protocol) in the patient in a cohort 
compared to the prior patient, the cohort would be expanded to an n=3 to obtain more data on 
this phenomenon.  If one of these 3 patients experience a DLT, the cohort will be expanded to six 
patients. Following amendment C, patients will be enrolled in cohorts 8-11, with the non-
myeloablative chemotherapy regimen, cells and low dose aldesleukin following a conventional 
3+3 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 metastatic melanoma and renal cancer, and cohort 2 will include patients with other 
types of metastatic cancer.  
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 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. 
x
The objective will be to determine if the combination of aldesleukin, lymphocyte depleting 
chemotherapy, and anti-VEGFR2 CAR-gene engineered CD8+ 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).


11-C-0013
4
UTable of Contents
1.0 
0BIntroduction............................................................................................................................... 6 
1.1. 
UStudy Objectives: ................................................................................................................ 6 
1.2. 
UBackground and Rationale: ............................................................................................... 6 
2.0 
1BEligibility Assessment and Enrollment................................................................................... 16 
2.1. 
UEligibility Criteria............................................................................................................. 16 
2.2. 
SScreening Evaluation .......................................................................................................... 18 
2.3. 
URegistration Procedures:.................................................................................................. 18 
3.0 
2BStudy Implementation............................................................................................................. 19 
3.1. 
UStudy Design:..................................................................................................................... 19 
3.2. 
UDrug Administration:....................................................................................................... 23 
3.3. 
UOn-Study Evaluation ........................................................................................................ 26 
3.4. 
URe-Treatment .................................................................................................................... 28 
3.5. 
Post Study Evaluation (Follow-up): ................................................................................ 29 
3.6. 
Criteria for Removal from Protocol Therapy and Off Study Criteria........................ 30 
3.6.1 Off Treatment Criteria: ................................................................................................... 30 
3.6.2 Off Study Criteria:............................................................................................................ 30 
4.0 
3BSupportive Care ...................................................................................................................... 31 
5.0 
4BData Collection and Evaluation .............................................................................................. 31 
5.1. 
UResponse Criteria.............................................................................................................. 31 
5.2. 
UToxicity Criteria................................................................................................................ 32 
5.3. 
UStatistical Considerations................................................................................................. 34 
5.4. 
UData and Safety Monitoring Plan.................................................................................... 36 
5.5. 
Principal Investigator/Research Team ........................................................................... 36 
5.6. 
Sponsor Monitoring Plan ................................................................................................. 37 
5.7. 
Safety Monitoring Committee (SMC)............................................................................. 37 
5.8. 
UHandling of Tissue Specimens for Research Purposes.................................................. 37 
6.0 
5BHuman Subjects Protections ................................................................................................... 38 
6.1. 
URationale for Patient Selection ........................................................................................ 38 
6.2. 
UParticipation of Children ................................................................................................. 38 
6.3. 
UEvaluation of Benefits and Risks..................................................................................... 38 
6.4. 
UConsent Document............................................................................................................ 39 
7.0 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN
39 
7.1 
UDefinitions.......................................................................................................................... 39 
7.2 
Routine Data Reporting:.................................................................................................. 41 
7.3 
NCI-IRB Reporting .......................................................................................................... 42 
7.4 
NIH Office of Biotechnology Activities (OBA)/Institutional Biosafety Committee 
(IBC) Reporting Criteria.............................................................................................................. 43 
7.5 
FDA Reporting Criteria:.................................................................................................. 43 
8.0 
Pharmaceutical Information.................................................................................................... 44 
8.1. 
Aldesleukin (Interleukin-2, Proleukin, Recombinant Human Interleukin 2)............. 44 
8.2. 
Fludarabine ....................................................................................................................... 44 
8.3. 
Cyclophosphamide............................................................................................................ 45 
8.4. 
Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex)................................. 46 
8.5. 
Filgrastim (Granulocyte Colony-Stimulating Factor, G-CSF, Filgrastim,  Neupogen)
47 


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8.6. 
Trimethoprim and Sulfamethoxazole Double Strength (TMP / SMX DS) ................. 47 
8.7. 
Herpes Virus Prophylaxis ................................................................................................ 47 
8.8. 
Fluconazole........................................................................................................................ 48 
8.9. 
Anti-VEGFR2 CAR transduced CD8+ PBL.................................................................. 48 
8.10. Support Medications......................................................................................................... 49 
9.0 
References............................................................................................................................... 50 
10.0 
Tables, Figures, and Appendices: ........................................................................................... 53 


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6
1.0
0BIntroduction
1.1.
UStudy Objectives:
1.1.1. Primary objectives:
x
To evaluate the safety of the administration of anti-VEGFR2 –CAR
engineered CD8+ peripheral blood lymphocytes in patients receiving the non-
myeloablative conditioning regimen, and aldesleukin
x
Determine if the administration of anti-VEGFR2 CAR-engineered CD8+ 
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.
1.1.2. Secondary objective:
x
Determine the in vivo survival of CAR gene-engineered cells.
1.2.
UBackground 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 VEGFR2 on tumor vasculature 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 VEGFR2
molecule on the tumor vasculature. 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 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 


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8
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 recently 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 2006 13.  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 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-myeloabalative 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 have now treated 24 patients with metastatic 
melanoma using autologous PBL transduced with an improved MART-1 F5 TCR 
following a non-myeolablative chemotherapy.  Six patients (25%) have achieved an 
objective partial response.  Toxicities were similar to those seen in the prior TCR gene 


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9
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 antibody receptor (CAR)genes.  We have studied 
PBL transduced with TCR genes targeting p53, CEA, NY-ESO-1 and TRAIL bound to the 
DR4 Receptor, and PBL transduced with CAR targeting Her-2 and CD19. 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 response.  
The clinical responses observed in seven of the fifteen evaluable patient receiving anti-
ESO TCR-engineered peripheral blood lymphocytes have been very encouraging.  Of the 
six synovial cell sarcoma, four patients have confirmed PRs. Of the nine patients with 
highly ESO expressing melanoma, there have been 2 confirmed complete responses and 2 
confirmed partial responses. In study 09-C-0082, an impressive ongoing partial remission 
at 7 months post treatment of the lymphoma occurred in the first patient treated on this 
study.  This patient had heavily pre-treated, progressive follicular lymphoma that involved 
all major lymph node areas.  Only three small lesions that were consistent with lymphoma 
persisted on positron emission tomography (PET) imaging post-treatment.
Most toxicities observed in these studies were expected toxicities of the 
chemotherapy and aldesleukin administration. However, in 2 studies, we have observed 
serious adverse events related to the transduced cells.  In 09-C-0051 (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 
clear. 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 colitis.  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.
1.2.3.
VEGFR2 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 


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10
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.
Antiangiogenic approaches can potentially have wide application to a variety of tumor 
types, though the cytostatic nature of the available treatments and the redundancy of 
angiogenic pathways have limited its effectiveness. Redirecting immune cells to target 
tumor vasculature can combine the advantages of cell transfer and antiangiogenic 
approaches and offers an alternative to overcome the  obstacles  confronting conventional 
tumor-specific immunotherapy and the use of cytostatic antiangiogenic inhibitors. 
Most solid tumors and some hematologic malignancies are characterized by an 
angiogenic phenotype which is an absolute requirement for their survival, progression, and 
metastasis16,17,18,19,20,21. Therapeutic approaches targeting molecules involved in tumor 
angiogenesis can inhibit tumor growth.
Proliferating endothelial cells in the vessels within solid tumors aberrantly express 
high levels of angiogenic growth factors, receptors and adhesion molecules  that are absent 
or barely detectable in established blood vessels, which are normally quiescent. Among 
these, vascular endothelial growth factor (VEGF) and its receptors appear to be the 
regulators of angiogenesis responsible for the vascularization of normal and neoplastic 
tissues21,22,23 . Over expression of VEGF and its receptors (VEGFR) are strongly 
associated with tumor angiogenesis, survival, invasion, metastasis, recurrence, and 
prognosis in human cancers. VEGF stimulates angiogenesis mainly through VEGFR 
receptor-2 (known as flk1 in mouse and KDR in human), a tyrosine kinase receptor that is 
overly expressed in tumor endothelial cells and on some tumor cells24,25,26,27.
Pharmacologic approaches to inhibit VEGF or VEGFR using monoclonal antibodies or 
small molecules are of value in cancer treatment though the cytostatic nature of these 
interventions and the redundancy of angiogenic pathways have limited the curative 
potential of these treatments.
Several immunotherapeutic approaches targeting VEGFR-2 on endothelial cells 
have been used to inhibit pathological angiogenesis and tumor growth, which include 
neutralization of VEGFR-228, immunization against VEGFR-2  or coupling VEGF to 
toxins to target and destroy VEGFR-2 positive cells29 as well as disruption of VEGFR 
genes though thus far these approaches have been of limited clinical value.
Chimeric T cell receptors utilize the variable regions of an antibody as a single 
chain attached to the intracellular signaling chains of a conventional T cell receptor to 
produce a molecule that when transduced into lymphocytes enables the cell to recognize 
targets based on the antigen recognition of the antibody. The availability of monoclonal 
antibodies against VEGFR2 and the antitumor potency of ACT suggested to us that the 
transduction of a chimeric receptor recognizing VEGFR2 into lymphocytes could produce 
self-replicating T cells capable of selectively destroying tumor vasculature.
1.2.4.
Preclinical Studies to Provide the Rationale for this Clinical Protocol
We generated a series of MSGV-based recombinant retroviral vectors encoding a 
chimeric antigen receptor comprised of the single chain variable regions (ScFv) of an anti-
mouse VEGFR-2 antibody DC101 linked to the intracellular mouse T cell signaling 
sequences derived from the mouse CD28 and 4-1BB molecules, and CD39 chains of the T 
cell receptor via the mouse CD8 hinge and transmembrane regions (referred as DC101-
CD828BBZ). We also constructed a  retroviral vector lacking the 4-1BB signaling domain
(referred to as DC101-CD828Z), a vector which retains DC101 ScFv sequence but lacked 


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11
the intracellular T cell signaling sequences (referred as DC101-CD8), a vector encoding a 
ScFv directed against a synthetic hapten TNP (termed SP6-CAR) and a vector lacking any 
of the CAR sequences (termed empty vector). All of these recombinant retroviral 
constructs described are schematically represented in Figure 1A.
Surface expression of the retrovirally encoded transgene products in transduced 
CD3+ primary mouse T cells was determined by flow cytometry. As shown in Figure 1B,  
the DC101 CAR expressing vectors efficiently and consistently transduced ConA/IL-7
activated mouse T cells (range 79-86%), which were mostly (~90%) CD8+ at 5 days post 
transduction (Fig. 1HCH). The intensity of CAR expression derived from the DC101 CAR 
comprising the 4-1BB signaling moiety was consistently lower than that derived from 
vectors lacking 4-1BB sequence in all the transduction experiments (n = 5). We then 
assessed the ability of DC101-CAR engineered T cells to respond to  immobilized 
VEGFR-2 protein. While there were no differences between T cells transduced with 
various vectors to respond to immobilized anti-mouse CD3 antibody, only the T cells 
engineered with DC101-CAR containing intact intracellular signaling sequences responded 
specifically to plate bound target VEGFR-2 protein, as measured by proliferation (Fig. 1D) 
and IFN-J secretion (Fig. 1E). 
Next we determined whether DC101 CAR modified T cells could specifically 
recognize mouse cell lines expressing VEGFR-2. Mouse endothelial cell lines and tumor 
lines from various tissue origins were examined for cell surface expression of VEGFR-2
by flow cytometry. The level of VEGFR-2 varied among the cell lines tested. While all the 
transformed mouse endothelial cell lines (SVEC4-10EHR1, bEND-3, SVR, and MS1) 
showed high levels of VEGFR-2 expression, most of the tumor lines showed either low 
(MC38, 4T1, P815, and MCA205), or undetectable levels  (MC17-51, EL-4, C4198, B16-
F10, MB49, and NIH-3T3) of cell surface VEGFR-2  protein (Fig. 2A). Two of the 
VEGFR-2 negative cell lines MB49 and NIH-3T3 were stably transduced with a lentiviral 
vector expressing VEGFR-2 (termed MB49-Flk1 and 3T3Flk1) and were used as positive 
controls in subsequent in vitro T cell functional assays.
A representative experiment showing the MHC nonrestricted IFN-J response of 
mouse T cells engineered to express DC101 CARs is presented in Figure 2B. IFN-J
response was specifically detected in response to most of the VEGFR-2 positive cell lines 
tested and the amount of IFN-J secretion was highly correlated with the level of VEGFR-2
expressed on target cells (Fig. 2B inset). Reactivity was restricted to T cells expressing 
DC101-CAR containing intact T cell signaling domains. There were no significant 
differences in the performance of DC101-CAR with or without 4-1BB signaling 
sequences.
To determine the therapeutic efficacy of our tumor vasculature targeting strategy, 
DC101 CAR transduced cells were used to treat mice bearing five different established 
vascularized subcutaneous tumors. Mice received 2 daily doses of exogenous rhIL-2 for 3 
days beginning with the first day of T cell therapy. 
Results presented in Figure 3A demonstrate that T cells expressing the DC101 CAR 
were capable of mediating a significant growth inhibitory effect against the poorly 
immunogenic B16-F10 (B16) melanoma, the MC38 colon cancer, the MCA-205 sarcoma 
in C57BL/6 mice and the CT26 colon and Renca renal cancer in BALB/C mice. There 
was no antitumor effect in mice treated with empty vector transduced T cells or IL-2 alone.  
T cells expressing DC101 ScFv but lacking the intracellular T cell signaling molecules 
(Fig. 3C) or the transfer of recombinant DC101 antibody at this dose (Fig. 3A) had no 
effect on controlling the growth of established B16 tumor.  Additionally, mice treated with 


11-C-0013
12
T cells engineered with an irrelevant CAR (SP6 CAR) or an empty vector failed to induce 
tumor inhibition. The treatment effect of DC101-CAR endowed T cells against the B16 
melanoma was a direct function of the number of cells administered (Fig. 3D). Delay in 
tumor growth could be achieved with as low as 2x106 DC101 CAR transduced T cells (P=
0.008). While, DC101 CAR transduced T cells on their own self evoked a moderate 
antitumor effect in the absence of exogenous IL-2, their tumor inhibitory effect was 
significantly enhanced by rhIL-2 coadministration (P= 0.009) (Fig.4A).  
T cells transduced with DC101 CAR vector containing the 4-1BB intracellular 
signaling sequences (DC101-CD828BBZ) or lacking those sequences (DC101-CD28Z) 
performed equally well and were statistically indistinguishable (P= 0.1) in delaying the 
growth of established bulky tumors (Fig. 4A). Further, T cells transduced with either of the 
vectors was significantly enhanced by exogenous IL-2 administration (P = 0.004, 0.009 
respectively). 
To define whether 4-1BB signaling could improve the long-term persistence of 
CAR modified T cells in vivo, tumor  from two mice from each of the treatment groups 
that received DC101 CAR engineered T cells and rhIL-2 (from experiment shown in Fig. 
4A) were harvested at 30 days post T cell transfer and individually analyzed for the 
presence of DC101 CAR expressing T cells by flow cytometry (Fig. 4B). Mice treated 
with DC101-CD828BBZ CAR engineered T cells had 4-5 fold more DC101 CAR 
expressing CD3+ in the tumor than mice treated with T cells carrying DC101-CD828Z 
CAR that lacked 4-1BB. These results suggested that 4-1BB influenced the persistence of 
adoptively transferred antigen-specific T cells at the tumor site. 
Trafficking of effector T cells to the tumor site is one of the important factors 
limiting an effective antitumor treatment of adoptively transferred T cells. To determine 
the extent and kinetics of trafficking of DC101 CAR T cells to tumor, we harvested tumors 
and spleens from mice treated with either empty vector or DC101 CAR transduced 
Thy1.1T cells at various times afer adoptive therapy and performed flow cytometric 
analysis. The percentage of infused T cells that specifically expressed Thy1.1 in the total 
population of single cell preparations of spleen and the lymphocyte gated population of 
cells from tumor was determined. Representative FACS data (Fig. 5A) and pooled data 
obtained from three different mice from independent experiments (Fig. 5B) are shown. By 
day 3 adoptively transferred T cells trafficked similarly to spleen and tumor irrespective of 
their genetic modification. However, at day 6 and 9, trafficking to the tumor was far 
greater for the DC101 CAR endowed T cells.
We also examined the toxicity resulting from the administration of CAR expressing 
VEGFR-2.  No signs of morbidity or mortality were seen in C57BL/6 mice treated with 
VEGFR-2 CAR transduced syngeneic T cells under conditions that mediated significant 
anti-tumor effects. Histopathologic analysis of various organs in C57BL/6 mice treated 
with maximum numbers (2 x 107) of VEGFR-2 CAR modified T cells showed no evidence 
of treatment related toxicity, despite reports of low levels of VEGFR-2 expression in 
vascularized tissues such as kidney, retina, and pancreas. In contrast, severe toxicity was 
seen in tumor bearing BALB/c mice treated similarly with 2 x 107 DC101-CAR transduced 
syngeneic T cells. However, in BALB/c mice comparable tumor inhibitory effect was 
achieved with minimal morbidity if the number of administered T cells was reduced to 5 x
106.
Histopathologic analysis of BALB/c mice treated with 2 x 107 DC101-CAR 
transduced T cells and IL-2 revealed findings characteristic of cytokine induced 
hypotension including multifocal mild coagulation necrosis in the liver and mild hepatic 
pericholangitis and pulmonary perivasculitis, villous atrophy, villous blunting, and crypt 


11-C-0013
13
epithelial hyperplasia of the small intestine and colon. No abnormalities in the gross 
appearance of the heart, lungs, liver, kidneys, spleen, pancreas, uterus, ovaries, or brain 
were seen. 
1.2.5.
Construction and testing of retroviral vectors expressing CAR against
Uhuman KDR 
The ability of the  T cell based antivascular targeting strategy to treat established, 
poorly immunogenic vascularized tumors in mouse tumor models led us to explore the 
translation of our preclinical findings to the treatment of human tumors.  We constructed a 
set of recombinant retroviral vectors expressing CAR against the human VEGFR-2 (KDR)
using the sequences from the 1121 monoclonal antibody from Imclone Corp.30 The first 
KDR CAR vector termed KDR1121-hCD828BBZ was comprised of a ScFv derived from 
a fully human antibody KDR1121(54) specific to the human KDR antigen linked to the 
hinge and transmembrane sequences from the human CD8D, which was in turn fused to the 
intracellular sequences derived from the human CD28, 4-1BB, and CD39 chain of TCR. A 
second vector referred to as KDR1121-hCD28Z was composed of KDR1121 ScFv linked 
to the hinge and transmembrane sequences and the intracellular signaling domain of the 
human CD28 molecule followed by cytoplasmic sequences from the  CD39 chain of TCR. 
The configuration and components of each of these vectors are illustrated in Figure 6A.  
The functional integrity of these KDR CAR encoding retroviral vectors 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 KDR CAR encoding retroviral vectors transduced human PBLs at a  
high frequency resulting in 79 to 85% of the CD3+ T cells expressing the CAR on the cell 
surface (Fig. 6B). Even though both vectors could transduce human PBLs at a comparable 
rate, the MFI of CAR expression driven from KDR-CD828BBZ CAR vector possessing 
the 4-1BB signaling sequence was slightly lower (Figure 6B and C), a result similar to our 
findings with the DC101 CAR in mouse T cells (Fig. 1C). However, no differences were 
seen between these two  KDR CAR vectors transduced into human T cells when 
measuring antigen-specific proliferation (Fig. 6D) and IFN-J secretion (Fig. 6E) when the 
cells were exposed to immobilized KDR protein in vitro. 
Next we tested the ability of KDR CAR modified T cells to recognize KDR 
positive human cells as measured by specific IFN-g secretion. KDR1121-CD828BBZ and 
KDR1121-CD28Z CAR transduced PBLs from 3 different donors, 8 days post 
transduction were cocultured for 24 hours with KDR negative 293 cells or 293-KDR cells, 
a stable transfectant expressing KDR protein (Fig. 6G).  Data presented in Figure 6F 
indicated that both the vectors conferred similar levels of specificity and functionality to 
the transduced human T cells as evidenced from their ability to specifically recognize only 
the KDR expressing 293-KDR cells and not the 293 cells. 
We also determined the activity of KDR1121-CD828BBZ CAR transduced T cells 
against various primary human cells. A  panel of normal primary human endothelial and 
epithelial cells from different tissue origins and muscle myoblasts, cultured in vitro for a 
short period, were examined for KDR expression by flow cytometry. The KDR protein 
was readily detectable only in cultured primary endothelial cells and not in any of the 
primary epithelial cells and myoblasts tested (Fig. 6G). The intensity of KDR expression 
was higher in skin-derived human dermal microvascular endothelial cells (HMVEC-D) 
compared to lung-derived HMVEC (HMVEC-L) or HUVECs. Notably, the KDR CAR 


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14
transduced T  cells secreted IFN-J only in response to KDR positive endothelial cells 
irrespective of their tissue origin and failed to recognize the other primary cells tested (Fig. 
6H).
Thus based on these experiments we selected the KDR1121-hCD828BBZ vector 
for use in our human clinical protocols.
The preclinical studies provides a rationale for the translation of VEGFR2 CAR 
transduced cells for the treatment of human cancer.
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 
systems7.
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 cancers.  
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 
cells31.  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 molecule.  Seven patients were treated and one achieved a 
partial response.  These 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 leukemia. 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 


11-C-0013
15
was concluded that the adverse event may have been due to an idiosyncratic reaction to the 
combination of the cyclophosamide and cell administration. In the Surgery Branch we 
have an approved clinical protocol to treat patients with B cell lymphomas utilizing their 
autologous lymphocytes transduced with a retrovirus encoding a CAR that recognizes the 
CD19 molecule.  Thus far two patients has been treated with between 2-4x108 cells.  The 
first patient treated has exhibited a near complete response ongoing at over 7 months with 
elimination of normal B cells.  No toxicity was seen.  The second patient treated on this 
study died prior to response evaluation from H1N1 influenza infection and a a fatal 
cerebral herniation following bilateral cerebral thromboembolism resulting in brain death
15 days after treatment. This death is possibly related to the research but is not related to 
the anti-CD19 CAR transduced cells.
In a second trial a 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 
clear.  It is for this reason that in the current protocol we have dropped the dose of CAR 
transduced cells by 10,000 fold and will start a dose escalation at 106 CAR transduced 
cells. This protocol has been discontinued.
Results of pre-clinical studies conducted in the Surgery Branch in C57BL/6 mice 
treated with the VEGFR2 CAR demonstrate no toxicity at a dose of 2 x 107 DC101-CAR 
transduced syngeneic T cells.  However, in BALB/c mice the transfer of 2 x 107 CAR 
transduced cells was lethal. In BALB/c mice a tumor inhibitory effect was achieved with 
minimal morbidity if the number of administered T cells was reduced to 5 x 106. When 
CD8 purified CAR transduced cells were administered there was no toxicity seen in 
BALB/c mice given  2 x 107 cells with no diminution of the therapeutic effect. 
Histopathologic analysis of BALB/c mice treated with 2 x 107 DC101-CAR transduced T 
cells and IL-2 revealed findings characteristic of cytokine induced hypotension including 
multifocal mild coagulation necrosis in the liver and mild hepatic pericholangitis and 
pulmonary perivasculitis, villous atrophy, villous blunting, and crypt epithelial hyperplasia 
of the small intestine and colon. No abnormalities in the gross appearance of the heart, 
lungs, liver, kidneys, spleen, pancreas, uterus, ovaries, or brain were seen. In the current 
protocol therefore we will be administering CD8 purified CAR tansduced cells.
Both C57BL/6 and BALB/c mice tolerated 2 x 107 CD8 purified CAR transduced 
cells without toxicity. In the current human protocol the starting cell dose will be 1 x 106
cells or 20 fold less cells than tolerated by the mice. Since a 70 Kg human is  3000 fold 
heavier than a mouse we estimate that our starting cell dose is  60,000 fold lower than that 
safely tolerated by mice.
Clinical toxicities attributed to VEGFR targeting angiogenesis inhibitors, such as 
monoclonal antibodies and tyrosine kinase inhibitors, include hypertension, proteinuria, 
cardiovascular and cerebrovascular thromboembolisms, bleeding, increased rate of 
gastrointestinal perforations, and hypothyroidism.  These toxicities are observed after 
prolonged administration of the VEGFR targeting angiogenesis inhibitors and are 
generally easily managed with standard treatments. In the current study, we will 
administer one dose of the anti-VEGFR2 CAR transduced 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 


11-C-0013
16
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.
Prior to amendment C, we accrued 12 patients to this study, with 4 patients accrued
to cohort 7 (109 cells). Three of the 4 patients accrued to cohort 7 experienced grade 3 or 4 
elevated liver enzymes, which are known toxicities of high-dose aldesleukin and are 
described in the informed consent. After discussions with the FDA, in order to address the 
possible contribution of aldesleukin to the severity of these events,  starting with 
amendment C the initial dose-escalation schema with high-dose aldesleukin will be 
stopped and a new dose-escalation schema will start at 109 cells (cohort 8), where 
aldesleukin will be administered intravenously at low doses (72,000 IU/kg every 8h for a 
maximum of 15 doses).
2.0
1BEligibility Assessment and Enrollment
2.1.
UEligibility Criteria
2.1.1.
UInclusion Criteria
a.
Metastatic cancer with evaluable disease.
b. Patients must have previously received at least one systemic standard care (or 
effective salvage chemotherapy regimens) for metastatic 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
f.
Clinical performance status of ECOG 0 or 1.
g. Life expectancy of greater than three months.
h. 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.
i.
Serology:
1. 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 


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17
thus be less responsive to the experimental treatment and more susceptible 
to its toxicities.) 
2. 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. 
j.
Hematology:
1. Absolute neutrophil count greater than 1000/mm3 without the support of 
filgrastim.
2. WBC (> 3000/mm3).
3. Platelet count greater than 100,000/mm3.
4. Hemoglobin greater than 8.0 g/dl.
k. Chemistry:
1. Serum ALT/AST less or equal to 2.5 times the upper limit of normal.
2. Serum creatinine less than or equal to 1.6 mg/dl.
3. 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). 
m. More than 4 weeks must have elapsed since an surgical procedure at the time 
the patient receives the preparative regimen due to the inhibition of  wound 
healing observed with VEGFR targeting angiogenesis inhibitors.
2.1.2.
UExclusion Criteria 
a.
Women of child-bearing potential who are pregnant or breastfeeding because 
of the potentially dangerous effects of the treatment on the fetus or infant.
b.
Patients with known brain metastases.
c.
Patients receiving full dose anticoagulative therapy.
d.
Active systemic infections, coagulation disorders or other major medical 
illnesses of the cardiovascular, respiratory or immune system, myocardial 
infarction, cardiac arrhythmias, obstructive or restrictive pulmonary disease.
e.
Any form of primary immunodeficiency (such as Severe Combined 
Immunodeficiency Disease).
f.
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).
g.
Patients with diabetic retinopathy.
h.
Concurrent Systemic steroid therapy.
i.
History of severe immediate hypersensitivity reaction to any of the agents 
used in this study.
j.
History of coronary revascularization or ischemic symptoms.
k.
Documented LVEF of less than or equal to 45% tested in patients with:
1. History of ischemic heart disease, chest pain, or clinically significant atrial 
and/or ventricular arrhythmias including but not limited to: atrial 
fibrillation, ventricular tachycardia, second or third degree heart block.
2. Age greater than or equal to 60 years old. 


11-C-0013
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2.2.
SScreening Evaluation
UWithin 4 weeks prior to starting the chemotherapy regimen: 
a.
Complete history and physical examination, including weight and vital signs, 
and eye exam noting in detail the exact size and location of any lesions that 
exist. (Note:  patient history may be obtained within 8 weeks)
b.
Chest x-ray
c.
EKG
d.
Baseline CT of the chest, abdomen and pelvis, and brain MRI to evaluate the 
status of disease. Additional scans and x-rays may be performed if clinically 
indicated based on patients’ signs and symptoms.
e.
Pulmonary Function Testing for patients with a prolonged history of cigarette 
smoking (20 pk/year of smoking within the past 2 years) or symptoms of 
respiratory dysfunction (may be performed within 8 weeks of treatment).
f.
Evaluation of LVEF in all patients over 60 years old. LVEF will be assessed 
by either echocardiogram or MUGA scan. Patients under the age of 60 who 
have cardiac risk factors may also undergo cardiac evaluations as noted above
(e.g., diabetes, hypertension, obesity).
g.
Patients with a LVEF of less than 45% will not proceed to treatment (may be 
performed within 8 weeks of treatment).
h.
HIV antibody titer and HbsAG determination, anti HCV (may be performed 
within 3 months of chemotherapy start date).
i.
Anti CMV antibody titer, HSV serology, and EBV panel (may be performed 
within 3 months of chemotherapy start date; patients who are known to be
positive for any of the above do not need to be retested).
UWithin 14 days prior to starting the chemotherapy regimen:
a.
Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium 
total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, 
ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein, 
Total CK, Uric Acid)
b.
Thyroid panel 
c.
CBC with differential and platelet count
d.
PT/PTT
e.
Urinalysis and culture, if indicated 
UWithin 7 days prior to starting the chemotherapy regimen: 
a.
ȕ-HCG pregnancy test (serum or urine) on all women of child-bearing 
potential
b.
ECOG performance status of 0 or 1 
2.3.
URegistration Procedures:
Patients will be registered on protocol 03-C-0277 (Cell Harvest and Preparation for Surgery 
Branch Adoptive Cell Therapy Protocols) prior to CD8 purification and transduction of CD8+ 
PBL cells (either fresh or cryopreserved samples), 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.


11-C-0013
19
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 faxed to 301-
480-0757.  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.
3.0
2BStudy Implementation
3.1.
UStudy Design:
3.1.1.
UTreatment Phase:
3.1.1.1.
UCell Preparation
CD8-enriched PBL will be grown, transduced with the anti-VEGFR2 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).  CD8-enriched 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-VEGFR2 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-VEGFR2 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.
3.1.1.2.U
Phase 1 - Dose Escalation: cohorts 1-7 – prior to amendment C (now closed)
Initially, the protocol was designed to evaluate patients who would be treated with a non-
myeloablative chemotherapy regimen, VEGFR2 CAR-transduced PBL and high-dose 
aldesleukin. A total of four patients were treated in cohort 7 and no additional patients will be 
accrued to this portion of the study. Patients accrued to the new cohorts will receive non-
myeloablative chemotherapy, transduced cells and low-dose intravenous aldesleukin. The total 
number of anti-VEGFR2 CD8+ engineered cells transferred for each cohort will be:
o
Cohort 1
106 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-VEGFR2 CAR genes is between 70-90% in PBL.
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.  


11-C-0013
20
In each cohort, if the patient experiences a DLT, five 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, five 
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 there are 1 or fewer DLTs in the first cohort, the study will proceed to the second 
cohort. If a dose limiting toxicity occurs in the first cohort, that cohort will be expanded to 
n=6 patients. If two DLTs occur in the first cohort, the study will be terminated.
If interferon-gamma levels post cell infusion increase 5 fold over background and are 
greater than 100 pg/ml in the patient treated in the current cohort compared to interferon-
gamma levels post cell infusion in the patient treated at the prior dose level, the current 
cohort will be expanded to n=3 to obtain more data on this phenomenon.  If one of these 3 
patients experiences a DLT, the cohort will be expanded to six patients.
The maximum tolerated cell dose is the highest dose at which
U<U 1 of 6 patients 
experienced a DLT or the highest dose level studied if DLTs are not observed at any of the 
three 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 CD8+ PBL plus IV aldesleukin (720,000 IU/kg q8h for a maximum 
of 15 doses). 
USafety Assessments:
A one week safety assessment period following regimen completion (defined as the last 
dose of IL-2) will be conducted between each patient in every cohort, except for the last 
patient as described below.
A two week safety assessment period following regimen completion (defined as the last 
dose of IL-2) will be conducted after the last patient is treated in each cohort prior to 
enrollment into the next 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.
3.1.1.3.U
Phase 1 - Dose Escalation
(opened with approval of amendment C)
Starting with cohort 8, all subsequent cohorts will proceed in a phase 1 dose-escalation 
design, with 4 additional cohorts of n=3.  However, should 2 DLTs occur in cohort 8, the cell 
dose will be reduced (cohort 8A).  All patient will received non-myeloablative 
chemotherapy, VEGFR2 CAR-transduced cells and low-dose intravenous aldesleukin.  The 
number of VEGFR2 CAR-transduced cells for each cohort will be:
o
Cohort 8
109 cells


11-C-0013
21
o
Cohort 8A 
3 X 108 cells (cell dose de-escalation if 2 DLTs in cohort 8)
o
Cohort 9
3 X 109 cells
o
Cohort 10
1010 cells
o
Cohort 11
3 X 1010 cells
Dosing is based on total cell number rather than % transduced cells since our transduction 
efficiency with the anti-VEGFR2 CAR genes is between 70-90% in PBL.
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.  
Following a standard 3+3 design  in each cohort, if the patient experiences a DLT, the cohort 
would be expanded to 6 patients 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, 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 (MTD) prior to starting the phase II portion. If there are 1 or fewer DLTs in 
the first cohort, the study will proceed to the second cohort.  If a dose limiting toxicity occurs 
in the first cohort of this portion of the study (cohort 8), that cohort will be expanded to n=6 
patients. If two DLTs occur in cohort 8, then accrual to cohort 8A (cell dose de-escalation) 
will start and no more than 1/6 patients may experience a DLT to establish the MTD. If 2 
DLTs occur in that cohort, the study will be terminated.
The maximum tolerated cell dose is the highest dose at which
U<U 1 of 6 patients experienced a 
DLT or the highest dose level studied if DLTs are not observed at any of the four 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 CD8+ PBL plus IV aldesleukin (72,000 IU/kg q8h for a maximum of 15 doses). 
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.  
Patients may undergo a second treatment as described in Section 3.4.
USafety Assessments:
A one week safety assessment period following regimen completion (defined as the last dose 
of IL-2) will be conducted between each patient in every cohort, except for the last patient as 
described below.
A two week safety assessment period following regimen completion (defined as the last dose 
of IL-2) will be conducted after the last patient is treated in each cohort prior to enrollment 
into the next 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.


11-C-0013
22
3.1.1.4.
UPhase 2 Portion:
Similar to the Phase 1 portion, prior to receiving the engineered CD8+ 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 CD8+ PBL plus IV 
aldesleukin (72,000 IU/kg q8h for a maximum of 15 doses). 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.  Patients may undergo a second 
treatment as described in Section 3.4.
The phase 2 portion of the protocol will proceed utilizing the MTD of anti-VEGFR2 CAR 
CD8+ 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 metastatic melanoma and renal 
cancer, and cohort 2 will include all other cancer types.
See section 3.1.1.2 for the safety assessment to be conducted for any incidence of  unexpected 
grade 4 or greater toxicity.
3.1.2.
UDefinition 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, decreased 
hemoglobin, neutropenia and thrombocytopenia, due to the chemotherapy 
preparative regimen that has resolved within 21 days from the day of cell 
transfer.
o
IL-2 expected toxicities as defined in Appendix 1 and 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
Alopecia, nausea, vomiting, non-hematologic lab abnormalities that persist 
for less than 96 hours, febrile neutropenia, infection,  pain and thrombus 
related to disease or VAD
o
Expected chemotherapy toxicities as defined in the Pharmaceutical 
Information section. 
o
Grade 3 Fever
3.1.3.
UProtocol 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
Two or more patients develop a grade 3 or greater toxicity at any point in the 
VWXG\JUDGHWR[LFLW\QRWDWWULEXWDEOHWRWKHFKemotherapy preparative 
regimen or aldesleukin therapy (or circumstances unrelated to this study).


11-C-0013
23
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 DLT due to autoimmune toxicity.
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. 
3.2.
UDrug Administration:
(see table 3.2)
3.2.1.
UPreparative Regimen with Cyclophosphamide and Fludarabine
(Times are offered as examples and may be changed as long as a similar time 
relationship between administration of the drugs is maintained. Study medication 
start times for drugs given once daily should be given within 2 hours of the scheduled 
time.  All other medications should be given  +/- one hour of the scheduled time;  the 
length of administration is all +/- 15 minutes.  Administration of diuretics, electrolyte 
replacement, and hydration and monitoring of electrolytes should all be performed as 
clinically indicated – the doses and times noted below are offered only as examples.
Chemotherapy infusions maybe slowed or delayed as medically indicated)
Days  -7 And -6
6 am
Hydrate: Begin hydration with 0.9% Sodium Chloride Injection containing 10 meq/L 
of potassium chloride at 2.6 ml/kg/hr (starting 11 hours pre-cyclophosphamide and 
continuing until 24 hours after last cyclophosphamide infusion). At any time during 
the preparative regimen, if urine output <1.5 ml/kg/hr or if body weight >2 kg over 
pre-cyclophosphamide value, furosemide 10-20 mg IV maybe administered. Serum 
potassium should be monitored and treated as indicated following administration of 
furosemide.
4 pm
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. 
5 pm
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 3.
6 pm 
Begin mesna infusion at 3 mg/kg/hour intravenously diluted in a suitable diluent (see 
pharmaceutical section) 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 3.
UDay -5 to Day -1:
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 3.


11-C-0013
24
3.2.2.
UCell Infusion and Aldesleukin Administration
The anti-VEGFR2 CAR CD8+ 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 via non-filtered tubing, gently agitating the bag 
during infusion to prevent cell clumping.
UDay 0 (one to four days after the last dose of the preparative regimen):U
x
Cells will be infused intravenously (i.v.) on the Patient Care Unit over 20 to 30 
minutes (between one and four days after the last dose of fludarabine).
x
With approval of amendment C, low dose aldesleukin will be administered as 
follows. Aldesleukin as described in section 3.2.5 below.
UDay 1-4 (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
With approval of amendment C, low dose aldesleukin will be administered as 
follows. Aldesleukin as described in section 3.2.5 below.
Table 3.2
Day
-7
-6
-5
-4
-3
-2
-1
1
1
2
3
4
Therapy
Cyclophosphamide (60 mg/kg)
X
X
Fludarabine (25 mg/m2)
X
X
X
X
X
Anti-VEGFR2 CAR CD8+ PBL
X1
Aldesleukin
X2
X
X
X
X
Filgrastim3  (5 mcg/kg/day)
X
X
X
X
TMP/SMX4
X
X
X
X
X
X
X
X
X
X
X
X
160mg/800mg (example)
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 preparative regimen
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
5Continue until ANC > 1000/mm3
6In patients positive for HSV continue until CD4 > 200 X 2 
3.2.3.
UInfection Prophylaxis


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25
Note:  Other anti-infective agents may be substituted at the discretion of the treating 
investigator. 
3.2.3.1.
UPneumocystis Jirovecii Pneumonia
All patients will receive the fixed combination of trimethoprim and sulfamethoxazole [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 between days – 5 and -8.
Pentamidine will be substituted for TMP/SMX-DS in patients with sulfa allergies. It will be 
administered aerosolized at 300 mg per nebulizer within one week of chemotherapy start date
and monthly thereafter.
3.2.3.2. Herpes Virus Prophylaxis
Patients with positive HSV serology will be given valacyclovir orally at a dose of 500 mg 
daily the day after chemotherapy ends, or acyclovir, 250 mg/m2 IV q 12 hrs 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 Pneumocystitis and Herpes will continue for 6 months post chemotherapy.  If 
the CD4 count is less that 200 at 6 months post chemotherapy, prophylaxis will continue 
until the CD4 count is greater than 200 for 2 consecutive measures.
3.2.3.3. Fungal Prophylaxis 
Patients will start Fluconazole 400 mg p.o. the day after chemotherapy concludes 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.
3.2.3.4.
UEmpiric Antibiotics 
Patients will start on broad-spectrum antibiotics, either a 3rd or 4th generation cephalosporin 
or a quinolone for fever of 38.3oC once or two temperatures of 38.0oC 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.
3.2.4.
UBlood 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 with the exception of the stem cell product 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.
3.2.5.
UAldesleukin: Intravenous Administration
With approval of amendment C, aldesleukin (based on total body weight) will be 
administered at a dose of 72,000 IU/kg as an intravenous bolus over a 15 minute 


11-C-0013
26
period beginning within 24 hours of the 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. Doses 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. In addition, 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.
3.3.
UOn-Study Evaluation
Note: Please refer to section 3.3.5 for research evaluations.
3.3.1.
UPrior to starting the preparative regimen
x
Apheresis as indicated (blood specimens may be collected via venipuncture in 
patients who refuse or are unable to undergo apheresis)
x
Within 14 days prior to starting the preparative regimen, patients will have a 
complete blood count, serum chemistries performed including electrolytes, 
BUN, creatinine, liver function tests, and TBNK.  If any results are beyond 
the criteria established for eligibility, the patient will not proceed until the 
abnormalities can be resolved. 
3.3.2.
UDuring the preparative regimen: DAILY
x
Complete Blood Count
x
Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium 
total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, 
ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein, 
Total CK, Uric Acid)
x
Urinalysis
3.3.3.
UAfter 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.
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).  Please refer 
to section 3.3.5 for additional post cell infusion evaluations. 
3.3.4.
During Hospitalization:
Every 1-2 days
x
Review of systems and physical examination as clinically indicated


11-C-0013
27
x
CBC
x
Chem 20 (Sodium (Na), Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), 
Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium 
total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, 
Total Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, Uric Acid )
x
Other tests will be performed as clinically indicated.
3.3.5.
UAdditional research evaluations:
3.3.5.1 Prior to Chemotherapy
x
5 CPT tubes (8 ml each)
x
1 SST tube (8 ml)
x
1 SST tube (4 ml) daily: starting day of chemotherapy
3.3.5.2 Prior to Cell Infusion (one 8 ml SST) 
x
Blood samples for analysis for detection of RCR by PCR
x
Blood samples for analysis of the cytokines IL-6, TNF-Į, IFN-J and GM-CSF.
3.3.5.3 Post cell infusion evaluations:
x
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.
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, then weekly
(while the patient is hospitalized):
x
5 CPT tubes (8 ml each)
x
1 SST tube (8 ml)
x
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 1) cryopreserved for immunological monitoring of cell function and 2) 
subjected to DNA and RNA extraction for PCR analysis of TCR and vector copy 
number estimation.
3.3.5.4
UBiopsies
Biopsies of tumor tissue or lymph nodes may be performed but are not required during the 
course of therapy.  A maximum of three biopsies will be performed.  These biopsies will only 
be performed if they are superficial and extracavitary.
Biopsy tissue will be processed in the 
Surgery Branch Cell Production Facility in the presence of a Pathology Laboratory pathologist 
and part of all biopsy tissue will go to the Laboratory of Pathology. Studies will be performed 
to evaluate the antigen expression by the tumor and to evaluate the reactivity of lymphocytes 
grown from these biopsies. In addition the presence of transduced cells will be quantitated 
using RT-PCR for vector sequences.
3.3.5.5
UImmunological 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:
1.
Cryopreserved for immunological monitoring of cell function,


11-C-0013
28
2.
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 VEGFR2 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.
x
Blood and tissue specimens collected in the course of this research project may be 
banked and used in the future to investigate new scientific questions related to this study 
if the subject provides permission on the optional studies section of the consent 
document for protocol 03-C-0277.
3.3.5.6
UMonitoring 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. 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.
3.4.
URe-Treatment
Patients will be evaluated 4 to 6 weeks after the initial treatment regimen (defined as 
the end of the last aldesleukin dose).  If patients have a partial response to treatment 
or stable disease that subsequently progresses, they may be re-treated when 
progression by RECIST criteria is documented after evaluation by principal 
investigator with the same schedule that they had been given safely (grade III toxicity 


11-C-0013
29
due to cell infusion which is reversible within 24 hours with supportive measures may 
be retreated, but patients who develop grade IV toxicity due to cell infusion will not 
be retreated.) The duration of a partial response or stable disease to treatment must 
be greater than or equal to 2 months, as indicated by RECIST criteria, in order to be 
eligible for retreatment.  Patients must continue to meet the original eligibility criteria 
to be considered for retreatment. Retreatment will consist of the non-myeloablative 
chemotherapy regimen, cell infusion, and aldesleukin. Cells used for retreatment 
will consist of cells from the first treatment which have been cryopreserved, if 
available. If cells from the first treatment are not available, cells for retreatment will 
consist of a new lot of transduced CD8+ PBL.  Toxicity related to cyclophosphamide 
or fludarabine should be stable and resolved to less than grade 1 prior to retreatment.  
Re-treatment benefits and risks will be carefully explained to the patient.  A 
maximum of 1 retreatment course may occur. Thirty-six patients have been evaluated 
after retreatment with a non-myeloablative chemotherapy regimen and autologous 
cells in the Surgery Branch.  Five partial responses and 2 complete responses have 
been observed for an objective response rate of 19.4%.
3.5.
Post Study Evaluation (Follow-up):
3.5.1
All patients will return to the NIH Clinical Center for evaluation 6 weeks (+/- 2 weeks) 
following the administration of the cell product
3.5.2
Patients discharged with grade 3 or greater significant adverse events should be 
evaluated by the referring physician within 2 weeks of discharge. 
3.5.3
Patients who experience stable disease, a partial response, or a complete response or 
have unresolved toxicities will be evaluated as noted below:
•
Week 12 (+/- 2 weeks) 
•
Every 3 months (+/- 1 month) x3
•
Every 6 months (+/- 1 month) x 2 
•
As per PI discretion for subsequent years
Note: Patients may be seen more frequently as clinically indicated
3.5.4
At each evaluation specified above patients will undergo:
x
Physical examination 
x
Toxicity assessment, including a review of systems. 
x
Lab tests
o
Chem 20 equivalent: Sodium (Na), Potassium (K), Chloride (Cl), Total 
CO2 (bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, 
Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline 
Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin, LD, 
Total Protein, Total CK, Uric Acid 
o
Complete blood count
o
Thyroid panel as clinically indicated
o
TBNK, until CD4 > 200 X 2
x
CT of the chest, abdomen and pelvis.  This end of course evaluation will be used 
to determine tumor response. If clinically indicated, other scans or x-rays may be 
performed, e.g. brain MRI, bone scan.


11-C-0013
30
x
Visual symptoms will be evaluated and if changes have occurred from baseline, 
i.e. changes in visual acuity, an ophthalmologic consult will be performed.
x
A 5 liter apheresis may be performed at the first follow up visit. If the patient is 
unable to undergo pheresis approximately 96 ml of blood may be obtained.
Subsequently, approximately 60 ml of blood will be obtained at follow up visits 
for at least 3 months. Peripheral blood mononuclear cells will be cryopreserved so 
that immunologic testing may be performed.  
x
Detection  of RCR and persistence of CAR gene transduced cells: (see section 
3.3.6.4)
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.
3.5.5
Patients who are unable or unwilling to return for follow up evaluations will be 
followed via phone or e-mail contacts.  Patients may be asked to send laboratory, 
imaging and physician exam reports performed be their treating physician.
3.6.
Criteria for Removal from Protocol Therapy and Off Study Criteria
3.6.1
Off Treatment Criteria:
Patients will be taken off treatment (and followed until progression of disease) for the 
following
x
Completion of the treatment period
x
Dose limiting toxicity (DLT) due to cell infusion.  The definition of DLT is in 
Section 3.1.2.
x
Grade 3 autoimmunity that involves vital organs (heart, kidneys, brain, eye, 
liver, colon, adrenal gland, lungs).
3.6.2
Off Study Criteria:
Patients will be taken off study for the following:
x
The patient voluntarily withdraws
x
There is significant patient noncompliance
x
Radiographic or clinical disease progression, unless the patient is eligible for 
second treatment.
x
General or specific changes in the patient’s condition render the patient 
unacceptable for further treatment on this study in the judgment of the 
investigator.
x
Death
Note: patients must be followed until all adverse events have resolved to grade 2 or 
less with the exception of lymphopenia or alopecia.  If an event is not expected to 
resolve to grade 2 or less, this will be noted in the patient medical record and the 
patient may be taken off study. 
Note: Patients taken off study will be consented and registered on the Surgery Branch 
follow-up protocol 09-C-0161 (Follow up Protocol for Subjects Previously Enrolled 
in Surgery Branch Studies)


11-C-0013
31
4.0
3BSupportive Care
Concomitant medications to control side effects of therapy will 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 
g6h) 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 antiemetics will be administered as needed for nausea and vomiting 
uncontrolled by ondansetron.  Antibiotic coverage for central venous catheters may be 
provided at the discretion of the investigator.
5.0
4BData Collection and Evaluation
5.1.
UResponse Criteria
5.1.1.
UEvaluation 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.
5.1.2.
UEvaluation 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
-----------------------------------------------------------------------------------
U1
U
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.
U2
U
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.”
5.1.3.
UEvaluation 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.


11-C-0013
32
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
5.1.4.
UConfirmatory Measurement/Duration of Response
UConfirmation
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.
UDuration 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.
UDuration 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. 
5.2.
UToxicity Criteria
This study will utilize the CTCAE version 3.0 for toxicity and adverse event 
reporting.  A copy of the CTCAE v3.0 can be downloaded from the CTEP home page 
(http://ctep.cancer.gov).
Over 100 patients have been treated in the Surgery Branch, NCI with tumor 
infiltrating lymphocytes.  Early toxicities related specifically to the infusion of the 
cells (those which are seen immediately following cell infusion and prior to 
aldesleukin administration) are generally mild and include fevers, chills, headache, 
and malaise.  Toxicities which occur following administration of aldesleukin but are 
thought to be related to the cells include immune mediated events such as vitiligo, 
transient uveitis, hearing loss and vestibular dysfunction.  The use of the non-
myeloablative regimen prior to cell administration increases the toxicity of this 


11-C-0013
33
treatment as profound myelosuppression occurs in all patients.  In 93 patients treated 
with TIL using the non-myeloablative chemotherapy regimen with or without total 
body irradiation, there was one treatment related death (NMA + 200 cGY TBI) due to 
an unexpected but preexisting diverticular abscess.  
The standard approach to the administration of high-dose aldesleukin in all studies is 
to continue dosing until grade 3 or 4 events occur. The most commonly seen grade 4 
events are pulmonary, and renal impairment, and mental status changes.  These 
toxicities may sometimes require intubation for protection of the patient’s airway.  It 
is important to note that although these patients require significant supportive 
measures during this period, all toxicities are reversible and the overwhelming 
majority of patients have suffered no long term sequelae following this treatment 
regimen.  However, fatal complications are possible and it is therefore only 
appropriate to carry out this experimental treatment in the context of life threatening 
metastatic cancer.
To ensure safety using this treatment, the NCI SB will review safety data on all 
protocols semi-annually at the time of continuing review.  Data will be presented for 
both the recent 6 month period and for the entire length of time the protocol has been 
open.  The toxicity data for review will include all toxicities captured on the protocol 
and will be presented in individual tables as follows: 
x
all toxicities attributed to the cells, 
x
all incidences of intubation including the duration of and reason for 
intubation,
x
all grade 2 unexpected adverse events, and all grade 3 or greater events 
regardless of attribution.
Toxicities seen on protocols using this non-myeloablative regimen and aldesleukin, 
that occur during the follow up period are rare but have included EBV lymphoma 
following prolonged lymphopenia, herpes zoster infection, and sensory neuropathy 
likely related to fludarabine.  
The major discomforts of the research are those of nausea, mucositis, anorexia, 
diarrhea, fever and malaise.  Side effects of common drugs used in this 
nonmyeloablative regimen include:
Cyclophosphamide: Marrow suppression, nausea, mucositis, rash, hemorrhagic 
cystitis, myocardial damage, alopecia, infertility, nausea and vomiting, SIADH.  
Fludarabine: Myelosuppression, fever and chills, nausea and vomiting, malaise, 
fatigue, anorexia, weakness, neurologic toxicity including sensory neuropathies and 
blindness, and interstitial pneumonitis.  Serious opportunistic infections have 
occurred in CLL patients treated with fludarabine.
Antimicrobials in general: Allergic reactions, renal impairment, nausea, vomiting, 
hepatic damage, marrow suppression. 
Aldesleukin: A variety of side effects have been associated with high-dose 
aldesleukin administration in our experience and a listing of these side effects in 652 


11-C-0013
34
patients who received 1,039 treatment courses are listed in Appendix 1 and Appendix 
2. The side effects of low-dose IL-2 have been elucidated from treatment of 150 
patients in a Surgery Branch protocol (91-C-0094/T91-0053) from 1991-2001.  The 
most common side effects include nausea/vomiting, oliguria, malaise, changes in 
level of consciousness, diarrhea, hypotension, peripheral edema, elevated creatinine, 
and infection
Clinical toxicities attributed to VEGFR targeting angiogenesis inhibitors, such as 
monoclonal antibodies and tyrosine kinase inhibitors, include hypertension, 
proteinuria, cardiovascular and cerebrovascular thromboembolisms, bleeding, 
increased rate of gastrointestinal perforations, and hypothyroidism.  These toxicities 
are observed after prolonged administration of the VEGFR targeting angiogenesis 
inhibitors and are generally easily managed with standard treatments.  
DLT is defined in section 3.1.2.  
For safety purposes, the following early stopping rules will apply to this trial:
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
For additional stopping rules, see Section 3.1.3.
5.3.
UStatistical Considerations
The primary objectives of this trial is to determine safety and also to determine 
whether the combination of high-dose aldesleukin, lymphocyte-depleting 
chemotherapy, and an infusion of anti-VEGFR2 CAR-gene CD8+ 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, prior to amendment C, the protocol will enroll 1 patient in each of the first 2 
dose cohorts unless that patient experiences a dose limiting toxicity (DLT).
Following cohort 2, all subsequent cohorts will proceed in a phase 1 dose escalation 
design, with 8 cohorts of n=3. The total number of anti-VEGFR2 CD8+ engineered 
cells transferred for each cohort will be:
o
Cohort 1
106 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


11-C-0013
35
In each cohort, if the patient experiences a DLT, five 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, five 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 two DLTs occur in the first 
cohort, the study will be terminated.  If there are 1 or fewer DLTs in the first cohort, 
the study will proceed to the second cohort.  
If interferon-gamma levels post cell infusion increase 5 fold over background and are 
greater than 100 pg/ml in the patient treated in the current cohort compared to 
interferon-gamma levels post cell infusion in the patient treated at the prior dose 
level, the current cohort will be expanded to n=3 to obtain more data on this 
phenomenon.  If one of these 3 patients experience a DLT, the cohort will be 
expanded to six patients.
Following amendment C, patients will be enrolled onto cohorts 8-11, incorporating 
low dose aldesleukin and cells, as shown in 3.1.1.3. Since a standard 3+3 design will 
be used for these cohorts, up to 24 patients may be enrolled in this portion of the trial 
following the amendment approval.
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 melanoma
or renal cancer, and patients with all other histologies.
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 high dose 
aldesleukin, lymphocyte depleting chemotherapy, and anti-VEGFR2 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%.


11-C-0013
36
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 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 118 patients may be required (12 patients 
enrolled up through dose level 7, and up to 24 additional patients in 4 possible 
escalations for cohorts 8-11 after amendment C in the phase 1 portion for a 
maximumin of 36 patients in phase 1, and 2 phase 2 cohorts with a maximum of 41 
apiece). 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.  
5.4.
UData and Safety Monitoring Plan 
Careful evaluation to ascertain the toxicity, immunologic effects and anti-tumor 
efficacy of cell infusions will be performed. Due to the nature of these studies, it is 
possible that expansion of specific T-cell clones will be observed as tumor reactive T-
cell proliferate in response to tumor antigens.  Therefore, care will be taken to track 
T-cell persistence both immunologically and molecularly according to plan specified 
in Section 3.3.7.
The principal investigator will review all serious adverse events and will monitor the 
data and toxicities to identify trends monthly.  The principal investigator will be 
responsible for revising the protocol as needed to maintain safety.  The NCI IRB will 
review submitted adverse events monthly to also evaluate trends and will require a 
follow up plan from the principal investigator whenever a trend is identified.  
5.5.
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. 


11-C-0013
37
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.
5.6.
Sponsor Monitoring Plan
A detailed description of the clinical trial monitoring plan has been included in the 
initial IND submission as required.  
This trial will be monitored by personnel employed by Harris Technical Services on 
contract to the NCI, NIH. 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.
At least 25% of enrolled patients’ will be randomly selected and monitored at least 
quarterly, base on accrual rate. The patients selected will have 100% source document 
verification done. Additional monitoring activities will include: adherence to protocol 
specified study eligibility, treatment plans, data collection for safety and efficacy, 
reporting and time frames of adverse events to the NCI IRB and FDA, and informed 
consent requirements. Written reports will be generated in response to the monitoring 
activities and submitted to the Principal investigator and Clinical Director or Deputy 
Clinical Director, CCR, NCI.
Note: The collection and analysis of research labs will be monitored by the TIL lab 
and not by Harris.
5.7.
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.
5.8.
UHandling of Tissue Specimens for Research Purposes
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.  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.


11-C-0013
38
Subjects will be given the option to store biospecimens on this protocol, those 
specimens for which permission for permanent storage and future use has not been
granted will be destroyed. At the conclusion of this protocol, if additional studies are 
to be performed on any samples obtained during the conduct of this trial, a Request to 
Conduct Research for Stored Human Samples Specimens, or Data Collected in a 
Terminated NCI-IRB Protocol will be submitted.  Otherwise, specimens will be 
disposed of in accordance with the environmental protection laws, regulations and 
guidelines of the Federal Government and the State of Maryland.
Any loss or unintentional destruction of the samples will be reported to the IRB, the 
NCI Clinical Director, and the office of the CCR, NCI.
6.0
5BHuman Subjects Protections
6.1.
URationale 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.
6.2.
UParticipation 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.  
6.3.
UEvaluation 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 5.2.  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.  


11-C-0013
39
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.
6.4.
UConsent Document
Patients initially signs a consent when they agree to have PBMC obtained for study 
and growth either 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.
7.0 SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING PLAN
7.1
UDefinitions
7.1.1
UAdverse Event
An adverse event is defined as any reaction, side effect, or untoward event that occurs during 
the course of the clinical trial associated with the use of a drug in humans, whether or not the 
event is considered related to the treatment or clinically significant. For this study, AEs will 
include events reported by the patient, as well as clinically significant abnormal findings on 
physical examination or laboratory evaluation. A new illness, symptom, sign or clinically 
significant laboratory abnormality or worsening of a pre-existing condition or abnormality is 
considered an AE.  
All AEs, including clinically significant abnormal findings on laboratory evaluations, 
regardless of severity, will be followed until return to baseline or stabilization of event. 
Serious adverse events that occur more than 30 days after the last administration of 
investigational agent/intervention and have an attribution of at least possibly related to the 
agent/intervention should be recorded and reported as per sections 7.2, 7.3, 7.5.
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


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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
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:


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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
Suggests 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 Routine Data Reporting:
All information surrounding patient screening, diagnosis, tumor harvest, treatment, and follow 
up will be recorded in the patient medical record. Data will be reported via the NCI C3D 
reporting system. The investigators will be responsible for the collection, maintenance, quality 
control of the study data.  
7.2.1
Routine Adverse Event Reporting 
Following registration through 30 days after cell infusion, adverse events will be 
recorded in the source documents, reviewed by the designated data manager or research 
nurse or principle investigator and will be captured in C3D.  All events occurring during 
the treatment phase of the study will be followed until resolution to grade 2 or baseline.  
During the follow up period, only grade 3 and 4 and unexpected grade 2 events that are 
related to the treatment will be captured in C3D.
7.2.2
Exclusions to Routine Adverse Event Reporting:
Patients will be receiving multiple agents which include commercially available agents 
(fludarabine, cyclophosphamide and supportive medications) in combination with the 
investigational agents.  Therefore,  Grade 2 adverse events ‘unrelated’ or ‘unlikely 
related’ to the investigational agent, and ‘possibly’, ‘probably’ or ‘definitely’ related to 
the commercially available agents as specified in the package inserts do not require 
reporting/recording. 
During the Phase 2 component of the trial, all grade 1 and 2 events and all grade 2 events 
unrelated to the cell product events will not be reported/recorded.
7.2.3
Reporting of laboratory events
Laboratory results will be uploaded in C3D however only those grade 3 and 4 eventsthat 
support the diagnosis of a reportable adverse event or that reflect major organ function 
will be considered adverse events. For example grade 3 and 4, creatinine, liver function 
tests, hemoglobin, ANC, ALC, platelets, and lipase and amylase as indicated will be 
captured as adverse events; electrolytes, BUN, albumin, total protein, uric acid etc, and 
the remainder of the CBC differential will not be captured as adverse events. For 
hematological toxicities, the event will not be considered resolved until it reaches grade 2 
without the support of transfusions or growth factors. 
7.2.4
Reporting of non-laboratory events
For reportable expected adverse events: the adverse event start date will be the date the 
event reaches a grade 3; the event will be considered resolved once it reaches grade 2.  


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The highest grade the event reaches in that period will be considered the grade of the 
event.  
For unexpected adverse events, the adverse event start date will be the date the event 
reaches a grade 2; the event will be considered resolved once it reaches grade 1 or 
baseline. The highest grade the event reaches in that period will be considered the grade 
of the event. 
7.2.5
Reporting Infections
x
Febrile neutropenia will be captured as follows:  The start date will be the date the 
fever of 38.5 or greater was first recorded.  The end date will be the date the patient 
has been afebrile greater than 48 hours or the date the patient develops a clinically 
significant infection.
x
If a patient has a positive culture during the period of febrile neutropenia, the event 
will be captured as “infection with neutropenia” with the start date as the date the 
fever of 38.5 was first recorded.  
x
Infection will only be captured once in any given period regardless of the number of 
organisms cultured or sites involved.
x
Positive cultures seen on routine surveillance cultures with no clinical symptoms will 
not be captured as infections regardless of whether anti infective agents are given.
7.3 NCI-IRB Reporting
7.3.1
NCI-IRB Expedited Reporting of Adverse Events, Unanticipated Problems, and Deaths
The Protocol PI will report to the NCI-IRB:
x
All deaths, except deaths due to progressive disease.
x
All Protocol Deviations
x
All Unanticipated Problems
x
All serious non-compliance
Reports must be received by the NCI-IRB within 7 working days of PI awareness via iRIS.
7.3.2
NCI-IRB Requirements for PI Reporting at Continuing Review
The PI will provide a summary report of adverse events that have occurred on the protocol 
since the previous continuing review and in aggregate, and interpreted in relation to the 
risk:benefit of study participants in the narrative.
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; 


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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. 
7.3.3
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.
7.4 NIH Office of Biotechnology Activities (OBA)/Institutional Biosafety Committee (IBC) 
Reporting Criteria
7.4.1
Serious Adverse Event Reports to OBA/IBC
The Principal Investigator will notify OBA/IBC via email of any unexpected fatal or life-
threatening experience associated with the use of anti-VEGFR2 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-VEGFR2 
CAR PBL, but are not fatal or life-threatening, must be reported to NIH OBA/IBC as soon as 
possible, but not later than 15 calendar days after the sponsor’s initial receipt of the 
information. 
7.4.2
Annual Reports to OBA/IBC
The study Principal Investigator will submit a brief report annually of the progress of the trial 
within 60 days of the anniversary date that the IND went into effect unless the IND sponsor 
has been authorized to submit this report.
7.5 FDA Reporting Criteria:
7.5.1
IND Safety Reports to the FDA (Refer to 21 CFR 312.32)
7.5.1.1 Expedited reporting to the FDA
The Sponsor will notify FDA  via phone, fax, or email of any unexpected fatal or life-
threatening suspected adverse reactions as soon as possible but no later than 7 calendar days 
of initial receipt of the information using the MedWatch Form 3500a.
The Sponsor is also responsible for reporting any:
x any suspected adverse reaction that is both serious and unexpected
x any findings from clinical, epidemiological, or pooled analysis of multiple studies or 
any findings from animal or in vitro testing that suggest a significant risk in humans 
exposed to the drug
x clinically important increase in the rate of a serious suspected adverse reaction over 
that listed in the protocol or investigator brochure.
to the FDA and to all investigators no later than 15 calendar days after determining that the 
information qualifies for reporting using the MedWatch Form 3500a. If FDA requests any 
additional data or information, the sponsor must submit it to the FDA as soon as possible, but 
no later than 15 calendars days after receiving the request.


1 1- C- 0 0 1 3
4 4
7. 5. 2 
F D A A n n u al Re p orts ( R efer t o 2 1 C F R 3 1 2. 3 3 )
A re p ort will be s u b mitte d a n n uall y t o t he F D A o n t h e pr o gress of t h e tria l wit hi n 6 0 da ys of 
t he a n ni versar y date t hat t he I N D w e nt i nt o effect as i n dicate d i n 2 1 C F R 3 1 2. 3 3, a n d a n y 
ass ociate d F D A c orres p o n de nces re gar di n g t he I N D a n n ual re p ort.
8. 0 P h ar m a ce utic al I nf or m ati o n
8. 1.  
Al desle u ki n (I nterle u ki n- 2, Pr ol e u ki n, Rec o m bi n a nt H u m a n I nterle u ki n 2)
H o w S u p plie d: Al desle u ki n will be pr o vi de d b y t he NI H Cli nical P har mac y
De part me nt fr o m c o m mercial s o urces.
F or m ulati o n/ Rec o nstit uti o n:  Al desle u ki n,  is pr o vi de d as si n gl e- use vials 
c o ntai ni n g 2 2 milli o n I U (
- w hite  
l y o p hilize d ca ke pl us 5 0 m g ma n nit ol a n d 0. 1 8 m g s o di u m d o dec yl s ulfat e, b uffere d 
wit h a p pr o xi matel y 0. 1 7 m g m o n o basic a n d 0. 8 9 m g di basi c s o di u m p h os p hate t o a 
p H of 7. 5 (r a n ge 7. 2 t o 7. 8).  T he vial is  rec o nstit ute d wit h 1. 2 m L of Steril e Water 
f or I njecti o n, U S P, a n d t he res ulta nt c o nce ntrati o n is 1 8 milli o n I U/ ml or 1. 1 m g/ m L.  
Dil ue nt s h o ul d be directe d a gai nst t he si de of  t he vial t o a v oi d e x cess f oa mi n g.  S wirl 
c o nte nts ge ntl y u ntil c o m pletel y diss ol ve d.  D o n ot s ha ke.  Si nce vials c o nt ai n n o 
preser v ati ve, rec o nstit ute d s ol uti o n s h o ul d be use d wit h 2 4 h o urs.
St ora ge: I ntact vials are st ore d i n t he refri gerat or ( 2° - 8° C) pr otecte d fr o m 
li g ht.  Eac h vial b ears a n e x pirati o n date.
Dil uti o n/ Sta bilit y: Rec o nstit ute d al desle u ki n s h o ul d be f urt her dil ute d wit h 5 0 
m L of 5 % H u ma n Ser u m Al b u mi n ( H S A).  T he H S A s h o ul d be a d de d t o t he dil ue nt 
pri or t o t he a d diti o n of al desle u ki n.  Dil uti o ns of t he rec o nstit ute d s ol uti o n o ver a 
1 0 0 0-f ol d ra n ge (i.e., 1 m g/ m L t o 1 mc g/ m L) are acce pta ble i n eit her gl ass b ottles or 
p ol y vi n yl c hl ori de ba gs.  Al desle u ki n is c h e micall y sta ble f or 4 8 h o urs at refri gerat e d 
a n d r o o m te m per at ures, 2° – 3 0° C.
A d mi nistrati o n: T he fi nal dil uti o n of al desle u ki n will be i nf use d o ver 1 5 
mi n utes.  Al desle u ki n will be a d mi nistere d as a n i n patie nt.  
T o xicities: E x pecte d t o xicities of al desle u ki n are liste d i n t he pr o d uct la bel 
a n d i n A p pe n di x 1 a n d 2.  Gra de 3 t o xicities c o m m o n t o al desle u ki n i ncl u de diarr h ea, 
na usea, v o miti n g, h y p ote nsi o n, s ki n c ha n ges,  a n ore xia, m uc ositis, d ys p ha gi a, or 
c o nstit uti o nal s y m pt o ms a n d la b orat or y c ha n ges as detaile d i n A p pe n di x 1.
A d diti o nal gra d e 3 a n d 4 t o xicities see n wit h  al desle u ki n are det aile d i n A p pe n di x 2.
8. 2. 
Fl u d ar a bi ne
Descri pti o n:  ( Please refe r t o pac ka ge i nsert f or c o m plete pr o d uct I nf or mati o n)
Fl u dara bi n e p h os p hate is a s y nt hetic p uri ne n ucl e osi de t hat differs fr o m p h ysi ol o gic 
n ucle osi des i n t hat t he s u gar m oiet y is ar a bi n ose i nstea d of ri b ose or de o x yri b ose. 
Fl u dara bi n e is a p uri ne a nta g o nist a nti meta b olite. 
H o w S u p plie d:  It will be p urc hase d b y t h e NI H Cli nical P har mac y 
De part me nt fr o m c o m mercial s o urces. Fl u dara bi n e is s u p plie d i n a 5 0 m g vial as a 
fl u dara bi ne p h os p h ate p o w der i n t he f or m of a w hite, l y o p hilize d s oli d ca ke.  
Sta bilit y: F oll o wi n g rec o nstit uti o n wit h 2 m L of sterile water f or i njecti o n t o 
a c o nce ntrati o n of 2 5 m g/ ml, t he s ol uti o n ha s a p H of 7. 7.  T he fl u d ara bi n e p o w der is 
sta ble f or at least 1 8 m o nt hs at 2- 8° C; w he n rec o nstit ute d, fl u dara bi ne is sta ble f or at 


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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 3.
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 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.
8.3.
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 alkyating 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.  


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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 3.
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 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. 
8.4.
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 3.  Toxicities include nausea, vomiting 
and diarrhea. 


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8.5.
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.
8.6.
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 on day -7 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 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.
8.6.1.
Aerosolized Pentamidine in Place of TMP/SMX DS:  
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 
asthmatics; increased incidence of spontaneous pneumothorax in patients with 
previous PCP infection or pneumatoceles, or hypoglycemia. 
8.7.
Herpes Virus Prophylaxis
8.7.1.
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 the day after the last dose of fludarabine 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.      
8.7.2.
Acyclovir


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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. 
8.8.
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.
8.9.
Anti-VEGFR2 CAR transduced CD8+ 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 the Standard Operating Procedures for the growth of the PBL,
purification of the CD8+ cells and transduction with retroviral supernatant containing 
the chimeric anti-VEGFR2 CAR genes.
8.9.1.
Retroviral Vector Containing the anti-VEGFR2 CAR Gene 
The retroviral vector supernatant [PG13-KDR1121-hCD828BBZ (D11)] 
encoding a chimeric antigen receptor (CAR) directed against VEGFR2 was prepared 
and preserved following cGMP in the Surgery BranchVector Production Facility 
(SBVPF). The retroviral vector utilizes the MSGV retroviral vector backbone and 
consists of 7199 bps including the 5’ LTR from the murine stem cell virus 
(promoter), packaging signal including the splicing donor (SD) and splicing acceptor 
(SA) sites, KDR1121 (anti-KDR )-based  CAR protein  (KDR1121-CD828BBZ) 
containing a signal peptide (mouse immunoglobulin kappa chain), KDR1121 light 
chain variable region (KDR1121 VL), linker peptide, KDR1121 heavy chain 
(KDR1121 VH), CD8 (hinge and transmembrane regions), CD28 (cytoplasmic 


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49
region), 4-1BB (cytoplasmic region), and TCR zeta (cytoplasmic region), followed by 
the murine stem cell virus 3’LTR. The physical titer will be 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 Cryonix, 
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 Hhttp://bmbl.od.nih.gov/sect3bsl2.htmH.
8.10.
Support Medications
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, pruritis, constipation and urinary retention.  Consult the 
package insert for specific dosing instructions.
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 pruritis.  Consult the package insert for a complete list of all side effects.


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10.0
Tables, Figures, and Appendices:


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Table 1: Frequency and duration of objective responses in melanoma patients treated with 
standard Tumor Infiltrating Lymphocytes (TIL) in the Surgery Branch, NCI
(8/1/09)
            
Treatment*
No TBI#
Total
43
          
PR (%)
17 (39.5%)
Į   (84, 52+, 36, 29, 28, 
14, 13, 11, 8, 8, 7, 4, 3, 
3, 2, 2, 2)
CR (%)
4 (9.3%)
(80+,77+,
67+, 66+)
OR (%)
21
(48.8%) 
200cG TBI
25
11 (44.0%)
(52+, 48+, 42+, 14, 10, 
6, 5, 5, 4, 3, 3) 
2 (8.0%)
(56+, 45+)
13
(52.0%) 
1200cG TBI
25
11 (44.0%)
(33+, 26+, 26, 26+, 13, 
7, 6, 6, 5, 4, 3) 
7 (28.0%)
(36+, 19, 33+, 
32+, 27+, 26+, 
25+)
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,  21 had prior IL-2 + chemotherapy


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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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TABLE 3:
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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Figure 1: Construction and characterization of recombinant retroviral vectors expressing chimeric antigen 
receptor (CAR) targeted against VEGFR-2
A) Schematic representation of recombinant retroviral vector constructs expressing chimeric antigen receptors 
(CAR) used in this study. DC101, a single chain antibody (ScFv) comprising the heavy chain (VH) and light chain 
(VL) variable regions of a rat IgG specific to mouse VEGFR-2, (DC101; Imclone Systems Inc.) fused by a 218 bp 
linker; SP6, ScFv of Sp6 mAb directed against a synthetic hapten TNP SP6. LS, leader sequence; CD8, hinge and 
transmembrane regions from mouse CD8D; CD28, 4-1BB, and CD39, intracellular T cell signaling domains derived 
from mouse CD28, 4-1BB, and CD3 molecules.  DC101-CD8, a binding-control vector encoding DC101 ScFv fused 
to the hinge and transmenbrane regions from mouse CD8D.
B) Retroviral mediated expression of various CARs in primary mouse T cells. Enriched splenic CD3+ T cells were 
trasduced with various CAR encoding retroviral vectors shown in Figure 1A. On day 4 post transduction, cells were 
costained with anti-mouse CD8 antibody and CAR-specific reagents and FACS analysed to determine the 
expression of transgene products in CD8+ and CD8- T cell subsets. Transduction efficiency and level of expression 
of CAR in transduced mouse T cells were represented as percent transgene positive cells (% Tdxn) and mean 
fluorescent intensity of expression (MFI) respectively. Data were represented as mean ± SEM derived from 5 
different experiments.  
C) A representative FACS data of 5 experiments showing the percentage of DC101 CAR or SP6 CAR transduced 
CD8+ and CD8- T subsets in the upper right and left quadrants respectively. 
D) Antigen-specific proliferation of DC101 CAR transduced primary mouse T cells in vitro in response to 
immobilized VEGFR-2 protein. Microtiter plates were coated with BSA, soluble mouse VEGFR2-hIgG.FC fusion 
protein, or mouse anti-mouse CD3 mAb as described in material and methods. CD3 enriched mouse T cells were 
transduced with indicated retroviral vectors as described in material and methods. Four days later cells were cultured 
on antigen coated micro titer plates for 3 days. At the end of culture period, cells were pulsed with 3[H]-thymidine 
for 18 hours and analysed for thymidine incorporation as a measure of proliferation. The assays were performed in 
triplicate and values are represented as mean ± SEM.  
E) DC101 CAR transduced mouse T cells specifically secreted IFN-J in response to immobilized VEGFR-2 protein.
Transduced mouse T cells were cultured for 2 days on antigen coated microtiter plates as described above. Culture 
supernatants were harvested and analysed by ELISA for the secretion of IFN-J. The assays were performed in 
triplicate and values are represented as mean ± SEM.


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Figure 2: DC101 CAR transduced mouse T cells are functionally competent in 
generating immune response against VEGFR-2 expressing mouse cell lines.
A) Expression of VEGFR-2 (Flk-1) on mouse endothelial cells and tumor cells.
Indicated mouse cell lines were incubated sequentially with recombinant DC101 
antibody (a rat anti mouse IgG specific for mouse VEGFR-2) or isotype control 
antibody (rat IgG1), mVEGFR2-hIgG-Fc fusion protein, and PE-conjugated anti-
hIgG-FC and analysed by FACS. Filled histogram VEGFR-2 specific staining; open 
histogram, staining with isotype control antibody. Results shown are representative of 
two experiments. 
B) Primary mouse T cells transduced with DC101 CAR generated antigen-specific 
immune response when exposed to VEGFR-2 expressing mouse cell lines. Primary 
mouse T cells were transduced with various retroviral vector constructs shown in 
Figure 1A. Four days later cells were cocultured with indicated mouse cell lines. 
Culture supernatants were harvested at 24 hours post coculture and assayed for IFN-J
by ELISA. Results are presented as the mean values ± SEM of triplicate wells. 
Correlation between the IFN-J secretion by DC101 CAR engineered effector T cells 
and the mean fluorescent intensity of VEGFR-2 expression on the target cells is 
shown (inset).


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Figure 3
Figure 3:  Adoptively transferred DC101-CAR engineered mouse T cells inhibit multiple 
types of established syngeneic tumors in vivo. (A) Mice bearing established subcutaneous
tumors were sublethally irradiated at 5 Gy TBI and injected with 2 x 107 DC101-CAR (closed 
triangle) or empty vector (closed squares) transduced syngeneic mouse T cells in conjunction 
with rhIL-2. Control groups received rhIL-2 alone (closed diamond) or no treatment (closed 
circle). Mice bearing CT26 or Renca tumors were treated with 5 x 106 lymphocytes.
(B) Anti-tumor effect of DC101-CAR transduced T cells was cell-mediated and not due to an 
antibody effect. One group in this experiment received a single dose of 800 μg/mouse DC101 
antibody (open triangle) or rat IgG1 control antibody (open diamond) plus rhIL-2.
(C) C57BL/6 mice bearing subcutaneous B16-F10 tumor were treated with T cells transduced 
with DC101-CAR (closed triangle) or SP6-CAR vector (closed diamond) containing intact 
mouse intracellular signaling sequences 28BBZ, DC101-CD8 vector that lacked all the 
signaling domains (open triangle), or an empty vector (closed square) plus rhIL-2 or were 
untreated (closed circle). 
(D) C57BL/6 mice bearing B16-F10 tumor were treated with 2 x 107 (solid line with closed 
triangle), 1 x 107 (solid line with open triangle), 5 x 106 (dashed line with closed triangle), or 2 x 
106 (dashed line with open triangle) syngeneic T cells transduced with DC101-CAR (DC101-
CD828BBZ) plus rhIL-2. Control groups received 2 x 107 T cells transduced with an empty 
vector plus rhIL-2 (solid square), rhIL-2 alone (solid diamond), or no treatment (solid circle). 


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Figure 4: Impact of exogenous rhIL-2, 4-1BB signaling and host lymphodepletion on the 
tumor treatment effect of DC101 CAR transduced T cells. (A) DC101-CAR transduced T 
cells required exogenous rhIL-2, but not 4-1BB signaling for effective tumor treatment.
C57BL/6 mice bearing subcutaneous B16-F10 tumor received 2 x 107 syngeneic T cells 
transduced with DC101-CAR containing the 4-1BB intracellular signaling domain (DC101-
CD828BBZ, diamond) or lacking 4-1BB (DC101-CD828Z; triangle) or an empty vector 
(square). Control groups received no T cell therapy (circle). Groups shown in closed symbols 
received exogenous rhIL-2 administration and those represented in open symbols did not 
receive rhIL-2.
(B) 4-1BB signaling enhanced the persistence of DC101-CAR modified T cells in vivo. Tumor 
samples from two mice treated with DC101-CAR transduced T cells plus rhIL-2 shown in Fig. 
4A were harvested on day 30 post T cell transfer. The low density cell fraction was prepared 
from each of the tumor samples and the cell surface expression of DC101-CAR was 
determined by FACS. The percent CD3+ T cells expressing DC101-CAR in the lymphocyte 
gated region of the forward and side scatter profiles is shown in the upper right quadrants. 
(C) Impact of host lymphodepletion on in vivo tumor therapeutic effect of DC101-CAR 
engineered T cells. C57BL/6 mice bearing subcutaneous B16-F10 tumors received 2 x 107
syngeneic T cells transduced with DC101-CAR (triangle), SP6-CAR (diamond), or an empty 
vector (square) plus rhIL-2, or not treated with T cells (circle). Mice in groups represented in 
closed symbols received 5 Gy TBI prior to T cell transfer.


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Figure 5
Figure 5: Enhanced tumor infiltration of adoptively transferred DC101 CAR 
transduced T cells in mice bearing established B16-F10 tumor 
C57BL/6 mice were subcutaneously injected with 2 × 105 B16-F10 tumor cells. On 
day 10 or 12, mice were irradiated at 5 Gy and treated intravenously with 20 × 106
DC101-CD828BBZ CAR or empty vector transduced Thy1.1+ syngeneic T cells in 
conjunction with rhIL-2. At indicated time points, tumors and spleens from individual 
mouse from each group were excised and processed to obtain single cell suspensions 
and analysed by flow cytometry. The percentage of CD3+ Thy1.1+ cells in the 
lymphocyte gated population of leukocyte fraction of tumor and total splenocytes was 
determined. 
A) A representative FACS data from 3 experiments with similar results.
B) Pooled data obtained from three different mice from independent experiments 
represented as mean ± SEM. 
C-E) Tumor samples were obtained from C57BL/6 mice bearing B16-F10 tumor 
treated with DC101-CD828BBZ CAR or empty vector transduced T cells in 
conjunction with rhIL-2 on day 4 post T cell therapy. Tumors sections were stained 
with FITC conjugated antibody against Thy1.1 antigen expressed on transferred T 
cells or PE-conjugated antibody against the endothelial cell marker CD31 together 
with DAPI to show the nucleuos and analysed using fluorescence microscopy. 


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Figure 6: Construction and characterization of retroviral vectors encoding CAR 
targeted against human VEGFR-2
A) Schematic representation of KDR CAR expressing retroviral vectors. KDR1121, a single 
chain antibody (ScFv) comprising the variable regions of light chain (VL) and heavy chain 
(VH) of fully human IgG specific to human KDR antigen (64) fused by a 218 bp linker; LS, 
leader sequence from human GM-CSF; CD8, hinge and transmembrane regions from human 
CD8D; CD28, 4-1BB, and CD39, intracellular T cell signaling domains derived from human 
CD28, 4-1BB, and CD3 molecules respectively.  
B) Retroviral mediated expression of various CARs in primary human T cells. OKT3 
activated human PBLs were transduced with various CAR encoding retroviral vectors shown 
in Figure 6A or mock transduced. On day 5 post transduction, cells were costained with anti-
human CD3 antibody and KDR CAR-specific reagents and FACS analysed. KDR CAR 
expression was detected by staining with soluble KDR-hIgG-Fc fusion protein followed by 
PE-conjugated anti human IgG-FC (DhIgG.FC). Transduction efficiency and level of 
expression of KDR CAR in transduced primary human T cells are represented as percent 
transgene positive cells (% Tdxn) and mean flurescent intensity of expression (MFI),
respectively. Data are represented as mean ± SEM of data derived from 3 different donors.  
C) A representative FACS data from 3 different donors transduced similarly with KDR1121-
CD828BBZ or KDR1121-hCD28Z CAR encoding retroviral vectors showing the percentage 


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of KDR CAR+CD3+ T cells and KDR CAR-CD3+ T cells in the upper right and lower right 
quadrants respectively. 
D) Antigen-specific proliferation of KDR CAR transduced primary human T cells in vitro in 
response to immobilized KDR protein. Microtiter plates were coated with BSA, soluble 
KDR-hIgG.FC fusion protein, or anti-human CD3 mAb. OKT3 stimulated human PBLs were 
transduced with indicated retroviral vectors or mock transduced as described in Figure 6B. 
Seven days later cells were cultured on antigen coated microtiter plates for 3 days. At the end 
of culture period, cells were pulsed with 3[H]-thymidine for 18 hours and analysed for 
thymidine incorporation as a measure of cell proliferation. The assays were performed in 
triplicate and values are represented as mean ± SEM.
E) KDR CAR transduced human T cells specifically secreted IFN-J in response to 
immobilized KDR protein. Transduced human PBLs were cultured for 2 days on antigen 
coated microtiter plates as described above. Culture supernatants were harvested and 
analysed by ELISA for the secretion of IFN-J. The assays were performed in triplicate and 
values are represented as mean ± SEM. 
F) KDR CAR transduced human T cells specifically secreted IFN-J in response to KDR 
expressing 293 cells. Primary human lymphocytes obtained from 3 different donors were 
transduced with KDR1121-hCD828BBZ or KDR1121-hCD28Z encoding retroviral vectors 
or mock transduced. Eight days later, 105 transduced T cells were cocultured with 105 
indicated human cells in 96 well microtiter plates in 200 Pl culture volume/well. Culture 
supernatants were harvested at 24 hours post coculture and assayed for IFN-J by ELISA. 
Results are presented as the mean values of duplicate wells.
G) KDR expression on various normal primary human cells. Indicated human cell types were 
stained with PE- conjugated mouse anti-human KDR antibody or isotyped matched IgG1 
control antibody and analysed by FACS. Filled histogram KDR-specific staining; open 
histogram, staining with isotype control antibody. Results shown are representative of two 
experiments. 
H) Primary human T cells transduced with KDR CAR generated antigen-specific immune 
response when exposed to KDR expressing human cells. Primary human T cells were mock 
transduced or transduced with KDR1121-hCD828BBZ CAR expressing retroviral vector.
Eight days later cells were cocultured with indicated human cells. Culture supernatants were 
harvested at 24 hours post coculture and assayed for IFN-J by ELISA. Results are presented 
as the mean values ± SEM of triplicate wells.


11-C-0013
64
Appendix 1:
$'9(56((9(1762&&855,1*,12)3$7,(17675($7(':,7+
ALDESLEUKIN (n=525)1
Body System
% Patients 
Body System
   % Patients
Body as a Whole
Metabolic and Nutritional Disorders
Chills 
52
Bilirubinemia 
40
Fever 
29
Creatinine increase 
33
Malaise 
27
Peripheral edema 
28
Asthenia 
23
SGOT increase 
23
Infection 
13
Weight gain 
16
Pain 
12
Edema 
15
Abdominal pain 
11
Acidosis 
12
Abdomen enlarged 
10
Hypomagnesemia 
12
Cardiovascular 
Hypocalcemia
11
Hypotension 
71
Alkaline phosphatase incr
10
Tachycardia 
23
Nervous
Vasodilation 
13
Confusion 
34
Supraventricular tachycardia 12
Somnolence 
22
Cardiovascular disordera
11
Anxiety 
12
Arrhythmia 
10
Dizziness 
11
Digestive 
Respiratory
Diarrhea 
67
Dyspnea 
43
Vomiting 
50
Lung disorderb
24
Nausea 
35
Respiratory disorderc
11
Stomatitis 
22
Cough increase 
11
Anorexia 
20
Rhinitis 
10
Nausea and vomiting 
19
Skin and Appendages
Hemic and Lymphatic
Rash 
42
Thrombocytopenia 
37
Pruritus 
24
Anemia 
29
Exfoliative dermatitis 
18
Leukopenia
16
Urogenital
Oliguria 
63
a Cardiovascular disorder: fluctuations in blood pressure, asymptomatic ECG changes, 
CHF.
b Lung disorder: physical findings associated with pulmonary congestion, rales, rhonchi.
c Respiratory disorder: ARDS, CXR infiltrates, unspecified pulmonary changes.
1Source: Proleukin® Prescribing Information – June 2007


11-C-0013
65
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


11-C-0013
66
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.


11-C-0013
67
Appendix 3:  Interleukin-2 toxicities observed in patients treated at the NIH Clinical Center


11-C-0013
68
Appendix 4
Certificate of Analysis:
Infused T cells transduced with anti-VEGFR2 CAR
Date of preparation of final product:
Patient:
Tests performed on final product:
Test
Method
Limits
Result
Initials/
Date
Cell viability1
trypan blue exclusion
>70%
Total viable cell 
number1
visual microscopic count
Between 106 and 
5 x 1010 cells
Identity
FACs 
> 80 % CD3+CD8+ 
after transduction on 
REP cells
Tumor reactivity2
J-IFN release vs. 
VEGFR2 cell line
>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
<5 E.U./kg
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.
Prepared by:
_________________________________________
Date: ____________________
QC sign-off:
__________________________________________
Date:  ____________________
Qualified Clinical or Laboratory 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: 
11-C-0013 
PRINCIPAL INVESTIGATOR: 
Steven A. Rosenberg, M.D., Ph.D. 
STUDY TITLE:  
Phase I/II Study of Metastatic Cancer Using Lymphodepleting Conditioning Followed by 
Infusion of Anti-VEGFR2 Gene Engineered CD8+ Lymphocytes  
Continuing Review Approved by the IRB on 03/09/15 
Amendment Approved by the IRB 07/15/15 (L) 
Date posted to web:  07/21/15 
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?  
Why are you being asked to take part in this study?  
 
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 


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: 11-C-0013 
CONTINUATION: page 2 of 15 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 
 
 
grow them in the laboratory.  We will be using the anti-VEGFR2 gene and a type of virus 
(retrovirus) in making these special cells (anti-VEGFR2 CD8+ cells). The anti-VEGFR2 CD8+ 
cells target the vascular endothelial growth factor receptor 2.  This receptor is located in cells in 
tumors that are involved in supplying blood to your tumor which may result in the growth of 
your tumor.  By blocking VEGFR2, 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- VEGFR2 CD8+ 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. 
 
This highly experimental regimen is explained below. 
 
How many people will take part in this study? 
Up to 118 patients may participate in this study. 
 
Description of Research Study 
 
This study has 5 stages outlined below: 
 
What 
Timeframe 
Location 
Comments & Instructions 
Work up 
1-2 weeks 
Inpatient and 
out patient   
Scans, x-rays, labs leukapheresis other tests 
as needed 
Chemotherapy 
(day -7 to -1) 
1 week 
Inpatient 
Receive IV chemotherapy to prepare your 
immune system for the cells 
Cells and IL-2 
(Day 0) 
1-5 days 
Inpatient and 
possibly ICU 
Receive anti-VEGFR2 CD8+ cells, and 
then IL-2 about every 8 hours for up to 15 
doses 
Recovery 
1-2 weeks 
Inpatient unit 
Recover from the effects of chemotherapy 
and IL-2. 
Follow -up 
Ongoing until 
disease 
progression 
Outpatient 
Return to clinic for physical exam, review 
of side effects, labs, scans every 1-6 
months 
 
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-VEGFR2 CD8+ cells.  There will be 11 dose levels of anti-VEGFR2 CD8+ cells. 
The first patients enrolled get the smallest dose and the dose is increased when a level has been 
determined to be safe.  Discuss with your doctor which dose of anti- VEGFR2 CD8+ cells you 
will be receiving.  The first twelve patients treated on this study received high dose IL-2 as part 


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: 11-C-0013 
CONTINUATION: page 3 of 15 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 
 
 
of the treatment.  However, in 3 of the 4 patients treated at the highest dose of cells, blood tests 
indicated that liver function was abnormal.  Since high dose IL-2 can cause abnormal liver 
function, we have decided to reduce the dose of IL-2 to see if this prevents abnormal liver 
function.  You will receive a low dose of IL-2 as part of your treatment.   
 
The major side effects of this experimental regimen (described in detail on pages 8 and 9) 
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.   
What will happen if you take part in this research study?   
Before you begin the study 
 
Cell harvest and growth 
You will undergo apheresis to obtain certain types of blood cells from you. These cells (anti 
VEGFR2 CD8+ cells) will be grown in the lab and genetically modified to recognize a protein 
on your tumor cells. If your cells do not grow, 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. 
 
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 will also have a large catheter inserted into a vein and 
leukapheresis will be performed.  You may be admitted to the hospital for these tests. 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 


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: 11-C-0013 
CONTINUATION: page 4 of 15 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 
 
 
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. In addition to the leukapheresis you will undergo as part of your work up, we will also 
ask you to undergo one additional pheresis procedure between 4 and 6 weeks after your cell 
regimen to see the impact of this therapy on the immune system and see if cells we gave you are 
still active. 
 
Chemotherapy Regimen (Day -7 through Day -1) 
After we have grown the anti-VEGFR2 CD8+ 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-CD8+ 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 -5 through Day -1).  The side effects of these medicines are 
described on the following pages. 
 
After you have completed the chemotherapy regimen you will receive the cell infusion 
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-VEGFR2 CD8+ 
cells.  The anti- VEGFR2 CD8+ cells will be given in your catheter over 20-30 minutes.  Within 
24 hours after the anti-VEGFR2 CD8+ 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 you active for as long as possible so they will fight your tumor.  The IL-2 will be given 
as a 15-minute infusion about 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. 
 


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: 11-C-0013 
CONTINUATION: page 5 of 15 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 
 
 
The day after you receive the anti- VEGFR2 CD8+ 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.  
 
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 5 to 10 days; however, you may need to stay in the hospital for longer 
than 10 days 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. 
 
We may ask you to allow us to perform a biopsy (remove a small piece) of your tumor or lymph 
node after receiving the regimen to look at the effects of the regimen on the immune cells in your 
tumor.  However, this biopsy is not required for you to participate in this experimental study.  To 
obtain cells by a biopsy, a small area of skin is numbed with an anesthetic and a small piece of 
your tumor is removed, either with a needle or by a small cut in the tumor.  The area is covered 
with a bandage for a day or two, during which time we will ask you to keep it dry.  
 
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 4 – 6 weeks after completing your regimen for 
evaluation.  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.  
 
Retreatment 
 
If your tumor shrinks or disappears following the initial regimen and then recurs you may 
receive one additional regimen if you tolerated the regimen well and if all the side effects have 
resolved.  If you are retreated, you will receive the same medications on the same schedule as 
with the first regimen.   
 
 
 


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: 11-C-0013 
CONTINUATION: page 6 of 15 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 
 
 
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 study 
treatment and for four months after you finish study treatment.  If you think that you or your 
partner is pregnant, you should tell your study doctor or nurse at once. 
 
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 transfer, 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 the regimen 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 will ask 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 regimen approach.  Please advise your 
family of this request. 
 
Alternative Approaches or Treatment 
What other choices do I have if I do not take part in this study?  
 
If there are effective salvage regimens (regimens used when standard regimens have failed), you 
will be directed to undergo these regimens before participating in this protocol. 


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: 11-C-0013 
CONTINUATION: page 7 of 15 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 
 
 
Other options for treatment of your cancer include: 
 
• Standard therapies (such as hormone therapy and/or systemic chemotherapy with or 
without Herceptin for treatment of metastatic breast cancer); 
• experimental vaccines;  
• experimental chemotherapies; or biotherapies (such as ipilimumab or tremelimumab);  
• other combination therapies;  or 
• getting no treatment; getting comfort care, 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.  
 
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.  
 
During the leukapheresis procedure, you may have some tingling in your face and lips due to the 
medicine used to keep your blood from clotting during the procedure.  The nurses may give you 
a calcium containing antacid, like TUMS to chew that takes away this tingling.  Rarely, people 
may experience lightheadedness or dizziness.  We ask that you eat prior to the procedure to 
prevent this.  Rare complications of this procedure are lowered blood pressure or bleeding. 
 
Discomfort due to a biopsy may include pain at the site of the biopsy, swelling, bruising, and 
infection. 
 
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-VEGFR2 CD8+ Cell Infusion (gene transfer)  
 
The cells we will be giving you have a type of virus (retrovirus) put into them along with the 
anti-VEGFR2 protein.  Although this retrovirus is not active, there is the rare possibility that it 


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: 11-C-0013 
CONTINUATION: page 8 of 15 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 
 
 
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-VEGFR2 CD8+ 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  
• 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 another clinical trial with a similar type of cell, a patient died due to an immune 
type reaction from the cells.   
 
• Several drugs and antibodies which target VEGFR2 have been used to treat patients 
in other studies.  Some of the side effects seen in these studies include high blood 
pressure, blood clots, stroke, increased bleeding, abdominal pain, hoarseness, and 
fatigue.  
  
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. 
 
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 


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: 11-C-0013 
CONTINUATION: page 9 of 15 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 
 
 
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. 
 
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 (Two patients on 
other studies in the 
Surgery Branch developed 
EBV lymphoma, and both 
died as a result of this 
disease.) 
 
Loss of fertility 
 
 
 


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: 11-C-0013 
CONTINUATION: page 10 of 15 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 
 
 
Support Medications – side effects 
Common 
Less common 
Rare 
Filgrastim (To increase production of white blood cells) 
 
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 
 
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 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: 11-C-0013 
CONTINUATION: page 11 of 15 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.  
 
Potential Benefits of Participation 
Are there benefits to taking part in this study? 
It is possible that your tumors may shrink as a result of this experimental regimen, but it is not 
possible to predict whether this will occur.  It is likely that there will be no long-term benefit for 
participating in this study but that the information derived from this study could benefit future 
patients.  
 
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: 11-C-0013 
CONTINUATION: page 12 of 15 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 (Steven A. Rosenberg, M.D., Ph.D.)  
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 the Study  
Your doctor may decide to take you off this study under the following circumstances: 
 
• if he/she believes that it is in your best interest 
• if your disease comes back during treatment 
• if you experience side effects from the treatment that are considered too severe 
• if new information becomes available that shows that another treatment would be better for 
you 
 
In this case, you will be informed of the reason for that decision. 
 
You can stop participating at any time. However, if you decide to stop participating in the study, 
we encourage 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. 
 
Conflict of Interest 
 
The National Institutes of Health 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 additional information or a copy of the Protocol Review Guide. Members of the research 


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: 11-C-0013 
CONTINUATION: page 13 of 15 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 
 
 
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 have developed the cell 
product being used in this study.  This means it s possible that the results of this study could lead 
to payments to NIH scientists and to the NIH.  By law, government scientists are required to 
receive such payments for their inventions.  You will not receive any money from the 
development of the cell product.     
 
Optional Biopsy 
The biopsy to be performed is exclusively for research purposes and will not benefit you.  It 
might help other people in the future.  Even if you sign “yes” to have the biopsy you can change 
your mind at any time.  Please read each sentence below and think about your choice.  After 
reading each sentence, circle and initial the answer that is right for you.  The decision to 
participate in this part of the research is optional, and no matter what you decide to do, it will not 
affect your care.   
 
I agree to have the tumor biopsy for research tests in this study 
 
Yes 
 
 
No 
 
 
Initials__________  
 


MEDICAL RECORD 
CONSENT TO PARTICIPATE IN A CLINICAL RESEARCH STUDY 
• 
Adult Patient or 
• Parent, for Minor Patient 
STUDY NUMBER: 11-C-0013 
CONTINUATION: page 14 of 15 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:  301-496-4164. You may also call the Clinical Center Patient Representative at 301-
496-2626. 
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: 11-C-0013 
CONTINUATION: page 15 of 15 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 MARCH 9, 2015 THROUGH MARCH 8, 2016 
 
 
 
 
 
 
 
 
Signature of Investigator 
Date 
Signature of Witness 
Date 
 
 
Print Name 
 
 
Print Name