Conditions: Metastatic Melanoma
Keywords: Adoptive transfer therapy Dendritic cell vaccines
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 14
Healthy volunteers: no

Brief summary:
The purpose of this phase 2 study is to find the best way to give this new experimental regimen and determine if it can treat metastatic melanoma in humans. In this phase 2 study, the experimental products are given initially to a group of 8 people. If safe and found to have significant anti-tumor activity, it will be given to up to 14 other people, for a total of 22 people in this study. Physicians watch subjects carefully for any harmful side effects. Although the experimental regimen has been well tested in laboratory and animal studies, and a similar regimen has been given to a group of patients at the National Cancer Institute in Bethesda, MD, the side effects in people cannot be completely known ahead of time. This protocol is offered only to people whose condition cannot be helped by other known treatments.

The study procedures will start with the collection of white blood cells through apheresis (a procedure in which blood is drawn from a patient and separated into its components, some of which are retained, such as white blood cells, and the remainder returned by transfusion to the patient).

Subjects will be asked to undergo two aphereses, one to make the gene-modified MART-1 TCR CTLs (cytolytic T lymphocyte) and the dendritic cell vaccines, and a second one after the subject receives the gene modified cells to later study them in the blood.

On the day of the first apheresis, subjects will be admitted to the hospital and will receive chemotherapy over the next five days which decreases the risk of rejection of the transferred cells by the subject's immune system and facilitates their expansion and attack of the melanoma lesions. During this time, the gene-modified MART-1 TCR CTLs and the dendritic cells will be manufactured in the laboratory from the apheresis product and will be extensively tested to assure that they express the appropriate TCR and that they do not contain any contaminating bacteria or virus. Then the gene-modified MART-1 TCR CTLs will be given back to the subject through a vein in the arm. It will be followed by vaccination with the dendritic cells under the skin. During the next fourteen days, subjects will also receive interleukin 2 (IL-2), which is a standard treatment for patients with metastatic melanoma. During the next 2 to 3 weeks, subjects will stay in the hospital until the study investigators determine that the subject has fully recovered from all of the procedures, and it is safe for the subject to go home. Chemotherapy frequently causes a decrease in the platelet or red blood cells, and therefore subjects may require platelet and/or red blood cell transfusions.

Detailed description:
This is a two-stage phase II clinical trial with the combined primary endpoints to determine the safety, feasibility and anti-tumor activity of adoptive transfer of peripheral blood mononuclear cells (PBMC) genetically engineered to express the alpha and beta chains of a high affinity T cell receptor (TCR) specific for the HLA-A\*0201-restricted MART-1 melanoma tumor antigen to patients with locally advanced or metastatic melanoma. This gene transfer will be facilitated by a retroviral vector pseudotyped with a gibbon ape leukemia virus (GaLV) envelope. The two transgenes are linked by a picornavirus 2A sequence. Their expression is driven by the retroviral long terminal repeat (LTR).

Patients with MART-1-positive locally advanced or metastatic melanoma who are HLA-A\*0201-positive, and HIV, hepatitis B and C seronegative, will receive a non-myeloablative but lymphocyte depleting chemotherapy conditioning regimen consisting of cyclophosphamide and fludarabine, and then receive the adoptive transfer of autologous PBMC transduced with the MSGV1-F5AfT2AB retroviral vector, which expresses a high affinity TCR for the MART-1 melanoma antigen (MART-1 F5 TCR). The cell dose will be up to 10\^9 autologous PBMC transduced with the MSGV1-F5AfT2AB retroviral vector. The transgenic T cells will be infused fresh on the day of harvest as done in the last three patients within this protocol, prior to which, thawed cryopreserved cells were infused. Following adoptive cell transfer, patients will receive MART-1.26-35 peptide-pulsed dendritic cell (DC) vaccines and low dose interleukin-2 (IL-2).

The MART-1 F5 TCR was provided by Dr. Stephen A. Rosenberg from the Surgery Branch, National Cancer Institute (NCI). The MART-1 F5 TCR is derived from the DMF5 tumor infiltrating lymphocyte (TIL) clone, and was selected from several MART-1-specific TCRs because of its high affinity and biological activity. This TCR delivered by the same retroviral vector is currently in clinical testing at the Surgery Branch/NCI. Both the NCI clinical trial and the trial at University of California at Los Angeles (UCLA) are based on the same retrovirus expressing the MART-1 F5 TCR used to transduce whole PBMC and re-infused to patients after a non-myeloablative but lymphodepleting chemotherapy conditioning regimen. Major differences between both clinical trials include the shorter ex vivo expansion of TCR transduced PBMC, the use of MART-126-35 peptide pulsed DC and the use of positron imaging tomography (PET) for non-invasive imaging of adoptively transferred TCR transgenic cells in the UCLA clinical trial.

The primary endpoints will be safety, feasibility and objective tumor response. The phase II clinical trial design will have two treatment stages following a Simon optimal two-stage clinical phase II clinical trial design 1. The clinical trial will have an initial stage with 8 patients followed by a second stage with up to 22 patients.

Safety will be determined in stage one, and if 3 out of 8 patients have MART-1 F5 TCR-induced dose limiting toxicities (DLT), then further accrual will not be warranted. Feasibility will be also determined in the first stage, and if 3 out of 8 patients cannot receive the intended cellular therapies, or if they result in suboptimal TCR transgenic cell in vivo persistence, further accrual will not be warranted to the protocol as currently designed. Objective tumor responses will be determined by RECIST objective response criteria with a design to rule out a 10% response rate as the null hypothesis, and a 35% response rate as the alternative hypothesis. With this statistical design, if 2 or more of 8 patients in stage one have an objective response, the study will proceed to stage two and accrue a total of 22 patients. If 5 or more patients in the overall study have a complete response (CR) or partial response (PR), which combined result in the objective response rate, the study will be declared positive.

Secondary study endpoints are transgenic T cell persistence in humans and their ability to home to MART-1 positive melanoma metastasis. Analysis will be performed by sampling of peripheral blood and tumor deposits for T cell persistence and by non-invasive metabolic imaging using PET scans.

Arms:
arm 1: F5 TCR transgenic cell adoptive transfer therapy

Interventions: intervention 1: F5 TCR transgenic cells and MART-1 peptide pulsed dendritic cells intervention 2: non-myeloablative conditioning chemotherapy
Intervention descriptions: intervention 1: After chemotherapy, patients receive up to 1 x 10(9) MART-1 F5 TCR transgenic T cells infused i.v., 1 x 10(7) MART-1 peptide pulsed dendritic cells intradermally, and low dose IL-2 500,000 IU/m2 s.c. twice daily for 14 days. intervention 2: Patients receive non-myeloablative conditioning chemotherapy with Cyclophosphamide 60 mg/kg/day x 2 days and Fludarabine 25 mg/m2/day i.v. over 30 minutes for 4 days

Protocol document:
Prot_SAP_000.pdf: 


MART-1 F5 TCR Protocol 01/10/2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2 
TABLE OF CONTENTS 
 
 
 
1.0 
Study Synopsis 
1.1 Overview 
1.1.1 Cell Dose Escalation within Amendment 6 after Meeting the Safety and Feasibility 
Criteria But Not Meeting the Initial Antitumor Criteria  
1.1.2 Cell Dose De-escalation and Change to Low Dose IL-2 with Amendment 11 After 
Exceeding the Safety Criteria Under Amendments 6-10 
1.2 Rationale and Hypothesis 
1.3 Study Objectives 
1.4 Study Population 
1.5 Number of Patients   
1.6 Study Duration 
1.7 Study Schematic  
1.8 Experimental Study Agents  
1.8.1 Retrovirus vector MSGV1-F5AfT2AB 
1.8.2 MART-126-35 Peptide Pulsed Dendritic Cells 
1.9 Timing of Events 
 
2.0 
Schedule of Events 
2.1 Schedule of Events for Repeated Dendritic Cell Vaccination 
 
3.0 
Table of Abbreviations 
 
4.0 
Study Objectives 
4.1 Primary: 
4.1.1 Safety  
4.1.2 Feasibility 
4.1.3 Clinical Response 
4.2 Secondary 
4.2.1 MART-1 F5 TCR Transgenic T Cell Persistance  
4.2.2 In Vivo Imaging 
 
5.0 
Background 
5.1 Current management of surgically-incurable melanoma 
5.2 Rationale for immune-based therapies for metastatic melanoma.   
5.3 MART-1 melanoma antigen as target for melanoma immunotherapy 
5.4 Why Gene Therapy? 
5.5 TCR gene therapy 
5.6 Retroviral Vector-based Human Gene Medicine 
5.7 Safety and Regulatory Issues Regarding Retroviral-based Gene Transfer Approaches.  
5.8 Rationale for the Use of 2A Picornavirus Sequences. 
5.9 PET-based Molecular Imaging  
5.10 MART-1 TCR-engineered T cell Experience at NCI Surgery Branch 
5.10.1 Use of Other TCRs in Humans 
5.10.2 Clinical Use of the MART-1 F5 TCRs in Humans 
 
6.0 
Preliminary Data 
6.1 Origin and Functionality of the MART-1 F5 TCR. 
6.2 Preliminary Data Using the MART-1 F5 TCR Retroviral Vector (MSGV1-F5AfT2AB) in Human 
Subjects at the Surgery Branch/NCI 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3 
7.0 
Eligibility Criteria 
7.1 Inclusion Criteria 
7.2 Exclusion Criteria 
7.3. Definition of Study Enrollment Date 
 
8.0 
Study Procedures  
8.1 Baseline Screening Assessments  
8.2 Initial Protocol Procedures 
8.3 Leukapheresis, Cell Cultures, and Admission to the Hospital 
8.4 Conditioning Chemotherapy Regimen with Cyclophosphamide and Fludarabine 
8.5 MART-1 F5 TCR PBMC Infusion 
8.6 MART-126-35 peptide pulsed DC administration 
8.7 Low Dose IL-2 
8.8 Hospital Discharge 
8.9 MART-1 F5 TCR Cell Tracking After Infusion 
8.10 Follow Up Study Visits 
8.11 Permissible Systemic Therapies During the Protocol On-study Period 
8.12 Days 75 to 90: Re-staging Exams and Procedures 
 
8.13 Day 90: Office Visit for Re-staging Evaluation 
 
8.14 Long-term Follow-up Plan 
8.15 Optional Delayed Dendritic Cell Vaccination 
  
  
9.0 
Study Agents 
9.1 PBMC Expressing MART-1 F5 TCR 
9.1.1 Retrovirus vector MSGV1-F5AfT2AB 
9.1.2 PBMC Transgenic for MART-1 F5 TCR 
9.2 MART-126-35 Peptide Pulsed Dendritic Cells  
9.2.1 MART-126-35 Peptide 
9.2.2 MART-126-35 Peptide Pulsed Dendritic Cells (DC): 
9.3 Interleukin-2 (aldesleukin, Proleukin, recombinant human Interleukin 2) 
9.4 Fludarabine 
9.5 Cyclophosphamide  
9.6 Laboratory Reagents 
9.6.1 OKT3  
9.6.2. Interleukin-2 
9.6.4 GM-CSF 
9.6.5 Interleukin-4 
9.6.6. Retronectin 
 
10.0 
Generation of Experimental Cell Therapies:  MART-1-TCR Transduced CD8 Cells and 
MART-126-35 Peptide Pulsed Dendritic Cells  
10.1 Generation of MART-1-TCR Transduced PBMC 
10.2 Generation of MART-126-35 Peptide Pulsed Dendritic Cells  
10.3 Samples for Transgenic TCR Cell Persistence and Immune Monitoring 
 
11.0 
Evaluation of Study Endpoints 
11.1 Primary Study Endpoints:  
11.1.1 Safety 
11.1.2 Feasibility  
11.1.3 Antitumor Activity 
11.2 Secondary Study Endpoints 
11.2.1 MART-1 TCR/sr39 Transgenic Cell Persistence 
11.2.2 In Vivo Imaging 


MART-1 F5 TCR Protocol 01/10/2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4 
 
12.0 
Adverse Event Reporting 
12.1 Definition of Serious Adverse Events (SAE) 
12.2 Definition of an Adverse Event (AE) 
12.3 Causality Assessment 
12.4 Withdrawal Due to Adverse Events  
 
13.0 
Compliance with Good Clinical Practice, Ethical Considerations and Informed Consent  
13.1 Compliance with Good Clinical Practice and Ethical Considerations 
13.2. Regulatory Approvals 
13.3 Subject Recruitment  
13.4 Gender and Minorities 
13.5 Children 
13.6 Informed Consent Procedure  
13.7 Subject Enrollment  
 
14.0 
Qualifications of Personnel and Study Facilities  
14.1 Qualifications of Investigators 
14.2 Personnel Training 
14.3 HGMP/JCCC Cell and Gene Therapy GMP Suite at UCLA 
14.4 Gene Therapy Viral Bank 
14.5 Clinical Facilities 
14.6 Compliance with Protocol Procedures and Rights and Welfare of Study Participants 
 
15.0 
Data Safety Monitoring Board, Study Oversight, and Data Collection and Analysis 
15.1 Data Safety Monitoring Board (DSMB) 
15.2 JCCC DSMB Internal Monitoring Plan 
15.3 Monitoring/Auditing Activities 
15.4 Detailed Reporting Mechanism for Adverse Events  
15.5 Data Recording and Retention of Study Data 
 
16.0 
Statistical Methods 
16.1 Sample Size Determination 
16.2 Definition of Evaluable Patients 
16.3 Efficacy Analysis 
16.4 Study Stopping and Non-stopping Rules  
 
17.0 
References 
 
Appendix: Summary of Protocol Amendments 
 


MART-1 F5 TCR Protocol 01/10/2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5 
1. STUDY SYNOPSIS 
 
1.1 Overview 
 
This is a two-stage phase II clinical trial with the combined primary endpoints to determine the safety, 
feasibility and antitumor activity of adoptive transfer of peripheral blood mononuclear cells (PBMC) 
genetically engineered to express the alpha and beta chains of a high affinity T cell receptor (TCR) 
specific for the HLA-A*0201-restricted MART-1 melanoma tumor antigen to patients with locally 
advanced or metastatic melanoma. This gene transfer will be affected by a retroviral vector pseudotyped 
with a gibbon ape leukemia virus (GaLV) envelope. The two transgenes genes are linked by a 
picornavirus 2A sequence. Their expression is driven by the retroviral long terminal repeat (LTR).  
 
Patients with MART-1-positive locally advanced or metastatic melanoma who are HLA-A*0201-positive, 
and HIV, hepatitis B and C seronegative, will receive a non-myeloablative but lymphocyte depleting 
chemotherapy conditioning regimen consisting of cyclophosphamide and fludarabine, and then receive 
the adoptive transfer of autologous PBMC transduced with the MSGV1-F5AfT2AB retroviral vector, 
which expresses a high affinity TCR for the MART-1 melanoma antigen (MART-1 F5 TCR). The cell 
dose will be up to 109 autologous PBMC transduced with the MSGV1-F5AfT2AB retroviral vector. The 
transgenic T cells will be infused fresh on the day of harvest as done in the last three patients within this 
protocol (since Amendment 8), prior to which, thawed cryopreserved cells were infused. Following 
adoptive cell transfer, patients will receive MART-126-35 peptide-pulsed dendritic cell (DC) vaccines and 
low dose interlukin-2 (IL-2) under Amendment 11.  
 
The MART-1 F5 TCR was provided by Dr. Stephen A. Rosenberg from the Surgery Branch, National 
Cancer Institute (NCI). The MART-1 F5 TCR is derived from the DMF5 tumor infiltrating lymphocyte 
(TIL) clone, and was selected from several MART-1-specific TCRs because of its high affinity and 
biological activity. This TCR delivered by the same retroviral vector is currently in clinical testing at the 
Surgery Branch/NCI. Both the NCI clinical trial and the trial at UCLA are based on the same retrovirus 
expressing the MART-1 F5 TCR used to transduce whole PBMC and re-infused to patients after a non-
myeloablative but lymphodepleting chemotherapy conditioning regimen. Major differences between both 
clinical trials include the shorter ex vivo expansion of TCR transduced PBMC, the use of MART-126-35 
peptide pulsed DC and the use of positron imaging tomography (PET) for non-invasive imaging of 
adoptively transferred TCR transgenic cells in the UCLA clinical trial. 
 
The primary endpoints will be safety, feasibility and objective tumor response. The phase II clinical trial 
design will have two treatment stages following a Simon optimal two-stage clinical phase II clinical trial 
design 1. The clinical trial will have an initial stage with 8 patients followed by a second stage with up to 
22 patients.  
 
Safety will be determined in stage one, and if 3 out of 8 patients have MART-1 F5 TCR-induced dose 
limiting toxicities (DLT), then further accrual will not be warranted. Feasibility will be also determined in 
the first stage, and if 3 out of 8 patients cannot receive the intended cellular therapies, or if they result in 
suboptimal TCR transgenic cell in vivo persistence, further accrual will not be warranted to the protocol 
as currently designed. Objective tumor responses will be determined by RECIST objective response 
criteria with a design to rule out a 10% response rate as the null hypothesis, and a 35% response rate as 
the alternative hypothesis. With this statistical design, if 2 or more of 8 patients in stage one have an 
objective response, the study will proceed to stage two and accrue a total of 22 patients. If 5 or more 
patients in the overall study have a complete response (CR) or partial response (PR), which combined 
result in the objective response rate, the study will be declared positive.  
 
Secondary study endpoints are transgenic T cell persistence in humans and their ability to home to 
MART-1 positive melanoma metastasis. Analysis will be performed by sampling of peripheral blood and 
tumor deposits for T cell persistence and by non-invasive metabolic imaging using PET scans.  


MART-1 F5 TCR Protocol 01/10/2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6 
 
1.1.1. Cell Dose Escalation within Amendment 6 after Meeting the Safety and Feasibility Criteria 
But Not Meeting the Initial Antitumor Criteria  
 
Under the initial protocol 8 patients were accrued to stage 1 of this clinical trial. The criteria for safety 
and feasibility were met, with no dose limiting toxicities (DLTs) and feasibly delivering the intended cell 
therapies. However, the response rate was less than required to meet the minimal antitumor activity of 
35% response rate, with less than 2 patients with an objective response at day 90 of the protocol among 
the first 8 patients. Therefore, the protocol as initially planned was halted and Amendment 6 was 
generated administering a cell dose escalation up to 1010 F5 TCR transgenic cells, or the maximum 
feasible for each patient, in a new start of this stage 2 clinical trial with an initial stage enrolling up to 8 
patients following the same statistical design as the original protocol. 
 
1.1.2. Cell Dose De-escalation and Change to Low Dose IL-2 with Amendment 11 After Exceeding 
the Safety Criteria Under Amendments 6-10 
 
The safety of increasing the F5 TCR transgenic cell dose to 1010 within the basic F5 TCR ACT protocol 
from UCLA was assessed under Amendments 6 to 10. Dose escalation to up to 1010 cryopreserved cells 
was undertaken in 2 patients, and then the protocol was additionally modified to administer cells that had 
not undergone cryopreservation (Amendments 8 to 10) in three additional patients. However, two of 
these patients developed SAEs of respiratory distress requiring intubation. Thereafter, the total 
adoptively transferred cell dose was decreased back to a maximum of 109 cells and the regimen of 
administration of IL-2 was changed to LD IL-2 (Amendment 11). 
 
1.2. Rationale and Hypothesis 
 
Rationale for TCR Transgenic Cell Adoptive Transfer Therapy. The adoptive transfer of large 
numbers of clonally-expanded antigen-specific T cells into patients with melanoma that have received a 
conditioning regimen to deplete endogenous lymphocytes (non-myeloablative but lymphodepleting), 
together with high doses of interleukin-2 (IL-2), results in the highest rate of melanoma responses (50%) 
reported to date 2,3. This approach provides a proof-of-principle for adoptive transfer immunotherapy for 
metastatic melanoma, but it is difficult to implement outside of pilot studies given its requirement for 
extensive ex vivo manipulations. The ability to generate large numbers of tumor antigen-specific T cells 
by a single ex vivo manipulation consisting of the transduction of melanoma-specific TCR genes may 
result in a more broadly applicable approach for patients with melanoma. In this project we will test the 
hypothesis that the engineering of human PBMC to express a high affinity TCR specific for the human 
melanoma antigen MART-1 is safe, feasible and can induce objective tumor responses in human 
subjects with advanced melanoma. 
 
Rationale for the Clinical Trial Study Design and Primary Endpoints. This two-stage phase 2 clinical 
trial is designed to allow for the simultaneous testing of 3 primary endpoints. Safety and feasibility are 
being tested during stage one. The clinical trial will not proceed to stage two if one third or greater 
numbers of patients develop DLTs. This is the standard level for DLT assessment as safety criteria in 
phase 1 dose-escalation clinical trials. In addition, if the study drugs and procedures cannot be delivered 
as planned in over one third of patients, assessed by not meeting the study feasibility criteria (see 
below), then enrollment to stage two will not be warranted. The protocol would need to be amended to 
meet the feasibility criteria before further enrollment. Antitumor activity is assessed in the overall phase 2 
clinical trial, but the study will only proceed to enroll in stage two if a minimal antitumor activity level is 
evident in stage one (see below). 
 
 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
7 
1.3 Study Objectives: 
 
Primary:  
 
• 
SAFETY. This TCR and retroviral vector at a cell dose up to 1011 have been already in clinical 
testing within a reported phase 2 clinical trial at the Surgery Branch/NCI. We started with a 
maximum of up to 109 MART-1 F5 TCR transgenic cells for infusion. Since we did not meet 
the minimal hurdle of antitumor activity to proceed to stage 2 of this research after having 
entered the first 8 patients, we increased the dose to a maximum of up to 1010 (Amendment 
6) which is still one log lower than the maximum cell dose within the NCI protocol. Therefore, 
there was no need for a standard phase1 dose escalation clinical trial design. After enrolling 
two patients under this increased dose, we further changed to infusion of freshly harvested 
transgenic cells in place of cryopreserved cells, keeping the same increased dose of up to 
1010 (Amendment 8). Two out of three patients enrolled under this regimen required 
intubation for respiratory distress. Thereby, we propose to decrease the dose to 109 freshly 
harvested MART-1 F5 TCR transgenic cells and change to low dose IL2 in this amendment 
(Amendment 11). Safety continues to be a major concern and a clinical endpoint of this 
clinical trial. Therefore, the study is designed with an early stopping rule based on 
development of DLTs in one third or higher among patients enrolled in the initial stage of this 
clinical trial. Safety will be determined in stage one, and if 3 out of 8 patients have MART-1 
F5 TCR-induced DLT, then further accrual will not be warranted.  
 
• 
FEASIBILITY. This clinical trial intends to deliver two patient-specific cell therapies, the TCR 
transgenic PBMC and MART-126-35 peptide pulsed DC, within a study design that requires 
other significant interventions, like a lymphodepletive conditioning regimen and post-infusion 
high dose IL-2 (up to Amendment 10), and then low dose IL-2 (after Amendment 11). This 
protocol has significant technical challenges, making feasibility a primary endpoint and 
requiring a clinical trial design with a feasibility early stopping rule. Therefore, feasibility will 
be determined in stage one, and if 3 out of 8 patients cannot receive the intended cellular 
therapies further accrual will not be warranted.   
 
• 
CLINICAL RESPONSE. The overall phase 2 clinical trial statistical design has objective 
tumor response as the main criteria for determining the number of study subjects. Objective 
tumor responses will be determined by RECIST objective response criteria, which are defined 
as adding the rate of complete response (CR) and partial response (PR) at restaging exams 
on day 90 of the protocol. To account for the frequently observed delayed responses with 
immunotherapy, patients with disease stabilization at day 90 who do not receive further 
therapy but go onto meet RECIST criteria for CR or PR will also be considered as having 
objective response on study. The clinical trial statistical design is aimed at ruling out a 10% 
response rate as the null hypothesis, and has a 35% response rate as the alternative 
hypothesis. The minimum hurdle to proceed from stage one to stage two of this clinical trial 
will be observing at least 2 objective responses out of the 8 initial patients. The study will be 
considered a positive study ruling out the null hypothesis if 5 patients have an objective 
response among 22 evaluable patients enrolled in the two stages of this phase II clinical trial. 
 
Secondary:  
 
• 
MART-1 F5 TCR TRANSGENIC CELL PERSISTENCE. To determine the persistence of 
MART-1 F5 TCR-engineered PBMC in serial peripheral blood samples and in biopsies of 
accessible metastatic melanoma lesions.  
 
• 
IN VIVO MOLECULAR IMAGING. To explore the use of PET-based imaging using the PET 
tracer [18F]FDG with the goal of determining if the adoptively transferred MART-1 F5 TCR-


MART-1 F5 TCR Protocol 01/10/2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
8 
engineered PBMC home and expand in secondary lymphoid organs and melanoma tumor 
deposits. Findings with non-invasive molecular imaging will be compared with results from 
immune monitoring assays in blood samples and tumor biopsies at different intervals after 
MART-1 F5 TCR PBMC adoptive transfer.   
 
1.4 Study Population 
 
Subjects must be HLA-A*0201, have locally advanced (stage IIIc) or metastatic melanoma (stage IV) 
with no standard therapeutic options with a curative intent, that is MART-1-positive, age greater than or 
equal to 18, clinical performance status of ECOG 0 or 1, life expectancy greater than 3 months, and be 
seronegative for HIV, Hepatitis B and C (seropositive subjects would be more likely to have toxic effects 
from the conditioning chemotherapy or may not tolerate it). Study subjects should have accessible 
lesions amenable to outpatient tumor biopsies that still allow an adequate assessment of the primary 
endpoint of antitumor activity and clinical response assessment. 
 
1.5 Number of Patients 
 
The phase 2 clinical trial design will have two treatment stages, an initial one with 8 patients, followed by 
a second stage with 14 additional patients (for a total of 22 patients). All patients will receive the same 
combined therapy of: 1) a lymphodepleting preparative regimen, 2) autologous PBMC retrovirally-
transduced with a MART-1 F5 TCR, followed by 3) IL-2 and 4) MART-126-35 peptide–pulsed DC.   
 
1.6 Study Duration: 
 
Total duration of the study is estimated to be approximately 18-24 months, if recruitment proceeds 
optimally with 2 patients screened each month, resulting in a mean of 1 to 2 patients entered in the trial 
every month. 
 
The initial screening period will be 30 days, but patients will be allowed to enroll after the 30-day initial 
screening period, for up to approximately 60 days, using the same screening procedure test results to 
avoid unnecessary assay duplication. A pregnancy test must be completed within 14 days prior to 
dosing, if applicable. 
 
1.7 Study Schematic  
 
Patients will undergo an initial leukapheresis to collect PBMC. The manufacture of both transgenic T 
cells and DC will be started on the day of leukapheresis. Immediately after processing the PBMC, 
1.5x109 PBMC will be put in activation media containing OKT3 (anti-CD3 antibody) and IL-2 for two 
days, 12x108 PBMC will be set up for DC culture and the rest will be cryopreserved. The next day after 
leukapheresis, patient will start receiving the non-myeloablative conditioning chemotherapy. Following 
activation, cells are transduced twice on two consecutive days with the MSGV1-F5AfT2AB retrovirus in 
retronectin-coated plates. Transduced cells will be expanded ex vivo for 4 days after the first 
transduction and then put into an infusion bag to be infused i.v. fresh on the day of harvest as soon as 
the lot release is cleared. The following day, after a week of ex vivo differentiation culture in GM-CSF 
and IL-4, DC is harvested, peptide pulsed and administered i.d. Patient will also receive systemic low 
dose IL-2 for 7 days for up to 14 doses.  
 
In the event the physician decides that the patient will not be clinically fit to receive the gene-modified 
MART-1 TCR CTL infusion within 24 hours of the scheduled day of cell harvest, the cells will be 
cryopreserved on the harvest day for later use. 
 
 
 
 




 
 
 
10 
2.0 Schedule of Events 
 
 
Screening 
Day   
-6 
Day 
–5 
and 
-4 
Day 
–4 to 
-1 
Day 
0 
Day 
1 
Day 
1 to 
7 
Day 
14 
Day 
20 
to 
40 
Day 
30 
Day 
45 
Day 
60 
Day 
75 
Day 
75-
90 
Day 
90 
Q2-3 
mo up 
to 
Year 
2a 
Q6 o 
up to 
Year 
5f 
Q12 
mo up 
to 
Year 
15g 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Visit and Physical  
X 
X 
 
 
 
 
 
 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
HLA Typing 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PCR 
or 
IHC 
for 
MART-1 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBCb 
X 
X 
X 
X 
X 
 
 
 
 
X 
X 
X 
 
X 
 
 
 
 
Metabolic Panelb 
X 
X 
X 
X 
X 
 
 
 
 
X 
X 
X 
 
X 
 
 
 
 
LDH 
X 
  
 
 
 
 
 
  
 
X 
X 
X 
 
X 
 
 
 
 
CRP 
x 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Uric Acid 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Magnesium 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Phosphorous 
x 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Lipase 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Amylase 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Coagulation 
Tests: 
PT/INR, PTT 
x 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Autoantibody Panel 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Thyroid Hormones 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Pregnancy Testc 
X 
  
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Infectious 
disease 
serologies 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CT or MRI Scan of 
the Brain 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Otologic Visit 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Ophthalmologic Visit 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Leukapheresis 
 
X 
 
 
 
 
 
 
X  
 
 
 
 
 
 
 
 
 
Plasmapheresis 
 
X 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
Blood 
for 
plasma 
(cytokines)d 
  
 
 
 
  
  
X 
X 
 
  
 
 
 
 
 
 
 
 
Blood 
for 
cell 
analyses 
(immune 
monitoring)e 
 
X  
 
 
 
 
 
 
X 
  
X 
X 
X 
X 
 
X 
X 
X 
X 
Biopsy 
X 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
ECHO or MUGA  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PFT 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


MART-1 F5 TCR Protocol 8/1/2016 
                                          
 
 
 
11 
 
Screening 
Day   
-6 
Day 
–5 
and 
-4 
Day 
–4 to 
-1 
Day 
0 
Day 
1 
Day 
1 to 
7 
Day 
14 
Day 
20 
to 
40 
Day 
30 
Day 
45 
Day 
60 
Day 
75 
Day 
75-
90 
Day 
90 
Q2-3 
mo up 
to 
Year 
2a 
Q6 o 
up to 
Year 
5f 
Q12 
mo up 
to 
Year 
15g 
PET/CT Scans 
X 
  
 
 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
Inpatient admission 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cyclophosphamide 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fludarabine 
 
 
 
X 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
MART-1 
F5 
TCR 
PBMC 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Low dose IL-2 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
MART-126-35/Dendritic 
Cell Vaccine 
 
 
 
 
 
X 
  
X 
 
X 
 
 
 
 
 
 
 
 
aLong term follow up is described in section 8.15.  For the first 2 years, office visits and blood collection will be at least every 3 months. 
bWhile patients are in the hospital, physical, CBC and metabolic panel will be per J-Medicine protocol. 
cFemales only. 
dAdditional blood for protein analyses in plasma or serum will only be collected after day 30 if there is a suspicion of a cytokine storm or an acute event 
requiring additional analyses in blood. The plasma or serum may be collected as 5-10 ml blood samples in anticoagulant or stored as the liquid phase of a 
Ficoll gradient when storing PBMCs. 
eBlood draws on these days should be performed if considered safe due to the pancytopenia and hemodynamic state of the patient. Can be done +/- 6 days. 
fAfter 2 years, office visits or phone follow-up at least every 6 months.  Collection of blood for immune monitoring analysis and for RCR testing annually. 
gAfter 5 years, office visits or phone follow-up at least annually. Collection of blood for immune monitoring analysis and for RCR testing annually.  
In general, changes in the range of 7 days up or down from the scheduled event will not be considered significant deviations from the study timeline. 
 
 
 


 
 
 
12 
3.0 TABLE OF ABBREVIATIONS 
 
ACT: Adoptive cell transfer 
AE: Adverse event 
ATG: Anti-Thymocite Globulin 
CBC: Complete blood count 
CFR: Code of federal regulations 
CMV: Cytomegalovirus 
CR: Complete response 
CRF: Case report form 
CRP: C reactive protein 
CT: Computer tomography. 
CTLs: Cytotoxic T lymphocytes 
DC: Dendritic cells 
DLT: Dose limiting toxicity 
DSMB: Data Safety and Monitoring Board  
EBV: Epstein-Barr virus 
FDA: Food and Drug Administration 
[18F]FLT: [18F] fluoro-L-thymidine 
[18F]FDG: [18F]fluorodeoxy-glucose 
GaLV: Gibbon ape leukemia virus 
GCP: Good Clinical Practices 
GCV: Ganciclovir  
GM-CSF: Granulocyte-macrophage colony stimulating factor 
GMP: Good Manufacturing Practices 
GVHD: Graft-versus-host disease  
GVL: Graft-versus-leukemia 
HBV: Hepatitis B virus 
HCV: Hepatitis C virus 
HCT: Hematopoietic cell transplantation  
H&E: Hematoxilin eosin staining 
HEPA: High-efficiency particulate air 
HGMP: Human Gene Medicine Program 
HIV: Human immunodeficiency virus 
HLA: Human leukocyte antigen 
IBC: Institutional Biosafety Committee 
ICF: Informed consent form 
ICS: Intracellular Cytokine Staining  
i.d.: Intradermal 
IHC: Immunohistochemistry 
IU VPF: Indiana University Virus Production Facility  
i.v.: Intravenous 
IQAC: Internal Quality Assurance Committee  
IRB: Institutional Review Board 
ISPRC: Internal Scientific Peer Review Committee 
IL: Interleukin 
JCCC: Jonsson Comprehensive Cancer Center 
LDH: Lactate dehydrogenase 
LTR: Long terminal repeat 
MHC: Major histocompatibility complex 
MLV: Molonely Leukemia Virus 
MRI: Magnetic resonance imaging 
MSCV: Murine stem cell virus 
N.C.I.: National Cancer Institute 
NGVL: National Gene Vector Laboratory 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
13 
N.I.H.: National Institutes of Health 
PBMC: Peripheral blood mononuclear cells 
PCR: Polymerase chain reaction 
PD: Progressive disease 
PET: Positron emitting tomography 
PR: Partial response 
RAC: Recombinant Advisory Committee 
RCR: Replication competent retrovirus 
RECIST: Response Evaluation Criteria in Solid Tumors 
SAE: Serious adverse event 
S.C.: Subcutaneous 
SD: Stable disease 
SUV: Standardized uptake value 
TSH: Thyroid stimulating hormone 
TCR: T cell receptor 
UCLA: University of California Los Angeles 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
14 
4.0 STUDY OBJECTIVES 
 
4.1 Primary: 
 
4.1.1 Safety  
 
The MART-1 F5 TCR and the retroviral vector MSGV1-F5AfT2AB have already been tested in two 
clinical experiences, including a completed and reported phase 2 clinical trial by investigators at the 
Surgery Branch/NCI 7, and the second experience being the 8 patients treated at UCLA under this 
same IND with the F5 TCR transgenic cell dose up to 109. Overall, the ACT of F5 TCR transgenic 
cells after a lymphodepletion protocol with high dose IL-2 and DC vaccination was well tolerated with 
no dose limiting toxicities (DLTs) developed in the first 8 patients. The safety of increasing the F5 TCR 
transgenic cell dose to 1010 within the basic F5 TCR ACT protocol from UCLA was assessed in stage 
1 of the protocol described in Amendment 6. Dose escalation to up to 1010 cells was undertaken in the 
following 2 patients, and after that the protocol was additionally modified to administer cells that had 
not undergone cryopreservation (Amendment 8) in three additional patients. However, two of these 
patients had serious adverse events (SAE) of respiratory distress requiring intubation. Therefore, the 
total adoptively transferred cell dose was decreased back to a maximum of 109 cells and the regimen 
of administration of IL-2 was changed to LD IL-2 (Amendment 11). 
 
Safety evaluations will continue into the second stage of the study and the same early stopping rules 
for DLTs will apply to the second stage of this study. Safety remains the priority in all phases and 
stage of the investigational studies. 
 
4.1.2 Feasibility 
 
This protocol attempts to administer two cell-based therapies that require laboratory manipulation 
within the setting of Good Manufacturing Practices (GMP). Each cell therapy, the MART-1 F5 TCR 
engineered T cells and the MART-126-35 peptide pulsed DC, will require strict lot release criteria of the 
final product before administration. Feasibility will be determined in the first stage, and if 3 out of 8 
patients cannot receive the intended cellular therapies, or there is suboptimal TCR transgenic cell 
persistence in vivo, further accrual will not be warranted. The feasibility assessment will be based on:  
 
i) 
Potential problems in the manufacturing of MART-1 F5 TCR engineered PBMC or the MART-
126-35 peptide–pulsed DC vaccines.  
ii) 
Suboptimal number of MART-1 F5 TCR engineered T cells recovered from patients at pre-
specified time points.  
 
4.1.3. Clinical Response 
 
Objective tumor responses will be determined by RECIST objective response criteria. The rate of CR 
plus PR will be used to assess the primary endpoint of antitumor activity with a clinical trial design to 
rule out a 10% response rate as the null hypothesis, and 35% response rate as the alternative 
hypothesis. Response assessment will be performed by comparing standard CT imaging scans and 
photographs of target lesions from baseline with repeated imaging tests obtained at day +90 after the 
TCR transgenic PBMC adoptive transfer.  
 
4.2 Secondary 
 
4.2.1 MART-1 F5 TCR Transgenic T Cell Persistence.  
 
The persistence of MART-1 F5 TCR-engineered cells will be determined by analyzing serial 
peripheral blood samples for the presence of T cells with the MART-1 F5 TCR by tetramer or 
dextramer analysis. In addition and when technically feasible, samples from metastatic melanoma 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
15 
sites will be biopsied and assessed for the presence of the MART-1 F5 transgenic cells by IHC, PCR 
and/or immune monitoring assays in non-adherent TIL. 
 
4.2.2 In Vivo Imaging. 
 
The adoptive transfer of MART-1 redirected TCR transgenic cells has the goal of targeting a tumor 
antigen expressed by melanoma cells. We plan to conduct serial PET molecular imaging using the 
tracer [18F]FDG to determine changes in the metabolic rate in secondary lymphoid organs and tumor 
metastasis. Accumulation of immune cells in secondary lymphoid organs and tumor lesions will result 
in an increase in glucose uptake and retention, which will result in an increase in the standardized 
uptake value (SUV) of [18F]FDG. The images obtained by whole-body PET will be matched with 
analysis of tumor biopsies to attempt to quantify the number of tumor-infiltrating MART-1 F5 TCR 
transgenic T cells.  
 
5.0 BACKGROUND 
 
5.1 Current Management of Surgically-incurable Melanoma 
 
Locally advanced and metastatic melanoma is notoriously resistant to standard forms of therapy. Five 
agents are approved by the U. S. Food and Drug Administration (FDA) for the treatment of stage IV 
melanoma. The chemotherapy drugs dacarbazine (DTIC) and hydroxiurea, and the administration of 
high doses of IL-2 8,9 were approved based on single arm studies. . These have response rates below 
15%, and neither form of therapy has been shown to increase survival in a randomized trial. Two new 
agents have been approved for the treatment of metastatic melanoma based on a demonstrated 
impact on overall survival, the anti-CTLA4 antibody ipilimumab (Yervoy) and the BRAF inhibitor 
vemurafenib (Zelboraf). Ipilimumab induces low frequency of response rates (in the range of 10%) 
that tend to be durable and have translated into improved overall survival in two randomized clinical 
trials 10,11. Vemurafenib gives a high response rate in BRAF V600 mutant metastatic melanoma, but 
median duration of response is 6-7 months 12,13. In a randomized clinical trial it improved overall 
survival compared to dacarbazine 14. 
 
5.2 Rationale for Immune-based Therapies for Metastatic Melanoma.   
 
Progress in our understanding of how the immune system recognizes and kills cancer cells has led to 
the notion that high levels of circulating, tumor-antigen-specific T cells may be required for effective 
antitumor responses. Active immunotherapy with several forms of cancer vaccines has shown that 
antigen-specific T cells can be activated and occasionally lead to antitumor responses 15,16. However, 
the circulating levels of antigen-specific T cells and tumor response rates are much lower than 
desirable 17. 
 
Adoptive transfer of clonally-expanded, tumor antigen-specific lymphocytes to lymphopenic hosts after 
nonmyeloablative conditioning chemotherapy has resulted in cell proliferation and persistent clonal 
repopulation 2. This approach has resulted in an objective response rate over 50% in patients with 
metastatic melanoma, that holds up in larger groups of patients 3. The major limitation for the broad 
clinical applicability of this approach is the requirement for large-scale ex vivo lymphocyte culture 
expansion (up to 1011 cells) 2, which restricts this approach to a highly select group of patients. 
 
5.3 MART-1 Melanoma Antigen as Target for Melanoma Immunotherapy 
 
Multiple lines of evidence suggest that most tumor responses to immunotherapy involve the activation 
of tumor antigen-specific CD8+ cytotoxic T lymphocytes (CTL) 18. These immune cells have a high 
affinity receptor, the TCR that specifically recognizes its ligand, a complex of MHC molecules with an 
8 to 10 amino acid peptide that provides the antigen specificity. When the antigen specificity of T cell 
clones obtained from TIL was assessed, over 80% of them were restricted to the MART-1/Melan-A 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
16 
melanoma antigen 19,20. Over the past 10 years we have conducted prior experimental immunotherapy 
clinical trials attempting to induce melanoma regression by activating MART-1-specific T cells. These 
experiences demonstrated that a subset of patients have immune mediated tumor regressions to 
MART-1-based immunotherapy that are extremely long lived, lasting years without relapses 4,5.  
 
5.4. Why Gene Therapy? 
 
The adoptive transfer of large numbers of antigen-specific T cells leads to the highest percentage of 
tumor regressions reported in patients with melanoma, but this approach is only feasible in a minority 
of patient where TIL can be cloned and expanded from melanoma metastasis 2,3. Genetic engineering 
of T cells with optimal TCRs would make this approach more broadly applicable. There is no other 
approach that would allow generating large numbers of T cells with uniform specificity for melanoma 
tumor antigens with a short (less than one week) ex vivo cell manipulation. Therefore, gene transfer 
techniques are the only approach that would allow testing this concept in human subjects, as 
supported by preliminary data from a published clinical trial at the Surgery Branch/NCI 21.  
 
5.5 TCR Gene Therapy 
 
The transfer of TCR genes is necessary and sufficient to endow recipient T cells with the specificity of 
donor T cells 22. Genetically modified T cells carrying foreign TCRs respond to target antigen 
recognition through the transgenic TCR both in vitro and in vivo, leading to effective immune 
responses to viral and tumor challenges in murine adoptive transfer models 23. T cells redirected by 
TCR gene transfer are fully functional after transfer into mice, and have been shown to expand 
dramatically (over three logs) after encounter with their cognate antigen in vivo 24. Preliminary data in 
humans provides evidence that the same is true in patients with metastatic melanoma 21. 
 
5.6 Retroviral Vector-based Human Gene Medicine 
 
Retroviral-mediated gene transfer is an efficient means for the expression of transgene into actively 
dividing primary cells. Up to 50-60% of hematopoietic stem cells (HSC) and T lymphocytes can be 
transduced if cells are non-specifically activated, usually using a cytokine cocktail for HSC and either 
immobilized anti-CD3 and anti-CD28 or cytokines for T cells 25-27.  
 
5.7 Safety and Regulatory Issues Regarding Retroviral-based Gene Transfer Approaches.  
 
Ten infants with X-linked severe combined immune deficiency (SCID) resulting from a mutation in the 
γc gene (a surface cytokine receptor) were successfully treated with bone marrow transplantation of 
autologous HSC genetically modified to express the missing gene using retroviral vectors. Two of the 
infants developed a T cell leukemia approximately 3 years later. In both cases, the retrovirus carrying 
the γc gene had inserted near LMO2, an oncogene that is activated in some forms of acute leukemias 
28,29. This event highlights the risks of insertional mutagenesis induced by DNA-integrating viral 
vectors. However, multiple lines of evidence suggest that this may be an isolated event linked to the 
possible oncogenic capacity of the γc gene when under the control of a retroviral promoter, with a 
second hit being the activation of an endogenous oncogene like LMO2 28-30.  
 
Retroviral vectors have a bias towards preferential integration in gene-rich regions, particularly near 
transcribed genes 31. The theoretical concern of insertional mutagenesis had been recognized early in 
the use of retrovirus as gene therapy vectors, since retrovirus cause tumors in mice, and their ability 
to transactivate normally silent genes has been a useful technique for the description of multiple 
oncogenes 32. However, retroviral vector oncogenesis from insertional mutagenesis has proven to be 
extremely rare in human subjects. Over 250 patients in over 40 clinical trials have received stem cells 
genetically modified with retroviral vectors 30, and the only cases of malignant transformation are the 
two infants in the SCID study 28,29. Therefore, particularities of this study (the γc gene functioning as a 
first hit for leukemogenesis, a predilection for vector insertion near the LMO2 oncogene as second hit, 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
17 
the baseline immune deficiency of the patients not being able to control transformed clones) seem to 
have played a major role in these two cases 33. 
 
5.8 Rationale for the Use of 2A Picornavirus Sequences 
 
Problems with expressing multiple genes in a single vector become apparent with the simultaneous 
use of multiple promoters and IRES sequences. The different encoded proteins are usually not 
expressed at the same level. This is a critical issue for this protocol, since the correct surface 
expression of a functional transgenic MART-1 F5 TCR requires both proteins to pair, and in addition 
try to decrease the heterodimerization with other endogenous TCR chains. For example, a MART-1 
F5 TCR α chain, produced in excess of the MART-1 TCR β chain, may pair with an endogenous TCR 
β chain, resulting in the generation of TCR with specificity other than MART-1 34. This problem has 
been circumvented by using picornavirus-derived “self-cleaving” 2A-like sequences. Using this 
approach, Szymczak et al. 35 have efficiently generated multicistronic retroviral vectors (with efficient 
translation of at least 4 genes) to restore TCR expression in CD3-deficient mice.  
 
The original description of the 2A self-cleaving peptides was done by Martin Ryan and colleagues at 
the University of St. Andrews, U.K., who were working on the Foot and Mouth Disease Virus (FMDV) 
and other picornavirus 36. The FMDV encores some of its proteins in a long single open reading frame 
(ORF) that is cleaved post-translationally by viral proteinases. At one position of this polyprotein there 
is a rapid co-translational self-processing due to the presence of an 18 amino acid peptide (termed 
2A) that directed its own separation from the growing polyprotein 36-38. The use of 2A-based vectors to 
express the two MART-1 TCR genes would be predicted to allow for the coordinated expression of 
the 2 transgenes. 
 
5.9 PET-based Molecular Imaging  
 
PET provides quantitative measurements of the 3D distribution of probes labeled with positron 
emitting radio-nuclides. PET scanners routinely measure radioactivity concentrations in the 10-12 M 
range, making PET the most sensitive imaging technology applicable to both preclinical and clinical 
studies. MicroPET scanners approach a spatial resolution of ~1 mm3 while clinical scanners have an 
intrinsic resolution of ~4-5 mm3 39. Currently, PET imaging of metabolic activity is extensively used in 
cancer diagnosis, with more than 3 million PET scans administered in clinical centers worldwide in 
2006. A majority of these scans use the glucose analog 2-[18F]-fluoro-2-deoxy-d-glucose ([18F]FDG) to 
measure the rate of glycolysis, a metabolic process that is unregulated in the majority of cancers. 
[18F]FDG PET allows early detection and staging of cancer, as well as evaluation of therapeutic 
interventions. In addition to cancer, [18F]FDG also accumulates at sites of inflammation or active 
immune responses. Similar to malignant cells, T cells and other immune cells rapidly induce glycolysis 
following activation. Increased [18F]FDG retention has been reported in several immune and 
inflammatory 
processes 
including 
autoimmune 
hepatitis, 
viral 
infections, 
systemic 
lupus 
erythematosus  and rheumatoid arthritis.  
 
The concept of PET measurements of metabolic processes exemplified by [18F]FDG has been applied 
to the development of PET imaging probes sensitive to the rate of DNA synthesis and cell 
proliferation. The prototype for these probes is 3’-18fluorothymidine ([18F]FLT), a nucleoside analog 
developed by Shields and colleagues 40. The accumulation of [18F]FLT in proliferating cells reflects the 
enzymatic activity of thymidine kinase 1, a cytosolic deoxynucleoside kinase which catalyzes the 
transfer of a γ-phosphate group from a nucleoside triphosphate to the 5’-OH group of thymidine. TK1 
expression is regulated during cell cycle: it starts increasing in late G1 cells, reaches a maximum in 
the S phase coinciding with DNA synthesis, and markedly decreases during mitosis. 
 
 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
18 
5.10 MART-1 TCR-engineered T cell Experience at NCI Surgery Branch 
 
5.10.1 Use of Other TCRs in Humans. The Surgery Branch/NCI has initiated two similar TCR gene 
transfer protocols using other TCRs, which as of the Fall of 2007 have enrolled a total of 54 patients:  
 
i) 
NCI trial 04-C-0251 (OBA# 0308-599, IBC# RD-03-XII-01) has treated 35 patients using a 
similar retroviral vector backbone and packaging cell line proposed in the current study. This 
clinical trial used a TCR that had much lower avidity for MART-1, which was termed F4 21. 
There were no adverse events associated with these patient treatments that were attributed to 
the gene transfer therapy. There have been four clinical responses (PR) observed during the 
trial, and two were long-term PRs (>12 months).  
 
ii) 
NCI trial 07-C-0175A (OBA# 0703-840) has treated 20 patients using the same retroviral 
vector backbone and packaging cell line proposed in the current study. The protocol was 
feasible and overall safe, with major toxicities attributed to the F5 TCR transgenic cells being 
anterior uveitis in 55% of patients and hearing loss in 50% of patients 7. Antitumor activity was 
demonstrated with 6 partial responses (PR) out of 20 patients (30%), with 3 of the responses 
being durable beyond 1 year 7. 
 
iii) 
NCI trial 07-C-0003 (OBA#0602-759, IBC # RD-06-II-10) has treated 7 patients using the 
same retroviral vector backbone and packaging cell line, the only difference being the TCR 
protein is specific for p53. There have been no adverse events associated with these patient 
treatments that were attributed to the gene transfer therapy. There has been no clinical 
response observed to date. 
 
5.10.2 Clinical Use of the MART-1 F5 TCRs in Humans. NCI trial 07-C-0175A (OBA# 0703-840) 
uses the same MART-1 F5 TCR and retroviral vector construct proposed in the current protocol 7,41. 
This clinical trial has initiated patient accrual and data is being generated currently. Below we list the 
major similarities and differences between both clinical trials: 
 
i) 
Major similarities: 
 
- 
TCR: Both clinical trials use the same MART-1 F5 TCR, derived from TIL clone DMF5, the 
highest affinity MART-1 TCR is a large screen at the Surgery Branch/NCI 7,41. 
- 
Retrovirus: Both clinical trials use the same retrovirus vector backbone, construct and 
master cell bank, and produced at the same facility, the Indiana University Vector 
Production Facility (IU VPF).  
- 
Target cells: Both experiences include the transduction of activated PBMC. Since a TCR 
can only be expressed and functional on the cell surface in cells that are CD3 positive, this 
allows the TCR engineering of both CD8+ CTL and CD4+ T helper cells. Therefore, CD4+ 
T cells acquire a TCR that redirects them to recognize antigen presented by MHC class I 
molecules. 
- 
Clinical trial design: Both are phase 2 clinical trials with primary endpoints of safety and 
antitumor activity.  
- 
Conditioning regimen: Both use the same non-myelodepleting but lymphodepleting 
chemotherapy conditioning regimen based on the administration of cyclophosphamide and 
fludarabine to allow homeostatic proliferation of the adoptively transferred TCR transgenic 
cells. 
 
ii) 
Major differences: 
 
- 
Rapid Expansion Protocol (REP): The F5 clinical trial at the Surgery Branch/NCI 7 included 
a 2-week ex vivo expansion after TCR engineering of lymphocytes following the REP 
protocol described by Riddell and colleagues 42. The clinical trial at UCLA will not include 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
19 
this forced ex vivo T cell expansion. The main reason is that several preclinical models 
suggest that extended ex vivo expansion of lymphocytes before adoptive transfer results in 
terminally differentiated cells with limited in vivo proliferation ability 43,44. Therefore, at 
UCLA the TCR transduced PBMC will only be kept for up to 96 hours in culture after the 
first transduction before re-infusing into patients. 
- 
Cell dose for adoptive transfer: Given that the UCLA study will not use REP, the target cell 
dose for adoptive transfer is one to two log lower at UCLA than in the clinical trial 
conducted at the Surgery Branch/NCI 7, where up to 1011 TCR transgenic cells were 
infused to patients. 
- 
Dendritic cell vaccination: Provision of antigen in the form of a vaccine is required in some 
animal models to support the antitumor activity of adoptively transferred T cells, most 
notably with the pmel-1 adoptive transfer model 45,46. This is because exposure to antigen 
while undergoing homeostatic proliferation stimulates further T cell expansion 47,48. We 
propose to use MART-126-35 peptide-loaded GM-CSF/IL-4 differentiated DC, a vaccine that 
has resulted 2 durable complete responders out of 18 patients with metastatic melanoma 
treated, currently ongoing beyond 5 and 6 years, respectively 4,5. 
- 
PET imaging: We include molecular imaging using the standard [18F]FDG PET probe, 
which allows non-invasive imaging of a key feature of activated lymphocytes, their high 
uptake of glucose due to aerobic glycolysis. 
- 
Low dose IL-2: The UCLA clinical trial initially used high dose IL-2 post-adoptive transfer 
as means to provide helper cytokines to support the proliferation of adoptively transferred 
TCR transgenic cells. After Amendment 11 this will change to low dose IL-2. 
 
6.0 PRELIMINARY DATA 
 
6.1 Origin and Functionality of the MART-1 F5 TCR. 
 
The retroviral vector, MSGV1-F5AfT2AB, expressing the MART-1 F5 TCR, has been provided by Dr. 
Steven A. Rosenberg from the Surgery Branch/NCI. This high affinity TCR specific for the MART-127-35 
epitope in the context of HLA-A*0201 has been selected based on its high affinity for its antigen and 
because it confers high functionality when genetically engineered into CD3 positive cells. 
 
The low response rate in the prior MART-1 TCR gene transfer protocol at the Surgery Branch/NCI 21 
led these investigators to attempt to identify MART-1 reactive TCR with higher avidity than the MART-
1 F4 TCR used in the prior gene therapy clinical trial. They generated multiple MART-1 reactive 
clones from a variety of MART-1 reactive tumor infiltrating lymphocyte cultures. Twenty four clones 
were isolated and tested for reactivity against the MART-1 peptide and against A2+ MART-1 
expressing melanoma lines as well as the 888 A2- melanoma line 41,49.  
 
The TCR utilized in their prior clinical trial came from the DMF4 TIL clone. When compared to many 
other MART-1 reactive clones this clone had only medium avidity for recognition of MART-1 peptide 
as well as MART-1 expressing tumors. The highest avidity T cell clone identified in this analysis was 
the DMF5 clone. In an overnight co-culture assay measuring interferon-gamma release the DMF5 
clone had the highest recognition of T2 cells pulsed with the native MART-127-35 peptide. This clone 
secreted 17,161pgm/ml of interferon gamma in an overnight co-culture assay compared to 
1,987pgm/ml secreted by the DMF4 clone used in prior trials. In addition, the recognition of the 
mel526 and the mel624 A2+ MART-1+ cell lines was also higher utilizing the DMF5 clone than the 
DMF4 clone (5806 and 10,865 pgm/ml compared to 1780 and 2397 pgm/ml). Neither clone had 
significant recognition of the control A2- MART-1+ mel 888 line 41,49,50. 
 
Investigators at the Surgery Branch/NCI next cloned the genes encoding the alpha and beta chains of 
the TCR from many of these cloned TIL populations. The genes encoding these alpha and beta 
chains were then electroporated into the same activated donor CD8+ PBMC. The DMF5 
electroporated TCR was again the most reactive TCR when assessed against the MART-1 peptide 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
20 
pulsed onto T2 cells as well as to A2+ MART-1+ melanoma cell lines. The reactivity of the TCRs 
recapitulated the reactivity seen in the original clones. They next checked lysis by these 
electroporated TCR in a 4-hour chromium release assay. Again the DMF5 TCR outperformed all of 
the other TCR and was approximately 10 times more avid compared to the DMF4 TCR used in their 
prior gene therapy clinical trial 41,51. 
 
In summary, the anti-MART-1 F5 TCR appears to have excellent reactivity against both the MART-1 
peptide as well as melanoma cell lines. Investigators at the Surgery Branch/NCI have developed a 
GMP quality retroviral vector encoding the alpha and beta chains of the MART-1 F5 TCR for use in an 
ongoing phase 2 clinical trial 51.  
 
6.2 Data Using the MART-1 F5 TCR Retroviral Vector (MSGV1-F5AfT2AB) in Human Subjects at 
the Surgery Branch/NCI 
 
Investigators at the Surgery Branch led by Dr. Steven A. Rosenberg have reported on 20 patients 
treated with the ACT of lymphocytes expressing the F5 TCR 7. The Surgery Branch protocol differs 
with the UCLA protocol in that up to 1011 TCR transgenic cells were administered, that these cells are 
generated and infused fresh (without cryopreservation as at UCLA), and that no DC vaccine is 
included. The protocol was feasible and overall safe, with major toxicities attributed to the F5 TCR 
transgenic cells being anterior uveitis in 55% of patients and hearing loss in 50% of patients 7. 
Antitumor activity was demonstrated with 6 partial responses (PR) out of 20 patients (30%), with 3 of 
the responses being durable beyond 1 year 7. 
 
7.0 ELIGIBILITY CRITERIA 
 
7.1 Inclusion Criteria 
 
a) Histologically confirmed melanoma that is considered surgically incurable with either: 
a. Stage IIIc melanoma including locally relapsed, satellite, in-transit lesions or bulky draining 
node metastasis. 
b. Stage IV melanoma (M1a, M1b or M1c). 
b) At least 1 lesion amenable for outpatient biopsies; this should be a cutaneous or palpable 
metastatic site or a deeper site accessible by image-guided biopsy that is deemed safe to access 
by the treating physicians and interventional radiologists. Patients without accessible lesions for 
biopsy but with prior tissue available from metastatic disease would be eligible at the investigator’s 
discretion. 
c) MART-1 positive melanoma by RT-PCR or IHC. 
d) HLA-A*0201 (HLA-A2.1) positivity by molecular subtyping*. 
e) Age greater than or equal to 18 years old.  
f) Life expectancy greater than 3 months assessed by a study physician. 
g) A minimum of one measurable lesion defined as:  
a. Meeting the criteria for measurable disease according to Response Evaluation Criteria in 
Solid Tumors (RECIST).  
b. Skin lesion(s) selected as non-completely biopsied target lesion(s) that can be accurately 
measured and recorded by color photography with a ruler to document the size of the 
target lesion(s).   
h) No restriction based on prior treatments.  
i) ECOG performance status (PS) 0 or 1. 
j) Adequate bone marrow and hepatic function determined within 30-60 days prior to enrollment, 
defined as: 
a. Absolute neutrophil count ≥ 1.5 x 109 cells/L. 
b. Platelets ≥ 100 x 109/L. 
c. Hemoglobin ≥ 10 g/dL. 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
21 
d. Aspartate and alanine aminotransferases (AST, ALT) ≤ 2.5 x ULN (≤ 5 x ULN, if 
documented liver metastases are present). 
e. Total bilirubin ≤ 2 x ULN (except patients with documented Gilbert’s syndrome). 
f. Creatinine < 2 mg/dl (or a glomerular filtration rate > 60). 
k) Must be willing and able to accept at least two leukapheresis procedures. 
l) Must be willing and able to accept at least two tumor biopsies. 
m) Must be willing and able to provide written informed consent. 
 
*Patients with HLA-A*0205 (HLA-A2.5) positivity by molecular subtyping may be eligible if there is 
demonstration that they can correctly present the MART-126-35 epitope as stimulators for IFN-gamma 
production by MART-1 F5 TCR transgenic cells. 
 
7.2 Exclusion Criteria 
 
a) Previously known hypersensitivity to any of the agents used in this study. 
b) Received systemic treatment for cancer, including immunotherapy, within one month prior to 
initiation of dosing within this protocol. However, cell harvesting by leukapheresis may be 
performed before one month from prior therapy if the study investigators consider that it will not 
have a detrimental impact on the generate of the two cell therapies in this protocol.  
c) History of, or significant evidence of risk for, chronic inflammatory or autoimmune disease (eg, 
Addison’s disease, multiple sclerosis, Graves disease, Hashimoto’s thyroiditis, inflammatory bowel 
disease, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, hypophysitis, pituitary 
disorders, etc.). Patients will be eligible if prior autoimmune disease is not deemed to be active 
(e.g. fibrotic damage of the thyroid after thyroiditis or its treatment, with stable thyroid hormone 
replacement therapy). Vitiligo will not be a basis for exclusion. 
d) History of inflammatory bowel disease, celiac disease, or other chronic gastrointestinal conditions 
associated with diarrhea or bleeding, or current acute colitis of any origin. 
e) Potential requirement for systemic corticosteroids or concurrent immunosuppressive drugs based 
on prior history or received systemic steroids within the last 4 weeks prior to enrollment (inhaled or 
topical steroids at standard doses are allowed). 
f) HIV seropositivity or other congenital or acquired immune deficiency state, which would increase 
the risk of opportunistic infections and other complications during chemotherapy-induced 
lymphodepletion. If there is a positive result in the infectious disease testing that was not 
previously known, the patient will be referred to their primary physician and/or infectious disease 
specialist. 
g) Hepatitis B or C seropositivity with evidence of ongoing liver damage, which would increase the 
likelihood of hepatic toxicities from the chemotherapy conditioning regimen and supportive 
treatments. If there is a positive result in the infectious disease testing that was not previously 
known, the patient will be referred to their primary physician and/or infectious disease specialist. 
h) Dementia or significantly altered mental status that would prohibit the understanding or rendering 
of informed consent and compliance with the requirements of this protocol.   
i) Clinically active brain metastases. Radiological documentation of absence of active brain 
metastases at screening is required for all patients. Prior evidence of brain metastasis 
successfully treated with surgery or radiation therapy will not be exclusion for participation as long 
as they are deemed under control at the time of study enrollment. 
j) Pregnancy or breast-feeding. Female patients must be surgically sterile or be postmenopausal for 
two years, or must agree to use effective contraception during the period of treatment and 
6 months after. All female patients with reproductive potential must have a negative pregnancy 
test (serum/urine) within 14 days from starting the conditioning chemotherapy.  The definition of 
effective contraception will be based on the judgment of the study investigators. 
k) Since IL-2 is administered following cell infusion: 
a. Patients will be excluded if they have a history of clinically significant ECG abnormalities, 
symptoms of cardiac ischemia or arrhythmias and have a left ventricular ejection fraction 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
22 
(LVEF) < 45% on a cardiac stress test (stress thallium, stress MUGA, dobutamine 
echocardiogram, or other stress test)  
b. Similarly, patients who are 50 years old with a baseline LVEF < 45% will be excluded. 
c. Patients with ECG results of any conduction delays (PR interval >200ms, QTC > 480ms), 
sinus bradycardia (resting heart rate <50 beats per minute), sinus tachycardia (HR>120 
beats per minute) will be evaluated by a cardiologist prior to starting the trial. Patients with 
any arrhythmias, including atrial fibrillation/atrila flutter, excessive ectopy (defined as >20 
PVCs per minute), ventricular tachycardia, 3rd degree heart block will be excluded from the 
study unless cleared by a cardiologist. 
d. Patients with pulmonary function test abnormalities as evidenced by a FEV1/FVC< 70% of 
predicted for normality will be excluded.   
 
7.3. Definition of Study Enrollment Date 
 
Enrollment will be considered the time when the eligibility criteria is being entered into the study 
eligibility form after the screening tests have been completed to declare if the patient is able or not 
able to be considered for this research. 
 
8.0 STUDY PROCEDURES 
 
This is a phase 2 study to determine the safety, feasibility and antitumor activity of retrovirus-
transduced T cell immunotherapy in patients with metastatic melanoma. The retrovirus vector 
encodes the MART-1 F5 alpha and beta TCR chains. The retrovirus vector MSGV1-F5AfT2AB will be 
used to transduce autologous PBMC. Infusions of a target minimum cell dose of 108 TCR transgenic 
cells will be performed at day 0. Subjects will be followed for laboratory and clinical adverse events, 
for evidence of replication competent retrovirus (RCR), and for survival of transduced T cells. The 
most intensive monitoring of research volunteers will be made over 24 months, but subjects will be 
followed for life.  
 
Given the pilot nature of this study, the multiple procedures involved and the use of two cell-based 
therapies manufactured from patients’ blood samples, the timeline of the study is aimed at defining 
their approximate administration to patients. In general, changes in the range of 7 days up or down 
from the scheduled event will not be considered deviations from the study timeline. If there is a clear 
trend of procedures that need to be performed at different times in study subjects, the protocol will be 
amended to attempt to adjust the study timeline to feasible procedures in the clinical study schedule. 
 
8.1 Baseline Screening Assessments  
 
Patients will be screened to determine whether they meet full eligibility criteria after the informed 
consent form (ICF) is signed. Standard of care medical history, pertinent laboratory data and 
significant medical problems of the patient will form the basis for proceeding to evaluate the candidate 
in more detail. Following signing of the ICF and enrollment into the study, the remainder of screening 
procedures and tests will be completed.  
 
An attempt will be made to complete all tests within 30 days of enrollment. However, given the 
multiple screening tests and the pre-study procedures to assess patient eligibility, tests older than 30 
days may be acceptable and will limit unnecessary test repetition. Key tests will be repeated at the 
discretion of the treating physicians if older than 30 days. 
 
Screening tests: 
 
• 
Office visit, vital signs and physical exam.  The physical exam will include weight, height, and vital 
signs.  Weight measurement will only be obtained at subsequent physical exams if clinically 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
23 
indicated.  Determination of HLA-A*0201 (HLA-A2.1) or HLA-A*0205 (HLA-A2.5) positivity by 
subtyping using at least intermediate resolution techniques (this testing may already be available 
from prior analysis).  
• 
There is data that patients who are HLA-A*0205 (HLA-A2.5) positive by molecular subtyping can 
correctly present the MART-126-35 epitope 52. To enroll patients who are HLA-A*0205 positive, 
patient-derived PBMC pulsed with MART-126-35 epitope will be used as stimulators for IFN-gamma 
production by MART-1 F5 TCR transgenic cells. If at least > 30,000 pg/ml/million cells of IFN-γ 
production upon MART-126-35 peptide stimulation using MART-126-35 peptide-pulsed HLA-A*0205 
PBMC. 
• 
Determination of MART-1 positivity in melanoma cells by immunohistochemistry (IHC) or PCR 
analysis of melanoma (this can be done in archived tissue, in a new tumor biopsy or may already 
be available from prior analysis).  A new biopsy may not be required if an acceptable biopsy was 
performed within 60 days of prior to screening. 
• 
Tumor Assessments (Imaging/Clinical): Documentation of baseline target and non-target lesions 
by imaging techniques or by measurement of clinical lesion(s) must be performed. Documentation 
of skin lesion(s) that can be clearly visualized must be established by color photography, including 
a ruler to document size.  
• 
Radiological Assessment of the Brain: All patients are required to have a baseline CT scan with 
i.v. contrast or MRI scan of the brain. Patients found to have active brain metastases are excluded 
from enrollment until the brain metastases are under control after neurosurgery or radiation 
therapy. 
• 
Collect blood for: 
 
WBC with differential count and absolute neutrophil count (ANC), red blood cell (RBC) count, 
hemoglobin, hematocrit, and platelet count. 
 
Blood Chemistries including: Calcium, chloride, total protein, potassium, random glucose, 
sodium, blood urea nitrogen (BUN), creatinine, AST (SGOT), ALT (SGPT), alkaline 
phosphatase (ALP), lactic acid dehydrogenase (LDH), total bilirubin, C-reactive protein, 
Magnesium, Phosphorous, Lipase, Amylase, Uric acid.  
 
Coagulation tests: PT/INR, PTT. 
 
Infectious disease serologies: HIV, CMV, EBV, HBV and HCV serotests (unless already tested 
within the past 1 year). 
 
Thyroid Function: Thyroid stimulating hormone/TSH, T3, T4. 
 
Autoantibody Panel: Anti-nuclear antibodies (ANA), antineutrophil cytoplasmic antibodies 
(ANCA), antimicrosomal, antithyroglobulin, anti liver-kidney microsomal (LKM), anti-islet cell, 
antibodies to Ro (SSA) and to La (SSB), and antiphospholipid antibodies. 
 
A trial investigator or a clinical study staff member will complete an Eligibility Checklist based on these 
results. The checklist will be reviewed by the HGMP compliance officer. If eligible to proceed, the 
leukapheresis will be scheduled and the pertinent laboratory staff will be notified of the anticipated 
dates of cell processing.  
 
8.2. Initial Protocol Procedures 
 
• 
Ophthalmologic Exam. Patients will undergo a formal ophthalmologic exam by a qualified 
ophthalmologist. The minimally required procedures will be an ophthalmic history and a 
comprehensive ophthalmic examination. 
 
• 
Otological Exam. Patients will undergo a formal otological exam by a qualified 
otorhynolaryngologist. The minimally required procedures will be an otological history and a 
comprehensive otologic examination. 
 
• 
Tumor Biopsy. A biopsy of a melanoma lesion accessible to outpatient sampling will be collected 
whenever feasible. A target minimum of 5 x 5 x 5 mm tumor tissue will be collected if feasible.  .  A 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
24 
new biopsy may not be required if an acceptable biopsy was performed within 60 days of prior to 
screening. 
 
• 
Baseline [18F]FDG PET Scan. A baseline PET scan using [18F]FDG as tracer will be used to 
determine the background against which the assessment of MART-1 F5 TCR transgenic T cell 
accumulation will be compared to. An attempt will be made for the baseline PET to be performed 
in conjunction with the baseline tumor assessment by CT scan to minimize radiation exposure. 
 
• 
Cardiac Function Tests. An electrocardiogram (ECG) will record potential pre-existing 
conduction abnormalities for future comparison if needed. A MUGA or echocardiogram will be 
performed to determine if the patient is fit to receive low dose IL-2. A LVEF lower than 45% would 
preclude participation. 
 
• 
Pregnancy Test. All females of childbearing potential must have a negative serum or urine 
Pregnancy Test within 14 days from the initial dose of conditioning chemotherapy in order to be 
eligible for participation. Females who have undergone surgical sterilization or who have been 
postmenopausal for at least 2 years are not considered to be of childbearing potential. 
 
• 
Central Venous Access Catheter. A central venous access catheter (CVC) will be placed during 
the screening period or upon hospital admission. This should ideally be a double lumen central 
catheter, but it may be a single lumen catheter at the discretion of the treating physicians. 
Examples may include double lumen Groshong or PICC catheters. 
 
• 
Pulmonary Function Tests (PFT): Tests to be performed in all patients, and if FEV1/FVC< 70% 
of predicted patient will be excluded from participation. 
 
8.3. Leukapheresis, Cell Cultures, and Admission to the Hospital 
 
       a. Day -6 in the Morning. Leukapheresis and Cell Cultures. In the morning of the day a 
leukapheresis will be performed to collect blood to produce the MART-1 TCR transgenic cells and the 
dendritic cell vaccines. 
 
        b. Day -6 in the Afternoon. Admission to the Hospital. Subject will be admitted to the 
Hematology/Oncology unit at the Ronald Regan UCLA Medical Center.  
 
• 
Subject will be asked about illnesses, injuries, side effects, and any medications subject has taken 
or medical procedures that have been done to subject since the prior visit.  
• 
Physical exam. 
• 
A catheter will be placed in a vein through which subject will receive liquids in preparation for the 
chemotherapy regimen. 
• 
Subject will have blood drawn to check health. Tests include complete blood counts; the ability of 
blood to clot; blood chemistries. Most of these blood tests will be repeated daily or every other day 
during subject’s stay in the hospital. 
• 
Subject will start to receive one or two drugs to prevent complications with infections, the antibiotic 
Bactrim to decrease the risk of Pneumocistis carinii pneumonia (or PCP), and the drug ganciclovir 
to decrease the risk of cytomegalovirus (or CMV). Gancyclovir will only be given to subjects who  
have evidence of prior exposure to the CMV virus. Both of these drugs will be stopped before 
administering the gene-modified MART-1 TCR CTLs, since both drugs would interfere with their 
function in the body. 
 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
25 
8.4 Days –5 to –1: Conditioning Chemotherapy Regimen with Cyclophosphamide and 
Fludarabine 
 
The conditioning chemotherapy will be initiated the next day following leukapheresis.  
 
Supportive Therapy During Chemotherapy Administration: 
 
This therapy will be started on day –5, counting as day 0 the day of engineered TCR transgenic cell 
infusion. The i.v. infusions, procedures and medications will be modified as indicated based on the 
Attending physician and clinical team taking care of patients. 
 
• 
Hydrate: Begin hydration with 0.9% Sodium Chloride Injection containing 10 meq/L of potassium 
chloride at 150 ml/hr, starting approximately 12 hours pre-cyclophosphamide and continuing 
hydration until 24 hours after last cyclophosphamide infusion. 
• 
Daily CBC, and basic chemical panel as per standard procedures in the J-Medicine ward 
• 
Ondansetron (Zofran) 8-16 mg i.v. on the days of chemotherapy (or similar antagonist of serotonin 
type 3 -5-HT3- receptors as per standard practices), lorazepam (Ativan) 1 mg i.v. q6h prn and 
prochlorperazine (Compazine) 10 mg. i.v. q6h prn on the days of chemotherapy conditioning, and 
po afterwards. 
• 
Trimethoprim-Sulfamethoxazole (Bactrim) 1 tablet po tid from days –6 to day –2, restarted once 
the absolute neutrophil count (ANC) is above 1 x 103/µL until day +100. 
• 
Ganciclovir (Cytovene) 6 mg/kg on days –6 to day –2 and then discontinued. Gancyclovir will only 
be given to patients that are CMV seropositive at baseline or following standard practices. 
• 
Posaconazole 200 mg po tid (or similar prophylactic antifungal based on standard practice) 
starting on the day of reinfusion and continued until the ANC is above 3 x 103/µL for 3 consecutive 
days. 
• 
MESNA at equidoses with cyclophosphamide, administered by continuous intravenous infusion for 
24 hours on the days of administration of cyclophosphamide. 
• 
Furosemide dosed as clinically indicated during the hydration period. 
 
Dexamethasone 10 mg may be added as an adjunct to other anti-nausea medications, but it should 
be discontinued before day –1 to avoid its immune suppressive effect against MART-1 F5 TCR PBMC 
adoptive transfer, MART-126-35/DC administration and low dose IL-2. 
 
Chemotherapy Administration: 
 
Days –5 to –4: 
 
• 
Cyclophosphamide 60 mg/kg/day x 2 days i.v. in 250 ml D5W with equidoses of mesna. Maximum 
dose calculated based on actual body weight should not exceed 140% of doses calculated based 
on ideal body weight.  
 
Day -4 to Day -1: 
 
• 
Fludarabine 25 mg/m2/day i.v. daily over 30 minutes for 4 days. Maximum dose calculated based 
on actual body weight should not to exceed 140% of doses calculated based on ideal body 
weight. 
 
Prophylactic Supportive Care Throughout the Pancytopenic Period.  
 
In order to closely monitor patient care and decrease study-related toxicities, patients will receive 
prophylactic therapy throughout the pancytopenic period. The intravenous infusions, procedures and 
medications will be modified as indicated based on the attending physician and clinical team taking 
care of patients. 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
26 
 
• 
During the preparative and pancytopenic period, patients will have a complete blood count and a 
comprehensive metabolic panel including a hepatic panel on a daily basis and periodic urine 
analysis (daily during cyclophosphamide administration days) following the standard J-Medicine 
protocol and the clinical judgment. 
• 
G-CSF (Neupogen) 300 or 480 µg will be used following the standard J-Medicine protocol and the 
clinical judgment. 
• 
Imipenem at 500 mg i.v. q6h. in case of fever > 38.2oC (other antibiotic in case of history of allergy 
to penicillin and derivates; exact antibiotic combination should be decided by the treatment team). 
• 
Packed RBC transfusion to maintain a Hb > 8.0 g/dL, following the standard J-Medicine protocol 
and the clinical judgment. 
• 
Platelet transfusion to attempt to maintain a platelet count of > 10 x 103/µL, > 20 x 103/µL if there 
is concomitant fever, and > 50 x 103/µL if there is evidence of bleeding, following the standard J-
Medicine protocol and the clinical judgment. 
• 
Norethindrone (Norlutate) 10mg PO daily will be used for menstruating women to prophylaxis 
against menorrhagia. 
 
8.5 Day 0: MART-1 F5 TCR PBMC Infusion 
 
Premedication  
 
Side effects following T cell infusions include transient fever, chills, and/or nausea. For this reason, 
patients will receive pre-medication with:  
 
• 
Approximately one hour prior to the infusion, an i.v. infusion of Dextrose 5%-Normal Saline, 250 
ml/hr will begin and continue for 3 hours.  
• 
Briefly (approximately 10 minutes) prior to the infusion, acetaminophen 650 mg po, 25 mg 
diphenhydramine and 12 g mannitol will be administered i.v. These medications may be repeated 
every six hours as needed. 
• 
A course of non-steroidal anti-inflammatory medication may be prescribed if the patient continues 
to have fever not relieved by acetaminophen.  
• 
It is recommended that patients not receive systemic corticosteroids such as hydrocortisone, 
prednisone, prednisolone, or dexamethasone, at any time, except in the case of a life-threatening 
emergency, since this may have an adverse effect on T cell survival. If corticosteroids are required 
for an acute infusion reaction, an initial dose of hydrocortisone 100 mg is recommended. Such 
subjects will not be removed from the study or replaced but will be followed to document the 
effects of such treatment.  
 
Transgenic MART-1 F5 TCR PBMC Cell Infusion 
 
The TCR genetically engineered PBMC product will be transported to the in-patient unit on ice in a 
rigid-walled container with a tight fitting lid (i.e. Tupperware; Nalgene container etc.) and labeled with 
a biohazard label. A target minimum of 108 and up to 109 transduced PBMC will be infused at a rate of 
~10 ml/min through standard IV tubing without a filter.  
 
Patient Monitoring After MART-1 F5 TCR PBMC Infusion 
 
• 
Patients will receive the TCR genetically modified PBMC in an individual use room equipped with 
a high-efficiency particulate air (HEPA) filters that meets the precautions normally used for 
patients undergoing HSC transplantation. 
• 
Patients will be in a monitored bed during cell infusion, with vital signs recorded. 
• 
Sterile saline, acetaminophen and emergency drugs, including epinephrine [0.3-0.5 mg (0.3-0.5 ml 
of a 1:1000 solution), diphenhydramine (25-50 mg i.v.), and methylprednisolone (30-60 mg i.v.), 
should be readily available in the amount and formulation as per the standard treatment of a 
potential hypersensitivity/anaphylactic reaction 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
27 
• 
If there is an adverse event (AE) characterized by life-threatening hypoxia or hypotension, or any 
other infusion reaction qualifying as a DLT as described in section 11.1.1.1, then further infusion of 
the TCR transgenic cells will be canceled and no other immune stimulating approaches, such as 
IL-2, or DC vaccines, will be administered under this protocol.  
 
Febrile Reaction  
 
In the event that a subject develops sepsis or systemic bacteriemia following PBMC infusion, 
appropriate cultures and medical management will be initiated. If a contaminated PBMC product is 
suspected, the product will be retested for sterility using archived samples that are stored in the UCLA 
HGMP/JCCC GMP Suite.  
 
8.6 Days 1, 14 and 30: MART-126-35 Peptide Pulsed DC Administration 
 
Patients will receive a target cell number of 107 MART-126-35 peptide pulsed DC administered 
intradermally (i.d.) close to a lymph node basin not known to be involved with melanoma. 
 
8.7 Days 1-7: Low Dose IL-2 
 
Patients will receive IL-2 (Prometheus) at 500,000 IU/m2 s.c. twice daily for 7 days. Doses may be 
skipped depending on patient tolerance. Doses will be skipped if patients reach grade III or IV toxicity 
due to IL-2 except for the reversible grade III toxicities common to IL-2 such as diarrhea, nausea, 
vomiting, medically manageable hypotension, skin changes, anorexia, mucositis, dysphagia, or 
constitutional symptoms and laboratory changes. Patients receiving IL-2 will have a CBC, electrolytes, 
BUN, creatinine, liver function tests, and serum chemistries evaluated at least once during the week.  
IL-2 administration will be held per the parameters below: 
 
A) Hypotension <80 systolic or hypertension >160 systolic 
B) Sinus tachycardia > 120, sinus bradycardia <60, or any other arrythmias 
C) Syncope  
D) Chest pain  
E) Seizures  
F) Respirations of <10 or >30, or O2 Sat < 90% Room Air 
G) Urine output of less than 100ml q 4 hr  
H) Change in mental status  
I) Temperature more than 103° C, unresponsive to medication  
 
Fevers During IL-2 Administration 
 
Fevers are a known side effect of IL-2 administration. Since patients at the time of IL-2 dosing will also 
be recovering from the conditioning chemotherapy and will likely be pancytopenic, the supportive care 
during this period includes empiric treatment for infections and IL-2-based toxicities without resulting 
in a definition of DLT. 
 
Supportive Care During Low Dose IL-2 Administration 
 
Concomitant medications to control side effects of therapy will be given following the standard UCLA 
practices for IL-2-based therapy. Meperidine (25-50 mg) will be given intravenously if severe 
chills/rigors develop. Other supportive therapy will be given as required and may include 
acetaminophen (650 mg q4h), indomethacin (50-75 mg q6h) and ranitidine (150 mg q12h). 
Ondansetron 0.15 mg/kg/dose i.v. every 8 hours (or an equivalent antiemetic) may be administered 
for nausea or vomiting. Additional antiemetics may be administered as needed for nausea or vomiting. 
Antibiotic coverage for central venous catheters may be provided at the discretion of the treating 
physicians. These side effects are more commonly seen with high doses of IL-2, not the low-doses 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
28 
which is about 15 fold lower in total amount than the high dose IL-2 regimen, plus lasting over a 
longer period, that’s used in this study.  
 
Blood Collection for Immune Monitoring Assays  
 
Collection of 100 ml of peripheral blood is scheduled on study days 0, 14, 30, 45, 60, 75, 90 and at 
each long term follow up visit as described in section 8.14. Blood draws on these days should be 
performed if considered safe, and may be skipped or delayed because patients are likely to have 
chemotherapy-induced pancytopenia and may be hemodynamically unstable after IL-2. These blood 
draws can be done +/- 7 days from the intended days. 
 
Blood Collection for Plasma Cytokine Analyses 
 
Although evidence indicates that it is not directly related to the F5 MART-1 TCR transgenic T cells, 
considering the two recent SAE cases requiring intubation, five to 10 ml of blood in anticoagulant will 
be collected daily for plasma during the first week after ACT routinely for cryopreservation/archiving 
and then as needed thereafter in case of an adverse event that warrants further collection of samples 
for cytokine/chemokine analyses.  
 
8.8 Hospital Discharge 
 
Patients will be discharged from the hospital when the following criteria are met and following the 
clinical judgment of the treating physicians: 
 
• 
Absolute neutrophil count (ANC) > 500/µL. 
• 
Platelet count > 20,000/µL. 
• 
Hemodynamically stable. 
• 
Creatinine on downward trend after L-2. 
• 
Liver function tests stable. 
• 
Not requiring daily blood product infusion.  
 
8.9 Days 20 to 40: MART-1 F5 TCR Cell Tracking After Infusion 
 
Between study days 20 and 40 (or when the treating physicians deem that the patient is stable 
enough to undergo these procedures), either as an inpatient or outpatient, the following will be 
repeated to attempt to detect the MART-1 F5 TCR transduced PBMC: 
 
• 
[18F]FDG PET scan with an attenuated restaging CT of the chest, abdomen and pelvis. 
• 
Tumor biopsy whenever feasible.  
• 
Leukapheresis and plasmapheresis: This will be a partial (5 liter) leukapheresis for PBMC 
collection and 100 ml of plasma.  
 
8.10 Days 30, 45, 60 and 75: Follow Up Study Visits 
 
On study days 30, 45, 60 and 75 patients will be seen as outpatients in the oncology clinic for office 
visit, vital sign determination, safety blood tests and collection of blood for immune monitoring 
analysis. 
 
8.11 Permissible Systemic Therapies During the Protocol On-study Period 
 
The on-study period is defined as one year from the date of the TCR engineered ACT infusion unless 
there is overt tumor progression or DLTs leading to patient discontinuation. Systemic immune 
suppressive therapies will be avoided during this time, but may be permissible in the following 
situations: 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
29 
 
• 
Systemic corticosteroids: Used in case there is a suspicion or evidence of a DLT (as described in 
section 11.1.1.1) that may be related to autoimmune or inflammatory reactions, or cytokine storm. 
In this case, administration of IL-2 and DC vaccines will not be continued. 
 
8.12 Days 75 to 90: Re-staging Exams and Procedures 
 
Patients will undergo restaging CT, PET and/or MRI scans of the brain, chest, abdomen and pelvis 
between study days 75 and 90. Restaging exams every 2-3 months are standard of care in the 
management of patients with metastatic melanoma. In addition, a follow up ophthalmologic and 
otological tests will be scheduled. The results of this testing will be reviewed in a study visit on day 90, 
which will be the last visit during the most intensive part of the study protocol.  
 
8.13 Day 90: Office Visit for Re-staging Evaluation 
 
On study day 90 patients will be evaluated for antitumor activity after undergoing re-staging exams 
and laboratory tests. Patient with SD, PR or CR will be offered to continue on protocol after discussion 
of the study results. Patients with PD will be offered to terminate participation. However, given the 
phenomenon of delayed responses with tumor immunotherapy approaches, the final decision to 
continue or not on study will be at the discretion of the treating physician and the patient, regardless of 
the tumor response assessment. 
 
8.14 Long-term Follow-up Plan 
 
Regardless if followed on-study or off-study, patients will be asked to comply with the following long 
term plan:  
 
• 
For the first 2 years, office visits at least every 3 months. 
• 
After 2 years, office visits or phone follow-up at least every 6 months. 
• 
After 5 years, office visits or phone follow-up at least annually. 
• 
Collection of 60-100 ml of blood for immune monitoring analysis and for RCR testing at 3 and 6 
months, and at one year post cell administration (as described in section 11.2.1.1). 
 
 
9.0 STUDY  AGENTS 
 
9.1 PBMC Expressing MART-1 F5 TCR 
 
Overview. A leukapheresis product obtained from study patients will be transferred to the 
HGMP/JCCC GMP Suite, on the 14th floor of the Factor Building. PBMC will be isolated by gradient 
centrifugation, and an aliquot of these cells will be cultured for 48 hours in the presence of anti-CD3 
(OKT3) and IL-2 in order to stimulate T-cell growth to prepare for viral vector transduction. PBMC will 
be transduced with a clinical grade retrovirus vector expressing MART-1 F5 TCR (MSGV1-F5AfT2AB) 
using retronectin-coated plates in two consecutive days and kept in culture for 96 hours from the time 
of the first retroviral transduction. Transduced cells will be harvested and infused fresh as soon as the 
lot release criteria are cleared. On the infusion day, patients will have completed the non-
myeloablative but lymphocyte depleting preparative regimen consisting of cyclophosphamide and 
fludarabine. 
 
In the event the patient is not clinically fit to receive the gene-modified MART-1 TCR CTL infusion 
within 24 hours of the scheduled day of cell harvest, the cells will be cryopreserved on the harvest day 
for later use. 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
30 
9.1.1 Retrovirus Vector MSGV1-F5AfT2AB 
 
Product Description. The retroviral vector, MSGV1-F5AfT2AB, was generated at the Surgery 
Branch/NCI, and consists of 4,196 bps including the 5' LTR from the murine stem cell virus 
(promoter), packaging signal including the splicing donor and splicing acceptor sites, alpha chain and 
beta chain genes of the anti-MART-1 F5 TCR from TIL clone DMF5 7,41, and murine stem cell virus 
3'LTR. The alpha and beta TCR chains are linked by a T2A picornavirus peptide sequence.  
Classification: Immunotherapeutic. 
Active Ingredient. This retroviral vector has two active transgenes, the alpha and beta chains of a 
high affinity TCR that jointly recognizes the MART-127-35 epitope in the context of HLA-A*0201. 
• 
MART-1 F5 TCR: This high affinity TCR pair specific for the melanoma antigen peptide MART-127-
35 presented by HLA-A*0201 was cloned from a patient with metastatic melanoma. The TCR chain 
genes have not been modified to alter their amino acid sequence, and the affinity for the MART-
126-35 peptide presented by MHC tetramers is identical between the original cells obtained from 
this patient and the affinity of the MART-1 F5 TCR when expressed as a transgene in primary 
human T cells 7,41. 
Mode of Action: One functional complex will be generated by the active transgenes: 
• 
MART-1 F5 TCR: The MART-1 F5 TCR is expected to redirect the specificity of the TCR 
transgenic T cells to the MART-127-35 peptide presented by HLA-A2*0201 on the surface of 
melanoma cells. 
Manufacturing Information for the Clinical Grade Vector: The clinical grade MSGV1-F5AfT2AB 
was generated at the Indiana University Vector Production Facility (IU VPF). The retroviral vector 
supernatant (PG13/F5af2aB C162D1) was prepared and preserved following cGMP conditions at IU 
VPF. The physical titer was determined by RNA dot blot according to the manufacturer’s certificate. 
The supernatant was stored upon the completion of production at least at –70o C at IU VPF. Upon 
shipment on dry ice, the supernatant is stored at –80o C at the HGMP/JCCC GMP suite. This facility is 
equipped with around-the-clock temperature monitoring. Supernatant is thawed and used for in vitro 
transduction of activated PBMC. There will be no re-use of the same unit of supernatant for different 
patients. The retroviral titer has been shown to be stable after immediate thawing and immediate 
administration (tissue culture wells previously coated with retronectin). Handling of the vector should 
follow the guidelines of Biosafety Level-2 (BSL-2).  
How Supplied: The clinical grade vector is supplied by IU VPF following their Standard Operating 
Procedures (SOP) for retroviral vector suspension, labeling and cryopreservation. 
Storage: Single use aliquots are stored in a viral bank established in a dedicated –80oC freezer with 
central, computerized monitoring system, with alarm and recording of all GMP storage systems 
located at the restricted access JCCC GMP Suite, 14th floor of the Factor Building at UCLA. 
Stability: Under the conditions of continuous storage at –80oC without freeze-thawing, it is expected 
that the retroviral vector is stable for at least 5 years. Aliquots of this vector will be recertified annually 
throughout the study period with selected assays for purity, identity and potency. 
Dose and Schedule: For ex vivo use only. 
Route of Administration. All use of this retrovirus vector is ex vivo, and no direct injection will be 
done in human subjects.  
Prior Human Experience. This same retrovirus vector expressing the MART-1 F5 TCR and similarly 
used to transduce human PBMC has already been administered to humans within a phase 2 clinical 
trial at the Surgery Branch/NCI 7. The prior human experience is as follows: 
• 
Retrovirus Vectors: Retroviral vectors have undergone extensive testing in humans. Their main 
safety caveat is the development of insertional mutagenesis, which seems to be a rare event and 
may be related to the expressed transgene and the immune competency of the host. Over 250 
patients in over 40 clinical trials have received stem cells genetically modified with retroviral 
vectors, and the only cases of malignant transformation are the two infants in a clinical trial 
inserting the gammaC gene (a surface cytokine receptor) to HSC of infants with X-linked severe 
combined immune deficiency (SCID) 28,29. 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
31 
• 
MART-1 F5 TCR: The same MART-1 F5 TCR alpha and beta gene pair is being tested at the 
Surgery Branch/N.C.I. (Steven A. Rosenberg, P.I.) for its ability to redirect antigen specificity of 
human PBMC adoptively transferred to lymphopenic hosts in a protocol design similar to the one 
proposed herein 7. If we gain knowledge of the development of serious adverse events (SAE) 
related to the MART-1 TCR transgenic T cell administration, our protocol procedures and informed 
consent form will be modified accordingly.  
Reported Adverse Events and Potential Risks: The potential adverse events are related to the viral 
vector, transgenes and genetically modified cells, as discussed in the following section. 
 
9.1.2 PBMC Transgenic for MART-1 F5 TCR 
 
Product Description. PBMC obtained from leukapheresis and activated for 48 hours with OKT3 (anti-
CD3 antibody) and IL-2, infected by the clinical grade MSGV1-F5AfT2AB retroviral vector supernatant 
in two consecutive days, maintained in culture for 4 days from the start of transduction in IL-2. 
Aliquots of these cells will be used to fulfill the lot release criteria.  
Classification: Immunotherapeutic. 
Active Ingredient. As described for the retrovirus vector MSGV1-F5AfT2AB. 
Mode of Action: The genetically modified PBMC expressing a transgenic surface MART-1 F5 TCR will 
be expected to be redirected to the MART-1 antigen expressed by HLA-A2*0201 and MART-1 positive 
melanoma cells. Recognition of the cognate antigen will result in the release of effector molecules from T 
cells, like the cytotoxic granules perforin and granzyme B, and activating cytokines like interferon-
gamma, IL-2 and TNF-alpha. 
Manufacturing Information: Genetically modified PBMC expressing transgenic MART-1 F5 TCR will 
be manufactured by transduction of previously activated PBMC using the clinical grade retroviral vector 
MSGV1-F5AfT2AB.  
Storage: PBMC transgenic for MART-1 F5 TCR will be harvested within 96 hours from the first 
transduction and infused fresh. If cryopreserved, they will be stored in a centrally-monitored liquid 
nitrogen freezer in the JCCC GMP suite located on the 14th floor of Factor Building, UCLA. 
Stability: These genetically modified PBMC will be infused fresh. When stored in liquid nitrogen, 
PBMC are viable for over 2 years with minimal viability loss if not subject to freeze-thawing. Cells will 
be used within 6 months of manufacture. 
Dose and Schedule: Administered once on day 0 of this protocol, receiving a minimum target cell 
dose of 108 and up to 109 PBMC transgenic for MART-1 F5 TCR. 
Administration: Transgenic cells will be transported from the GMP suite to the hospital bed in an 
infusion bag with over-wrap, on ice in a rigid-walled container with a tight fitting lid (i.e. Tupperware; 
Nalgene container etc.) labeled with a biohazard label. A detailed explanation for the change of PBMC 
protocol from Amendment 7 is provided at the end of the protocol in the Appendix: Amendment 8. 
Unused Transgenic Cells. Transgenic cells generated but not administered to patients will be 
cryopreserved and banked for up to 5 years. Reinfusion of cells has to be done within 6 months of 
their cryopreservation. Cells may be used for additional in vitro testing beyond this period of time. 
Disposal of transgenic cells remaining in the bag and i.v. tubing after administration to patients will be 
done in a biohazard container that will be autoclaved before disposal. 
Prior Human Experience: As described above, PBMC transduced with the same retrovirus vector 
MSGV1-F5AfT2AB, is currently in clinical testing 7. 
Reported Adverse Events and Potential Risks: Potential risks are derived from the viral vector, the 
transgenes it will express and the infusion of genetically modified cells: 
• 
Retroviral Vectors: There are two potential serious anticipated risks associated with retroviral 
vectors. One is related to its ability to randomly and permanently integrate in the genome of target 
cells, which may lead to altered expression of genes in these cells potentially inducing oncogenic 
changes. The second one is related to the possibility of its recombination and generation of 
replication competent virus that could potentially lead to a HIV-like disease. 
o Risk of Insertional Mutagenesis with Retroviral Vectors. The risks of insertional 
oncogenesis with integrating gene delivery vectors is likely a complex function of several 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
32 
factors, related to the type of vector used, the promoter and other transcriptional control 
elements of the vector, the transgene per se, as well as properties of the target cells. 
Moloney Leukemia Virus (MLV)-based gamma-retroviral vectors have been shown to have 
a high predilection for insertion into the 5’ region of actively transcribed genes, which may 
increase the risks for trans-activation of transcription from the cellular gene promoter 55. 
Genes that modulate cell proliferation, such as the gammaC gene used to treat X-linked 
SCID may provide one component of a proliferative signal that plays a cooperative role in 
cellular transformation with cellular genes involved in cellular proliferation, such as LMO-2, 
when activated by insertional trans-activation 28. In contrast, effector proteins such as TCR 
would not, a priori, be expected to confer an unregulated proliferative stimulus, although 
they may play a role in clonal expansion upon encounter with the cognate antigen. 
o Risk of Replication Competent Retrovirus (RCR) with Retroviral Vectors. Another risk 
is that genetic recombination within components of the vector system could lead to the 
generation of RCR. This event would have the potential of generating infective retrovirus 
that may lead to oncogenesis or immune deficiency. Our patients will be screened at 
baseline to be HIV seronegative. Since there will be no wild type HIV virus present, the 
possibility of providing the missing genes for the generation of infective retrovirus in vivo 
would be lower than when retroviral vectors are administered to HIV positive subjects.  
• 
MART-1 TCR: The main potential toxicities from the transgenic alpha and beta MART-1 TCR 
genes expressed in PBMC are derived from the MART-1 specificity of the TCR, or the mispairing 
with endogenous TCR genes leading to cells with autoimmune specificity.  
o Autoimmune Toxicity against MART-1 Positive Cells. Toxic events could be potentially 
derived from cytotoxic activity of the transgenic PBMC against MART-1 positive cells 7. 
This could result in disappearance of pigmented cells in the skin and development of 
vitiligo, pigmented cells in the uvea of the eye inducing uveitis, in the retina with the 
potential development of blindness, and in the inner ear resulting in vestibular problems. In 
particular, in the NCI Surgery Branch experience, anterior uveitis was noted in 55% of 
patients and hearing loss in 50% of patients receiving F5 TCR transgenic cells. These 
cases responded to local injection of corticosteroids 7. These toxicities were not noted in 
the original UCLA experience with this F5 TCR with cell doses up to 109. With the F5 
infusion cell number going back down to 109, We will keep prospectively monitoring 
ophthalmologic toxicity following standard eye exam evaluation for patients receiving tumor 
immunotherapy 56,57, and potential otological toxicities by serial otological exams. 
o Autoimmune Toxicity from Mispairing of TCR Chains. The alpha and beta chains of the 
MART-1 TCR could undergo heterologous pairing with endogenous alpha and beta chains 
of TCR expressed by the transduced T cells, leading to misdirected T cells with newly 
acquired TCR specificities. These would be unpredictable and not subjected to the thymic 
selection process, potentially resulting in T cells with autoreactivities. If evidence of 
autoimmunity developed, patients would receive immune suppressive therapy as clinically 
indicated based on the severity of symptoms, using medications like corticosteroids, 
cyclosporin-A, mycophenolate mofetil, anti-TNF-alpha antibodies or anti-thymocite globulin 
(ATG). 
• 
MART-1 F5 TCR Transgenic T Cells. The adoptive transfer of an ex vivo modified autologous 
cellular product may lead to potential toxicities related to the cell manipulation. 
o Allergic Reaction and Cytokine Release Syndrome. The culture of cells ex vivo in 
media products to maintain lymphocyte viability, the use of reagents to activate cells, the 
retroviral transduction process, and the expression of foreign genes may lead to alterations 
in the cell product resulting in immediate untoward effects. The most serious would be 
infusion reactions leading to severe allergic reactions, or the triggering of a cytokine 
release syndrome. Therefore, the genetically modified cells will be re-infused in an 
inpatient setting, with continuous monitoring of vital signs and by experienced personnel 
within the Hematology-Oncology HSC unit. 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
33 
o Contaminated Cell Product. Ex vivo culture and manipulation of cells may result in a cell 
product contaminated by bacteria, fungus, mycoplasma or virus. With the goal of detecting 
potential contaminants, the genetically modified cells will undergo intensive lot release 
testing prior to infusion. 
o Autoimmune Toxicity from Mispairing of TCRs. The chains of the MART-1 TCR could 
pair with chains of TCR previously expressed by the T cells, leading to T cells that could 
then attack normal organs inducing a condition similar to autoimmune diseases. If 
evidence of autoimmunity developed, patients would receive immune suppressive therapy 
as clinically indicated based on the severity of symptoms, using medications like 
corticosteroids, cyclosporin-A, mycophenolate mofetil, anti-TNF-alpha antibodies or anti-
thymocite globulin (ATG). 
 
9.2 MART-126-35 Peptide Pulsed Dendritic Cells  
 
9.2.1 MART-126-35 Peptide 
 
Product Description: MART-126-35 is an anchor-modified decamer derived from the immunodominant 
epitope derived from the well-characterized MART-1 melanoma lineage antigen in the context of HLA-
A*0201 20,58-60. This HLA haplotype corresponds to greater than 30% of the general population in major 
ethnicities. This decamer anchor-modified peptide derived from the native MART-127-35 peptide with 
the amino-acid sequence ELAGIGILTV (MART-126-35) has more favorable HLA-A*0201 binding 
kinetics, with a longer off-rate resulting in greater ability to interact with T cells specific for MART-127-35 
58-60. This peptide has shown ability to activate MART-127-35-specific T cells in human subjects with a 
very good toxicity profile 6,61.  
Amino Acid Sequence: ELAGIGILTV (H-Glu-Leu-Ala-Gly-Ile-Gly-Ile-Leu-Thr-Val-OH). 
Other Names: Melan-A. 
Classification: Antitumor vaccine. 
MART-126-35 Peptide Molecular Weight: 985.2. 
Chemical Formulation: C45H80N10O14. 
Active Ingredient: MART-126-35 Peptide: An immunodominant anchor-modified peptide in the HLA-
A*0201 haplotype derived from the melanoma tumor antigen MART-1. 
Mode of Action: MART-126-35 is recognized in vivo by the immune system and induces tumor-specific 
CTL, by initiation of a MHC class I-restricted cytotoxic response against tumor cells bearing this antigen.  
MART-126-35 Clinical Grade Peptide Manufacture: Clinical grade peptides will be purchased from 
Biosynthesis, and provided with a Certificate of Analysis (COA). 
How Supplied: MART-126-35 peptide is supplied as a lyophilized powder by Biosynthesis (Lewisville, 
TX) and provided with a COA. 
Storage: MART-126-35 peptide for injection 10 mg per vial: Store intact vials refrigerated at -20o C. 
Working stocks of peptide solubilized in DMSO can be stored at – 80oC.  
Stability:  The peptide is stable at the recommended storage temperature for at least one year.  
Dose and Schedule: For ex vivo use only. 
Administration: The peptide will be used ex vivo to pulse DC, will not be directly administered to 
human subjects. 
Prior Human Experience: Administration of DC pulsed with the anchor-modified MART-126-35 peptide 
has shown to be safe and able to activate T cells specific for the native MART-127-35 peptide in human 
subjects 6,61. 
Reported Adverse Events and Potential Risks: Multiple clinical trials of tumor antigen-based peptide 
immunization have been conducted and published, either using biochemical adjuvants or pulsing DC 
62. Immunization with the native MART-127-35 peptide pulsed onto DC has proven to be a generally safe 
approach able to stimulate antigen-specific T cells in human subjects 4,5. A recently completed phase I 
clinical trial at UCLA administered DC pulsed with this same modified MART-126-35 peptide together 
with the anti-CTLA4 antibody tremelimumab (CP-675,206, Pfizer Inc.) to 16 patients with metastatic 
melanoma, with no untoward adverse events attributed to the modified MART-126-35 peptide. Four 
patients in this series have ongoing objective responses and are melanoma-free at 9+ to 42+ months. 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
34 
 
9.2.2 MART-126-35 Peptide Pulsed Dendritic Cells (DC): 
 
Product Description: Dendritic cells will be differentiated from autologous adherent PBMC in cultures 
containing GM-CSF and IL-4. After one week, loosely-adherent cells are harvested and pulsed with the 
anchor-modified MART-126-35 peptide, and then prepared for i.d. administration to patients as fresh cells. 
Active Ingredient: The biologically active ingredient is the MART-126-35 peptide, which is presented by 
HLA-A2*0201 complexes on the cell surface of DC together with costimulatory molecules and activating 
cytokines. 
Mode of Action: Dendritic cells are the central cells for initiating a cellular immune response. They 
are the most efficient cells for processing and presenting antigen in a MHC class I and II-restricted 
fashion. Antigenic epitopes are presented together with a high level of cell surface costimulatory 
(B7.1, B7.2, CD40) molecule expression. 
Tissue Source: Autologous adherent PBMC differentiated in vitro in GM-CSF (Genzyme) and IL-4 
(Cell Genix or the current supplier of clinical grade IL-4) to generate DC. 
Manufacturing Information: Dendritic cells are generated from PBMC obtained from a single, 
unmobilized leukapheresis procedure by in vitro culture for 7 days in media containing 5% autologous 
heat-inactivated plasma, supplemented with GM-CSF and IL-4. The in vitro generation of DC at the 
JCCC GMP Facility for MART-1 peptide DC-based clinical trials is covered by INDs BB # 7122 and 
11579 4,5. The DC manufacture in the current study will be conducted following the same procedures.  
Storage: MART-126-35 peptide pulsed DC will not be stored; they will be administered fresh after lot 
release testing. During the time between peptide pulsing and injection to patients after lot release 
testing, the cells will be kept loaded in the syringe used for the injection in the final formulation, and 
kept at 4oC or on ice in an ice bucket. 
Stability:  MART-126-35 peptide pulsed DC are stable and viable up to 24 hours from the time they are 
loaded onto a syringe for injection.  
Dose and Schedule: A target cell number of 107 MART-126-35 peptide pulsed DC i.d. days 1, 14 and 
30. 
Route of Administration: Intradermal, close to a lymph node basin not known to be infiltrated by 
melanoma. 
Prior Human Experience: Our completed phase I/II clinical trial (IND #7122) 4,5, where patients with 
melanoma were treated with DC pulsed with the native MART-127-35 immunodominant peptide, has 
allowed us to gain ample experience in this cell-based immunization strategy. This experience has 
been incorporated to an ongoing clinical trial administering the DC pulsed with the anchor-modified 
MART-126-35 anchor-modified peptide together with the anti-CTLA4 antibody tremelimumab (formerly 
CP-675,206) manufactured by Pfizer Inc. (Groton-New London, CT) (IND # 11579). We have 
optimized our laboratory technique to minimize the contamination risk and allow detection of any 
adventitious component in the vaccine before injection into patients. Pharmacokinetics of DC-based 
vaccines in humans have been conducted by others. When DC are injected i.d., as proposed in the 
current trial, the cells can be detected in the draining lymph nodes, supposedly at the site where they 
are able to stimulate the host immune system 63,64. The pharmacodynamic activity of i.d.-injected DC 
can be demonstrated by the generation of antigen-specific immune responses detected in PBMC 4,5.   
Reported Adverse Events and Potential Risks: Several phase I/II clinical trials have been published 
where DC have been administered to human subjects 62,65. This has proven to be a generally safe 
treatment with significant activity in previously treated low grade lymphomas, metastatic melanomas, 
renal cell carcinoma, prostate cancer and colon cancer, among other malignancies. A potential 
adverse event induced by the administration of ex vivo cultured DC is the administration of a product 
contaminated by pathogens. In an attempt to avoid this event, DC will undergo strict lot release criteria 
of the final product before administration to human subjects, and the administration route has been 
chosen to minimize the possibility of systemic toxicities. Otherwise, in our experience of 63 patients (25 
with MART-127-35/DC, 22 with AdVMART1/DC and 16 with MART-126-35/DC and concomitant anti-
CTLA4 antibodies), we have not observed SAE attributable to the MART-1-expressing DC. 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
35 
9.3 Interleukin-2 (aldesleukin, Proleukin, recombinant human Interleukin 2) 
 
Description: Please refer to package insert for complete product Information. Human recombinant 
interleukin-2 is a highly purified protein with a molecular weight of approximately 15,300 daltons. It is a 
lymphokine produced by recombinant DNA technology using a genetically engineered E. coli strain 
containing an analog of the human interleukin-2 gene. 
Mode of Action: Immunotherapeutic agent which stimulates T and NK cell responses. 
How Supplied: Interleukin-2 (IL-2) is manufactured by Prometheus.   
Formulation/Reconstitution: IL-2 is provided as single-use vials containing 22 million IU (~1.3 mg) 
IL-2 as a sterile, white to off-white lyophilized cake plus 50 mg mannitol and 0.18 mg sodium dodecyl 
sulfate, buffered with approximately 0.17 mg monobasic and 0.89 mg dibasic sodium phosphate to a 
pH of 7.5 (range 7.2 to 7.8). The vial is reconstituted with 1.2 mL of Sterile Water for Injection, USP, 
and the resultant concentration is 18 million IU/ml or 1.1 mg/mL. Diluent should be directed against 
the side of the vial to avoid excess foaming. Swirl contents gently until completely dissolved. Do not 
shake. Since vials contain no preservative, reconstituted solution should be used within 24 hours. 
Storage: Intact vials are stored in the refrigerator (2o – 8oC) protected from light. Each vial bears an 
expiration date. 
Dilution/Stability: Reconstituted IL-2 should be further diluted with 50 mL of 5% Human Serum 
Albumin (HSA). The HSA should be added to the diluent prior to the addition of recombinant IL-2. 
Dilutions of the reconstituted solution over a 1000-fold range (i.e., 1 mg/mL to 1 mcg/mL) are 
acceptable in either glass bottles or polyvinyl chloride bags. IL-2 is chemically stable for 48 hours at 
refrigerated and room temperatures, 2o to 30oC. 
Dose and Schedule: 500,000 IU/m2 q12 hours for a maximum of 14 doses.  
Administration: The final dilution of IL-2 will be given subcutaneously. 
Toxicities: Expected toxicities of IL-2 are listed in the product label. Grade III toxicities common to IL-
2 include diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, or 
constitutional symptoms and laboratory changes.   
 
9.4 Fludarabine 
 
Description: Please refer to package insert for complete product Information. Fludarabine phosphate 
is a synthetic purine nucleoside that differs from physiologic nucleosides in that the sugar moiety is 
arabinose instead of ribose or deoxyribose. Fludarabine is a purine antagonist antimetabolite.  
Mode of Action: Chemotherapy agent. 
How Supplied: Fludarabine is supplied by Bayer in a 50 mg vial as a fludarabine phosphate powder 
in the form of a white, lyophilized solid cake.  
Stability: Following reconstitution with 2 mL of sterile water for injection at a concentration of 25 
mg/ml, the solution has a pH of 7.7. The fludarabine powder is stable for at least 18 months at 2-8oC; 
when reconstituted, fludarabine is stable for at least 16 days at room temperature. Because no 
preservative is present, reconstituted fludarabine will typically be administered within 8 hours.  
Specialized references should be consulted for specific compatibility information. Fludarabine is 
dephosphorylated in serum, transported intracellularly and converted to the nucleotide fludarabine 
triphosphate; this 2-fluoro-ara-ATP molecule is thought to be required for the drug’s cytotoxic effects. 
Fludarabine inhibits DNA polymerase, ribnucleotide reductase, DNA primase, and may interfere with 
chain elongation, and RNA and protein synthesis. 
Storage: Intact vials should be stored refrigerated (2-8oC). 
Dose and Schedule: 25 mg/m2/day for 4 days. The dose of fludarabine will be adjusted based on 
estimation of the creatinine clearance (CrCl). For CrCl 30-70 the dose should be reduced by 20%, and 
for CrCl <30 – not given. 
Administration: Fludarabine is administered as an i.v. infusion in 100 ml 0.9% sodium chloride, USP 
over 15 to 30 minutes. To prevent undue toxicity the dose will be based on body surface area (BSA), 
but will not exceed a dose calculated on surface areas based on body weights greater than 140% of 
the maximum ideal body weight per height and weight.   


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
36 
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 patients with chronic lymphocytic leukemia 
(CLL) 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/NCI have caused 
persistently low (below 200) CD4 counts, and one patient developed polyneuropathy manifested by 
vision blindness, and motor and sensory defects 3. 
 
9.5 Cyclophosphamide  
 
Description: Please refer to package insert for complete product Information. 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 i.v. 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.   
Mode of Action: Chemotherapy agent. 
How Supplied: Cyclophosphamide will be obtained from commercially available sources.  
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 2-80C.   
Dose and Schedule: 60 mg/kg/day x 2 days i.v. 
Administration: It will be diluted in 250 ml D5W and infused i.v. over one hour. The dose will be 
based on the patient’s body weight, but to prevent undue toxicity, it will not exceed a dose greater 
than 140% of the maximum ideal body weight per height and weight.    
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 
allopurinal induction of hepatic microsomal enzymes). At high doses, cyclophosphamide can result in 
a syndrome of inappropriate antidiuretic hormone (SIADH) secretion; hyponatremia with progressive 
weight gain without edema occurs. At high doses, cyclophophamide 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 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
37 
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.  
 
9.6 Laboratory Reagents 
 
9.6.1 OKT3. OKT3 (Miltenyi Biotec or other supplier of clinical grade OKT3), is an anti-CD3 antibody 
used to activate T cells in vitro by engaging the CD3 complex. In the current clinical trial, OKT3 will be 
strictly used ex vivo for the activation of PBMC for retroviral transduction. A clinical grade OKT3 
(Miltenyi) is a therapeutic monoclonal antibody approved by the FDA to treat rejection of transplanted 
organs, including the heart, kidneys and liver. However, this antibody is currently not being produced 
by the clinical grade manufacture. When used in vivo, OKT3 antibodies eliminate CD3 positive 
lymphocytes which is beneficial to treat graft rejections. It can induce anaphylactic or anaphylactoid 
reactions, cytokine release syndrome and a variety of constitutional symptoms when administered 
systemically. The source of OKT3 antibody for the current studies is obtained with a certificate of 
analysis providing its manufacturing and product characterization. In addition, after aliquoting, the 
batches used within this study are further characterized by performing the following tests: Gram stain, 
fungal stain, bacterial culture, fungal culture, mycoplasma culture, endotoxin assay. 
 
9.6.2. Interleukin-2. The IL-2 used for in vitro stimulation of PBMC will be the same as described for 
systemic IL-2 treatment in patients. All pertinent information is described in that section. 
 
9.6.3 GM-CSF. Leukine® (Genzyme) is a recombinant granulocyte-macrophage colony-stimulating 
factor (rhu GM-CSF) approved by the FDA for use following induction chemotherapy to shorten time 
to neutrophil recovery. When used in vivo, it can induce constitutional symptoms and bone pain. 
When used in vitro, it can sustain the differentiation of monocytes to DC. In the current clinical trial, 
GM-CSF will be strictly used ex vivo for the generation of DC. 
 
9.6.4 Interleukin-4. Clinical grade recombinant human IL-4 is purchased from Miltenyi Biotec or the 
current supplier of clinical grade IL-4. This cytokine has been previously tested systemically in human 
subjects 66, but has not been licensed by the FDA for human use. When used in vitro, IL-4 increases the 
differentiation of monocytes to DC. This same cytokine source has been used in prior DC-based clinical 
trials at UCLA (IND # 11579 and 11053) and was safe and biologically active when used for DC 
generation 67. 
 
9.6.5. Retronectin. RetroNectin® (CH-296) is purchased from Clontech Laboratories or the current 
supplier of clinical grade RetroNectin®. RetroNectin® is a chimeric peptide of recombinant human 
fibronectin fragments produced in E. coli, consisting of three functional domains: a central cell-binding 
domain (type III repeat, 8-10), heparin-binding domain II (type III repeat, 12-14), and a CS1 site within 
the alternatively-spliced IIICS region. It is a 574 amino acid protein with a molecular weight of 63 kDa. 
When coated on the surface of containers such as culture dishes, petri dishes, flasks or bags, 
retronectin significantly enhances retrovirus-mediated gene transduction into mammalian cells. This 
enhancement is hypothetically due to co-localization of retroviral particles and target cells on the 
molecules of retronectin. Virus particles bind retronectin via interaction with heparin-binding domain II, 
and target cells bind mainly through the interaction of the cell surface integrin receptor VLA-4 with the 
fibronectin CS1 site. Additionally, cells may also bind through the interaction of another fibronectin 
ligand (RGDS in repeat 10) within the central cell-binding domain with a corresponding integrin 
receptor VLA-5 on the cell surface. 
 
 
 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
38 
10.0 GENERATION OF EXPERIMENTAL CELL THERAPIES: MART-1-TCR TRANSDUCED CD8 
CELLS AND MART-126-35 PEPTIDE PULSED DENDRITIC CELLS 
 
Feasibility is one of the primary endpoints of this protocol. Repetitive deviations to the procedures 
described in this section will be recorded in the SOPs and the protocol modified accordingly and 
highlighted in the next submission to the IRB and FDA. 
 
10.1 Generation of MART-1 F5 TCR Transduced PBMC  
 
10.1.1 Leukapheresis Procedures  
 
Patients will undergo a 12 liter leukapheresis procedure to obtain PBMC for retroviral transduction and 
monocytes for DC generation. Any signs and symptoms of citrate toxicity due to apheresis (such as 
perioral paresthesia and muscle cramps) will be treated according to the UCLA Hemapheresis center 
standard procedures. The leukapheresis product will be transferred to the UCLA HGMP/JCCC GMP 
Suite for cell processing.  
 
10.1.2 PBMC Isolation  
 
Following leukapheresis, the product will be separated into mononuclear fraction for PBMC isolation 
using Ficoll and layering the leukapheresis product over this liquid and centrifuging at 500g for 30 
minutes.  
 
10.1.3 PBMC Activation 
 
An aliquot of PBMC obtained from leukapheresis will be cultured in AIMV with 5% heat-inactivated AB 
serum at 106 cells/ml and activated for 48 hours in the presence of OKT3 (50 ng/ml, anti-human CD3 
antibody) and human IL-2 (300 IU/ml, aldesleukin) in order to stimulate T-cell growth to prepare for 
viral transduction. 
 
10.1.4 Clinical Grade Retrovirus MSGV1-F5AfT2AB Lot Release Testing 
 
Prior to use, the clinical grade retroviral vector will require fulfilling the lot release criteria included in 
Table 1. 
 
Table 1. Clinical Grade MSGV1-F5AfT2AB Retrovirus Lot Release Criteria: 
 
Table 1a. Certificate of Analysis of the MSGV1-F5AfT2AB Master Cell Bank (MCB). 
 
Test 
Methods 
Limits 
Results 
Date 
Contaminants 
Aerobic and anaerobic 
culture for bacteria and 
fungus 
No growth within 14 
days 
No growth within 14 
days 
Apr-10-2007 
 
Mycoplasma culture 
and vero indicator cells 
Negative for the 
presence of 
mycoplasma 
Negative 
Apr-04-2007 
 
In-vitro viral assay 
utilizing 3T3, MRC-5 
and Vero cells 
No CPE or 
hermadsorption 
No CPE or 
hermadsorption 
Apr-10-2007 
 
Bovine viral assay* 
Negative for cyto-pathic 
effect, hermadsorption 
and the presence of 7 
specific bovine viruses 
using IFA  
Negative for cyto-pathic 
effect, hermadsorption 
and the presence of 7 
specific bovine viruses 
using IFA 
Dec-11-2006 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
39 
 
Porcine viral assay* 
Negative for cytopathic 
effect, hemadsorption 
and the Presence of 
porcine Viruses, BVDV 
Reovirus and rabies 
Negative for cytopathic 
effect, hemadsorption 
and the Presence of 
porcine Viruses, BVDV 
Reovirus and rabies 
Dec-06-2006 
 
In-viro viral assay* 
No evidence of 
contamination with 
adventitious viral 
agents 
No evidence of 
contamination with 
adventitious viral 
agents 
Dec-21-2006 
 
MAP/LCM* 
No evidence of viral 
contamination 
No evidence of viral 
contamination 
Dec-21-2006 
 
Transmission E.M.* 
No Vital particles other 
than retro-virus like 
particles and no other 
identifiable microbial 
agents. 
No Vital particles other 
than retro-virus like 
particles and no other 
identifiable microbial 
agents  
Nov-15-2006 
 
SV40Tag (PCR)* 
Negative 
Negative 
Nov-14-2006 
 
E1a (PCR)* 
Negative 
Negative 
Jan-22-2007 
Human Vital 
Contaminants 
HTLV-1/11 (PCR)* 
Negative 
Negative 
Nov-16-2006 
 
HIV-1/2 (PCR)* 
Negative 
Negative 
Nov-16-2006 
 
HEP B (PCR)* 
Negative 
Negative 
Nov-14-2006 
 
HEP C (PCR)* 
Negative 
Negative 
Nov-08-2006
 
 
 
HHV 6 (PCR)* 
Negative 
Negative 
Nov-15-2006 
 
 
HHV 7 (PCR)* 
Negative 
Negative 
Nov-14-2006 
 
HHV 8 (PCR)* 
Negative 
Negative 
Nov-15-2006 
 
CMV (PCR)* 
Negative 
Negative 
Nov-15-2006 
 
EBV (PCR)* 
Negative 
Negative 
Nov-14-2006 
 
Human parvovirus B19 
Negative 
Negative 
Apr-14-2006 
Replication 
Competent 
Retrovirus: 
GAL-V  
S+L- (PG-4/293) 
amplification) 5% of 
MCB supernatant 
No Evidence of RCR 
No Evidence of RCR 
Apr-10-2007 
Replication 
Competent 
Retrovirus: 
GAL-V 
S+L- (PG-4/293 co-
culture) 1% of MBC 
No Evidence of RCR 
No Evidence of RCR 
Apr-10-2007 
Replication 
Competent 
Retrovirus: 
ECO 
Marker rescue  
No Evidence of RCR 
No Evidence of RCR 
Apr-10-2007 
Identity  
ADA Isoenzyme 
Cells confirmed to be 
Of murine origin 
Cells confirmed to 
of murine origin  
Apr-10-2007 
 
Vector insert stability 
Vector size/sequence 
consistent with 
expectant fragment 
size 
Vector size/sequence 
consistent with 
expectant fragment size 
Apr-10-2007 
Vector 
Sequencing 
Sequencing of the 
insert in the MCB 
> 98% homology with 
the expected sequence 
> 99.9% homology 
Sep-26-2008 
 
Table 1b. Certificate of Analysis of the MSGV1-F5AfT2AB Final Product. 
 
Test 
Methods 
Limits 
Results 
Date 
Contaminants 
Aerobic and anaerobic 
culture for Bacteria and 
No growth 14 days 
No growth 14 days 
March-18-
2008 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
40 
Fungus  
 
Mycoplasma culture 
and Vero indicator cells 
Negative for the 
presence of 
Mycoplasma 
Negative for the 
presence of 
Mycoplasma 
Apr-04-2008 
 
In-Vitro viral assay 
utilizing 3T3, MRC-5 
And Vero cells 
No CPE or 
hermadsortion 
No CPE or 
hermadsortion 
Mar-18-2008 
Endotoxin 
Limulus amebocyte 
lysate  
Less than 0.33 EU/mL 
Harvest 1-7: less than 
0.06 EU/mL 
Mar-18-2008 
Replication 
competent 
retrovirus: GAL-V 
S+L-(PG-4) 
(293 infection) 5% of 
vector supernatant 
No Evidence of RCR 
No Evidence of RCR 
Mar-18-2008 
 
S+L-(PG-4) (293 co-
cuture) 108 cells from 
production run 
No Evidence of RCR 
No Evidence of RCR 
Mar-18-2008 
Transgene 
expression 
MART-126-35 tetramer 
or dextramer staining 
in activated PBMC 
> 10% MART-126-35 
tetramer or dextramer 
positive cells among 
CD3+ T lymphocytes 
> 75% MART-126-35 
tetramer positive cells 
among CD3+ T 
lymphocytes 
Sept-02-2008 
Potency 
MART-126-35 peptide 
and antigen-specific 
IFN-γ production by 
ELISA 
> 30,000 pg/ml of IFN-γ 
production upon MART-
126-35 peptide 
stimulation using 
MART-126-35 peptide-
pulsed K562/A2.1 cells 
> 100,000 pg/ml of IFN-
γ production upon 
MART-126-35 peptide 
stimulation using 
MART-126-35 peptide-
pulsed K562/A2.1 cells 
Sept-05-2008 
 
10.1.5 Clinical Grade Retrovirus MSGV1-F5AfT2AB Annual Recertification 
 
Single-use aliquots of the clinical grade retrovirus vector will be subjected to annual recertification, 
and the results of this testing will be included in the annual progress report for the FDA (Table 2).  
 
Table 2a. MSGV1-F5AfT2AB Retrovirus Annual Recertification: 
 
Test Item 
Method 
Limit of Detection 
Acceptable Criteria 
Physical viral vector titer 
qRT-PCR 
5 x 103 copies/µl 
Report value 
Infectious viral vector titer 
(Transgene expression) 
MART-126-35 
tetramer 
or dextramer staining in 
activated PBMC 
0.03% 
MART-126-35 
tetramer 
or 
dextramer 
positive 
cells 
among 
CD3+ T lymphocytes  
> 
10% 
MART-126-35 
tetramer 
or 
dextramer 
positive cells among CD3+ 
T lymphocytes 
Sterility  
Aerobic and anaerobic 
culture for bacteria and 
fungus 
No growth within 14 days 
No growth within 14 days 
Mycoplasma  
MycoAlert test 
 20 cfu/ml   
Negative (<1 ratio)   
PCR/ELISA 
mycoplasma assay if 
the MycoAlert results 
are 
equivocal 
or 
positive 
10 cfu.ml  
Negative by PCR 
Endotoxin  
Limulus 
amebocyte 
lysate or Endosafe - 
MCS System 
0.1 EU/ml 
<0.33 
EU/ml 
for 
each 
sample 
Legend: EU: endotoxin units. 
 
 
 
 




MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
42 
HSA, put into an infusion bag and kept in a 4 degrees refrigerator until lot release clearance and i.v. 
infusion. Lot release tests are performed on aliquots of this final product. In the event patient is not 
able to receive the infusion within 24 hours of the scheduled harvest, cells will be re-suspended in 
cryopreservation medium and put into an infusion bag. Cryopreservation will be at < -130oC in a 
monitored liquid nitrogen freezer at the JCCC GMP suite.  
 
10.1.8 Product Labeling 
 
The final MART-1 F5 TCR transgenic PBMC will be labeled using a preprinted study label with the 
following information: 
 
• 
Patient UCLA ID. Number. 
• 
Patient’s initials. 
• 
Subject laboratory clinical trial code. 
• 
Date of cell preparation, with an expiration date of 24 hours if fresh and 6 months if 
cryopreserved. 
• 
Initials of person who prepared the vaccine. 
• 
Labeled with “For autologous use only”. 
• 
Labeled as “Not Tested for Biohazards” (due to no specific testing for HIV, HepC, HepB and 
other adventitious viruses in the final product).  
 
10.1.9 In-process Testing and Final Product Lot Release Testing  
 
In Process Testing. We will conduct the following in-process testings (Table 3a and 3b).  
 
Table 3a. Activated PBMC (Day 2) In-process Testing: 
 
Test Item 
Acceptability Criteria 
Results 
Gram Stain 
Negative 
Negative 
Fungal Stain 
Negative 
Negative 
Bacterial Culture 
No growth 
No growth x 14 days 
Fungal Culture 
No growth 
No growth x 14 days 
Mycoplasma Culture 
No growth 
No growth x 14 days 
 
Table 3b. Transduced PBMC (Day 5) In-process Testing: 
 
Test Item 
Acceptable Criteria 
MycoAlert test 
Negative ;ratio<1 (readingB/readingA) 
PCR/ELISA mycoplasma assay if the MycoAlert 
results are equivocal or positive 
Negative by PCR   
TCR transgene expression  
>10% of T cells by MART-126-35 tetramer or 
dextramer staining  
TCR transgene functionality  
> 30,000 pg/ml/million cells of MART-1 specific 
IFN-γ production by ELISA 
Final Product Testing and Lot Release Criteria. In Table 4a the results that will be required before 
administering the cells to the patients are described, and in Table 4b the results that need to be in 
process at that time but results may not be available at the time of administration to patients are 
described. 
 
Table 4a. MART-1 F5 TCR Transgenic PBMC Lot Release Testing with Results Available Before 
Administration to Patients: 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
43 
 
Test Item 
Acceptable Criteria 
Viability  
> 70%  
Gram and fungal stains
a 
Negative  
Endotoxin Assay b 
≤ 5 EU/ kg body weight per dose  
MycoAlert test
c 
Negative ;ratio<1 (readingB/readingA) 
PCR/ELISA mycoplasma assay if the 
MycoAlert results are equivocal or positive 
Negative by PCR   
TCR transgene expression  
>10% of T cells by MART-126-35 tetramer or 
dextramer staining  
a. Performed on the final product by the UCLA Clinical Microbiology Laboratory. All sterility cultures 
and mycoplasma culture will be followed up to 14 days. If the cultures become positive after the 
patient has received the cells, the patient will be started on empiric antibiotics. 
b. Performed on the final product using the Limulus amebocyte lysate read using the Quantitative 
Chromogenic Procedure analyzed using an OCL-1000 (Bio Whittaker), or the Endosafe - PTS 
System (Charles River) assays.  
 c. If first line MycoAlert test becomes positive, it will be repeated with a second sample from the final    
product. Moreover, we will perform PCR/ELISA mycoplasma assay if the MycoAlert results are 
equivocal or positive. The release specifications for the mycoplasma test should be "negative by 
PCR". 
 
Table 4b. MART-1 F5 TCR Transgenic PBMC Lot Release Testing with Results Available After 
Administration to Patients: 
 
Test Item 
Acceptable Criteria 
Bacterial and fungal culture 
No growth x 14 days 
Mycoplasma culture 
No growth x 14 days  
GalV S+  /L- RCR assay  
Sample archived for later use if required. Not tested 
prospectively in all samples. 
 
Plan in Case of Positive Results of Tests Not Available at the Time of TCR Transgenic Cell 
Administration to Patients. The following will be our plan in case of results different from our 
proposed acceptable criteria: 
 
- 
Bacterial or Fungal culture: An antibiogram will be obtained on the culture to typify the 
contaminant. The patient will be contacted, two peripheral blood draws will be collected for culture, 
and the patient will be started on therapy, first with broad spectrum antibiotics or antifungals, and 
then adapted to the antibiogram of the cultures. 
- 
Mycoplasma culture: If the culture is positive after the rapid mycoplasma test had been negative 
and the cells were released for administration to patients, then the patient will be contacted, two 
peripheral blood draws will be collected for mycoplasma culture, and the patient will be started on 
therapy with erythromycin, clarithromycin or azithromycin. 
- 
RCR: Since the TCR transgenic cell manufacture process will include ex vivo culture for up to 4 
days from the first transduction, RCR will not be tested prospectively in the final product following 
the Guidance for Industry document by CBER, FDA from November 2006. If a sample from a 
patient tested at 3, 6 or 12 months or later is positive for RCR by PCR test, the stored sample of 
the final product will be tested. The patient will be contacted, two peripheral blood draws will be 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
44 
collected for confirmation, and the patient will be started on therapy with combination 
antiretrovirals used to treat HIV infection. 
 
Notification of Regulatory Agencies for Positive Results of TCR Transgenic Cell 
Administration to Patients. If a preparation with bacterial, fungal, mycoplasma or RCR 
contamination has been administered to a patient, this event should be reported to the following 
agencies and committees within 48 hours of first knowledge: 
 
- 
Human Gene Medicine Program compliance officer. 
- 
UCLA IRB. 
- 
UCLA ISPRC. 
- 
Study DSMB. 
- 
RAC. 
- 
FDA. 
 
Additional Optional Testing of TCR Transgenic Cells. The release tests will be used to determine 
whether the final cell product can be released for infusion (see Tables above). In addition, the cell 
product may undergo further characterization apart from the release testing. The vector-specific 
proviral copy number per cell may be determined. We will perform additional testing for the number of 
vector copies and if higher than 5 we will propose a new dilution of the vector supernatant before 
using it for cell transduction. The sites of integration may be defined as the structure of the integrated 
transgene. Detailed flow cytometry and TCR chain usage analysis may be performed. The product 
may be further characterized for measurable transgene products (Table 5).   
 
Table 5. MART-1 F5 TCR Transgenic PBMC Optional Additional Testing: 
 
Characterization Assays 
Comments 
Vector proviral copy per 
transduced cell  
Quantification of the copy number of provirus by qRT-PCR  
Final product phenotyping  
CD3, CD4, CD8, CD27, CD28, CD45RA/RO, CD62L, CCR7, PD-1  
Transgene integration site 
analysis  
Determination of integration site(s)  
Structure of integrated 
transgene  
Stability of integrated transgene  
Detailed flow cytometry  
Phenotype of cells  
TCR transgene functionality  
> 30,000 pg/ml/million cells of MART-1 specific IFN-γ production by 
ELISA 
Other characterizations  
Telomere loss, telomerase expression, Th1 vs Th2 pattern, FoxP3 
expression in pre/post expansion T cells  
 
 
10.2 Generation of MART-126-35 Peptide Pulsed Dendritic Cells 
 
10.2.1 MART-126-35 Peptide: 
 
MART-126-35 is an anchor-modified immunodominant epitope 58-60 derived from the well-characterized 
MART-1 melanoma lineage antigen in the context of HLA-A*0201 20. This HLA haplotype present in 
greater than 30% of the general population in major ethnicities. 
 
10.2.2 Peptide Manufacturing 
 




M A R T -1 F 5 T C R Pr ot o c ol  0 1 / 1 0/ 2 0 18   
                                           
 
 
 
4 6  
 
P ati e nt s will u n d er g o a l e u k a p h er e si s t o o bt ai n P B M C f or M A R T -1 F 5 T C R tr a n s d u c e d c ell s a n d D C 
m a n uf a ct ur e.  A ut ol o g o u s  pl a s m a  ( 1 0 0  ml)  will  b e  o bt ai n e d  b y  pl a s m a p h er e si s  o n  t h e  d a y  of  
l e u k a p h er e si s. A ut ol o g o u s pl a s m a i s h e at-i n a cti v at e d at 5 6o C f or 1/ 2 h o ur a n d k e pt at -2 0 o C i n st eril e 
c o ni c al t u b e s l a b el e d wit h t h e p ati e nt' s a s si g n e d tri al i d e ntifi c ati o n. E a c h  b at c h of c o m pl et e m e di u m i s 
filt er e d ( 0. 2 0 µ m) pri or t o u s e.  
 
1 0. 2. 7 L e u k a p h er e si s L ot R el e a s e Crit eri a:  
 
B ef or e  a d di n g  D M S O -c o nt ai ni n g  fr e e zi n g  m e di a  t o  t h e  P B M C  o bt ai n e d  fr o m  t h e  l e u k a p h er e si s  
pr o d u ct,  a n  ali q u ot  of  t h e s e  c ell s  i s  s e nt  f or  gr a m  st ai n,  st erilit y  c ult ur e s  f or  b a ct eri a,  f u n gi  a n d  
m y c o pl a s m a ( T a bl e 8). All c ult ur e s ar e k e pt f or at l e a st 1 4 d a y s, b ut t h e pr o d u ct c a n b e u s e d aft er 
n e g ati v e st ai n s ar e a s s e s s e d a n d b ef or e fi n al r e s ult s of t h e c ult ur e s.  
 
T a bl e 8. L e u k a p h er e si s L ot R el e a s e Crit eri a:  
 
T e st It e m  
A c c e pt a bilit y Crit eri a  
Gr a m St ai n, F u n g al St ai n  
N e g ati v e  
B a ct eri al C ult ur e   
N o gr o wt h  
F u n g al C ult ur e   
N o gr o wt h  
M y c o pl a s m a c ult ur e  
N o gr o wt h  
 
 
1 0. 2. 8 Pl a s m a L ot R el e a s e Crit eri a:  
 
E a c h b at c h of a ut ol o g o u s pl a s m a will b e t e st e d f or gr a m st ai n, st erilit y c ult ur e s f or b a ct eri a, f u n gi a n d 
m y c o pl a s m a aft er h e at i n a cti v ati o n ( T a bl e 9). All c ult ur e s ar e k e pt f or at l e a st 1 4 d a y s, b ut t h e pr o d u ct 
c a n b e u s e d aft er n e g ati v e st ai n s ar e a s s e s s e d a n d b ef or e fi n al r e s ult s of t h e c ult ur e s.  
 
T a bl e 9.  Pl a s m a L ot R el e a s e Crit eri a:  
 
T e st It e m  
A c c e pt a bilit y Crit eri a  
Gr a m St ai n, F u n g al St ai n  
N e g ati v e  
B a ct eri al C ult ur e   
N o gr o wt h  
F u n g al C ult ur e  
N o gr o wt h  
M y c o pl a s m a C ult ur e  
N o gr o wt h  
 
1 0. 2. 9 D e n driti c C ell M a n uf a ct ur e:  
 
D C ar e c ult ur e d at t h e U C L A C ell a n d G e n e T h er a p y C or e F a cilit y, 1 4 t h fl o or F a ct or B uil di n g, a c ell 
c ult ur e  f a cilit y  m e eti n g  c G M P  st a n d ar d s,  s p e cifi c all y  d e si g n at e d  f or  t h e  c o n d u ct  of  cli ni c al  tri al s  
r e q uiri n g t h e pr o d u cti o n of bi ol o gi c al c ell-b a s e d pr o d u ct s. D C ar e pr e p ar e d fr o m a d h er e nt, a ut ol o g o u s 
p eri p h er al bl o o d m o n o n u cl e ar c ell s e x p o s e d f or 1 w e e k i n ti s s u e c ult ur e t o G M -C S F a n d I L -4.  
 
M o n o n u cl e ar c ell s ar e i s ol at e d fr o m a l e u k a p h er e si s pr o c e d ur e b y Fi c oll -H y p a q u e c e ntrif u g ati o n, a n d 
u s e d fr e s h or cr y p or e s er v e d i n at l e a st 2 0 % h u m a n  A B s er u m, a n d 1 0 % D M S O. D C V a c ci n e # 1 will 
b e  c ult ur e d  fr o m  fr e s h  P B M C  a n d  t h e  s u b s e q u e nt  D C  v a c ci n e s  will  b e  c ult ur e d  b y  t h a wi n g  
cr y o pr e s er v e d P B M C.  O n e w e e k b ef or e t h e pl a n n e d D C v a c ci n ati o n, c ell s ar e w a s h e d i n s ali n e a n d 
pl at e d at 2. 5 – 5 x 1 0 6  vi a bl e c ell s/ ml i n R P MI 1 6 4 0 + 1 -5 % h e at -i n a cti v at e d a ut ol o g o u s pl a s m a ( D C 
c ult ur e  m e di u m).  T h e  c ult ur e  c a n  al s o  b e  d o n e  i n  cli ni c al  gr a d e  h u m a n  A B  s er u m  i n st e a d  of  
a ut ol o g o u s pl a s m a, w hi c h m a y b e d o n e if t h e a m o u nt of a ut ol o g o u s pl a s m a  i s li miti n g, or if t h er e i s 
f ail ur e t o g e n er at e g o o d D C pr e p ar ati o n s wit h t h e m a n d t h e pr o bl e m i s a s si g n e d t o t h e a ut ol o g o u s 
pr o d u ct. Aft er all o wi n g a d h er e n c e f or 2 hr at 3 7 oC, n o n -a d h er e nt c ell s ar e g e ntl y r e m o v e d b y w a s hi n g 


M A R T -1 F 5 T C R Pr ot o c ol  0 1 / 1 0/ 2 0 18   
                                           
 
 
 
4 7  
wit h st eril e s ali n e s ol uti o n. A d h er e nt c ell s ar e c ult ur e d i n D C c ult ur e m e di u m f or 7 d a y s at 3 7
o C i n 5 % 
C 0 2  i n t h e pr e s e n c e of r h G M-C S F ( 7 6 8 I U/ ml) a n d r hI L -4 ( 4 8 0 I U/ ml).  
 
1 0. 2. 1 0 I n-Pr o c e s s T e sti n g  
 
I n-Pr o c e s s B a ct eri al a n d F u n g al T e sti n g. O n d a y 4 of D C c ult ur e, a n ali q u ot of 0. 5 ml of s p e nt 
c ult ur e m e di u m i s r e m o v e d fr o m e a c h ti s s u e c ult ur e fl a s k c o nt ai ni n g D C c ult ur e s, a n d i s s u bj e ct e d t o 
gr a m st ai n, f u n g al st ai n a n d st erilit y c ult ur e s f or b a ct eri a, f u n gi a n d m y c o pl a s m a. All c ult ur e s ar e k e pt 
f or at l e a st 1 4 d a y s. 
 
M y c o pl a s m a T e sti n g.  O n d a y 6 of D C c ult ur e, a s a m pl e i s t a k e n o ut fr o m e a c h fl a s k f or m y c o pl a s m a 
d et e cti o n u si n g a c o m m er ci al P C R -E LI S A kit fr o m R o c h e ( c at al o g N o. 1 6 6 3 9 2 5) or t h e M y c o Al ert 
M y c o pl a s m a D et e cti o n Kit ( L o n z a I n c, All e n d al e, N J), w hi c h i s b a s e d o n d et e cti n g s el e cti v e a cti vit y of 
m y c o pl a s m al e n z y m e s i n a bi o c h e mi c al t e st, wit h t h e r e a d o ut b y bi ol u mi n e s c e n c e ( or a n ot h er r a pi d 
a s s a y wit h si mil ar s e n siti vit y t o d et e ct M y c o pl a s m a c o nt a mi n ati o n). R e s ult s will b e r e p ort e d b ef or e 
v a c ci n e i nj e cti o n i nt o t h e p ati e nt. Crit eri a f or a c c e pt a n c e  of t h e D C pr e p ar ati o n a s m y c o pl a s m a -fr e e 
will b e < 1 r ati o a n d 0. 2 b a s e d o n t h e bi ol u mi n e s c e nt a s s a y a n d P C R -E LI S A a s s a y, r e s p e cti v el y.  
 
1 0. 2. 1 1 Pr e p ar ati o n of M A R T - 12 6 -3 5  P e pti d e P ul s e d D C f or I m m u ni z ati o n:  
 
M A R T - 12 6 -3 5  P e pti d e  P ul si n g.  O n  t h e  d a y  of  i m m u ni z ati o n,  D C  ar e  h ar v e st e d,  w a s h e d  i n  st eril e  
s ali n e a n d r e s u s p e n d e d at a c o n c e ntr ati o n of 1 0 7  i n 1 ml s er u m-fr e e R P MI 1 6 4 0 a n d 1 0 µ g/ ml of 
M A R T - 12 6 -3 5 p e pti d e. Aft er 1 -2 h o ur of i n c u b ati o n at r o o m t e m p er at ur e, M A R T - 12 6 -3 5 / D C ar e pr e p ar e d 
f or i m m u ni z ati on.  
 
Fi n al  M A R T - 12 6 -3 5 / D C  V a c ci n e  Pr e p ar ati o n. Aft er  o n e  h o ur  of  i n c u b ati o n,  M A R T - 12 6 -3 5 / D C  ar e  
p ell et e d a n d w a s h e d t hr e e ti m e s i n st eril e s ali n e s ol uti o n. C ell s ar e c o u nt e d i n tr y p a n bl u e a n d u p t o 1 
x 1 0 7  D C ar e r e s u s p e n d e d i n st eril e s ali n e ( o pti o n all y s u p pl e m e nt e d wit h 1 -5 % of h e at -i n a cti v at e d 
a ut ol o g o u s  pl a s m a),  l o a d e d  o nt o  t h e  s yri n g e  a n d  k e pt  i n  a  s e al e d  b a g  o n  i c e  or  at  4 o C  i n  a  
r efri g er at or, w aiti n g fi n al t e sti n g f or l ot r el e a s e. U p t o 1 x 1 07 D C will b e a d mi ni st er e d, a s f e a si bl e 
b a s e d o n t h e n u m b er of D C i n e a c h pr e p ar ati o n. If a b at c h c o nt ai n s l e s s t h a n t h e m a xi m u m all o w e d 
c ell s, it will still b e a d mi ni st er e d t o p ati e nt s a n d t h e fi n al n u m b er of D C a d mi ni st er e d r e c or d e d i n t h e 
S O P a n d C R F s.  
 
1 0. 2. 1 2 Fi n al M A R T - 12 6 -3 5 / D C L ot R el e a s e T e sti n g:  
 
Fl o w C yt o m etr y.  Aft er t h e D C ar e p e pti d e -p ul s e d, a n ali q u ot of 0. 6 x 1 0 5  t o 1. 2 x 1 06 c ell s will b e 
u s e d  f or  p h e n ot y p e  c h e c k.  N o  si n gl e  m ar k er  c a n  s u c c e s sf ull y r e c o g ni z e  D C,  a n d  t h u s  a p a n el of  
m ar k er s will b e u s e d t o d et er mi n e t h e D C c o m p o siti o n. C ell s will b e  st ai n e d wit h a n a nti -M H C cl a s s II 
a nti b o d y  a n d  a nti -C D 8 6  c o sti m ul at or y  m ol e c ul e  a nti b o d y  a n d  a s s a y e d  b y  F A C S  a n al y si s  t o  
d e m o n str at e t h at  t h e  c ell s  ar e  e q ui p p e d  t o f u n cti o n  a s  a nti g e n pr e s e nti n g  c ell s.  C ell s  will  al s o  b e  
st ai n e d  wit h  a n  a nti -C D 1 4  a nti b o d y  t o  a s s e s s  t h e  D C  m at ur ati o n  l e v el.  Aft er  g ati n g  t h e l ar g e  a n d  
gr a n ul ar c ell s i n t h e p o p ul ati o n, t h e p er c e nt a g e of D C str o n gl y d o u bl e p o siti v e f or M H C cl a s s II a n d 
C D 8 6 a n d w e a kl y p o siti v e f or C D 1 4 i s d et er mi n e d. A c c e pt a bl e D C c o nt e nt d efi n e d a s c ell s d o u b l e 
p o siti v e f or M H C cl a s s II a n d C D 8 6 > 1 0 % fr o m t h e t ot al c ell p o p ul ati o n ( > 3 0 % of t h e g at e d l ar g e a n d 
gr a n ul ar c ell s).  
 
T e sti n g of t h e Fi n al Pr o d u ct.  Aft er t h e M A R T - 12 6 -3 5 / D C h a v e b e e n l o a d e d o nt o a s yri n g e a n d pl a c e d 
i n a s e al e d b a g o n i c e, a n ali q u ot of t h e fi n al pr o d u ct c o nt ai ni n g a p pr o xi m at el y a mi ni m u m of 1 x 1 0 5 
D C f or e a c h st u d y i s s u bj e ct e d t o t h e f oll o wi n g t e sti n g:  
 
a.  Vi a bilit y d et er mi n ati o n b y tr y p a n bl u e e x cl u si o n. Li mit of a c c e pt a n c e: > 7 0 % vi a bilit y of t h e l ar g e 
gr a n ul ar c ell s b y tr y p a n bl u e e x cl u si o n.  


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
48 
 
b. Gram and fungal stains, with negative results to be confirmed before administration of the vaccine 
to the patient. The sensitivity of the Gram stain is 105 cells/ml. 
 
c. Repeated culture for sterility, maintained in culture for a total of 14 days. Culture is done by 
inoculating the sample on Thioglycollate and Tryptic soy broths and Sheep Blood agar plates for 
each sample, and the cultures are examined for 14 days for evidence of growth. The 
Thioglycollate broth will detect aerobic, facultative anaerobic and anaerobic bacteria, and the 
Tryptic soy broth and Blood agar plates detect aerobic and facultative anaerobic bacteria and 
fungi. If results of this or the in process cultures become positive at any time, the patient will be 
immediately contacted and started on empirical broad spectrum intravenous antibiotic treatment 
with Imipenem and Vancomycin, with adjustment of the antibiotic treatment according to the 
sensitivities obtained from the cultures. 
 
d. Mycoplasma Culture. A sample from the DC final product is sent for mycoplasma culture. If the 
mycoplasma culture becomes positive after the patient has been injected with MART-126-35/DC, 
the patient will be contacted and started on Azithromycin. 
e. Evaluation of endotoxin contamination by Limulus Amebocyte Lysate (LAL, Quantitative 
Chromogenic Procedure) or the Endosafe - PTS System (Charles River). The assays have a 
sensitivity of 0.01 and 0.05 endotoxin units (EU)/ml. Acceptance Criteria for Test Article: <5.0 
EU/Kg weight of the study subject. 
 
Additionally, a 0.05-0.1 ml sample of the final MART-126-35/DC preparation from each vaccination time 
will be sterily cryopreserved and stored at < –130oC in a monitored liquid nitrogen tank for possible 
ancillary testing in the future. These samples will be stored a minimum of one year and a day after 
vaccine administration. 
 
10.2.13 MART-126-35/DC Lot Release Criteria: 
 
The criteria for lot release with all results available at the time of injection to the patient is described in 
Table 10. 
 
Table 10. MART-126-35/DC Lot Release Criteria: 
 
Culture Day 
Testing 
Lot Release Criteria 
Leukapheresis Gram stain 
Sterility cultures 
Negative 
Negative to date* 
Plasma 
Gram stain 
Sterility cultures 
Negative 
Negative to date* 
0 
Fresh/ Thawed PBMC  
> 70% Viability  
+4 
Gram stain 
Sterility cultures 
Negative 
Negative to date 
+6 
Mycoplasma Rapid Test 
Negative (Ratio < 1) 
+7 
DC Viability 
DC Phenotype 
Gram stain 
Fungal stain 
Sterility cultures 
Mycoplasma culture 
Endotoxin 
Label 
> 70% Viability 
CD86 + MHC Class II > 30% LGC 
Negative 
Negative 
Negative to date* 
Negative to date* 
< 5 EU/Kg body weight/dose 
“Not Tested for Biohazards” 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
49 
 
*All sterility cultures and mycoplasma culture will be followed up to 14 days and the final results will 
not be available at the time of vaccination.  After injection of the MART-126-35/DC into the patient, if 
positive culture is reported at any time, the patient will be started on empiric antibiotics. 
 
10.2.14 Product Labeling.  
 
The final MART-126-35/DC vaccine will be labeled using a preprinted study label with the following 
information: 
 
• 
Patient UCLA ID. Number. 
• 
Patient’s initials. 
• 
Subject laboratory clinical trial code. 
• 
Date of cell preparation, with an expiration time of 24 hours (since it is administered fresh). 
• 
Initials of person who prepared the vaccine. 
• 
Labeled with “For autologous use only”. 
• 
Labeled as “Not Tested for Biohazards” (due to no specific testing for HIV, HepC, HepB and 
other adventitious viruses in the final product).  
 
10.2.15 MART-126-35/DC Administration.  
 
Patients will receive up to 1 x 107 MART-126-35/DC that meet the lot release criteria injected 
intradermally (i.d.) in 0.1-0.5 ml normal saline close to a lymph node basin not known to be involved 
with melanoma.  
 
10.3 Samples for Transgenic TCR Cell Persistence and Immune Monitoring 
 
Samples of peripheral blood are collected for the secondary endpoints of MART-1 F5 TCR transgenic 
T cell persistence, RCR analysis, retrovirus insertion sites in dominant clones and immunological 
monitoring assays. These PBMC will be obtained from the following: 
 
• 
PBMC obtained from the baseline leukapheresis. 
• 
A second leukapheresis scheduled between study days + 20-40 processing 5 liters of blood 
(partial leukapheresis). 
• 
Peripheral blood collections in EDTA (from which PBMC are separated through Ficoll gradient 
centrifugation. 
 
11.0 EVALUATION OF STUDY ENDPOINTS  
 
11.1.1 Safety and Definitions of DLT.  
 
Criteria for safety evaluation. Safety will be assessed by monitoring and recording potential adverse 
effects of the treatment using the Common Toxicity Criteria at each study visit. Subjects will be 
monitored by medical histories, physical examinations, ophthalmologic exams, and blood studies to 
detect potential toxicities from the treatment. 
 
11.1.1.1 Definition of Dose Limiting Toxicities (DLT).   
 
If 3 patients in the initial 8 patients experience DLTs, the study will not proceed to the next phase.  
 
Adverse events are defined following NCI CTCAE v3.0. DLTs are defined as:  
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
50 
• 
Grade 2 or greater allergic reaction/hypersensitivity (except for fever or rash). 
• 
Grade 2 or greater autoimmune toxicity not associated with anti-tumor response or which 
threatens vital organ (heart, lung, kidney, bowel, bone marrow, musculoskeletal and central 
nervous system,) function with continued treatment. 
• 
Clinical evidence of grade 3 or higher autoimmune reaction in the eye:  
 New subjective patient symptoms in comparison to the data obtained on day 1 of the study 
protocol: 
o Nyctalopia. 
o Loss of peripheral visual field.  
o Photopsias. 
o New floaters. 
o Color desaturation. 
o Blurry vision.   
 New ophthalmologic findings in comparison to the data obtained on day 1 of the study 
protocol: 
o Loss of greater than the equivalent of 1 line of visual acuity. 
o Reproducible decreases in visual field of greater than 3 MD deviation.  
o New onset defined scotoma, depigmentation of the retinal pigment epithelium.  
o Appearance of subretinal fluid. 
o Onset of uveitis not controlled with corticosteroid eye drops. 
• 
Clinical evidence of grade 2 or higher autoimmune reaction in the ear: 
o New subjective patient symptoms in comparison to the data obtained on day 1 of the study 
protocol: 
 
Deafness. 
 
Oscilopia. 
o New otological findings in comparison to the data obtained on day 1 of the study protocol: 
 
Loss of greater than the equivalent of 1 line of ear acuity. 
• 
Grade 3 non-hematologic toxicity due to cell infusion, not easily reversed by standard measures to 
less than a grade 2 within 48 hours. 
• 
Grade 3 skin toxicity at the DC injection site (pain or swelling with inflammation or phlebitis) will 
not be a reason for therapy withholding, as long as it has improved to grade 1 by the time of 
retreatment. However, grade 4 local skin toxicity (ulceration or necrosis) will be a reason for 
treatment withholding. 
• 
Any Grade of skin rashes related to Toxic Epidermal Necrolysis/ Steven Johnson’s Syndrome  
• 
Hypersensitivity reaction, Grade 3. 
• 
Hepatitis, Grade 3. 
• 
Unexpected Grade 3 or greater skin ulceration and skin rashes with desquamation. 
• 
Unexpected ≥ Grade 3 toxicity due to cytokines that does not decrease to ≤ Grade 2 in 96 hours. 
• 
Grade 4 non-laboratory toxicity due to cell infusion  
• 
Intubation due to respiratory distress after receiving the TCR transgenic ACT therapy. 
 
The known toxicities and side effects of the chemotherapy preparative regimen, as listed in the 
protocol or package insert, or IL-2 (Proleukin) or G-CSF (Neupogen) cytokine administration adverse 
event, as listed in the protocol or package insert, will not be considered for the assessment of DLTs.  
 
11.1.1.2 Ocular Examinations.  
 
Since MART-1 positive pigmented cells are present in the uvea and retina, careful evaluation of the 
eye will be conducted throughout the study period.  
 
Qualifications of the Examiners: The ophthalmologic evaluation will be performed by a qualified 
ophthalmologist with a faculty appointment at UCLA. The primary ophthalmologists performing these 
examinations will be the study co-investigators Dr. Bradley R. Straatsma, M.D., J.D., Professor of 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
51 
Ophthalmology and Director Emeritus of the Jules Stein Eye Institute, and Dr. Tara McCannel, M.D., 
Ph.D., Assistant Professor of Ophthalmology. 
 
Procedures: The following will be the procedures performed in the comprehensive ophthalmologic 
visits at screening and post treatment Day 75-90: ophthalmic history, comprehensive ophthalmic 
examination, psychophysical visual function assessment and fundus photography. If any abnormality 
is detected, subjects will undergo an additional fluorescein angiography and electrophysiological 
visual function assessment. For other ophthalmologic visits, the minimally required procedures will be 
an ophthalmic history and a comprehensive ophthalmic examination. 
 
Ocular DLTs: Ocular DLTs consist of new ophthalmologic findings or subjective patient symptoms in 
comparison to the data obtained during screening. The new symptoms of concern include nyctalopia, 
loss of peripheral visual field, photopsias, new floaters, color desaturation, or blurry vision. On 
examination, several findings would consist of an ocular DLT. These include loss of greater than the 
equivalent of 1 line of visual acuity, reproducible decreases in visual field of greater than 3 MD 
deviation or new onset defined scotoma, depigmentation of the retinal pigment epithelium, 
appearance of subretinal fluid, or onset of uveitis. 
 
Procedures to follow if an ocular DLT is determined: Subjects with findings of an ocular DLT as 
defined above will receive either topical or systemic prednisone (100 mg qday for 5 days) as 
recommended by the ophthalmologist, with repeated ophthalmologic assessment. If a favorable 
course is noted, then patients will be followed every week for one month, and monthly afterwards until 
complete resolution of the findings. If the patient does not have a favorable course after the initial 
prednisone treatment, then the patient will be admitted for intravenous corticosteroids therapy with the 
possibility of adding cyclosporin A, OKT3, micophenolate or other immune suppressant.  
 
11.1.1.3 Otologic Examinations.  
 
Since MART-1 positive pigmented cells are present in the inner ear, careful evaluation of the ear 
function will be conducted throughout the study period.  
 
Qualifications of the Examiners: The otologic visit will be performed by a qualified 
otorhinolaryngologist with a faculty appointment at UCLA. The primary otorhinolaryngologist 
performing these visits will be the study co-investigator Dr. Akira Ishiyama. 
 
Procedures: The following will be the procedures performed in the comprehensive ophthalmologic 
visits on day 1 and 75-90: otologic history, comprehensive otologic examination. If any abnormality is 
detected, subjects will undergo additional targeted examinations. 
 
Otologic DLTs: Otologic DLTs consist of new ophthalmologic findings or subjective patient symptoms 
in comparison to the data obtained on day 1 of the study protocol.  
 
Procedures to follow if an otologic DLT is determined: Subjects with findings of an otologic DLT 
as defined above will receive either topical or systemic prednisone (100 mg qday for 5 days) as 
recommended by the otorhynolaryngologist, with repeated otologic assessment. If a favorable course 
is noted, then patients will be followed every week for one month, and monthly afterwards until 
complete resolution of the findings. If the patient does not have a favorable course after the initial 
prednisone treatment, then the patient will be admitted for intravenous corticosteroids therapy with the 
possibility of adding cyclosporin A, OKT3, micophenolate or other immune suppressant.  
 
11.1.2 Feasibility  
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
52 
After entering 8 patients to the first stage of this study, followed up for a minimum of 3 months after 
the last subject has received the infusion of the MART-1 F5 TCR transgenic cells, an assessment of 
protocol feasibility will be done by the study investigators (Table 9). The feasibility assessment will be 
based on: i) the number of MART-1 F5 TCR engineered cells that can be recovered from patients at 
pre-specified time points, and ii) potential problems in the manufacturing of MART-1 F5 TCR 
engineered cells and/or the MART-126-35 peptide–pulsed DC vaccines (Table 11). 
 
Table 11. Criteria to Declare the Study Unfeasible. 
 
Parameter 
Criteria for Unfeasibility 
Feasibility of generating MART-1 transgenic 
cells that meet the lot release criteria. 
3 or more preparations not meeting the lot release 
criteria in stage 1. 
Feasibility of generating MART-126-35/DC that 
meet the lot release criteria. 
3 or more preparations not meeting the lot release 
criteria in stage 1. 
 
11.1.3 Antitumor Activity 
 
11.1.3.1 Malignancy status 
 
Objective tumor responses are aimed at ruling out a 10% response rate as the null hypothesis, and 
35% response rate as the alternative hypothesis within a phase II two-stage clinical trial design. 
Therefore, if 2 out of 8 patients in the first stage have objective responses and the criteria for safety 
and feasibility are met, the study will proceed to a second stage entering 14 additional patients for a 
total of 22 patients in the clinical trial. 
 
Potential objective responses to this combinatorial immunotherapy will be recorded following the 
RECIST (Response Evaluation Criteria in Solid Tumors) criteria 68. Appropriate evaluations, including 
physical exam, pictures of visible lesions and imaging exams, will be evaluated at screening and then 
between study days 75-90, or at discontinuation of study. The duration of patient evaluation may be 
extended if additional outpatient visits are required to assess duration of tumor response or time to 
progression. 
 
All patients who have received an infusion of MART-1 F5 TCR transgenic T cells and have tumor 
assessments at baseline and during the study follow up will be considered evaluable for tumor 
response.  
 
11.1.3.2 Definition of Measurable and Non-measurable Lesions 
 
Measurements of all lesions should be recorded in metric units. All baseline evaluations must be 
performed as close as possible to the first day of study treatment. The same method of assessment 
and the same technique should be used to characterize each identified and reported lesion at 
baseline and during follow-up. 
a. Measurable Lesions: 
• 
Lesions that can be accurately measured in at least 1 dimension (longest diameter to be 
recorded) as ≥2.0 cm with conventional imaging techniques, ≥1.0 cm with spiral CT scan 
with slices of 5 mm thickness, or no less than double the slice thickness using modern CT 
scanners with thinner slices.   
• 
Skin nodules that can be clearly documented by color photography, including a ruler, to 
document the maximum diameter of the target lesion(s), regardless of their size.  


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
53 
• 
If the measurable disease is restricted to a solitary lesion, its neoplastic nature should be 
confirmed by cytology/histology, or by positivity by [18F]FDG PET scan if the lesion is not 
easily accessible for tissue sampling. 
b. Non-measurable Lesions:  
• 
All other melanoma lesions: Previously irradiated lesions are non-measurable except in 
cases of documented progression of the lesion since the completion of radiation therapy. 
 
11.1.3.3 Definition of Target and Non-Target Lesions 
 
Patients must have at least one measurable lesion at baseline to be included in the analysis of this 
endpoint. Baseline documentation of tumor sites may include imaging assessment of disease in the 
chest, abdomen and pelvis. A baseline imaging study of the brain is required. 
a. Target Lesions:  
• 
Up to 10 total lesions, a maximum of 5 lesions per organ, that are representative of all 
involved organs may be selected and recorded as target lesions 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). 
The sum of the longest diameter for all target lesions will be calculated and reported as the 
baseline sum longest diameter. The baseline sum longest diameter will be used as 
reference to further characterize the objective tumor response of the disease. 
• 
Since at least 2 biopsies will be performed, at least one of the baseline target lesions 
should not be completely excised throughout the study period. 
b. Non-Target Lesions:  
• 
All other lesions (or sites of disease) should be identified as non-target lesions and 
recorded as non-target lesions at baseline. Measurement of non-target lesions at baseline 
is not required and should be recorded as “present.” Each non-target lesion should be 
documented as either present, absent or new in each subsequent evaluation. 
 
11.1.3.4 Objective Response Classifications 
 
The following RECIST criteria will be the primary method utilized in this study for the assessment and 
reporting of tumor response data: 
• 
Complete Response (CR): Disappearance of all evidence of melanoma in target and non-
target lesions. CR must be confirmed by repeat assessments performed no less than 4 weeks 
after the criteria for response are first met to qualify as CR. This definition includes a 
pathological CR when hyperpigmented lesions visible by exam are biopsied and demonstrate 
no viable measurable melanoma and pigment accumulated in phagocytic cells. 
• 
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. Non-target lesions may persist provided 
there is no unequivocal progression in these lesions. PR must be confirmed by repeat 
assessments performed no less than 4 weeks after the criteria for response are first met to 
qualify as PR; 
• 
Progressive Disease (PD): At least a 20% increase in the sum LD of the target lesions from 
the smallest sum LD recorded since the beginning of therapy or the appearance of one or 
more new lesions or unequivocal progression of existing non-target lesions; and 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
54 
• 
Stable Disease (SD): Measurements demonstrating neither sufficient shrinkage to qualify for 
PR nor sufficient increase to qualify as PD after the start of treatment taking as reference the 
smallest sum LD since the treatment started. During this time, non-target lesions may persist 
provided there is no unequivocal progression in these lesions.  
 
11.1.3.5 Frequency of Tumor Measurements 
 
Tumor assessments for response will be performed starting at the end of 3 months (Day 90) and 
every 2-3 months subsequently. Tumor assessment will also be performed at discontinuation from the 
study, unless these have been performed within the last 4 weeks prior to discontinuation. CR and PR 
must be confirmed by repeat assessments performed no less than 4 weeks after the criteria for 
response are first met to qualify as such response. Additional tumor measurements may be 
completed as needed. 
11.1.3.6 Assessment of Response for In-Transit Metastasis 
 
In patients with in-transit metastasis only, at least one in-transit metastatic lesion should not be 
excised. If there is only one measurable lesion, biopsies should not interfere with the measurement of 
the largest diameter of the lesion. Response will be assessed as above, taking the measurement from 
pictures with a built-in ruler. 
 
11.1.3.7 Duration of Response:  
 
The duration of overall complete response will be measured from the time measurement criteria has 
been first met for CR until the first date that recurrent or progressive disease is objectively 
documented.  
 
The duration of overall response will be measured from the time measurement criteria is 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).  
 
11.1.3.8 Time to Disease Progression 
 
Defined as the length of time from the date of enrollment to the date of progressive disease first 
documented, death, or the start of secondary antitumor therapy, whichever occurs first.  Subjects lost 
to follow-up will be censored at their last available follow-up date. 
 
11.1.4 Definition of Study Failure Due to Failure of Meeting the Primary Study Endpoints.  
 
Study failure according to safety and feasibility considerations is defined as follows:  
 
1) Unexpected SAEs that are related to the transduction process or research reagent and 
required that the study be stopped. 
2) 3 or more DLT in the first stage with 8 patients. 
3) Considered to be an unfeasible approach as defined above. 
4) 1 or less patients with CR or PR among 8 patients in stage 1. 
5) 4 or less patients with CR or PR among 22 patients in the completed study combining patients 
in stage 1 and 2. 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
55 
11.2 Secondary Study Endpoints:  
 
11.2.1 MART-1 TCR/sr39 Transgenic Cell Persistence 
 
11.2.1.1 Transgenic Cell Persistence.  
 
TCR and vector presence will be quantitated in PBMC samples, obtained before adoptive transfer and 
at 7, 14, 30, 45, 60, 75 and 90 days after transgenic cell adoptive transfer. Thereafter, sampling will 
be every 3 months during the first 2 years, and then every 6 to 12 months. Analysis will be performed 
both using immune monitoring and molecular techniques. Detection of surface expression of the 
MART-1 TCR transgenic protein will be analyzed both by MHC tetramer or dextramer analysis and 
staining for the specific region. Molecular analysis of the persistence of cells bearing the MART-1 F5 
TCR cDNA will be done by real time PCR techniques using primers specific for the transgenes and 
retroviral vector sequences. This testing will provide data to estimate the in vivo survival of 
lymphocytes derived from the infused cells. 
 
11.2.1.2 Long Term Monitoring for Replication Competent Retrovirus (RCR).  
 
Analysis for detection of RCR by PCR will be performed at the National Gene Vector Biorepository 
(NGVB) at the Indiana University Viral Production Facility (IU VPF) under the supervision of Dr. 
Kenneth G. Cornetta. Samples taken from cells prior to infusion will be archived and blood samples 
obtained from study patients at 3 and 6 months, and at one year post cell administration will be tested. 
Blood samples will be archived annually thereafter if all previous testing has been negative.  
 
If a patient dies or develops a neoplasm (other than melanoma recurrence) during this clinical trial, 
efforts will be made to assay a biopsy sample for RCR. If any post-treatment samples are positive, 
then the baseline final product will be tested for RCR. Further analysis of the RCR and more 
extensive patient follow-up will be undertaken, in consultation with the UCLA IRB, RAC and FDA.   
 
11.2.1.3 Analysis of Retroviral Insertion Sites in Long Term Persisting MART-1 F5 TCR Clones 
 
It is possible that expansion of specific T-cell clones will be observed as tumor reactive T cells 
proliferate in response to tumor antigens. Therefore, care will be taken to track T cell persistence both 
immunologically and molecularly. Blood samples for persistence of TCR gene transduced cells will be 
obtained at 3, 6, 12 months, and then annually thereafter. If any patient shows a high level of 
persistence of TCR gene transduced cells at month 6 (by semi quantitative DNA-PCR using primers 
specific for vector sequences) the previously archived samples will be subjected to techniques that 
would allow the identification of clonality of persisting TCR gene transduced cells. Such techniques 
may include T cell cloning or LAM-PCR. Clonality analysis is available through NGVL 69. 
 
If a predominant or monoclonal T cell clone derived from TCR gene transduced cells is identified 
during the follow-up, the integration site and sequence will be identified and subsequently analyzed 
against the human genome database to determine whether the sequences are associated with any 
known human cancers. If a predominant integration site is observed, the T cell cloning or LAM-PCR 
test will be used at an interval of no more than three months after the first observation to see if the 
clone persists or is transient. In all instances where monoclonality is persistent and particularly in 
instances where there is expansion of the clone, regardless of whether or not the sequence is known 
to be associated with a known human cancer, the subject should be monitored closely for signs of 
malignancy. 
 
11.2.1.4 Immunological Monitoring.  
 
Immunological monitoring will consist of quantifying T cells bearing surface MART-1 F5 TCR by 
MART-126-35 tetramer or dextramer analysis. The functionality of these cells will be tested in ELISA 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
56 
and/or multicytokine array assays, intracellular cytokine staining (ICS). Immunological assays will be 
compared between 1) pre-infusion PBMC, 2) an aliquot of the engineered PBL at the time of infusion, 
3) cells recovered from patients’ peripheral blood after adoptive transfer.  
 
Our published definitions for a positive or negative immunological response using standardized MHC 
tetramer assays will be used for these two assays 70. These definitions are based on the assay 
performance specifications by defining changes that are beyond the assay variability with a 95% 
confidence level. For the other assays, differences of 1 fold will be considered indicative of true 
biologic differences.  
 
Additional testing may be performed with other assays, for example intracellular phosphoprotein 
staining for signaling networks, multiplexed microfluidic and nanotechnology-based immune 
monitoring assays 71,72.  
 
11.2.2 Evaluation of MART-1 TCR Transgenic Cell Tumor Trafficking 
 
11.2.2.1 In Vivo Imaging 
 
The ability to non-invasively image T cell responses to cancer hinges upon the detection of metabolic 
changes related to TCR transgenic cell proliferation in vivo detectable with PET probes.  
 
Patients will be injected with 200 MBq [18F]FDG at baseline and approximately 20-40 days after ACT, 
with allowed variability of scheduling for 2-4 weeks of the intended dates. One hour post injection, a 
PET/CT scan covering the area from the base of the skull to the upper abdomen will be acquired. 
Scans of the extremities will be performed in patients with metastatic lesions in the arms or legs. The 
CT scan will be performed in a “low dose mode” (110 kVp, 30 mAs anode current) resulting in an 
effective dose of less than 1.3 mSv per scan 73. These doses are comparable with typical diagnostic 
CT or PET scans and below the limits set by federal regulations for research studies involving 
radioactive imaging agents (http://www.access.gpo.gov/nara/cfr/waisidx_01/21cfr361_01.html).  
 
Regional uptake of [18F]FDG within metastatic tumor sites and secondary lymphoid organs will be 
quantified by standardized uptake values (SUV) normalized to the body weight of the patient. As an 
internal quality control, SUVs will also be determined for several normal organs, such as muscle, liver 
and lungs. These measurements will allow us to identify technical problems in the SUV calculations, 
such as partially paravenous tracer administration 74. An attempt will be made to have all imaging 
studies will be performed on the same PET/CT system in order to eliminate confounding effects of 
differences in scanner sensitivity, spatial resolution or image processing. 
 
11.2.2.2 Tumor Biopsies 
 
To correlate the [18F]FDG signal with the numbers and functional phenotype of MART-1 F5 TCR 
transgenic T cells accumulating at the tumor site, percutaneous or image-guided biopsies of tumor 
lesions between study days 20-40, with allowed variability in this range, will be performed. The 
biopsies will be analyzed by H&E, IHC to quantify the numbers of T lymphocytes. If sufficient quantity 
of tissue is available, the study investigators will attempt to monitor the phenotype of the TIL obtained 
from tumor biopsy samples by MHC tetramer or dextramer, multicolor flow cytometry (FACS), and 
other immune monitoring assays.  
 
12.0 ADVERSE EVENT REPORTING  
 
12.1 Definition of Serious Adverse Events (SAE) 
 
A serious adverse event or serious adverse drug reaction is defined as any untoward medical 
occurrence at any dose that: 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
57 
 
• 
Results in death. 
• 
Is life-threatening (immediate risk of death). 
• 
Requires inpatient hospitalization or prolongation of existing hospitalization. 
• 
Results in persistent or significant disability/incapacity. 
• 
Results in congenital anomaly/birth defect. 
 
Progression of the malignancy under trial should not be reported as an adverse event. However, if the 
malignancy has a fatal outcome during the trial or within the safety-reporting period, then disease 
progression must be recorded as a Grade 5 serious adverse event. 
 
Medical and scientific judgment should be exercised in determining whether an event is an important 
medical event. An important medical event may not be immediately life threatening and/or result in 
death or hospitalization. However, if it is determined that the event may jeopardize the patient and 
may require intervention to prevent one of the other outcomes listed in the definition above, the 
important medical event should be reported as serious. 
 
SAE Reporting Period 
 
Events that meet the SAE definition will be reported within the following timelines based on the time of 
first knowledge of the SAE by a study investigator: 
 
• 
SAE other than resulting in unexpected death: 
o P.I.: Within 24 hours. 
o IRB, ISPRC, DSMB, IBC, NIH/RAC: Within 7 days. 
o FDA: Within 15 days. 
• 
Unexpected death: 
o P.I.: Within 24 hours. 
o IRB, ISPRC, DSMB, IBC, NIH/RAC: Within 24 hours. 
o FDA: Within 7 days. 
 
In general, any SAE will be reported as soon as it is known by the study investigators and enough 
data is gathered for report filing. All SAE reports will be managed through the UCLA SAE Compliance 
Officer, working under the JCCC Regulatory Affairs Office. Initial reports may be completed or 
amended with any new relevant additional information. 
 
Copies of all reports will be maintained in the study regulatory file. Copies of any report sent to the 
FDA will also be sent to the study sponsor. 
 
12.2 Definition of an Adverse Event (AE) 
 
An adverse event is any untoward medical occurrence in a clinical investigation patient administered a 
product or medical device; the event need not necessarily have a causal relationship with the 
treatment or usage.  
 
Abnormal Test Findings 
 
The criteria for determining whether an abnormal objective test finding should be reported as an 
adverse event are as follows:  
 
• 
Test result is associated with accompanying symptoms, and/or 
• 
Test result requires additional diagnostic testing or medical/surgical intervention, and/or 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
58 
• 
Test result leads to a change in trial schedule or discontinuation from the trial, significant 
additional concomitant drug treatment, or other therapy, and/or 
• 
Test result is considered to be an adverse event by the investigator or sponsor. 
 
Merely repeating an abnormal test, in the absence of any of the above conditions, does not constitute 
an adverse event. Any abnormal test result that is determined to be an error does not require 
reporting as an adverse event. 
 
12.3 Causality Assessment 
 
The investigator’s assessment of causality must be provided for all adverse events (serious and non-
serious). An investigator’s causality assessment is the determination of whether there exists a 
reasonable possibility that the investigational product caused or contributed to an adverse event. If the 
investigator’s final determination of causality is unknown and the investigator does not know whether 
or not investigational product caused the event, then the event will be handled as “related to 
investigational product” for reporting purposes. If the investigator's causality assessment is "unknown 
but not related to investigational product", this should be clearly documented on the study records. In 
addition, if the investigator determines a serious adverse event is associated with trial procedures, the 
investigator must record this causal relationship in the source documents and CRF, as appropriate, 
and report such an assessment in accordance with the serious adverse event reporting requirements, 
if applicable. 
 
12.4 Withdrawal Due to Adverse Events  
 
Withdrawal due to adverse event should be distinguished from withdrawal due to insufficient 
response, according to the definition of adverse event noted earlier, and recorded on the appropriate 
adverse event CRF page. When a patient withdraws due to a serious adverse event, the serious 
adverse event must be reported in accordance with the reporting requirements defined below. 
 
13.0 COMPLIANCE WITH GOOD CLINICAL PRACTICE, ETHICAL CONSIDERATIONS, 
INFORMED CONSENT 
 
13.1 Compliance with Good Clinical Practice and Ethical Considerations 
 
This study must be conducted in compliance with all local and federal regulatory requirements, in 
particular those which afford greater protection to the safety of the trial participants. This study will be 
conducted according to the current revision of the Declaration of Helsinki and with local and federal 
laws and regulations relevant to the use of new therapeutic agents. The ICF must contain a language 
readily understood by the potential study subjects. 
 
13.2 Regulatory Approvals 
 
The Protocol and ICF must be approved by the following committees before initiation of patient 
enrollment: 
 
Local Committees: 
 
o Institutional Review Board (IRB).  
o Institutional Biosafety Committee (IBC). 
o Institutional Scientific Peer Review Committee (ISPRC). 
 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
59 
Federal Committees: 
 
o Recombinant DNA Advisory Committee (RAC). 
o Food and Drug Administration (FDA). 
 
13.3 Subject Recruitment  
 
Study subjects will be invited to participate by the study investigators as part of the discussion of 
standard and experimental therapy for locally advanced or metastatic melanoma. Patients may 
access the study investigators through: 
a) Subjects may be referred from UCLA or outside clinics. No patient will be approached without a 
referral or agreement from the patient’s physician in order to uphold patient confidentiality. 
b) Subjects may be identified by the study investigators from their own patient pools while discussing 
standard and experimental options for advanced or metastatic melanoma.  
c) Study flyers will be posted in public places, including the oncology clinic waiting room, for subject 
self-referral.  
d) Subjects may self-refer to this study when inquiring about clinical trial options at UCLA. 
e) This study will be listed at the NIH web site listing active clinical trials, which also assures trial 
registration at a public site.  
 
13.4 Gender and Minorities 
 
There will be no discrimination based upon sex or race. Subjects of any gender and ethnicity with 
melanoma have been and will be considered in the proposed clinical trials. There has been and will 
certainly be no discrimination due to gender or ethnicity, but the epidemiology of melanoma and HLA 
haplotypes may result in uneven minority population distribution. Since melanoma is slightly more 
prevalent in males (2/3), we expect that approximately 30-35% of the study population will be females. 
 
Most subjects referred to the UCLA Melanoma Program come from the UCLA Oncology Network, with 
over 50 offices throughout the Los Angeles County and Western US, including areas where there is a 
majority of subjects of Asian, Hispanic and African American descent. Cutaneous malignant 
melanoma occurs less frequently among non-white populations than among whites. Data from the 
California Cancer Registry reveal that, between 1988 and 1993, 95.5% of subjects with melanoma in 
California were non-Hispanic whites, 3.5% were Hispanic, 0.5% Asian, and 0.5% non-Hispanic blacks 
75. The patients treated in previous melanoma clinical trials conducted over the past 5 years at the 
UCLA Melanoma Program mirror the gender and race characteristics of the malignant melanoma 
patient population in the Southern California area. Table 12 reflects the gender and minority 
distribution of subjects accrued to clinical trials in the UCLA Melanoma Program over the past 5 years. 
93% were white and 33% were female, which is in accordance with the incidence of melanoma in our 
population. 
 
Table 12. Gender and Minorities Accrued to Prior Melanoma Clinical Trials at the UCLA Melanoma 
Program (2002-2007): 
 
 
American 
Indian or 
Alaskan 
Native 
Asian or 
Pacific 
Islander 
Black, not 
of Hispanic 
Origin 
Hispanic 
White, not 
of Hispanic 
Origin 
Other or 
Unknown 
Total 
Female 
- 
1 
1 
5 
52 
1 
60 
Male 
- 
2 
- 
2 
117 
1 
122 
Unknown 
- 
- 
- 
- 
- 
- 
- 
Total 
- 
3 
1 
7 
164 
2 
182 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
60 
13.5 Children 
 
Metastatic melanoma is very rare in subjects younger than 18 years old. The use of the non-
myeloablative regimen in this protocol is a major procedure that 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 melanoma, which is not responsive to other standard forms of therapy, future research will 
be conducted in the pediatric population to evaluate potential benefit in that patient population. 
However, patients with age less than 18 will be considered on a case-by-case basis, and a specific 
pediatric consent will be developed if necessary.  
 
13.6 Informed Consent Procedure  
 
13.6.1 Informed Consent Form (ICF). This study must be conducted in compliance with UCLA IRB, 
IBC, ISPRC, the RAC and the FDA, and relevant informed consent regulations. The ICF must contain 
a language readily understood by the potential study subjects. The original consent form, signed and 
dated by the patient and by the person who conducted the informed consent discussion, will be 
included in the patient’s UCLA medical chart. A copy of the signed ICF will be provided to the patient. 
 
There will be one consent form describing all of the study procedures, including the administration of 
genetically modified cells and the procedures for gene transfer. The rationale for including gene 
transfer and non-gene transfer procedures in the same consent form is that their separation would 
result in unnecessary duplication of text and non-linear explanation of the study procedures.  
 
13.6.2 Procedure of Consent. Subjects will be seen by one of the study investigators who will 
explain the study in detail including procedures, potential risks and side effects. Subjects will be 
encouraged to take additional time to consider the study and will be given the opportunity to take 
home the ICF for review and consideration. They will then be invited to meet with one of the 
investigators at another time. Subjects will be encouraged to have significant others or relatives with 
them at the time of the interviews. Questions from subjects, significant others and relatives will be 
encouraged. If the subject is non-English speaking, the study investigators will have the informed 
consent translated in the subject’s native language. Ample time will be allowed for discussions and 
clarification of the goals of the research project with the trial investigators. Potential subjects will be 
asked if they wish to participate and told that it is voluntary and refusal will not affect their relationship 
with their physician or the care given to them at UCLA. No coercion will be used.  
 
13.7 Subject Enrollment  
 
Subjects will be enrolled at the time the ICF is signed. Fulfillment of the eligibility criteria for each new 
subject enrolled will be prospectively and independently monitored by the UCLA Human Gene 
Medicine Program (HGMP) Compliance Officer. 
 
14.0 QUALIFICATIONS OF PERSONNEL AND STUDY FACILITIES 
 
14.1 Qualifications of Investigators 
 
The UCLA Human Gene Medicine and Melanoma Programs: Antoni Ribas, James S. 
Economou, John A. Glaspy, William H. McBride, Bartosz Chmielowski, Arun Singh, Deborah 
Wong, Begonya Comin-Anduix, Richard C. Koya, Thinle Chodon, Donald Kohn. The first human 
gene transfer clinical trial conducted by Drs. Economou, Glaspy and McBride was initiated in 1992. 
This was a double retroviral vector gene marking of tumor infiltrating lymphocytes (TIL)76. Two 
adenovirus vector gene transfer-based clinical trials were conducted by Drs. Economou, Glaspy, 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
61 
McBride and Ribas at UCLA. These clinical trials involved the generation of genetically manipulated 
DC-based cellular vaccines using adenoviral vectors for melanoma and hepatocellular carcinoma 
(INDs 9908 and 11299). 
 
- 
Antoni Ribas, M.D., Professor of Medicine and Surgery, Director of the Tumor Immunology 
Program Area at the JCCC,. Dr. Ribas is a translational medical oncologist and tumor 
immunologist, conducting experimental clinical trials in patients with melanoma.  
 
- 
James S. Economou, M.D. Ph.D., Professor of Surgery and Microbiology, Immunology, and 
Molecular Genetics, Chief of the Division of Surgical Oncology, Deputy Director of the JCCC and 
the Director of the Human Gene Medicine Program (HGMP), and Vice Chancellor for Research at 
UCLA. Dr. Economou is an accomplished translational tumor immunologist and pioneer in clinical 
trials with retrovirus-modified tumor infiltrating lymphocytes (TIL).  
 
- 
John A. Glaspy, M.D., M.P.H., Professor of Medicine, Vice-Chair of the Division of Hematology-
Oncology, Director of the JCCC Clinical Research Unit Shared Resource, and the Associate 
Chief, UCLA 100 Medical Plaza Community Oncology Practice. Dr. Glaspy is a leading expert in 
clinical research, with a focus on melanoma and the development of cytokines and growth factors 
for human use.  
 
- 
William H. McBride, D.Sc. Professor and Vice Chair for Research, Director, Experimental 
Radiation Oncology. Dr. McBride is a recognized expert in animal models of tumor immunotherapy 
and radiation therapy.  
 
- 
Bartosz Chmielowski, M.D., Ph.D. Assistant Professor of Medicine in the Division of 
Hematology-Oncology. An expert in thymic development 77-79 and in the care of patients with 
malignant melanoma. 
 
- 
Arun Singh, M.D., Clinical Instructor in Medicine at the Division of Hematology-Oncology. Dr. 
Singh is a trained hematology-oncologist with expertise in high dose chemotherapy and IL-2 
administration, and an expert in the use of molecular imaging to study immune responses to 
cancer. 
 
- 
Deborah Wong, M.D., Ph.D. Clinical Instructor, Division of Hematology and Oncology.  Dr. Wong 
is a translational medical oncologist whose research and clinical interest is in improving the 
understanding and the treatment of patients with melanoma. 
 
- 
Siwen Hu-Lieskovan, M.D, Ph.D.  Clinical Instructor, Division of Hematology and Oncology at 
UCLA.  Dr. Hu-Lieskovan is a translational medical oncologist whose research and clinical 
expertise is in development of immune and targeted therapy for melanoma, lung cancer and other 
solid malignancies. 
 
- 
Daniel Shin, M.D.  Fellow, Division of Hematology and Oncology at UCLA.  Dr. Shin is a 
translational medical oncologist. 
 
- 
Begoña Comin-Anduix, Ph.D. Adjunct Associate Professor, Division of Surgical Oncology. An 
expert in immune monitoring and culture of human lymphocytes 70. Will lead the laboratory 
analysis of patient-derived samples. 
 
- 
Beata Berent-Maoz, Ph.D.  Assistant Project Scientist, Division of Hematology and Oncology at 
UCLA.  An expert in retroviral and lentiviral vector manufacture and transduction of target cells. 
Will be responsible for the GMP manufacture of TCR genetically modified cells and DC. 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
62 
- 
Paula Kaplan-Lefko, Ph.D. Project Scientist, Division of Hematology-oncology at UCLA. 
Experienced in writing industry-sponsored and investigator-initiated regulatory documents. Will 
coordinate the regulatory activities during the conduct of this clinical trial. 
 
- 
Richard C. Koya, M.D., Ph.D. Associate Professor of Oncology, Vector Development and 
Production Facility and Associate Director, Center for Immunotherapy at the Roswell Park Cancer 
Institute.,. An experienced molecular biologist and expert in gene therapy vectors, including 
retrovirus and lentiviral vectors.   
 
- 
Thinle Chodon, M.D., Ph.D.  Assistant Professor of Oncology, Center for Immunotherapy and 
Facility Director of CFI Translational Research Operations at the Roswell Park Cancer Institute, an 
expert in retroviral and lentiviral vector manufacture and transduction of target cells. Will be 
responsible for the GMP manufacture of TCR genetically modified cells and peptide pulsed DC. 
 
- 
Donald Kohn, M.D., Professor of Pediatrics and Molecular Microbiology and Immunology at 
UCLA. Dr. Kohn is an international leader in clinical trials of HSC genetically modified using 
retroviral vectors.  
 
The UCLA AIDS Institute Gene Therapy Program: Jerome A. Zack and Zoran Galic. Two clinical 
trials of HSC gene therapy in patients with HIV infection and AIDS, have been led by Dr. Zack (INDs 
7328 and 10183). These trials involve introduction of an anti-HIV ribozyme into autologous CD34+ 
hematopoietic progenitor cells from HIV+ adults.  
 
- 
Jerome A. Zack, Ph.D. Professor of Medicine and Vice Chair of Microbiology, Immunology, and 
Molecular Genetics. Dr. Zack is a thymocyte development, embryonic stem cells and HIV 
immunity expert.  
 
- 
Zoran Galic, Ph.D. Assistant Professor of Medicine. Dr. Galic is an embryonic stem cell and 
thymocyte development expert. 
 
The Caltech/USC Engineering Immunology Program: David Baltimore, Pin Wang, Lili Yang.  
 
- 
David Baltimore Ph.D., President of Caltech and Nobel Prize Laureate for the description of 
reverse transcriptase. Dr. Baltimore has a longstanding interest in the development of 
immunotherapy for cancer and infectious diseases, and has unique expertise in retroviral vectors 
for gene transfer.  
 
- 
Pin Wang, Ph.D. Associate Professor in Chemical Engineering and Material Sciences at USC. Dr. 
Wang is an expert in the genetic engineering of retroviral and lentiviral vectors.  
 
- 
Lili Yang, Ph.D., Project Leader at the Engineering Immunology Program at Caltech. Dr. Yang is 
an expert in TCR genetic cloning and the genetic engineering of HSC. 
 
The University of Connecticut Immunotherapy Program: Bijay Mukherji.  
 
- 
Bijay Mukherji, M.D. Professor of Medicine at the University of Connecticut. Dr. Mukherji is a 
pioneer in cloning human T cells specific for tumor antigens and in the study of their 
immunobiology. Dr. Mukherji conducted the first dendritic cell-based clinical trial in human 
subjects.  
 
 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
63 
The UCLA In Vivo Cellular and Molecular Imaging Center: Caius Radu, Johannes Czernin, 
Michael Phelps.  
 
- 
Caius G. Radu, M.D., Associate Professor of Molecular and Medical Pharmacology. Dr. Radu is 
an expert in molecular immunology and in vivo imaging of immune responses in murine models.  
 
- 
Owen N. Witte, M.D. Howard Hughes Medical Institute Investigator at UCLA, Professor of 
Microbiology, Immunology, and Molecular Genetics, President's Chair in Developmental 
Immunology, Professor of Molecular and Medical Pharmacology, and Director of the Institute of 
Stem Cell Biology and Medicine at UCLA. Dr. Witte is a recognized molecular biologist who has 
pioneered an approach to non-invasively image antitumor T cell responses in alive animals.  
 
- 
Johannes Czernin, M.D., Professor of Molecular and Medical Pharmacology. Dr. Czernin is a 
leading expert in the use of metabolic imaging for assessment of tumor response in patients.  
 
- 
Michael E. Phelps, Ph.D., Norton Simon Professor and Chairman of Molecular and Medical 
Pharmacology, Chief, Division of Nuclear Medicine. Dr. Phelps pioneered PET imaging to 
examine molecular and cellular functions in humans. 
 
NanoSystems Biology Cancer Center (NSBCC): James R. Heath. 
 
- 
James R. Heath, Ph.D., Elizabeth W. Gilloon Professor of Chemistry at Caltech and Director of 
the Caltech-UCLA-Institute for Systems Biology (ISB) NSB Cancer Center. Dr. Heath is a 
nanotechnology and microfluidics expert who has developed novel and highly multiplexed immune 
monitoring assays able to analyze limiting samples from tumor biopsies. 
 
Biostatistics, Analytical Support & Evaluation (BASE) Unit  
 
- 
David Gjertson, Ph.D. Professor of Biomathematics at UCLA. Dr. Gjertson is an experienced 
biostatistician who will provide overall statistical oversight. 
 
- 
Xiaoyan Wang, Ph.D. Assistant Professor of Medicine at UCLA. Dr. Wang is an experienced 
biostatistician who will work with Dr. Gjertson. 
 
The Jules Stein Eye Institute Ophthalmology Program: Bradley Straatsma, Tara McCannel. 
 
 
- 
Tara McCannel, M.D., Ph.D., Assistant Professor of Ophthalmology. Dr. McCannel is an expert 
in ocular melanoma who has been involved in the monitoring of potential ocular toxicities of 
experimental immunotherapies tested in clinical trials at UCLA. 
 
Otological Exams: Akira Ishiyama. 
 
- 
Akira Ishiyama, M.D. Professor of Head and Neck Surgery at UCLA. He is an expert in otological 
analysis, in particular in the study of autoimmune phenomena in the middle and inner ear. 
 
In conclusion, the study investigators are very familiar with the use of retroviral vectors to genetically 
modify primary lymphocytes, the requirements of GMP standards for ex vivo cell culture and genetic 
manipulation, the requirements for GCP standards for conducting human experimental clinical trials, 
and have fully equipped clinical facilities that have been successfully reviewed by regulatory agencies 
for the conduct of cellular therapy and gene transfer studies. 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
64 
14.2 Personnel Training 
 
a. Clinical Personnel: The following will be required courses, training sessions or certificates 
required for clinical study personnel: 
 
• 
UCLA IRB Human subjects certificate. 
• 
UCLA IRB HIPPA certificate. 
• 
Certificate in Good Clinical Practice. 
• 
UCLA Human Gene Medicine Program Educational Course. 
 
b. Laboratory Personnel: The following courses, training sessions or certificates will be required for 
study laboratory personnel involved in TCR transgenic cell manufacturing: 
 
• 
UCLA IRB Human subjects certificate. 
• 
UCLA IRB HIPPA certificate. 
• 
UCLA JCCC current Good Manufacturing Practice training. 
• 
UCLA Human Gene Medicine Program Educational Course. 
• 
UCLA IBC training on Biosafety Level 2. 
• 
UCLA IBC certificate in the use of biosafety cabinets. 
• 
UCLA IBC shipping of Biological Material 
• 
UCLA IBC training in biological waste management. 
• 
UCLA IBC training on Bloodborne Pathogens. 
 
14.3 HGMP/JCCC Cell and Gene Therapy GMP Suite at UCLA 
 
UCLA has a dedicated, fully equipped and functional GMP suite specifically designed for cellular and 
gene transfer clinical trials. This facility is located on the 14th floor of the JCCC Factor Building and is 
jointly managed by the JCCC and the HGMP. It consists of a 2395 square feet BioSafety level 2 
(BSL2) suite with 7 HEPA-filtered rooms. This secured area has centralized monitoring of air and 
equipment linked to the study investigators’ pagers. Access is restricted to investigators with certified 
training and is supervised by the JCCC Regulatory Affairs Office. The 4 positive pressure laboratories 
are designed for ex vivo cell manipulation of cells, since it minimizes the possibility of outside 
pathogen contamination. The 3 negative pressure laboratories are designed for gene transfer 
procedures, since it ensures that the recombinant vectors will not contaminate the surrounding 
environment. There have been 18 investigator-initiated clinical trials in which the cell or gene therapy 
product has been manufactured by the investigator team in the JCCC GMP suite within RAC and FDA 
compliance.  
 
14.4. Gene Therapy Viral Bank 
 
The clinical grade MSGV1-F5AfT2AB will be stored in a dedicated –80oC freezer with central, 
computerized monitoring system, with alarm and recording of all GMP storage systems (refrigerators, 
freezers and incubators). This freezer is located at the JCCC GMP Suite. This freezer is designed for 
exclusive use as a viral bank for ongoing gene therapy trials at UCLA, and is located in a secure area 
for containment to ensure proper storage. Access to the facility is guarded by two security doors with 
restricted access only to adequately trained and authorized individuals. Completion of a five-course 
training program is required to gain access to this GMP suite. This training program is provided by the 
JCCC Regulatory Affairs Office and the UCLA IBC. 
 
Under these conditions, retroviral vectors retain activity for a period exceeding 5 years if not subjected 
to freeze-thawing. Each aliquot of the final retroviral vector is calculated to be for a single use, 
therefore avoiding freeze-thawing cycles. All activities related to the GMP retroviral vector stored in 
the viral bank will be recorded in appropriate SOPs. This includes initial lot shipment, viral vector 
storage, viral vector dispensing, remaining samples after use to generate retrovirus gene-modified cell 
therapies, and yearly recertification. 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
65 
 
14.5. Clinical Facilities 
 
Chemotherapy conditioning and MART-1 F5 TCR cell adoptive transfer will be delivered in an 
isolation hospital bed in the Hematology/Oncology Hematopoietic Stem Cell (HSC) transplantation 
ward. This is a fully staffed inpatient ward with experienced nurses, house officers, clinical consultants 
and attending physicians. This ensures that, in the event of an adverse reaction or any other adverse 
event, treatment will be promptly available. Patients will be placed in individual use rooms equipped 
with a high-efficiency particulate air (HEPA) filters that meet the precautions normally used for 
patients undergoing hematopoietic stem cell transplantation. While receiving the TCR transgenic 
PBMC adoptive transfer, patients will be continuously monitored.  
 
This inpatient stay will be under the administrative responsibility of the UCLA General Clinical 
Research Center (Isidro B. Salusky, M.D., G-CRC Director, telephone: 310-206-6987). This will 
provide an additional level of administrative and scientific oversight. The G-CRC staff provides expert 
care for research subjects and collaborates with investigators for the coordination and performance of 
clinical research. The G-CRC includes a Computer Center under the supervision of the G-CRC 
Biostatistician Dr. Robert M. Elashoff, Ph.D. (telephone: 310-825-9421) to assist in data analysis. 
 
The UCLA Melanoma Program medical oncology outpatient clinic is located at the UCLA 100 Medical 
Plaza, Suite 550. This facility is staffed with an office manager, 3 front desk assistants, 6 nurses, 2 
phlebotomists, 2 insurance specialists and one nurse practitioner.  
 
The UCLA Hemapheresis Unit is located at the 6th floor of the 200 Medical Plaza building, Suite 660. This 
unit has 6 staff nurses on a one-to-one basis, a supervisor, a coordinator, and a Hematology/Oncology 
fellow and attending doctors.  
 
14.6 Compliance with Protocol Procedures and Rights and Welfare of Study Participants 
 
Human Gene Medicine Program (HGMP) Compliance Officer. Kit Shaw (contact phone: 310-267-
0584), Ph.D. Dr. Shaw worked on engineering and testing retroviral gene-transfer vectors in primary 
cells and small animal models.  She has been the study director for IND study of gene transfer to 
hematopoietic stem cells (HSCs) for adenosine deaminase (ADA)-deficient severe combined 
immunodeficiency (SCID), where she had to ensure trial compliance with the regulatory bodies (FDA, 
DSMB, RAC, IRB) overseeing the trial.  She also has experience with IND submissions and cGMP for 
cell-based products.  She is familiar with clinical laboratory operations in addition to research 
laboratory operations.  Dr. Shaw will oversee activities involving gene transfer in the UCLA GMP suite 
and will prospectively review adherence to the protocol, RAC and FDA procedures.  Dr. Shaw also 
serves on the Jonsson Comprehensive Cancer Center (JCCC) DSMB. For our investigator-initiated 
gene therapy trial, Dr. Shaw  will perform real time monitoring and auditing of the study.  
 
 
JCCC Office of Regulatory Compliance. The manager of this office is Terra Hughes. Ms. Terra 
Hughes recently joined the JCCC from the UCLA IRB where she managed the Medical IRB 
committee 2. She has a bachelor’s degree from the University of Southern California (USC) in Biology 
and a Masters degree from CSULA in Criminalistics. Ms. Hughes will oversee the adequate conduct 
of this clinical trial.  
 
Clinical Research Compliance Officer. Sujna Raval-Fernandes, Ph.D. (contact phone 310-794-
8693). Dr. Raval-Fernandes has a strong background in cancer research with more than 10 years of 
basic research experience. She has been in the Jonsson Comprehensive Cancer Center (JCCC)’s 
Office of regulatory Compliance for the past four and half years serving as the compliance officer and 
as a resource to investigators and clinical study staff in resolving quality assurance issues related to 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
66 
research procedures and documentation. She is responsible for monitoring and auditing of studies to 
ensure regulatory compliance. She also serves on the JCCC Data and Safety Monitoring Board 
(DSMB). Her responsibilities also include timely review of all serious adverse events submitted to the 
DSMB. She ensures that the investigators provide the JCCC DSMB with periodic summary reports 
that include accrual, safety and efficacy data. For our investigator-initiated gene medicine study, she 
will serve to ensure compliance to the JCCC DSMB.  
 
Office of Research Participant Advocacy (ORPA). The ORPA was established to assure the safety 
and well-being of all participants in clinical research studies conducted at the UCLA G- CRC while 
maximally facilitating the research process. Stanley Korenman, M.D. (telephone: 310-794-1818), 
Associate Dean of Ethics and Professor of Endocrinology, serves as the Clinical Director of the office. 
Laurie Shaker-Irwin, Ph.D. (telephone: 310-794-7504) serves as the Research Subject Advocate and 
manager of the office. ORPA fosters policies for the ethical conduct of clinical research, assuring 
autonomy and respect in the informed consent process, justice in the selection of participants, and 
beneficence in the initial and ongoing review and evaluation of risks and benefits. The office enhances 
patient safety by providing guidance and education to investigators and research personnel in the 
areas of ethics, research integrity, data and safety monitoring, adverse event reporting, and Good 
Clinical Practices (GCP).  
 
15.0 DATA SAFETY MONITORING BOARD, STUDY OVERSIGHT, DATA COLLECTION AND 
ANALYSIS 
 
15.1 Data Safety Monitoring Board (DSMB) 
 
The Jonsson Comprehensive Cancer Center (JCCC) Data and Safety Monitoring Board (DSMB) was 
constituted in January 2001. The DSMB membership consists of representatives of the Cancer Center 
clinical research community. The Director of the Cancer Center appoints members to a term of 2 
years and each member is selected for their professional expertise in oncology practice and research.  
 
The JCCC DSMB meets monthly to review all serious adverse event reports for JCCC institutional 
clinical trials at UCLA and those encountered in the UCLA TORI network where the JCCC DSMB has 
oversight. All serious adverse event reports, which have been filed since the previous meeting, are 
presented to the committee for review. 
 
For all JCCC and TORI studies, where the JCCC DSMB has primarily oversight for AE review, all AEs 
occurring within these studies shall be reported to the JCCC DSMB in a timely manner consistent with 
the UCLA IRB time requirements [ten days, two days for a death] regardless of relationship or 
seriousness.  The JCCC Office of Regulatory Compliance (ORC) will review all submissions and staff 
will enter the information into the JCCC Clinical Trials database.  Reports are generated for full JCCC 
DSMB review of those SAEs that have some component of relatedness to the study drug and may, at 
the discretion of the JCCC compliance officers, include SAE reports that may be incorrectly assessed 
by the PI. For institutional trials, where the JCCC DSMB has primary DSMB review responsibility, the 
office will request that the PI generate cumulative adverse event reports for biannual or annual review.  
 
The DSMB reviews each Serious Adverse Event reported and determines whether or not protocol 
modifications are warranted to ensure patient safety. In this review, prior occurrences of similar 
toxicity with the therapy under study are taken into consideration, as well as the seriousness of the 
event and the likelihood that it was related to a study drug. The DSMB may recommend no changes 
to the study if the event is expected or related to other causes such as the patient’s underlying 
condition.  The DSMB may request an expert’s advice of other non-UCLA Principal Investigator with 
national experience to support their deliberations and decisions.   The JCCC DSMB has the authority 
to recommend to the UCLA IRB the immediate halt to the study (i.e., discontinuation of any further 
treatment of enrolled patients and discontinuation of enrollment of new patients) should there be any 
serious unexpected toxicity that warrants further investigation. JCCC DSMB correspondences are 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
67 
addressed to the Principal Investigator and copied to the UCLA IRB. Minutes of the DSMB meetings 
are recorded and processed into the computer file. 
 
15.2 JCCC DSMB Internal Monitoring Plan. The NIH and NCI policy statements allow for variable 
monitoring and reporting plans, commensurate with the potential risks and with the size and 
complexity of the trial. The monitoring plan must be sufficiently rigorous and effective to ensure 
subject safety and to ensure protocol compliance and data validity and integrity.  
 
15.2.1 Level of Risk of a Study. For trials overseen by the JCCC DSMB, the JCCC DSMB will 
determine the degree of risk of the study and will ensure that there are procedures in place to ensure 
the safety of the subjects that are enrolled in the trial.  The intensity level of the monitoring is 
determined by the risk category.  Some of the factors that must be considered when assigning the 
Level of Risk category include: 
 
1. A biostatistical design and appropriate procedures for proper data management so that the 
information collected can be properly validated. 
2. Appropriate Serious Adverse Event reporting procedures must be in place. 
3. The study duration must be appropriate and must be based on a realistic rate of enrollment. 
4. Data collection and data management must be adequate to verify and ensure subject safety.  
 
15.2.2 Assignment of risk. Assigning risk ensures that the data and safety monitoring is based on 
the level of risk (low, medium, or high) to ensure that the data and safety monitoring activities are 
appropriate.  Below are some of the criteria used to make a decision regarding the assignment of risk: 
 
• 
Expected duration of the study based upon the estimated rate of one. 
• 
Type of study population (e.g., children, geriatric) 
• 
The procedures used in the trial are commensurate with the degree of risk. 
• 
Adequate data management systems in place and appropriate case report forms 
• 
Proper serious adverse event reporting procedures in place 
• 
Proper biostatistical design and data analysis procedures in place. 
 
15.3 Monitoring/Auditing Activities.   
 
The compliance officer of the JCCC Office of Regulatory Compliance [ORC] will monitor the clinical 
records for all human subjects enrolled onto JCCC institutional trials overseen by the JCCC DSMB.  
The JCCC compliance officer will perform real time review of informed consent form processes and 
the meeting of all inclusion and exclusion criteria at study entry.  Active monitoring will offer the JCCC 
study teams prospective information that can be used to enhance the quality of research being 
performed contemporaneously.  Auditing is a review of historic performance of the research effort and 
is performed on case report forms, regulatory files and source documents to measure the quality of 
the research effort in a retrospective manner.    
 
15.4 Detailed Reporting Mechanism for Adverse Events 
 
Each protocol is required to have a detailed description of the Adverse Event Reporting method.   The 
DSMB expects each protocol to abide by the reporting time line and definitions consistent with the 
NCI and FDA guidelines.   
 
An adverse event is any undesirable experience associated with the use of a medical product in a 
patient (any unfavorable and unintended sign, symptom or disease temporally associated with the use 
of a medical treatment or procedure regardless of whether it is considered related to the medical 
treatment or procedure).  Any clinical adverse event must be recorded on the case report form during 
the course of the study. The investigator must evaluate and document the adverse event for severity, 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
68 
grade it according to the NCI Common Toxicity Guideline, causal relationship to the study drug under 
study, take appropriate action to care for the patient and to document the outcome.  All information 
recorded in the case report form must be verifiably documented in the source (i.e., medical record or 
physician office chart). 
 
When an adverse event occurs, it is the responsibility of the investigator to evaluate and record into 
the source documents, the nature of the symptom, prescribe the appropriate remedy and to report the 
event.  The adverse event must be reported to the sponsor, the IRB, FDA and any other appropriate 
agency (e.g., IBC and NIH for gene medicine trials.)  The assessment and reporting must occur in 
writing within 10 days, 48 hours for death.    
 
If the event is Serious and Unexpected, the event should be reported immediately (in writing within 
10 days, 48 hours for death) by the Principal Investigator to the IND sponsor, UCLA IRB, JCCC 
DSMB and to any appropriate agency; the NIH and the UCLA IBC for gene medicine trial; directly to 
the FDA if it is an institutional IND trial; and to the NCI if it is an NCI sponsored trial.  For institutional 
trials and for gene medicine trials, the JCCC ORC and Gene Medicine Compliance Officers will assist 
the investigator to ensure that all serious adverse events are properly documented and reported in 
accordance to federal and institutional requirements. 
 
All adverse events that occur in a research study overseen by the JCCC DSMB must be submitted, 
regardless of relationship, expectedness, or seriousness. 
 
15.5 Data Recording and Retention of Study Data 
 
Data recording will be done in compliance with Good Clinical Practice. Medical records and notes 
should be clearly marked and permit easy identification of participation by an individual in the 
specified clinical trial. Medical records and source data should be completed within a reasonable 
amount of time for purposes of timely data collection and entry. 
 
The investigators are to record all data with respect to protocol procedures, drug administration, 
laboratory data, safety data and efficacy ratings on the case report forms (CRF). The CRF may be a 
printed, optical or electronic document. All corrections on a CRF and on source documents must be 
made in a way that does not obscure the original entry. The correct data must be inserted, dated and 
initialed/authorized by study site personnel. If the change is not obvious provide a reason. 
 
16.0 STATISTICAL METHODS 
 
This is a two-stage phase II clinical trial aimed at determining the safety, feasibility and antitumor 
activity of gene transfer using retrovirus MSGV1-F5AfT2AB transduced PBMC. UCLA will be 
responsible for registering patients and for maintaining a complete database of the study information 
excluding laboratory assay data. The clinical laboratory assay data will be obtained from source 
documents from the UCLA medical records (including both paper and electronic files and laboratory 
results) and transcribed into CRFs. A shadow chart may be kept with copies of the source documents 
in a locked room at the UCLA Clinical Research Unit or under direct control of the study P.I. Failure to 
complete the study due to the stopping rules being invoked will be the main basis for determining 
safety and feasibility of this study. This study is intended to provide data that might allow the 
investigators to conduct a preliminary assessment of safety and feasibility. 
 
16.1 Sample Size Determination 
 
The Simon optimal two-stage design1 is used to determine the sample size, using the co-primary 
endpoint of response rate as the criterion for the clinical trial statistical design.  
 




MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
70 
16.3 Efficacy Analysis 
 
The toxicities observed after each TCR transgenic cell infusion will be summarized in terms of type 
(organ affected or laboratory determination such as absolute neutrophil count), severity (by Toxicity 
Table) and nadir or maximum values for the laboratory measures, time of onset (i.e. course number), 
duration, and reversibility or outcome. Tables will be created to summarize these toxicities and side 
effects by dose and by course. Baseline information (e.g. the extent of prior therapy) and 
demographic information will be presented, as well, to describe the patients treated in this pilot study. 
All responses will be reported.  
 
16.4 Study Stopping and Non-stopping Rules.  
 
It is recognized that AEs can occur frequently in this population based on the underlying metastatic 
melanoma and these can be SAEs. The review of SAEs will form the basis for potential early stopping 
of the study. Only unexpected SAEs that are related to the transduction process/research reagent 
would define a stopping rule. The review of these adverse events, and any decision to prematurely 
stop subject enrollment, will be determined by the UCLA DSMB and reviewed by the IRB. 
 
Absolute stopping rules for this clinical trial will be: 
 
1. Any death that is possibly related to the investigational agents namely MART-1 F5 TCR PBMC or 
MART-126-35 peptide pulsed DC. 
2. Two or more grade 4 events that are possibly related to MART-1 F5 TCR PBMC or MART-126-35 
peptide pulsed DC. 
3. Any event of hematological malignancy must be considered study stopping criteria until 
oncogenesis related to retroviral mutagenesis, EBV lymphoma and Post Transplant 
Lymphoproliferative Disorder (PTLD) can be excluded. 
 
Premature termination of the clinical trial may occur because of a regulatory authority decision, 
change in opinion of the FDA, RAC, IRB, the DSMB, or determination that there are problems in the 
cell product generation or the safety of their administration as descried in the assessment of primary 
study endpoints. Additionally, recruitment may be stopped for reasons of particularly low recruitment, 
protocol violations, or inadequate data recording. 
 
17.0 REFERENCES 
 
 
1. 
Simon R: Optimal two-stage designs for phase II clinical trials. Control Clin Trials 
10:1-10, 1989 
 
2. 
Dudley ME, Wunderlich JR, Robbins PF, et al: Cancer regression and autoimmunity in 
patients after clonal repopulation with antitumor lymphocytes. Science 298:850-4, 2002 
 
3. 
Dudley ME, Wunderlich JR, Yang JC, et al: Adoptive cell transfer therapy following 
non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory 
metastatic melanoma. J Clin Oncol 23:2346-57, 2005 
 
4. 
Butterfield LH, Ribas A, Dissette VB, et al: Determinant Spreading Associated with 
Clinical Response in Dendritic Cell-based Immunotherapy for Malignant Melanoma. Clin Cancer Res 
9:998-1008, 2003 
 
5. 
Ribas A, Glaspy JA, Lee Y, et al: Role of dendritic cell phenotype, determinant 
spreading, and negative costimulatory blockade in dendritic cell-based melanoma immunotherapy. J 
Immunother 27:354-67, 2004 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
71 
 
6. 
Ayyoub M, Zippelius A, Pittet MJ, et al: Activation of human melanoma reactive CD8+ 
T cells by vaccination with an immunogenic peptide analog derived from Melan-A/melanoma antigen 
recognized by T cells-1. Clin Cancer Res 9:669-77, 2003 
 
7. 
Johnson LA, Morgan RA, Dudley ME, et al: Gene therapy with human and mouse T-
cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 
114:535-46, 2009 
 
8. 
Tsao H, Atkins MB, Sober AJ: Management of cutaneous melanoma. N Engl J Med 
351:998-1012, 2004 
 
9. 
Cochran A, Glaspy JA, Ribas A, et al: Malignat Melanoma of the Skin. Cancer 
Treatment, Fifth Edition Charles M. Haskell Editor. W. B. Saunders:179-198, 2000 
 
10. 
Hodi FS, O'Day SJ, McDermott DF, et al: Improved Survival with Ipilimumab in 
Patients with Metastatic Melanoma. N Engl J Med, 2010 
 
11. 
Robert C, Thomas L, Bondarenko I, et al: Ipilimumab plus dacarbazine for previously 
untreated metastatic melanoma. The New England journal of medicine 364:2517-26, 2011 
 
12. 
Flaherty KT, Puzanov I, Kim KB, et al: Inhibition of mutated, activated BRAF in 
metastatic melanoma. N Engl J Med 363:809-19, 2010 
 
13. 
Ribas A, Kim K, Schuchter L, et al: BRIM-2: An Open-label, multicenter Phase II study 
of RG7204 (PLX4032) in previously treated patients with BRAF V600E mutation-positive metastatic 
melanoma. Journal of Clinical Oncology 29:Abstr 8509, 2011 
 
14. 
Chapman PB, Hauschild A, Robert C, et al: Improved survival with vemurafenib in 
melanoma with BRAF V600E mutation. The New England journal of medicine 364:2507-16, 2011 
 
15. 
Ribas A, Timmerman JM, Butterfield LH, et al: Determinant spreading and tumor 
responses after peptide-based cancer immunotherapy. Trends Immunol 24:58-61, 2003 
 
16. 
Ribas A: Update on immunotherapy for melanoma. J Natl Compr Canc Netw 4:687-94, 
2006 
 
17. 
Rosenberg SA, Yang JC, Restifo NP: Cancer immunotherapy: moving beyond current 
vaccines. Nat Med 10:909-15, 2004 
 
18. 
Blattman JN, Greenberg PD: Cancer immunotherapy: a treatment for the masses. 
Science 305:200-5, 2004 
 
19. 
Kawakami Y, Eliyahu S, Delgado CH, et al: Cloning of the gene coding for a shared 
human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc Natl Acad Sci 
U S A 91:3515-9, 1994 
 
20. 
Kawakami Y, Eliyahu S, Sakaguchi K, et al: Identification of the immunodominant 
peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted 
tumor infiltrating lymphocytes. J Exp Med 180:347-52, 1994 
 
21. 
Morgan RA, Dudley ME, Wunderlich JR, et al: Cancer regression in patients after 
transfer of genetically engineered lymphocytes. Science 314:126-9, 2006 
 
22. 
Dembic Z, Haas W, Zamoyska R, et al: Transfection of the CD8 gene enhances T-cell 
recognition. Nature 326:510-1, 1987 
 
23. 
Schumacher TN: T-cell-receptor gene therapy. Nat Rev Immunol 2:512-9, 2002 
 
24. 
Kessels HW, Wolkers MC, Schumacher TN: Adoptive transfer of T-cell immunity. 
Trends Immunol 23:264-9, 2002 
 
25. 
Kasahara N, Dozy AM, Kan YW: Tissue-specific targeting of retroviral vectors through 
ligand-receptor interactions. Science 266:1373-6, 1994 
 
26. 
Weber E, Anderson WF, Kasahara N: Recent advances in retrovirus vector-mediated 
gene therapy: teaching an old vector new tricks. Curr Opin Mol Ther 3:439-53, 2001 
 
27. 
Lois C, Refaeli Y, Qin XF, et al: Retroviruses as tools to study the immune system. 
Curr Opin Immunol 13:496-504, 2001 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
72 
 
28. 
Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al: LMO2-associated clonal T cell 
proliferation in two patients after gene therapy for SCID-X1. Science 302:415-9, 2003 
 
29. 
McCormack MP, Rabbitts TH: Activation of the T-cell oncogene LMO2 after gene 
therapy for X-linked severe combined immunodeficiency. N Engl J Med 350:913-22, 2004 
 
30. 
Kohn DB, Sadelain M, Glorioso JC: Occurrence of leukaemia following gene therapy 
of X-linked SCID. Nat Rev Cancer 3:477-88, 2003 
 
31. 
Rohdewohld H, Weiher H, Reik W, et al: Retrovirus integration and chromatin 
structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites. J 
Virol 61:336-43, 1987 
 
32. 
Suzuki T, Shen H, Akagi K, et al: New genes involved in cancer identified by retroviral 
tagging. Nat Genet 32:166-74, 2002 
 
33. 
Berns A: Good news for gene therapy. N Engl J Med 350:1679-80, 2004 
 
34. 
Clay TM, Custer MC, Spiess PJ, et al: Potential use of T cell receptor genes to modify 
hematopoietic stem cells for the gene therapy of cancer. Pathol Oncol Res 5:3-15, 1999 
 
35. 
Szymczak AL, Workman CJ, Wang Y, et al: Correction of multi-gene deficiency in 
vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat Biotechnol 22:589-94, 2004 
 
36. 
Ryan MD, Belsham GJ, King AM: Specificity of enzyme-substrate interactions in foot-
and-mouth disease virus polyprotein processing. Virology 173:35-45, 1989 
 
37. 
de Felipe P, Martin V, Cortes ML, et al: Use of the 2A sequence from foot-and-mouth 
disease virus in the generation of retroviral vectors for gene therapy. Gene Ther 6:198-208, 1999 
 
38. 
Donnelly ML, Luke G, Mehrotra A, et al: Analysis of the aphthovirus 2A/2B 
polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a 
putative ribosomal 'skip'. J Gen Virol 82:1013-25, 2001 
 
39. 
Phelps ME: PET : molecular imaging and its biological applications. New York, 
Springer, 2004  
 
40. 
Shields AF, Grierson JR, Dohmen BM, et al: Imaging proliferation in vivo with [F-
18]FLT and positron emission tomography. Nat Med 4:1334-6, 1998 
 
41. 
Johnson LA, Heemskerk B, Powell DJ, Jr., et al: Gene transfer of tumor-reactive TCR 
confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and 
tumor-infiltrating lymphocytes. J Immunol 177:6548-59, 2006 
 
42. 
Riddell SR, Greenberg PD: The use of anti-CD3 and anti-CD28 monoclonal antibodies 
to clone and expand human antigen-specific T cells. J Immunol Methods 128:189-201, 1990 
 
43. 
Klebanoff CA, Gattinoni L, Torabi-Parizi P, et al: Central memory self/tumor-reactive 
CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proc Natl 
Acad Sci U S A 102:9571-6, 2005 
 
44. 
Klebanoff CA, Gattinoni L, Restifo NP: CD8+ T-cell memory in tumor immunology 
and immunotherapy. Immunol Rev 211:214-24, 2006 
 
45. 
Overwijk WW, Theoret MR, Finkelstein SE, et al: Tumor regression and autoimmunity 
after reversal of a functionally tolerant state of self-reactive CD8+ T cells. J Exp Med 198:569-80, 
2003 
 
46. 
Lou Y, Wang G, Lizee G, et al: Dendritic cells strongly boost the antitumor activity of 
adoptively transferred T cells in vivo. Cancer Res 64:6783-90, 2004 
 
47. 
Borrello I, Sotomayor EM, Rattis FM, et al: Sustaining the graft-versus-tumor effect 
through posttransplant immunization with granulocyte-macrophage colony-stimulating factor (GM-
CSF)-producing tumor vaccines. Blood 95:3011-9, 2000 
 
48. 
Cui Y, Kelleher E, Straley E, et al: Immunotherapy of established tumors using bone 
marrow transplantation with antigen gene--modified hematopoietic stem cells. Nat Med 9:952-8, 2003 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
73 
 
49. 
Hughes MS, Yu YY, Dudley ME, et al: Transfer of a TCR gene derived from a patient 
with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 
16:457-72, 2005 
 
50. 
Rosenberg SA: Phase II Study of Metastatic Melanoma Using Lymphodepleting 
Conditioning Followed by Infusion of Anti-MART-1 F5 TCR-Gene Engineered Lymphocyte. 
OBA/RAC # 0703-840, 2007 
 
51. 
Rosenberg SA: Current immunotherapies based on lymphodepletion and adoptive cell 
transfer. Keystone Symposia: The Potent New Anti-tumor Immunotherapies:Abstract 003, 2007 
 
52. 
Fleischhauer K, Tanzarella S, Wallny HJ, et al: Multiple HLA-A alleles can present an 
immunodominant peptide of the human melanoma antigen Melan-A/MART-1 to a peptide-specific 
HLA-A*0201+ cytotoxic T cell line. J Immunol 157:787-97, 1996 
 
53. 
Yee C, Thompson JA, Byrd D, et al: Adoptive T cell therapy using antigen-specific 
CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, 
migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci U S A 99:16168-73, 2002 
 
54. 
Mackensen A, Meidenbauer N, Vogl S, et al: Phase I study of adoptive T-cell therapy 
using antigen-specific CD8+ T cells for the treatment of patients with metastatic melanoma. J Clin 
Oncol 24:5060-9, 2006 
 
55. 
Wu X, Li Y, Crise B, et al: Transcription start regions in the human genome are favored 
targets for MLV integration. Science 300:1749-51, 2003 
 
56. 
Gordon LK, Ribas A, Nusinowitz S, et al: Surveillance of the eye and vision in a 
clinical trial of MART1-transformed dendritic cells for metastatic melanoma. Control Clin Trials 
25:400-7, 2004 
 
57. 
Straatsma BR, Nusinowitz S, Young TA, et al: Surveillance of the eye and vision in 
clinical trials of CP-675,206 for metastatic melanoma. Am J Ophthalmol 143:958-969, 2007 
 
58. 
Jaeger E, Bernhard H, Romero P, et al: Generation of cytotoxic T-cell responses with 
synthetic melanoma-associated peptides in vivo: implications for tumor vaccines with melanoma-
associated antigens. Int J Cancer 66:162-9, 1996 
 
59. 
Men Y, Miconnet I, Valmori D, et al: Assessment of immunogenicity of human Melan-
A peptide analogues in HLA-A*0201/Kb transgenic mice. Journal of Immunology 162:3566-73, 1999 
 
60. 
Valmori D, Dutoit V, Schnuriger V, et al: Vaccination with a Melan-A peptide selects 
an oligoclonal T cell population with increased functional avidity and tumor reactivity. J Immunol 
168:4231-40., 2002 
 
61. 
Schuler-Thurner B, Schultz ES, Berger TG, et al: Rapid Induction of Tumor-specific 
Type 1 T Helper Cells in Metastatic Melanoma Patients by Vaccination with Mature, Cryopreserved, 
Peptide-loaded Monocyte-derived Dendritic Cells. J Exp Med 195:1279-88., 2002 
 
62. 
Timmerman JM, Levy R: Dendritic cell vaccines for cancer immunotherapy. Annu Rev 
Med 50:507-29, 1999 
 
63. 
Morse MA, Coleman RE, Akabani G, et al: Migration of human dendritic cells after 
injection in patients with metastatic malignancies. Cancer Res 59:56-8, 1999 
 
64. 
Fong L, Brockstedt D, Benike C, et al: Dendritic cells injected via different routes 
induce immunity in cancer patients. J Immunol 166:4254-9, 2001 
 
65. 
Rosenberg SA: Progress in human tumour immunology and immunotherapy. Nature 
411:380-4, 2001 
 
66. 
Roth MD, Gitlitz BJ, Kiertscher SM, et al: Granulocyte macrophage colony-stimulating 
factor and interleukin 4 enhance the number and antigen-presenting activity of circulating CD14+ and 
CD83+ cells in cancer patients. Cancer Research 60:1934-41, 2000 
 
67. 
Bertinetti C, Zirlik K, Heining-Mikesch K, et al: Phase I trial of a novel intradermal 
idiotype vaccine in patients with advanced B-cell lymphoma: specific immune responses despite 
profound immunosuppression. Cancer Res 66:4496-502, 2006 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
74 
 
68. 
Therasse P, Arbuck SG, Eisenhauer EA, et al: New guidelines to evaluate the response 
to treatment in solid tumors [see comments]. J Natl Cancer Inst 92:205-16, 2000 
 
69. 
Patterson AP: Minutes of the Recombinant DNA Advisory Committee - 9/21/05. 
http://www4.od.nih.gov/oba/rac/minutes/RAC_minutes_09-05.pdf, 2005 
 
70. 
Comin-Anduix B, Gualberto A, Glaspy JA, et al: Definition of an immunologic 
response using the major histocompatibility complex tetramer and enzyme-linked immunospot assays. 
Clin Cancer Res 12:107-16, 2006 
 
71. 
Kwong GA, Radu CG, Hwang K, et al: Modular nucleic acid assembled p/MHC 
microarrays for multiplexed sorting of antigen-specific T cells. J Am Chem Soc 131:9695-703, 2009 
 
72. 
Ma C, Fan R, Ahmad H, et al: A clinical microchip for evaluation of single immune 
cells reveals high functional heterogeneity in phenotypically similar T cells. Nature medicine 17:738-
43, 2011 
 
73. 
Yaghoubi S, Barrio JR, Dahlbom M, et al: Human pharmacokinetic and dosimetry 
studies of [(18)F]FHBG: a reporter probe for imaging herpes simplex virus type-1 thymidine kinase 
reporter gene expression. J Nucl Med 42:1225-34, 2001 
 
74. 
Weber WA: Use of PET for monitoring cancer therapy and for predicting outcome. J 
Nucl Med 46:983-95, 2005 
 
75. 
Cress RD, Holly EA: Incidence of cutaneous melanoma among non-Hispanic whites, 
Hispanics, Asians, and blacks: an analysis of california cancer registry data, 1988-93. Cancer Causes 
Control 8:246-52., 1997 
 
76. 
Economou JS, Belldegrun AS, Glaspy J, et al: In vivo trafficking of adoptively 
transferred interleukin-2 expanded tumor-infiltrating lymphocytes and peripheral blood lymphocytes. 
Results of a double gene marking trial. J Clin Invest 97:515-21, 1996 
 
77. 
Chmielowski B, Muranski P, Ignatowicz L: In the normal repertoire of CD4+ T cells, a 
single class II MHC/peptide complex positively selects TCRs with various antigen specificities. J 
Immunol 162:95-105, 1999 
 
78. 
Chmielowski B, Muranski P, Kisielow P, et al: On the role of high- and low-abundance 
class II MHC-peptide complexes in the thymic positive selection of CD4(+) T cells. Int Immunol 
12:67-72, 2000 
 
79. 
Chmielowski B, Pacholczyk R, Kraj P, et al: Presentation of antagonist peptides to 
naive CD4+ T cells abrogates spatial reorganization of class II MHC peptide complexes on the surface 
of dendritic cells. Proc Natl Acad Sci U S A 99:15012-7, 2002 
 
Appendix: Summary of Protocol Amendments 
 
Amendment 1 
 
Date: March 16, 2009 
 
Main protocol and consent modifications; 
 
a) Protocol: 
 
While generating the forms to fill in the product lot release criteria we noted that the approved 
version of the study protocol did not specify which final product lot release tests should be 
received before administration of the cells to patients. The lot release criteria were detailed in 
Table 4 of the protocol, but both tests with immediate results and with delayed results were listed 
together. The key issues were the low likelihood of obtaining results of the copy number of viral 
insertions in the target cells, and the testing for replication competent retrovirus (RCR), which Dr. 
Reiser, the FDA product reviewer for this IND, had asked to be done at the Indiana University Viral 
Production Facility (IU VPF) under the direction of Dr. Ken Cornetta. 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
75 
 
Dr. Reiser at phone number 301-827-3420 who agreed that final results of the copy number and 
RCR were not required to be in hand before administering the TCR transgenic cells to patients. 
Dr. Reiser said that a plan should be in place in case the results were positive, including the 
contacting and treatment of study subjects and the reporting of the event to the FDA. 
 
With this clarification, we discussed that we would amend the protocol to clearly state which 
results would be in hand before TCR transgenic cell administration to patients, and which results 
would be available at a later time point. We also discussed that this protocol amendment would 
include a plan for patient and FDA notification. 
 
The lot release criteria have been broken down into two tables in the amended version of the 
protocol dated 03/16/09. Table 4a (page 37) details the results that need to be in hand before the 
release of the TCR transgenic cells for patient administration. Table 4b (page 37-38) details the 
results that may be available afterwards. After this table there is a specification of the plans to 
contact and treat the patients and how the local and federal regulatory agencies should be 
notified. 
 
b) Consent Form: 
 
No changes. 
 
c) HS-1 Form: 
 
No changes. 
 
Amendment 2 
 
Date: April 13, 2009 
 
Main protocol and consent modifications; 
 
a) Protocol: 
 
- 
After discussion with the FDA reviewers in a series of teleconferences and email exchanges 
on April 7 and 8 (see attached minutes of these information exchanges), we deleted the 
requirement for quantitation of the number of copy inserts of retroviral vector in the final 
product. The FDA reviewers agreed with us that the quantitation of surface TCR expression in 
transduced cells was a good quantitative assessment of transduction efficiency. In fact, the 
previously IRB approved protocol had this test listed twice, as lot release criteria and as 
optional additional testing in the TCR transduced cells. 
- 
We clarified the intent of the schedule of events as the intent to perform the protocol 
procedures on those days. However, since this is a pilot study and needs to account for 
variation on patient outcome and scheduling issues, we clarify in this second amendment that 
the dates are approximate. 
- 
The use of antiementics and prophylactic antifungals was updated to match the standard 
practice in the stem cell transplantation ward at UCLA. 
- 
The administration of MESNA was corrected to continuous intravenous infusion for 24 hours. 
 
b) Consent Form: 
 
- 
We received the NIH Certificate of Confidentiality (CoC). Therefore, we added back the CoC 
language to the consent form. 
- 
We corrected a typographical error in page 7. 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
76 
 
c) HS-1 Form: 
 
No changes. 
 
Amendment 3 
 
Date: May 26, 2009 
 
Main protocol and consent modifications; 
 
a) Protocol: 
 
- 
After new discussion with the FDA reviewers on April 27 (see attached minutes), we slightly 
modified our ex vivo culture plan after transduction to limit it to 4 days, which following the 
Guidance for Industry for Replication Competent Retrovirus (RCR) testing (November 2006) 
allows us to bank but not prospectively test for RCR in the final product. The FDA reviewers 
agreed with us that a reasonable plan would be to test samples from peripheral blood obtained 
at 3, 6 and 12 months after TCR transgenic adoptive transfer and only test the final product 
sample if one of the patient-derived samples is positive for RCR. The proposed S+/L- assay is 
a one-month assay with high costs that would not be able to provide results in time for patient 
safety. Therefore, we discussed with the FDA and went onto amend our protocol following the 
Guidance for Industry plan for studies where gene-modified cell therapies are cultured for less 
than 4 days. This was included in Table 4b and Sections 10.1.9 and 11.2.1.2. 
- 
The timing of sampling of blood of patients for RCR was modified to follow the Guidance for 
Industry document to samples at 3, 6 and 12 months, and stored samples annually thereafter 
(Section 11.2.1.2). 
- 
The reporting of the results of the mycoplasma PCR-based rapid test was changed, and we 
changed it in the protocol accordingly (Tables 4a and 10, and Section 10.2.10). 
- 
We deleted a 100 ml blood draw for immune monitoring analysis that was planned on day +1 
since in the first two patients this blood draw provided very few lymphocytes since the blood 
draw happens at a time when the patient is pancytopenic. Analysis of these lymphocytes by 
immune monitoring assays did not provide much information.  
- 
We added a note in the schedule of events (Section 2) and in the protocol description in 
Section 8.6 text to note that 100 ml blood draws for immune monitoring analysis being planned 
for days 7 and 14 after TCR transgenic adoptive transfer can be done with a wider window 
since in our first 2 patients we have noted the recovering pancytopenia and hemodynamic 
state after high dose IL-2 may not make it clinically wise to obtain these blood draws on these 
particular days. 
- 
We added statements to the supportive care plan for patients to follow the clinical judgment 
and the standard practices in J-Medicine (Section 8.3). 
- 
Minor typographical errors were corrected throughout and marked in tracking mode in one of 
the protocol copies. 
 
b) Informed Consent Form 
 
- 
Upon review of the ICF we noted that there was no good explanation of the amount and timing 
of blood draws for immune monitoring analysis. Therefore, we added a paragraph in Section 4 
of the Study Procedures to explain to patients that repeated blood draws are planned to obtain 
cells for immune monitoring analysis.  
- 
After a correspondence with the IRB, an additional change was made to the consent form to 
take out the explanation and checkbox for the request for autopsy. 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
77 
c) HS-1 Form: 
 
- 
A new grant from our collaborator Dr. Bijay Mukherji from the University of Connecticut, with a 
UCLA subcontract, was added as funding source for this research project. 
- 
The timing of immune monitoring assays was added to the experimental procedures. 
- 
The tables of cell therapy lot release criteria were updated to match the tables included in the 
protocol after amendments 1, 2 and 3. 
 
Amendment 4 
 
Date: July 16, 2009 
 
Main protocol and consent modifications; 
 
a) Protocol: 
 
- 
A new section (8.12.) was added to allow the administration of booster MART-1 peptide pulsed 
DC vaccinations when the levels of MART-1-specific T cells would fall lower than 5% of total T 
lymphocytes. The schedule of events was included in the new section 2.1. 
- 
The requirement for paired tumor biopsies as inclusion criteria was changed to performing 
biopsies when felt feasible by the study investigators. 
- 
An appendix at the end of the protocol detailing the sequential protocol amendments was 
added. 
- 
Minor typographical errors were corrected throughout and marked in tracking mode in one of 
the protocol copies. 
 
b) Informed Consent Form 
 
- 
The ICF was updated to include the administration of 3 delayed DC booster vaccinations. 
- 
The details on how the study was funded were updated in the ICF.  
- 
The change in the requirement for tumor biopsies was added, where patients without easily 
accessible lesions for surgical biopsy would still be eligible for this protocol either with no 
biopsy, or with an image-guided biopsy. 
- 
The contact information for study investigators was updated. 
 
c) HS-1 Form: 
 
- 
A new grant from our collaborator Dr. David Baltimore from the California Institute of 
Technology, with a UCLA subcontract, was added as funding source for this research project. 
- 
The changes in the eligibility not strictly requiring paired tumor biopsies were included. 
- 
The addition of booster DC vaccinations was included. 
 
Amendment 5 
 
Date: October 13, 2009 
 
Main protocol and consent modifications; 
 
a) Protocol: 
 
- 
Correction of the schedule of events to reflect the follow up of patients who are in response or 
without progression after 3 months.  


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
78 
- 
Added subjects who are HLA-A*0205 (HLA-A2.5) positive as potential participants in this 
research, provided that we demonstrate that MART-1 F5 TCR transgenic cells produce > 
30,000 pg/ml/million cells of IFN-γ production upon MART-126-35 peptide stimulation using 
MART-126-35 peptide-pulsed PBMC from the potential HLA-A*0205 (HLA-A2.5) subject 
candidate. 
- 
Added the Endosafe - MCS System as an alternative method for testing for endotoxin levels in 
TCR transgenic cells and dendritic cells.  
- 
Added to the pre-study procedures the placement of a double lumen central venous access 
catheter. 
- 
Added the possibility of using clinical grade human AB serum for the DC manufacture.  
 
b) Informed Consent Form 
 
- 
The dendritic cell vaccine booster part of the dendritic cell addendum consent was corrected to 
monthly vaccines. 
- 
The table at the end of the main consent form was updated to add the follow up visits, blood 
work and scans every 3 months for at least 2 years in patients who have no progression after 
the first 90 days of study participation. 
 
c) HS-1 Form: 
 
- 
The changes in eligibility and procedures included in the protocol were made in the HS-1 form. 
 
Amendment 6 
 
Date: April 6, 2010 
 
Main protocol and consent modifications; 
 
a) Protocol: 
 
- 
The major modification in Amendment 6 was the increasing of the F5 TCR transgenic cell 
dose to 1010 and restarting the stage 1 of this protocol since with the prior version 
administering a cell dose up to 109 we did not meet the minimal hurdle of antitumor activity to 
proceed to stage 2 of this research after having entered the first 8 patients.  
- 
The description and references to the NCI Surgery Branch F5 TCR adoptive cell transfer 
protocol have been updated based on the published data included in the manuscript by 
Johnson et al. in Blood 2009. 
- 
C reactive protein (CRP) testing was added to the baseline exams since there is emerging 
evidence that it may provide information about the immune permissively of the tumor 
microenviroment. 
- 
The timing of procedures related to prior therapy was clarified. 
- 
The eligibility/exclusion for patients with prior brain metastasis was clarified. 
- 
The sources of clinical grade IL-2 and GM-CSF were updated since they have recently 
changed due to pharmaceutical company agreements and cross-licensing. 
- 
We added a description of Retronectin to the detailed explanation of reagents used in this 
study. 
- 
We added the possibility of samples being tested with novel immune monitoring assays that 
have become available in the past several months. 
- 
We added Dr. James Heath from Caltech as a non-clinical investigator. 
- 
Several typographic errors and inconsistencies with the laboratory procedures were corrected 
and updated. 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
79 
b) Informed Consent Form 
 
- 
No modifications were made to the ICF. 
 
c) HS-1 Form: 
 
- 
The changes in eligibility and procedures included in the protocol were made in the HS-1 form. 
 
Amendment 7 
 
Date: October 5, 2010 
 
Main protocol and consent modifications; 
 
a) Protocol: 
 
- 
Deletion of Dr. Erika von Euw from the protocol roster since she is no longer involved in this 
research project. 
- 
Clarification of the enrollment of patients with a prior diagnosis of brain metastasis. 
- 
Updated information on the source of OKT3 for this protocol. 
- 
Updated details on the procedure of retroviral transduction of activated lymphocytes.. 
- 
The inclusion of the testing for C reactive protein (CRP) in the Schedule of Events. 
 
b) Informed Consent Form 
 
- 
Several typographical errors have been corrected in the ICF, and wording has been slightly 
modified to improve it. 
 
c) HS-1 Form: 
 
- 
The relevant changes in the main study protocol were made in the HS-1 form. 
 
 
Amendment 8 
 
Date: December 17, 2010 
 
Main protocol and consent modifications 
 
a) Protocol: The following is a description of the major changes in Amendment 8 and the reasoning 
behind them: 
 
1. Four days of Fludarabine at 25 mg/m2/day instead of 5 days: 
 
The available information based on the course, correlative studies and outcome of the SAE on 
patient F5-10 strongly suggest that it was due to direct marrow toxicity from the conditioning 
regimen as the most likely cause (see full description of the SAE). The most likely cause of a 
delayed marrow aplasia is fludarabine. Therefore, we have decreased the number of fludarabine 
doses at 25 mg/m2/day i.v. daily from 5 days to 4 days. 
 
2. Capping of the administration of high dose IL-2 to up to 9 doses instead of 14 doses: 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
80 
A potential adverse contributor to the marrow aplasia in patient F5-10 is the 14 doses of IL-2 that 
this patient received, which is higher than other patients treated in this protocol. The prior versions 
of the protocol included dosing IL-2 at 600,000 IU/kg every 8 hours for up to 14 doses based on 
tolerance. This patient tolerated all doses within the expected parameters and received all of 
them. In conversations with our colleagues at the NCI Surgery Branch, who have administered the 
same conditioning regimen followed by high dose IL-2 to over 200 patients, they feel that the 
dosing of IL-2 may be an adverse contributor. In addition, review of the literature shows that higher 
doses of IL-2 up to 15 (720,000 IU/kg, 8 hourly for 5 days) lead to grade 3/4 toxicities whereas 
decreasing it to a maximum of 9 (720,000 IU/kg, 8 hourly for 3 days) results in fewer toxicities and 
still have the anti-tumor activities in conjunction with the adoptive cell transfer (ACT) 7,21. In fact, 
there was a trend wherein less doses of IL-2 correlated with higher clinical responses. Therefore, 
we plan to cap the number of IL-2 doses to a maximum of 9 doses.  
 
3. Using freshly harvested F5 MART-1 TCR transgenic T cells: 
 
The major problem for patients treated within this protocol has been relapse of melanoma after an 
initial tumor response. Our ongoing experiments analyzing the functionality of the TCR transgenic 
cells recovered from patients demonstrate a progressive loss of ability to secrete multiple 
cytokines upon MART-1 antigen exposure. This is reminiscent of preclinical studies of T cell 
exhaustion and lack of secondary response to antigen. Therefore, improving the in vivo 
functionality of the TCR transgenic cells continues to be a major goal of our research plans.  
 
A recent experiment in a murine model of ACT performed by our collaborators Drs. Lili Yang and 
David Baltimore from the California Institute of Technology (Caltech) indicated that 
cryopreservation has a profound detrimental effect on the in vivo long-term survival of the 
adoptively transferred T cells and their ability to have a secondary response to antigen exposure. 
We noted a similar effect within our clinical trial, since in patient F5-3 received a round of three 
delayed MART-1 peptide pulsed dendritic cell vaccines and had no secondary expansion of 
MART-1-specific T cells. 
 
These results have prompted us to modify our protocol to avoid cryopreservation of the TCR 
transgenic cells administered to patients, which is included in Amendment 8. 
 
b) Informed Consent Form 
 
- 
The use of freshly harvested F5 MART-1 TCR transgenic T cells for infusion in place of 
cryopreserved cells as depicted in Amendment 8 
 
- 
The use of high dose IL-2 every 8 hours following transgenic T cell infusion for a maximum 9 
doses over 3 days has been adopted to replace the previous regimen of maximum 14 doses in 
Amendment 8 
 
c) HS-1 Form 
 
- 
As IRB submission at UCLA was converted to webIRB which is entirely done online, HS-1 
form was no longer required.  
 
 
 
 
 
 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
81 
Amendment 9  
 
Date: April 20, 2011  
 
Main protocol and consent modifications; 
 
a) Protocol: 
 
- 
Addition of a PCR/ELISA mycoplasma assay if the MycoAlert results are equivocal or positive 
both during in-process testing and before cells are administered to the patient. 
- 
Lowering of the lot release criteria to 10% of tetramer positive cells for MART-1(26-35) as long 
as all the other lot release criteria are met. 
- 
Indication of performing additional testing for the number of vector copies. If higher than 5 we 
will propose a new dilution of the vector supernatant before using it for cell transduction. 
- 
Cardiac stress test on Day 75-90 has been removed.  
- 
Addition of potential risks associated with autoimmune toxicity from mispaired TCRs 
- 
The use of Trimethoprim-Sulfamethoxazole and Ganciclovir will start from Day -6 
- 
Replacement of William Quan by Kit Shaw as the Human Gene Medicine Program (HGMP) 
Compliance Officer 
      -     More detailed description of Day -6 events 
-     Minor formatting and wording modification  
 
b) Informed Consent Form 
 
- 
Addition of potential risks associated with autoimmune toxicitiy from mispairing TCRs  
- 
Cardiac stress test on Day 75-90 has been removed 
- 
Addition of Day -6 events to the schedule table in the end of the consent  
- 
Addition of risk to include life-threatening infection and/or respiratory failure requiring     
mechanical ventilation 
 
Amendment 10 
 
Date: August 22, 2011  
 
Main protocol and consent modifications; 
 
a) Protocol  
 
- 
Addition of Sujna Raval-Fernandes as the compliance officer for the DSMB. 
- 
Addition of Arun Singh as a study investigator with patient contact. 
- 
Addition of Phyllis Wu as study coordinator and regulatory coordinator. 
- 
Change of manufacturer of OKT3 to Miltenyi: As per FDA recommendation on July 28, 2011, 
we replaced eBioscience OKT3 with Miltenyi OKT3.  
- 
Change of GM-CSF manufacturer to Berlex and IL-2 manufacturer to Prometheus only in 
accordance to FDA product reviewer recommendation on July 22, 2011. The sources of 
clinical grade IL-2 and GM-CSF were changed due to pharmaceutical company agreements 
and cross-licensing. 
- 
Added dextramer as an alternative option for tetramer for the purpose of quantification of F5 
TCR transgenic cells. We had tested by staining the same sample side-by-side at least three 
times and obtained comparable results repeatedly. Test results attached. 
- 
Corrected “Blood for Plasma/serum” procedure in the schedule of events to “Plasmapheresis”. 
Plasmapheresis is carried out at the time of leukapheresis for every patient on Day 0 and Day 30.  
- 
Correction of minor typographic errors. 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
82 
b) Informed Consent Form 
 
- 
Addition of Arun Singh as a study investigator. 
- 
Description of a day 20-40 ECHO/MUGA was deleted.  
 
Amendment 11 
 
Date: November 7 , 2011  
 
Main protocol and consent modifications; 
 
a) Protocol  
 
- 
Decrease the maximum cell dose for ACT to 109 total cells. 
- 
Changing the IL-2 administration from high dose (HD) intravenously to low dose (LD) 
subcutaneously for up to 14 days. 
- 
Require having pulmonary function tests in all patients and change the patient exclusion 
criteria to exclude patients with a FEV1/FCV< 70% of predicted for normality. 
- 
Electrocardiogram (ECG) is added as a heart test for patients.  
- 
List pancytopenia caused by bone marrow infiltration by melanoma cells as a risk factor.  
- 
Added to exclusion criteria the following: 
o History of, or significant evidence of risk for, chronic inflammatory or autoimmune 
disease (eg, Addison’s disease, multiple sclerosis, Graves disease, Hashimoto’s 
thyroiditis, inflammatory bowel disease, psoriasis, rheumatoid arthritis, systemic lupus 
erythematosus, hypophysitis, pituitary disorders, etc.). Patients will be eligible if prior 
autoimmune disease is not deemed to be active (e.x. fibrotic damage of the thyroid 
after thyroiditis or its treatment, with stable thyroid hormone replacement therapy). 
Vitiligo will not be a basis for exclusion. 
o History of inflammatory bowel disease, celiac disease, or other chronic gastrointestinal 
conditions associated with diarrhea or bleeding, or current acute colitis of any origin. 
- 
Added “Must be willing to perform two biopsies at least” to inclusion criteria.  
- 
Revised screening blood test details, reflected in the schedule table and section 8.1  
- 
In section 8.4, added “Norethindrone (Norlutate) 10mg PO daily will be used for menstruating 
women for prophylaxis against menorrhagia” under “Prophylactic Supportive Care Throughout 
the Pancytopenic Period”. 
- 
Added 8.11 Permissible Systemic Therapies During the Protocol On-study Period 
- 
Change GM-CSF source to Genzyme 
- 
Revisions in response to FDA inquires regarding cross-referenced IND 14760.  
- 
Correction of minor typographic errors. 
 
b) Informed Consent Form 
 
- 
Description of the LD IL-2 administration 
- 
Description of the SAE of intubation in a second patient. 
- 
Description of pancytopenia caused by bone marrow infiltration by melanoma cells as a risk 
factor.  
- 
Requirement of having pulmonary function tests in all patients and change the patient 
exclusion criteria to exclude patients with a FEV1/FCV< 70% of predicted for normality. 
- 
Electrocardiogram (ECG) is added as a heart test for patients.  
- 
Added MART-1 testing in the screening procedures.  
 
 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
83 
Amendment 12 
 
Date:  July 25, 2013 
 
Main Protocol and Consent Modifications: 
 
a)  Protocol 
 
- 
No changes were made to the protocol 
 
b)  Informed Consent Form 
 
- 
Description of an SAE in which the patient died due to melanoma progression with massive 
intraabdominal bleeding. 
 
Amendment 13 
 
Date:  September 9, 2014 
 
Main Protocol and Consent Modifications: 
 
a)  Protocol 
 
- 
Changed contact information for several co-investigators on page 1 
- 
Added Paula Kaplan-Lefko, Beata Berent-Maoz, Daniel Shin, Siwen Hu-Lieskovan and 
Xiaoyan Wang  
- 
Removed Bradley Straatsma and Phyllis Wu 
- 
Blood tests (Mg, Phos and uric acid) in table in Section 2.0 were changed from Day -5 through 
Day 0 to only on Day -6. 
- 
Modified days of blood collection and physical exams in table in Section 2.0 and 8.0. 
- 
Added to section 8.0 and section 2.0 that changes in the range of 7 days up or down from the 
scheduled event will not be considered deviations from the study timeline.  Removed the word 
“significant” referring to deviations. 
- 
On page 25, ECOG has been deleted from the list of tests to be performed in the hospital. 
- 
On page 26, “mineral panel” has been deleted from the blood tests to be monitored. 
- 
Clarification that physical exam at baseline will include weight, height, and vital signs and that 
weight measurement will only be obtained at subsequent physical exams if clinically indicated 
(Section 8.1) 
- 
Deleted that physical exam would include weight, height and vital signs on Day -6 (Section 
8.3) since weight measurement is not standard inpatient practice.  
- 
In section 8.1 and 8.2, added that a new biopsy may not be required if an acceptable biopsy 
was performed within 60 days of prior to screening per coverage analysis for other trials. 
- 
In section 8.2, modified cardiac function test details. 
- 
Reduced IL-2 administration from twice daily for 14 days to twice daily for 7 days per IRB due 
to SAEs in IRB #12-000153 and 13-001624. 
- 
In section 8.4, changed dose of Ativan from 2 mg to 1 mg 
- 
Added holding parameters for IL-2 and fevers due to IL-2 to section 8.7 
- 
Changed section 8.9 to indicate that CT will be attenuated. 
- 
In section 8.15, removed optional repeated dendritic cell vaccines.  
- 
In sections 9.6.4 and 9.6.5, updated information regarding IL-4 and retronectin. 
- 
In section 10.1.5, Table 2b Annual Recertification was updated. 
 
 
 


MART-1 F5 TCR Protocol 01/10/2018 
                                          
 
 
 
84 
b)  Informed Consent Form 
 
- 
Reduced IL-2 administration from twice daily for 14 days to twice daily for 7 days. 
- 
Removed optional repeated dendritic cell vaccines 
 
Amendment 14 
 
Date:  August 1, 2016 
 
a)  Protocol 
 
- 
Modified annual recertification tests required (Section 10.1.5) 
 
b) Informed Consent Form 
 
- 
No changes 
 
Amendment 15 
 
a) Protocol 
-  In section 10.1.5, Table 2b Annual Recertification was updated 
- 
In section 10.2.5 MART-126-35, Table 7 Peptide Annual Recertification was updated, and  
      modified annual recertification tests required. 

ICF_001.pdf: 
MART-1 F5 TCR ICF 5/12/2016 
 
Page 1 of 29 
UNIVERSITY OF CALIFORNIA, LOS ANGELES 
 
CONSENT TO PARTICIPATE IN RESEARCH 
 
TITLE: Adoptive Transfer of MART-1 F5 TCR Engineered Peripheral Blood Mononuclear 
Cells (PBMC) after a Nonmyeloablative Conditioning Regimen, with Administration of 
MART-126·35-Pulsed Dendritic Cells and Interleukin-2, in Patients with Advanced Melanoma  
 
 
Lay Title: A Phase 2 Study of Gene Modified Immune Cells in Patients with Advanced 
Melanoma 
 
You are being invited to participate in a research study conducted by Antoni Ribas, M.D., Bartosz 
Chmielowski, M.D., Ph.D., Arun Singh, M.D., Siwen Hu-Lieskovan, M.D., Ph.D. and Deborah 
Wong, M.D., Ph.D. and John A. Glaspy, M.D., M.P.H. from Division of Hematology/Oncology; 
James S. Economou, M.D., Ph.D. from the Division of Surgical Oncology; Johannes Czernin, M.D., 
from the Division of Nuclear Medicine; Akira Ishiyama, M.D. from the Division of Head and Neck 
Surgery, and Tara McCannel, M.D., Ph.D. from the Jules Stein Eye Institute at the University of 
California, Los Angeles (UCLA). The study is sponsored in part by the National Institutes of Health 
(NIH), the Melanoma Research Foundation, the Melanoma Research Alliance, the Caltech-UCLA 
Joint Center for Translational Medicine and the California Institute for Regenerative Medicine 
(CIRM), and the Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at 
UCLA. You are being asked to take part in this research study because you have melanoma that has 
spread to other areas of your body. This research study uses a new approach in treatment of cancer, 
i.e. gene-modified cells will be given intravenously to attempt to fight melanoma. The risks of 
participation are currently not entirely defined. 
 
Your participation in this study is entirely voluntary. Between 8 and 22 patients at UCLA will be 
enrolled in this research study. If you agree, you will be asked to participate in this protocol for at 
least 3 months, and the study investigators will follow you indefinitely. You should read all the 
information given to you in this consent form and ask questions about anything that you do not 
understand before you decide to participate or not. A copy will be given to you to take home. 
 
Disclosure Statement: 
 
Your health care provider may be an investigator of this research protocol. As an investigator, he or 
she is interested in both your clinical welfare and in the conduct of this study. Before entering this 
study or at any time during the research, you may ask for a second opinion about your care from 
another doctor(s) not associated with this project. You are not under any obligation to participate in 
any research project offered by your physician. 
 
• PURPOSE 
 
The purpose of this phase 2 study is to find the best way to give this new experimental regimen and 
determine if it can treat metastatic melanoma in humans. In this phase 2 study, the experimental 
products are given initially to a group of 8 people. If safe and found to have a significant antitumor 
activity, it will be given to up to 14 other people, for a total of 22 people in this study. Physicians watch 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 2 of 29 
subjects carefully for any harmful side effects. Although the experimental regimen has been well tested 
in laboratory and animal studies, and a similar regimen has been given to a group of patients at the 
National Cancer Institute in Bethesda, MD, the side effects in people cannot be completely known 
ahead of time. This protocol is offered only to people whose condition cannot be helped by other 
known treatments. 
 
The study investigators’ main aim is to obtain sufficient information about the effects of gene-modified 
cells in humans. The gene modification of cells is an attempt to direct your own immune cells to kill 
melanoma cancer cells. The gene-modified cells studied in this experimental protocol are your own 
white blood cells from your blood that will be modified in the laboratory using genetic techniques to 
express a specific receptor against melanoma cells. 
 
Gene modification of cells involves the transfer of foreign genetic material (DNA) into a cell, in this 
case your immune system cells, using a form of a crippled virus that has been modified to express the 
melanoma specific receptor called MART-1 T cell receptor (or TCR).  
 
Study Drugs. This clinical trial has two main study agents: 
 
• The first study agent is called gene modified MART-1 TCR T cells. T cells are a special type of 
white blood cells that have the ability to kill cancer cells. They are also called cytotoxic T 
lymphocytes (or CTL), which means that they can kill other cells. The CTLs will be obtained from 
your body and taken to the laboratory to grow them and modify their genes. The CTL cells will be 
gene modified with a virus vector similar to the HIV virus called a retrovirus vector. This retrovirus 
is called a vector because it has been extensively modified to make sure that it cannot make copies 
of itself or induce AIDS in patients. The retrovirus vector will allow expression of a protein called 
the T cell receptor (or TCR). This TCR is specific for a marker on the surface of melanoma cells 
called the MART-1 tumor antigen. The investigators expect that the insertion of the TCR specific 
for the MART-1 melanoma marker will allow the CTLs to be redirected to recognize and attack 
melanoma cancer cells. 
 
• The second study agent is called a dendritic cell vaccine loaded with the MART-1 melanoma 
protein. Dendritic cells are a type of blood cells that specialize in stimulating the immune system, 
and will be grown in the laboratory from your own blood cells. The dendritic cells do not directly 
fight cancer cells. Instead, they teach other cells in the body to look for cancer cells with the 
MART-1 protein and destroy them. Therefore they function as an “on switch” for the immune 
system and it is called a tumor antigen. MART-1 is a protein produced by your cancer, which may 
be recognized by cells of the immune system. The goal of giving the dendritic cell vaccines to you 
will be to further help the ability of the gene modified MART-1 TCR CTLs to attack melanoma 
lesions in your body. 
 
Study Design. The study participants in this phase 2 clinical trial will be divided into two groups, an 
initial group of 8 patients and a second group of 14 patients, to allow the study investigators to assess 
the safety and antitumor activity of this combination. If the first group of 8 patients does not show 
favorable outcomes in terms of safety and antitumor activity, the second group of patients will not be 
enrolled. If there is a favorable outcome in the first group of patients, the study will proceed to enroll a 
total of 22 patients with melanoma. No random assignment to a particular group is used in this trial; 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 3 of 29 
both groups will receive the same treatment. Therefore, all subjects in this study will receive both the 
same dose of gene modified immune system cells and the dendritic cell vaccines.  
 
• OVERVIEW OF THE STUDY PROCEDURES 
 
The study procedures will start with the collection of white blood cells through apheresis (a 
procedure in which blood is drawn from a patient and separated into its components, some of which 
are retained, such as white blood cells, and the remainder returned by transfusion to the patient.). 
You will be asked to undergo two aphereses, one to make the gene-modified MART-1 TCR CTLs 
and the dendritic cell vaccines, and a second one after you have received the gene modified cells to 
later study them in your blood.  
 
On the day of the first apheresis, you will be admitted to the hospital and will receive chemotherapy 
over the next five days that decreases the risk of rejection of the transferred cells by your immune 
system and facilitates their expansion and attack of the melanoma lesions. During this time, the 
gene-modified MART-1 TCR CTLs and the dendritic cells will be manufactured in the laboratory 
from the apheresis product and will be extensively tested to assure that they express the appropriate 
TCR and that they do not contain any contaminating bacteria or virus. 
 
Then the gene-modified MART-1 TCR CTLs will be given back to you through a vein in your arm. 
It will be followed by vaccination with the dendritic cells under the skin. During the next 7days you 
will also receive interleukin 2 (IL-2), which is a standard treatment for patients with metastatic 
melanoma. During the next 2 to 3 weeks you will stay in the hospital until the study investigators 
determine that you have fully recovered from all of the procedures and it is safe for you to go home. 
Chemotherapy frequently causes a decrease in the platelet or red blood cells, and therefore you may 
require platelet and/or red blood cells transfusions. 
 
In the event the treating physicians determine that you are not clinically fit to receive the gene-
modified MART-1 TCR CTL infusion within 24 hours of the scheduled day, the cells will be 
cryopreserved on the harvest day for later use. It is possible that the cells are manufactured but they 
do not meet the pre-specified criteria for safe re-administration to you. In that case the study 
investigators will not be able to administer the cells to you despite you having received the 
conditioning chemotherapy to be prepared to receive the MART-1 TCR CTL infusion. 
 
During this clinical trial we will perform imaging studies, called PET (or Positron-Emission-
Tomography) scans. They will allow the study investigators to analyze if the gene-modified 
MART-1 TCR CTLs travel to the tumor. Additionally, several biopsies of your tumor lesions will 
be performed. 
 
• STUDY PROCEDURES 
 
1. Screening Tests and Procedures. If you volunteer and are eligible to participate in this study, 
you will need to be seen by one of the participating study investigators. The study investigators will 
explain the study to you and if you agree to participate, you will need to give your written consent. 
Before you are enrolled in this study, you will be asked questions and tests will be done to see if 
you are eligible to enroll. This will include: 
 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 4 of 29 
• Medical history. 
• Physical exam, including ECOG performance status (checks how you are functioning in your 
day to day living), weight, height, and vital signs (temperature, blood pressure while sitting, 
heart rate). 
• CT, PET or MRI scans of your brain, chest, abdomen, and pelvis. These tests will either be 
reviewed if they have been performed recently, or will need to be repeated if older than one 
month. 
• If you have visible melanoma lesions on your skin, photographs may be taken. 
• Discussion about birth control. If you or your partner are able to have children and are sexually 
active, you will be asked to follow birth control measures during the participation in this study.  
• MART-1 protein testing. You will have your tissue sample tested through immunohistochemistry 
(IHC) or genetic analysis of melanoma (this can be done in archived tissue, in a new tumor 
biopsy or may already be available from prior analysis) to know whether you are MART-1 
antigen positive to be eligible for the study.  If we are able to obtain a tissue sample from a pre-
existing biopsy that was performed within 60 days of your screening visit, then an additional 
biopsy may not be required.  In the event that a screening biopsy is required to be performed, 
the study will cover this cost. 
• Blood tests:  
• The blood tests will require about 6 teaspoonfuls drawn from a vein in your arm. The 
blood tests will include a complete blood count, which checks for anemia and bone 
marrow function; clotting tests of the blood; and blood chemistries, which check for 
liver, kidney, and thyroid function.  
• You will be checked for antibodies to viruses including the human immunodeficiency 
virus (HIV), hepatitis B and C viruses, cytomegalovirus, and Epstein-Barr virus. You 
will be asked to sign a separate consent for the HIV testing. The reason for this testing is 
that a positive infection with any of these viruses would make it too risky to give you the 
chemotherapy included in this protocol, and therefore it would not be safe for you to 
participate. The California State Health Department requests that positive results of these 
tests be reported to them. Therefore, in the case that your tests are positive the study 
investigators will have to report them to the State Health Department, and this reporting 
will include your personal identifying information. In the case of positive tests, you will 
be referred for counseling to the appropriate infectious disease specialist designated by 
your insurance. 
• We will study if you have any evidence of your immune system being activated against 
your normal organs (that is, to rule out an “autoimmune condition”). The reason for this 
testing is that a positive test would make it too risky for you to receive the immune 
activating treatments in this protocol, and therefore it would not be safe for you to 
participate. 
• Unless tested on you before, blood will be sent for determination of your type of immune 
system (HLA testing). The reason for this testing is that this protocol can only have a 
chance of working if you have a particular HLA type called HLA-A2.1 (or HLA-A*0201). 
 
All of these tests need to be done within 60 days prior to receiving the study drug. These tests will 
help the study investigators to decide if you will be eligible and if it is safe for you to receive the 
experimental drugs.  
 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 5 of 29 
If you decide that you want to participate in the study and the study investigators determine that you 
are eligible, you will then be enrolled. It is possible that you may finish all of the screening tests but 
not be eligible to participate in this study.  
 
2. Procedures in Preparation for Your Participation in the Study. You will then be asked to 
undergo several tests before you have the first infusion of the study drugs. 
 
Tumor Biopsies. This protocol attempts to study the safety of administering the study drugs, and 
the ability of the gene modified MART-1 TCR CTLs to find melanoma lesions in humans, which 
requires the collection of samples from your tumor whenever feasible. Therefore, a surgeon or 
dermatologist will see you in the office, while in the hospital, or you will be asked to go to the 
outpatient surgery center (usually on the 1st or 6th floor of the 200 UCLA Medical Plaza). A biopsy 
is the removal of solid tissue by cutting. This involves the following steps: First cleaning the skin 
around the sites of the biopsy, then injecting numbing medicine (a local anesthetic) applied with a 
needle and a syringe. Following that, the surgeon will cut the skin, and take out a piece of your 
tumor. The surgeon will then stitch back the skin over this area. The stitches will be taken out one 
week later. Only tumor lesions in the skin or that can be felt under the skin will be eligible for 
biopsy. The biopsy of your tumor will help the study investigators determine how the 
investigational agents find your tumor deposits. Another way to obtain a biopsy would be by image-
guided biopsy, most frequently locating a lesion by ultrasound or CT scan imaging and placing a 
thick needle into the lesion to obtain a piece of tissue from it.  The biopsies performed during the 
study will be billed to you or your insurance.  In the event that a screening biopsy is required to be 
performed, the study will cover this cost. 
 
Maintenance of the Tumor Biopsy and Leukapheresis Tissue Banks. The samples will be 
processed and maintained in a dedicated laboratory space at the UCLA Factor building. The 
laboratory has restricted access. Only investigators specifically trained are allowed entry to that 
area. Your samples will be stored frozen in a freezer equipped with a centralized monitoring system 
that immediately notifies the investigators if the temperature changes to above or below the optimal 
range of temperatures. Your samples will be coded, and the samples will not contain personal 
information that allows directly identifying you. The code linking your sample with your personal 
information will be maintained in a secured cabinet under the care of the Principal Investigator of 
this study. The samples will be maintained in this tissue bank as long as the study investigators 
require tumor samples or blood samples for the study of immune responses to cancer and 
understand the mechanisms of action of the study drugs. 
 
Baseline PET/CT Scans. The study investigators want to determine if PET/CT scans using markers 
of tumor sugar consumption and tumor cell aggressiveness can help determine whether your 
immune system is activated after the administration of the study drug. The PET probe FDG 
measures tumor sugar use, and is labeled with a small amount of radioactive fluorine (18F 
radioactive compound), making them visible by PET scanning. 
 
PET/CT combines a PET scanner and a CT scanner into one device. The PET/CT device is in 
clinical use in more than 1000 hospitals worldwide. If you have undergone a recent (less than 30 
days) CT scan or complete PET/CT scan, then the research PET/CT scans will be done with a 
limited set of CT images to avoid excessive exposure to irradiation. Otherwise, the research 
PET/CT scans will include a complete CT to allow the study investigators to adequately find which 
organs are involved with melanoma in your body. 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 6 of 29 
 
 
PET is an established scanning technique that utilizes small amounts of radioactivity that are 
attached to a sugar. This sugar is called fluorodeoxyglucose (shortened as FDG). FDG serves as a 
“contrast agent” for PET and is injected in one of your hand or arm veins. The PET scan helps 
identify the sugar consumption of cancer tissue as well as activated immune cells, as compared to 
that of other normal tissues.  
 
Eye Exam. A thorough eye exam will be conducted by an ophthalmologist at the UCLA Jules Stein 
Eye Institute. The purpose of this eye exam is to detect if the immune system activation by the study 
drugs has any adverse effects on the eyes. This eye exam will include dilatation of your pupils, 
taking pictures of your eye, and may include injecting a dye through a vein in your arm.  
 
Ear Exam. A thorough ear exam will be conducted by an ear-nose-throat (ENT) specialist at the 
UCLA outpatient clinics. The purpose of this ear exam is to detect if the immune system activation 
by the study drugs has any adverse effects on the ears. This will include a history of ear-related 
problems and an examination of the ear function. 
 
Heart Test. Prior to enrolling in this study, you will undergo an electrocardiogram (ECG). This will 
be done to ensure that there are no conduction abnormalities in your heart and that there are no 
signs of ischemic heart disease, which could put you at risk with the therapies employed in this 
study. 
 
Cardiac Function Test.  A baseline MUGA scan or echocardiogram will be performed prior to 
enrolling in the trial. An LVEF<45% or severe diastolic dysfunction will preclude patients from 
enrolling in the trial. 
 
Pulmonary Function Test.  The administration of IL-2 within this protocol may result in leakage 
of liquid into the lung resulting in breathing problems. In two patients these problems were severe 
enough to require intubation (putting a breathing tube down the patient’s throat and hooking it into 
a breathing machine). Given this serious risk you will be asked to undergo breathing tests to analyze 
the function of your lungs. If these breathing tests are not within normal range then it will not be 
safe for you to participate in this protocol. 
 
Pregnancy Test. If you or your partner can be or become pregnant, you should discuss this with the 
study coordinators. The gene transfer approach used in this protocol may put a fetus at risk. 
Therefore, if you are a female with potential to become pregnant, a pregnancy test will be 
performed within 2 weeks of being admitted for the administration of the gene modified study cells. 
 
Need to Place a Temporary Catheter in Your Veins for the Hospital Admission. All patients 
need to have access of treatments delivered to big veins during the hospital admission. Some 
patients may already have a temporary or permanent catheter (a flexible tube) that allows nurses and 
study investigators to infuse treatments through a large vein, called Central Venous Catheter or 
CVC. Common examples of such catheters are tunneled catheters placed in a neck vein (Hickman 
type catheter) or a PICC (which stands for “peripherally inserted central catheter”) that is placed in 
an arm vein. A catheter is placed in a large vein in your arm or neck area by an experienced 
physician. This can be done at the bedside or in a surgical room. It is anticipated that the most 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 7 of 29 
common catheter placed will be a PICC catheter in an arm vein. This catheter may be placed before 
being admitted to the hospital or after being admitted. 
 
If there are problems with the making of the gene modified MART-1 TCR CTLs, it is possible that 
you could undergo all of these procedures and the study investigators then tell you that you cannot 
receive the study drugs. This may happen if the gene modified MART-1 TCR CTLs do not meet the 
standards set up by the study investigators before they are given to you. 
 
3. Hospital Stay for Leukapheresis and Study Drug Administrations. The following procedures 
will require that you be admitted to the UCLA hospital. The procedures are counted from the day 
when you will receive the gene modified MART-1 TCR CTLs, which is designed as day 0. 
 
Day -6. Leukapheresis. The leukapheresis will be planned on the day of admission to the hospital 
and will be done early in the morning before you are admitted. This protocol attempts to generate 
two study agents that are made up of cells collected from your body. The study investigators 
determined that they would require more blood cells than can safely be obtained by a regular blood 
draw. Therefore, you will be asked to undergo a procedure called apheresis (in this case, a 
leukapheresis, since only white blood cells are collected) once or twice. The apheresis will be used 
to collect cells to make the MART-1 TCR CTL and the dendritic cell vaccines. During the apheresis 
procedure, white blood cells are collected using a medical device called a blood cell separator. 
Blood will be drawn from a needle in your arm, and attached to the blood tubing set in the blood 
cell separator. The blood is then mixed with a blood thinner called citrate, and it is drawn into the 
machine. The purpose of this is to keep your blood from making blood clumps (clots) during 
processing in the blood cell separator machine. The white blood cells are separated in a centrifuge 
and pumped into a collection bag. The total amount of white blood cells collected is about ½ – 1 
cup. The remainder of your blood is returned to you through a needle placed in the other arm. The 
amount of blood outside your body at any one time is about 1-½ cups. Normal saline (salt water) 
will be used to fill the tubing, help return the red cells at the end of the procedure, and keep the 
needle or catheter from clotting. Additionally, a sample of the liquid part of the blood called plasma 
(10 teaspoonfuls) will also be drawn at the time of the leukapheresis procedure. This procedure 
usually takes 3-4 hours and will be done at the UCLA Hemapheresis Unit, on the 6th floor of the 
200 Medical Plaza building.  
 
Potential Need to Place a Temporary Catheter in Your Veins for the Leukapheresis 
Procedure. If the veins in your arm are too small or the blood flow is inadequate for the 
leukapheresis procedure, which is fairly common, a temporary catheter (a flexible tube) will be 
surgically placed in a large vein in your groin or neck area by an experienced physician. This can be 
done at the bedside or in a surgical room. If you need this procedure, you will be asked to sign a 
separate consent form for the procedure. 
 
Laboratory Culture of Gene-Modified Immune System Cells. The CTLs are grown from your 
white blood cells obtained from the first apheresis procedure. Two days later the CTLs will be 
isolated and gene modified with the retrovirus vector expressing the melanoma-specific MART-1 
TCR. These cells will be tested extensively to make sure that they are not contaminated with 
bacteria or other organisms, and that they express the appropriate TCR and function properly.  
 
Laboratory Culture of Dendritic Cells. The dendritic cells will be grown in the laboratory from 
your white blood cells obtained from the leukapheresis procedure. It takes one week to grow them. 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 8 of 29 
The MART-1 melanoma antigen will be added to these dendritic cells. You will receive three 
injections of dendritic cells expressing MART-1, one on the following day after the gene modified 
CTL infusion and then at two weeks interval. On each occasion, the dendritic cells expressing 
MART-1 will be injected into the skin close to where your lymph glands drain, usually in your 
lower belly. 
 
Day -6. Admission to the Hospital. After the leukapheresis, you will be admitted to the 
Hematology/Oncology unit at the Ronald Regan UCLA Medical Center. This unit is located on the 6th 
floor of the hospital building. This is so that any complications that might occur will be detected and 
cared for. The following will occur: 
 
• You will be asked about illnesses, injuries, side effects, and any medications you have taken or 
medical procedures that have been done to you since the prior visit.  
• Physical exam, including ECOG performance status (checks how you are functioning in your 
day to day living), weight, height, and vital signs (temperature, blood pressure while sitting, 
heart rate).  
• You will have a catheter placed in a vein through which you will receive liquids in preparation 
for the chemotherapy regimen. 
• You will have blood drawn to check your health. Tests include complete blood counts, which 
check for anemia and bone marrow function; the ability of your blood to clot; blood chemistries, 
which check for liver and kidney. Most of these blood tests will be repeated daily or every other 
day while you are in the hospital. 
• You will start to receive one or two drugs to prevent complications with infections, the 
antibiotic Bactrim to decrease the risk of a rare form of pneumonia called Pneumocistis carinii 
pneumonia (or PCP), and the drug ganciclovir to decrease the risk of activation of a virus called 
cytomegalovirus (or CMV). Gancyclovir will only be given to patients who have evidence of 
prior exposure to the CMV virus. Both of these drugs will be stopped before administering the 
gene-modified MART-1 TCR CTLs, since both drugs would interfere with their function in 
your body. 
 
Day –5 to –4: Administration of High Dose Cyclophosphamide. You will receive two daily 
doses of the chemotherapy agent cyclophosphamide (also called Cytoxan) through a vein in your 
arm, together with medications to prevent nausea and vomiting. You will have frequent vital sign 
evaluations, daily blood tests taken from a vein in your arm, and daily analysis of your urine. 
 
Day –4 to –1: Administration of High Dose Fludarabine. You will receive four daily doses of the 
chemotherapy agent fludarabine (also called Fludara) through a vein in your arm, together with 
medications to prevent nausea and vomiting. You will have frequent vital sign evaluations and daily 
blood tests taken from a vein in your arm. 
 
After High Dose Chemotherapy Administration. You will require very close observation in the 
hospital for the 2 to 3 weeks after receiving the chemotherapy.  
 
• Throughout the period after the chemotherapy and before you go home, you will be evaluated 
by your nurses and treating physicians frequently to detect any complication. During this time, 
you will have frequent evaluations of your vital signs, at least daily blood tests taken from a 
vein in your arm, and periodical urine analysis. 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 9 of 29 
• You will receive a medication to treat low white blood cell counts called Neupogen or G-CSF. 
This medication is usually given as a daily injection under the skin, and its goal is to help your 
white blood cells recover faster after the chemotherapy. 
• If you develop fevers during the period in the hospital, you may require receiving treatment with 
antibiotics or drugs that fight fungal infections. 
• You may require receiving blood or platelet transfusions, since the chemotherapy given in this 
protocol is likely to lower your red cells and platelets (blood cells that help in clotting) in the 
blood.  
 
Day 0: Administration of the Gene-Modified MART-1 TCR CTLs. The cells will be harvested 
fresh on the day of infusion and sealed in an infusion bag. After the clearance of the quality control 
tests, you will receive the gene-modified cells through a vein in your arm or a central venous 
catheter. While they are being given, you will have your vital signs checked constantly to make sure 
that you do not have a reaction to the cells. 
 
Days 1-7: Administration of Interleukin-2 (IL-2). You will receive IL-2 as an injection under the 
skin (subcutaneous) twice a day for 7 days. Before each dose, a nurse will assess how you are 
tolerating the treatment; you will have vital signs checked continuously and blood tests drawn. This 
information will be evaluated by one of your treating physicians, who will make an assessment of 
the safety of giving the next dose of IL-2. Doses of IL-2 will be given or skipped based on how your 
body is tolerating them. You will be given medications to prevent nausea and vomiting, medications 
for chills and for fever, as required by you. You will likely receive antibiotics during this period, 
since fevers are frequent with IL-2 administration, and the study investigators cannot differentiate 
fevers caused by IL-2 from those caused by an infection. 
 
Day 1, 14 and 30: Dendritic Cell Vaccine Administrations. On each one of these days you will 
receive an injection of the dendritic cells expressing MART-1 under the skin, close to a lymph node 
gland that is not known to be involved with melanoma (in most cases, these injections are given in 
the lower belly). The last dendritic cell administration may occur after you have been discharged from 
the hospital, depending on how rapidly your body recovers from the chemotherapy. 
 
Day 14 to 30: Discharge Home. You will be discharged from the hospital when your white blood 
cells and platelets have recovered from the chemotherapy given to you before administering the 
gene-modified MART-1 TCR CTLs. This usually takes 3 to 4 weeks, and it varies depending on 
each patient. Therefore, the date of release from the hospital cannot be fully predicted before 
initiating the study. 
 
4. Procedures after Receiving the Experimental Drugs. 
 
Days 14, 30, 45, 60, 75 and 90: On each of these days (or a day close to this day), the study 
investigators will draw approximately 7 tablespoons (100 ml) of blood from one of your veins. This 
blood will be used to analyze how your immune system has been repopulated with the gene-modified 
MART-1 TCR CTLs. 
 
Day 20-40. You will be scheduled to undergo procedures to determine the fate of the gene-modified 
MART-1 TCR CTLs after they were given to you. Each one of these procedures may be done at a 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 10 of 29 
different site, and the study investigators or study coordinators will be able to direct you to where 
they are being scheduled. It will include the following: 
 
• Research PET/CT Scan. A PET/CT scan will be done for research purposes between study days 
20 and 40 (although it can also be performed two weeks or up to one month after this period of 
time).  
• Tumor Biopsy. If feasible, a tumor lesion will be removed for research purposes following the 
same procedure as described in the Screening biopsy. This will help the study investigators 
determine if the gene-modified MART-1 TCR CTLs are able to find your melanoma lesions 
throughout your body. 
• Partial Leukapheresis. A partial leukapheresis to collect white blood cells and plasma (3.5 
tablespoons) will be performed that will take half as much time as the one described in the 
Screening procedures. The purpose of this leukapheresis is to collect blood cells for future research. 
It will take less time because the study investigator will plan to process half the amount of blood 
compared to the initial leukapheresis. This procedure will take approximately 2 hours and will be 
done at the hospital bed or at the UCLA Hemapheresis Unit, on the 6th floor of the 200 Medical 
Plaza building. This will help the study investigators determine if the gene-modified MART-1 TCR 
CTLs are able to expand in your blood. 
 
Days 30, 45, 60 and 75. You will be asked to go to the UCLA oncology outpatient clinic, either in 
the 100 or 200 Medical Plaza for follow up study visits. 
 
• You will be asked about illnesses, injuries, side effects, and any medications you have taken or 
medical procedures that have been done to you since the prior visit. 
• Physical exam, including ECOG performance status (checks how you are functioning in your 
day to day living), weight, height, and vital signs (temperature, blood pressure while sitting, 
heart rate). 
• About 4 teaspoonfuls of blood will be drawn to check your health.  
 
Between Days 75 and 90. You will be asked to undergo the following:  
 
• CT, PET and MRI scans of your brain, chest, abdomen, and pelvis to determine the potential 
effects of the study drugs on your melanoma tumors. 
• If you have visible melanoma lesions on your skin, photographs will be taken. 
• Eye Exam. This eye exam will be conducted by an ophthalmologist at the UCLA Jules Stein 
Eye Institute, and may include dilatation of your pupils, taking pictures of your eye, injecting a 
dye through your vein and placing a contact lens in your eye. 
• Ear Exam. This exam will be conducted by an ear-nose-throat (ENT) specialist at the UCLA 
outpatient clinics. It will include a history of ear-related problems and an examination of the ear 
function. 
 
Day 90. This will be the last visit during the most intensive part of the study protocol. The main 
goal of this visit is to determine if the study drugs had an effect against your melanoma, and if you 
have any side effect from your participation in the study. 
 
• You will be asked about illnesses, injuries, side effects, and any medications you have taken or 
medical procedures that have been done to you since the prior visit. 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 11 of 29 
• Physical exam, including ECOG performance status (checks how you are functioning in your 
day to day living), weight, height, and vital signs (temperature, blood pressure while sitting, 
heart rate). 
• About 5 teaspoonfuls of blood will be drawn to check your health.  
• The study investigators will go over the results of the tests to evaluate your melanoma, 
including CT, PET or MRI scan of your chest, abdomen and pelvis.  
• If you have visible skin tumors, these tumors will be photographed and may be biopsied.   
 
5. Follow-Up (or Post Study) Procedures. The federal agencies require that all patients that have 
received experimental gene transfer products be followed for up to 15 years. Therefore, the study 
investigators will want to see or contact you at least every 3 months during the first 2 years, and 
every 6 months during the next 3 years, and then at least once a year up to 15 years thereafter. You 
will be asked about your melanoma and whether you have received new therapy for melanoma. At 
those time points, blood will be taken from your vein to study how they persist and the potential 
long term effects of the gene modified cells given to you. If you die during this time, your family 
members or physician will be asked about the date and cause of death. If your melanoma has not 
progressed, your tumors will continue to be evaluated until they progress. If you have treatment-
related health problems, the study investigators will continue to follow them to make sure that you 
have recovered appropriately. Additional procedures and laboratory tests may be needed. 
 
• AUTOPSY REQUEST 
 
If you die, no matter what the cause is, the study investigators will ask for permission to perform an 
autopsy. The reason is to perform a thorough evaluation of all your organs due to your participation 
in this experimental gene transfer protocol. You should discuss this option with your family 
members and discuss with the study investigators at any time if you have questions or concerns 
about the future request for an autopsy procedure.  
 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 12 of 29 
Chart of Study Procedures 
 
The chart below shows what will happen to you as explained above.  The left-hand column shows 
the day of the study and the right-hand column tells you what will happen on that day.  Day 0 is the 
day that the gene-modified cells are infused. 
 
Day 
 
 
 
What will happen 
Day -6 (6 days before 
infusion of cells) 
• Get routine blood tests 
• Leukapheresis 
• Get admitted to the hospital 
• Physcial exam 
• Bacterim administration 
• Gancyclovir administration (if prior exposure to CMV) 
Days -5 to -4 
• Get routine blood tests 
• Cyclophosphamide treatment (chemotherapy administration) 
Days -4 to -1 
 
• Get routine blood tests 
• Fludarabine treatment (chemotherapy administration) 
Day 0 
• Get routine blood tests 
• Infusion of gene-modified MART-1 F5 TCR  
Day 1 
• Dendritic cell vaccine administration 
Days 1 to 7 
• Twice daily IL-2 administration 
• Get blood to test cytokine profile 
Day 14 
• Get blood to test gene-modified cells and cytokine profile 
• Dendritic cell vaccine administration 
Days 20 to 40 
• Leukapheresis 
• Biopsy of tumor 
• PET/CT scan 
Day 30 
• Get blood to test gene-modified cells  
• Get routine blood tests  
• Physical exam 
• Dendritic cell vaccine administration 
Day 45 
• Get blood to test gene-modified cells 
• Get routine blood tests  
• Physical exam  
Day 60 
• Get blood to test gene-modified cells 
• Get routine blood tests  
• Physical exam  
Day 75 
• Physical exam 
• Get blood to test gene-modified cells 
Day 75-90  
• Get routine blood tests 
• MRI or CT scan of the brain 
• Eye exam 
• Ear exam 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 13 of 29 
Day 90 
• Physical exam 
• Get blood to test gene-modified cells 
Every 2-3 months for 
two years 
• Physical exam 
• Get blood to test gene-modified cells 
Every 6 months up to 
five years 
• Physical exam 
• Get blood to test gene-modified cells 
Every 12 months up to 
fifteen years 
• Physical exam 
• Get blood to test gene-modified cells 
 
• POTENTIAL RISKS AND DISCOMFORTS  
 
You must tell the study investigators immediately if you have side effects, injuries or new 
symptoms or complaints, even if you do not think they are related to the study medication. Any 
medicine may have side-effects. As with any investigational drug, there may be risks or side effects 
with gene-modified cells that are currently unknown. These may possibly be serious, permanent or 
even life-threatening. It is particularly important that you tell the study investigators immediately if 
you experience new symptoms that may be related to your participation in this research protocol. 
 
A) Serious Risks Observed in Prior Subjects in this Protocol and in Similar Protocols at UCLA. 
 
1. Prolonged Drop in Blood Cells. One patient out of 14 in this protocol required two months of 
hospital stay due to a delayed recovery of blood cell counts. During this time the patient required being 
in an isolation room to prevent from infections, and had to receive frequent transfusions of red blood 
cells and platelets. The patient also received immune dampening medications like corticosteroids. The 
blood cell counts recovered approximately two months after starting in the protocol. 
 
2. Pneumonia Requiring Extended Hospital Stay. Two patients in this protocol experienced serious 
complications and died due to a lung infection (pneumonia) requiring mechanical ventilation and 
intensive care support. These patients experienced severe trouble breathing, impaired kidney function, 
and low blood pressure during these episodes leading to a prolonged hospital stay. Due to these 
complications the study protocol was amended to require a baseline lung function test within the 
normal range, the number of cells infused was decreased and the dosing of IL-2 was changed from 
high dose to low dose since it was felt it was a very likely cause of these severe complications. 
 
3. Tumor Related Bleeding.  One patient in this protocol died after serious complications.  The 
melanoma did not respond and as it got worse, it led to massive intra-abdominal bleeding while the 
patient was on a blood thinner.   
 
4. Visual Loss. One patient in a related trial (NY-ESO-1 protocol using a different tumor marker than 
MART-1 F5) developed visual loss in both eyes. On the second day of chemotherapy, the patient noted 
some blurry vision that improved and had a normal examination of the patient’s visual system. Three 
weeks later, this patient noted worsening of the vision with a severe decrement of the visual acuity 
which progressed to a complete loss of vision.  
 
5.  Tumor Related Bleeding.  One patient in a related protocol died after serious complications.  The 
patient had melanoma (an aggressive form of skin cancer), and this cancer did not respond to the 
treatment. As it got worse, the melanoma growth in the abdomen led to massive bleeding which 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 14 of 29 
happened while the patient was on a blood thinner.   
 
6. Arrhythmia.  One patient in the NY-ESO-1 protocol developed serious irregular heart beats called 
unsustained ventricular tachycardia that required intravenous medication to control the rhythm after 18 
doses of IL-2. This is a known complication of IL-2 and the symptoms were resolved after IL-2 was 
discontinued.  The number of doses of IL-2 have been reduced to no more than 14 doses for all similar 
trials. 
 
B) Potential Risks Related to this Protocol. 
 
1. Risks of Gene Transfer. The HIV-like virus used in this protocol is called a retroviral vector used 
for gene transfer. There is limited experience in using retroviral vectors in humans, so the potential for 
short-term and long-term side effects are not fully known. Below we describe the risks associated with 
two gene therapy vectors, one that is not part of this protocol (adenoviral vectors) and one that is part of 
this protocol (retroviral vectors), to inform you of the type of risks that may be associated with gene 
therapy in general. 
 
• Risk of Cancer with Retroviral Vectors. The vectors used to perform the gene transfer may 
cause cancer. When retroviral vectors enter a normal cell, the vector inserts itself into the 
normal genes in that cell in a process that is called integration. Most integrations cause no harm 
to the cells or to the subject. But, there is a chance that there may be some places of the normal 
human genes where integration of the viral vector may result in activation of nearby genes. 
 
If one of the genes that are activated by viral vector integration has the role of controlling cell 
growth, integration near it may result in cancer. This has occurred in animal studies in mice and 
monkeys using retroviral vectors. The retroviral vector integration seems related to cancer 
development in these animals.  
 
The development of cancer has also happened in human clinical trials using retroviral vectors. 
Twelve children with a severe form of immune deficiency received blood stem cells gene 
modified using a retroviral vector. Most of the children who participated in that clinical trial 
appear to have been cured of their immune deficiency disease. However, 3 children developed a 
form of cancer of the blood called leukemia approximately 3 years after receiving the gene 
transfer. One of the children died from leukemia. A group of experts looked at all the study test 
results, and found that the gene transfer caused the leukemia cancer.  
 
The retroviral vector used to make the gene modified MART-1 TCR CTLs is related but different 
from the retroviral vectors that caused the leukemia cancers. In animal models, there is little 
evidence that the type of retroviral vector used in the current protocol induces cancer from 
integration. Therefore, it is unlikely that you would be exposed to the same risks. However, the 
long-term risks of any form of gene transfer approach in humans are currently unknown. 
 
 
• Risk Associated with Replication Competent Retrovirus (or RCR) with Retroviral 
Vectors. The retroviral vector used to make the gene modified MART-1 TCR CTLs is related to 
(but different from) HIV, the virus that causes AIDS. To make it as safe as possible, this retrovirus 
vector does not have the parts that would allow it to behave like an HIV virus in your body. In 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 15 of 29 
particular, the genes that allow the HIV virus to make new virus and infect new cells are not 
present in the retroviral vector. However, there is the risk that the retroviral vector may mutate 
(change its genes) and grow as a virus after it has been inserted in your CD8+ cells. This would be 
called a replication competent retrovirus (or RCR).  
 
The risks of RCR are currently unknown, but it is possible that it may make you sicker than you 
are now. It is theoretically possible that you may develop a disease similar to HIV infection. To 
date, no patient has developed an RCR from retrovirus vectors. 
 
To minimize the risks of you receiving an RCR, the gene modified MART-1 TCR CTLs will be 
tested for RCR before they are administered to you. You will be monitored for the appearance of 
RCR in your blood during many of the follow up visits included in this protocol. If one of the tests 
for RCR is positive at any time, you will be contacted and asked to return to the clinic within 2 
weeks to provide more blood to confirm the test results. A positive test result does not mean that 
you have really developed RCR. Should your repeated tests show that you have RCR, there is no 
approved treatment for an RCR. Medical and research experts will work with you to define the best 
care available to attempt to treat the RCR, which most likely may include the type of drugs that 
lead to control of HIV infection. 
 
2. Risks of Turning the Immune System against Melanoma. This protocol is intended to turn on the 
immune system against melanoma cancer cells by guiding it to attack cells that express the MART-1 
marker. This melanoma marker MART-1 is not only expressed in melanoma cancer cells and may be 
expressed also in normal pigmented cells in the body.  
 
• Risk of Skin Toxicities. Turning on the immune system against MART-1 may result in loss of 
color of moles or large areas of the skin. This may result in increased sensitivity to sunlight and 
predispose to sunburns. Patients treated with a similar regimen at the National Cancer Institute, 
Bethesda, MD, have developed skin rashes that feel like sunburns throughout the body, and normal 
pigmented cells have disappeared from large parts of their body. These toxicities are likely to 
happen to you if you participate in this research. 
 
• Risk of Eye Toxicities. A serious toxicity would be if the immune system starts attacking MART-
1 positive pigmented cells in the eye. This may result in blindness. Therefore, we request that you 
comply with the visits with the eye doctors scheduled in this study to allow the study investigators 
to detect any adverse effect in your eyes and start a treatment for it. 
 
• Risk of Ear Toxicities. Some patients treated with a similar regimen at the National Cancer 
Institute, Bethesda, MD, have developed problems in the ear and equilibrium. This has presented as 
problems keeping the balance and sensation that things around the person are moving when they 
are not. This condition is known as autoimmune vestibular toxicity and it is due to an attack of the 
gene modified MART-1 TCR CTLs against MART-1 positive cells in the middle and inner ear. 
These toxicities may happen to you if you participate in this research. If this happens to you, you 
should contact the study investigator immediately and you would likely receive injections of 
corticosteroids into your ear. 
 
• Risk Associated with Autoimmune Toxicity from Mispairing of TCRs. The chains of the 
receptor on the transgenic lymphocytes could mispair with the chains on your own lymphocytic 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 16 of 29 
cells, which could result in conditions such as loss of weight, muscle atrophy, fatigue, weakness, 
significant loss of appetite, bone marrow failure,  lymphocytic depletion, pancreatitis, and 
colitis, etc.. If any of the above occurs or there is evidence of other autoimmunity development, 
you would receive immune suppressive therapy as clinically indicated based on the severity of 
symptoms, using medications like corticosteroids, cyclosporin-A, mycophenolate mofetil, anti-
TNF-alpha antibodies or anti-thymocite globulin (ATG). 
 
3. Risks of Dendritic Cell Administration: Vaccination with dendritic cells expressing MART-1 
could potentially result in an allergic reaction, which could include redness and swelling at the 
injection site, itching, hives, low blood pressure, difficulty breathing, or in the most extreme situation, 
death. Receiving acetaminophen (Tylenol ) and diphenhydramine (Benadryl ) before each injection 
of the vaccine reduces the possibility of these reactions occurring. In addition, if your immune system 
becomes overly stimulated, you might develop pain, redness and swelling at the injection site over the 
course of several days. However, the only harmful effects noted in other studies where dendritic cells 
were administered to human subjects with cancer were occasional mild fever or swelling at the 
injection site, lasting for 1-2 days, and loss of color of moles and skin. 
 
A serious complication may arise if the vaccine preparation is contaminated with bacteria. This can 
happen since this vaccine is cultured in the laboratory for one week in a medium where bacteria may 
grow. This may lead to fever, chills and lowering of the blood pressure, and may require 
hospitalization. The vaccine is tested for the presence of bacteria during the culture and at the time of 
injection in an attempt to prevent this complication, with results reported and verified before 
administering the vaccine to you. However, final results of this testing may not be available for up to 
two weeks, and may show that the vaccine is contaminated with bacteria. If this should occur, you will 
be contacted to begin treatment with antibiotics. 
 
4. Risks of High Dose Chemotherapy with Cyclophosphamide and Fludarabine. Below are the 
major toxicities associated with high doses of chemotherapy, describing the common side effects of the 
combined chemotherapy, and then listing some of the unique toxicities associated with each of the two 
chemotherapy drugs being administered in preparation for the administration of the gene modified 
cells: 
 
• 
Risks of High Dose Chemotherapy. 
- 
A common side effect of high dose chemotherapy is nausea and vomiting. Your treating 
physicians will try to prevent this side effect by giving you modern antinausea medications, like 
a drug called Zofran.  
- 
We expect to see moderate to severe decreases of white blood cells, red blood cells and 
platelets. If serious decreases occur, you may have to be given blood cells or platelets.  
- 
Fever may be a result of infection that develops when white blood cells are low after 
chemotherapy. This will require treatment with antibiotics and medications to fight fungal or 
viral infections.  
- 
Some patients may develop headache and insomnia, which would be treated with medication, if 
needed.  
- 
A universal side effect of high dose chemotherapy is complete disappearance of the hair (or 
alopecia) within one month of receiving the chemotherapy. The hair usually reappears over the 
next several months. 
- 
Other side effects may include diarrhea (lasting up to 3 weeks), and abnormal function of liver, 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 17 of 29 
which may be mild, moderate or potentially life threatening.  
- 
It is possible that chemotherapy drugs may cause sterility and you may not be able to bear or 
father a child.  
- 
A rare but potential side effect of high dose chemotherapy is an increased risk of developing 
cancer several years later. 
- 
In rare cases, bone marrow failure may result following high-dose chemotherapy indicated by 
drop in the number of white blood cells and platelets.    
- 
There also could be life-threatening infection and/or respiratory failure requiring mechanical 
ventilation. These risks could happen to you.  
- 
During the immuno-compromised status after chemotherapy, any kind of infection may flare 
up giving rise to unexpected complications.  
 
• 
Risks of High Dose Chemotherapy with Cyclophosphamide.  
- 
Common risks: 
o Cyclophosphamide (or Cytoxan) can lead to bleeding in the urine. You will receive a 
medication called Mesna to prevent this potential side effect, and your urine will be 
checked daily for the presence of blood while you receive this chemotherapy drug.  
o Weakness and muscle pain.  
o Fatigue.  
- 
Uncommon risks (less than 1%): 
o Seldom, cyclophosphamide can induce serious cough and shortness of breath within a 
condition known as interstitial pulmonary fibrosis. 
o Seldom, cyclophosphamide can induce serious loss of salt from the blood resulting in 
serious accumulation of liquid in the body, which is a resultant of a condition known as 
syndrome of inappropriate antidiuretic hormone secretion (SIADH). This condition is 
due to an effect of this drug on a gland in the brain.  
o Seldom, cyclophosphamide can induce serious problems in the heart, which can be 
inflammation (myopericarditis), death of heart cells (diffuse hemorrhagic myocardial 
necrosis) and inflammation of the lining of the heart (pericarditis). 
 
• 
Risks of High Dose Chemotherapy with Fludarabine. 
- 
Common risks: 
o Weakness and muscle pain.  
o Fatigue.  
o Tingling in hands or feet (paresthesias). 
o Transient visual or hearing disturbances.  
o Transient sleep disorders. 
- 
Uncommon risks (less than 1%): 
o Seldom, fludarabine can induce break down of red blood cells resultant from an 
immune response, which is called hemolytic anemia. If this happens, patients feel 
extreme fatigue and become very pale, and require immediate medical attention and 
treatment with corticosteroids to decrease immune responses. 
o Seldom, fludarabine can induce serious cough and shortness of breath with a condition 
known as idiopathic interstitial pneumonitis. 
o Seldom, fludarabine can induce a reaction in the place where blood cells are made, the 
bone marrow, known as bone marrow fibrosis. This would lead to severe decrease in 
cells in the blood resulting in fatigue, propensity to bleeding and propensity to 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 18 of 29 
infections. 
o Seldom, fludarabine can induce severe changes in the brain known as progressive 
encephalopathy, which may lead to blindness and coma.  
 
5. Risks of Low Dose Interleukin-2 (IL-2). High dose IL-2 has been approved by the Food and Drug 
Administration (FDA) for the treatment of melanoma and kidney cancer since it results in long term 
disappearance of metastatic melanoma in a small percentage of patients. In the current protocol you 
will receive low dose IL-2 (approximately 15 times less IL-2 in total than the FDA approved regimen 
for melanoma and kidney cancer, over a longer time period.). Injection site reactions have been noted 
with the subcutaneous form of IL-2, including transient injection site nodules and induration. 
Furthermore, some patients develop a rash, itching and dermatitis. This treatment can result in 
moderate nausea and vomiting, and moderate fevers and chills in most of the patients. Some patients 
develop moderate to severe pain in the joints that requires treatment with morphine-like drugs. It has 
the potential to induce moderate to severe heart, lung, kidney and liver problems. These side effects 
improve rapidly (within days) after the treatment is stopped, and it is very infrequent that it may result 
in long-term problems. There are reports of patient deaths due to IL-2 toxicities.  
 
Due to these potential toxicities, patients that receive IL-2 need to be checked if their heart and lungs 
can stand the stress imposed by this treatment by performing heart tests. You will be asked to undergo 
such testing before you are allowed to participate in this study. Despite these known toxicities and 
risks, the great majority of patients are able to safely receive low dose IL-2, including it as an adjunct 
in stem cell transplant settings, and permanent problems are extremely rare. 
 
6. Risks of Leukapheresis: The most common side effect from white blood cell collection is caused 
by the medication (called citrate) used to prevent clumping of the blood. Citrate binds to calcium in the 
body and may temporarily lower the calcium level. The first symptoms of this occurring are tingling, 
vibrating, or a numb feeling of the face, lips, teeth, hands, or feet. You will be instructed to tell the 
nurse immediately if you have any of these symptoms. All patients are continuously observed during 
the procedure. The nurse will give you calcium replacement to prevent or treat these symptoms, and 
when necessary slow down the speed at which the anticoagulant is given. Most patients are very 
comfortable throughout the procedure. There are rarely serious complications resulting from use of the 
blood cell separator. However, as in any medical procedure, there are certain risks involved. These 
include but are not limited to air entry into the blood stream, infection, shock, irregular heartbeat or 
heart failure. These complications are rare. Also, as in any donation of blood, there are a variety of 
minor reactions, which may occur such as fainting, dizziness, nausea, and bruising/swelling around the 
needle site. In rare instances, the small amount of blood remaining in the tubing cannot be returned to 
the patient. Any feeling of discomfort experienced during or after should be brought to the attention of 
the Hemapheresis Unit staff. If any complications arise, the Hemapheresis nurse and medical staff will 
provide immediate treatment. 
 
It is possible that you require the placement of a catheter in a neck vein (central line) if the veins in 
your arms do not allow to correctly perform the leukapheresis procedure. This will be evaluated by an 
experienced leukapheresis nurse. Complications of placing a central line in a neck vein include pain, 
bruising and rarely, infection, at the site where blood is drawn. It is also possible to feel lightheaded or 
faint. A rare complication is puncturing your lung resulting in shortening of breath, which would 
require that you are admitted to the hospital for the placement of a tube in your chest to drain the air 
and allow your lung to re-expand.  
 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 19 of 29 
7. Risks of Tumor Biopsies: A biopsy may result in local bruising, swelling or bleeding at the site 
where the skin is opened, lightheadedness, and fainting. Rarely, an infection may develop. These risks 
are reduced by having the biopsy performed in a clean environment by an experienced surgeon, and by 
close monitoring of the stitched wound after the procedure. 
 
8. Risks of Research PET/CT Scans.  
 
• Intravenous Injection: Administration of PET dyes through the vein can cause some slight 
discomfort when the needle is inserted into the arm vein. However, this is no more than the 
discomfort encountered when the blood samples are taken during a periodical medical 
examination or when blood is donated at the blood bank. The potential side effects of placing an 
intravenous line may include momentary discomfort during the puncture, lightheadedness, 
fainting, soreness and/or bruising for several days. In very rare circumstances, either bleeding or 
infection can develop at the needle puncture site. However, since the procedure is performed 
using sterile and standard medical practices, the chance of that occurring is very unlikely. 
 
• Confinement: Being in the PET/CT scanner can cause claustrophobia (anxiety), in about 5% of 
patients. If you develop this feeling of claustrophobia, you should tell the study doctor or the 
technologist who is in the room that you cannot continue the study, and it will be terminated. 
The scanning procedure takes place in a quiet room. The scanner itself does not make any noise. 
 
• Radiation Exposure:  We are exposed to radiation on a daily basis, both from natural (sun and 
earth) and man made sources.  In addition to the radiation that you may be exposed to as part of 
your clinical care (if you are receiving clinical care), you may receive one PET/CT scan while 
participating in this research study.  The estimated radiation dose that you will receive as a 
result of this additional scan is 1436 millirem, or 29% of the 5,000 millirem annual limit 
allowed radiation workers 
 
If you participate again within 12 months in another study involving radioactivity or x-rays, you 
should inform the person ordering the scans that you were a participant in this study. The 
investigator will ensure through accurate record keeping at UCLA that the total amount of 
radioactivity administered for research purposes will remain small, and again, is not expected to 
cause any adverse effects.  
 
• For Women of Childbearing Potential: Current knowledge indicates no confirmed 
detrimental effects to a developing fetus of radiation doses below 1000 mSv. The radiation dose 
that you will receive from the study procedure is very small in comparison. However, the study 
doctors wish to minimize any potential for exposure of a fetus to even these low levels of 
radioactivity. Because of exposure to radiation, it is important for you to practice effective 
methods of birth control, as discussed in the section below entitled “Risk to the Unborn Child 
(Men and Women)”. You should indicate to the study doctors any possibility that you are 
pregnant or that you have not used an effective and medically acceptable method of birth control 
in the recent past.   
 
You will be asked for a urine specimen to have a pregnancy test done before the research PET 
CT scans are done. If your pregnancy test is positive you cannot participate in this research 
study. 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 20 of 29 
 
9. Risks of Regular PET/CT Scans and MUGA Scans.  The amount of radiation received by you 
during these scans is the same as that for similar patients who are not on protocol; therefore, there is 
no increased risk of radiation exposure by participating in the study. You will be asked not to move 
during the test, but relax and breathe normally. 
 
A liquid solution known as a contrast, given through a vein, is frequently used during CT scans. The 
contrast is a solution used to make certain organs, blood vessels and tissues stand out in greater 
contrast to better image your disease. You may experience nausea, flushing, warmth and a salty 
taste.  
 
Intravenous contrast can cause allergic reactions. Allergic reactions to contrast may include itching, 
rash or hives. These usually disappear quickly. If needed, antihistamines can be given to help 
relieve the symptoms. Please inform the study physician about any bad previous experience with 
intravenous contrast administration.  
 
Anaphylactic reaction is a severe allergic reaction. When this occurs, you may experience severe 
hives and/or difficulty in breathing. This reaction is quite rare, but is potentially life-threatening if 
not treated. Medications that may reverse this adverse reaction include corticosteroids, 
antihistamines, and epinephrine.  
 
Toxicity to the kidneys that can result in kidney failure is an extremely rare complication of the 
intravenous contrast used in CT scans. Patients who already have impaired kidney function are most 
prone to this reaction.  
 
It is estimated that the overall frequency of adverse reactions is 5 to 10 percent. Thus, 5 to 10 of 100 
patients experience allergic reactions. Most of these are very mild and may consist of only a few 
hives. However, in one of every 1,000 to 2,000 examinations, a moderate or severe reaction can 
occur. The risk of death from a contrast agent is estimated to be 0.3 to 2.6 per 100,000 uses 
(comparable in magnitude to the risk of death from receiving a dose of penicillin). 
 
10. Risks of MRI. An MRI uses magnetic energy and radio waves rather than x-ray radiation. A 
few patients may experience anxiety due to claustrophobia, or an inability to be in a confined space 
without being given a sedative (a drug used to calm them). In addition, you will hear loud, knocking 
noises. Occasionally, subjects will report a mild headache from the "knocking" sound. There are no 
other reported side effects. If you have any metal implants within the body (cardiac pacemaker, 
orthopedic pins, plates, or screws) you will not be able to undergo an MRI since metal implants 
interfere with the MRI system's magnetic field. You will still be able to participate in this study, but 
would have a CT scan instead of an MRI. 
 
Recently, it has been reported that a rare but serious adverse reaction called nephrogenic systemic 
fibrosis (NSF) may occur after exposure to the gadolinium-based contrast agent gadodiamide 
(Omniscan®, GE Health Diagnostic, Amersham, United Kingdom). Nephrogenic systemic fibrosis 
is a condition wherein patients develop large areas of hardened skin with lesions called plaques and 
papules with or without skin discoloration. In some cases, NSF could lead to physical disability and 
may involve not only the skin, but also the liver, lungs, muscles and heart. The typical patient in 
whom this has occurred is middleaged and has end-stage kidney disease.” 
 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 21 of 29 
11. Risks of Ophthalmologic Eye Exam: Dilatation of your pupils may result in sensitivity to daylight 
requiring wearing sunglasses for several hours after the visit. Taking pictures of your eye may also 
result in sensibility to the light and local pain. Injecting a dye through a vein in your arm may result in 
local pain, a burning sensation in the arm and in extreme cases an allergic reaction. Placing a contact 
lens in your eye may result in local pain in the eye. A rare risk associated with pupil dilatation during 
the eye exam is the development of a condition known as acute angle closure glaucoma, which would 
manifest as severe eye pain due to sudden increase in pressure in the eye. This would require treatment 
to reduce the pressure in the eye. 
 
12. Risk to the Unborn Child (Men and Women). The study drug used in this study may involve 
currently unforeseeable risks to pregnant women, the embryo or fetus, or to breast-feeding infants, 
and for this reason you must not become pregnant or father a baby while on this study, or for six 
months following your participation in the study. If you are sexually active, you must use a 
medically acceptable method of birth control while on this study. Examples of accepted birth 
control measures are: birth control pills, a condom with spermicidal jelly, a diaphragm with 
spermicidal jelly, or abstinence. You must continue using birth control for six months after your last 
dose of study drug. The study investigators must approve the form of birth control. 
 
If you are a woman of childbearing potential (not surgically sterile or post-menopausal), you must 
have a negative pregnancy test before beginning treatment. Additionally, you must not be nursing 
an infant during this study. 
 
If you or your partner miss a period or become pregnant while you are on this study, you must tell 
the study investigators immediately. You or your partner will be referred to counseling about the 
options available to you. The study investigators will not be part of any decision resulting from this 
counseling. If you become pregnant, the study treatments will be stopped and we will request to 
follow you for the pregnancy outcome. 
 
13. Risks of Central Venous Catheter Insertion. The most common risks of placement of CVC 
catheters in an arm vein like a PICC line are local pain and redness in the area where the catheter 
goes through the skin and reaches the vein. Other problems may be bleeding or oozing from the site 
where the catheter is placed. These risks are minimized by their placement by specially trained 
nurses called PICC nurses, or by their placement under radiology guidance to correctly find a vein. 
Central line insertion in a neck vein may additional serious complications like a pneumothorax 
(rupture of a lung), which would be experienced as an immediate feeling of shortness of breath. 
This risk is minimized by the placement of this type of catheters by highly trained physicians that 
are specifically approved to do place this type of catheters. If the tip of either a PICC or neck 
catheter reaches the heart and comes in contact with the heart wall it may induce changes in heart 
rate called arrhythmia, which you may feel as palpitations. This would be managed by pulling out 
the catheter a bit. Another complication may be if the tip of the catheter goes towards the neck veins 
as opposed to going to the large vessels in the heart area. In this case there would also be a need to 
pull out the catheter a bit. The site where the catheter tip is located is checked with an X-ray of the 
chest after the catheter has been placed, and the catheter is not used until a Doctor has confirmed 
that this tip is in a good location. With both types of catheters there may be complications of 
infection, which would be due to introducing bacteria into the blood with the use of the catheter. If 
you develop fevers or other signs of infection while having a catheter in place, blood cultures will 
likely be taken from both the catheter and from a vein elsewhere in the body. If the culture from the 
central line grows bacteria, the line is the likely source of the infection. In this case, the Doctors 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 22 of 29 
may consider pulling this line out and placing another one later, or treating it with antibiotics 
without pulling the line out. 
 
14. Risks of Blood Draws. The most common side effects of drawing blood that have been seen in 
humans or animals are pain, bruising and rarely, infection, at the site where blood is drawn. It is 
also possible to feel lightheaded or faint. Please tell the study investigators or study staff if you do 
not feel well after having your blood drawn. Some patients may require the insertion of a device 
into your vein in order to minimize the amount of discomfort and trauma to your veins. 
 
15. Risks of HIV Testing. Testing for the HIV virus, which is the cause of AIDS, may include 
anxiety while waiting for the test results, emotional distress upon learning of the test results, risks of 
social stigmatization and discrimination in employment, housing and insurance. 
 
16. Risks of Electrocardiogram (ECG) Test.  You may feel some discomfort similar to pulling off 
an adhesive bandage when the technician removes the electrodes placed on your chest during the 
procedure.  
 17. Risks of Pulmoanry Function Test (PFT). Side effects from PFT testing are extremely rare 
and have usually occurred in patients with severe underlying cardiac disease. Some potential 
complications include: 
- 
Potential for syncope with breathing in deeply (valsalva) 
- 
Extremely rare but possibility of myocardial infarction (heart attack) with the valsalva 
maneuver (bearing down) 
- 
Pain and low risk of infection from arterial blood gas drawing   
 
18. Risks of Echocardiogram (ECHO) or MUGA scan. Echocardiography testing is very safe and 
it does not involve radiation. There is a small risk of dermatologic irritation from the gel used 
during the procedure. Another method of acquiring the LVEF is called a MUGA scan – this scan 
involves the injection of tin and Technetium-99 – but these are given at very low doses so the side 
effects are minimal. There is an extremely small amount of radioactivity with the test. For all 
patients, an ECHO will be preferentially ordered. 
19. Unknown Risks. The study treatments and procedures may involve risks that are currently 
unforeseeable. 
 
• ANTICIPATED BENEFIT TO SUBJECTS 
 
Taking part in this study may or may not improve your health. While doctors hope the MART-1 
TCR CTLs will be more useful against cancer compared to the usual treatment, there is no proof of 
this yet.  You have the option to refuse to participate in this study. 
 
 
 
 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 23 of 29 
• ANTICIPATED BENEFITS TO SOCIETY 
 
This study may lead to a better understanding of how the immune system fights cancer. Such an 
understanding may lead to the development of improved methods for treating human cancer. 
Therefore, the knowledge gained may benefit others. 
 
• ALTERNATIVES TO PARTICIPATION 
 
Your participation is voluntary and you may choose not to participate in this study. The study 
investigators are very willing to discuss the benefits and side effects of alternative treatments.  
 
There are seven treatments approved by the FDA for metastatic melanoma: 
• The anti-PD-1 antibody pembrolizumab (or Kytruda®)  
• The anti-Pd-1 antibody nivolumab (or Opdivo®) 
• The anti-CTLA4 antibody ipilimumab (or Yervoy ®)  
• BRAF V600E inhibitors vemurafenib (or Zelboraf ®) or dabrafenib (or Tafinlar ®) 
• MEK inhibitor trametinib (or Mekinist ®) 
• The chemotherapy agent DTIC (or Dacarbazine)  
• The chemotherapy agent hydroxyurea  
• The immunotherapy agent Interleukin-2 (or IL-2 or Proleukin ®) 
 
Pembrolizumab (Kytruda) has recently demonstrated an improvement in survival compared to other 
standard of care therapies in two randomized clinical trials. Vemurafenib, dabrafenib, and 
trametinib showed an effect on melanoma patients with BRAF V600E mutation. Neither DTIC, 
hydroxyurea, nor IL-2 has demonstrated an improvement in survival for patients with metastatic 
melanoma.  IL-2 is part of this protocol.  You should be aware that you have the option of receiving 
these treatments without participating in this protocol.  
 
Your other choices may include: 
• Getting treatment or care for your cancer without being in a study 
• Taking part in another study with chemotherapy, radiation therapy or experimental 
anticancer agents 
• 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 the 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.   
 
You also have the option to receive no treatment at this time. Other investigational protocols with 
chemotherapy, radiation therapy, or experimental anticancer agents may be available for your 
disease. The study investigators can provide you with detailed information about your disease and 
the risks and benefits of different treatment options. At any time, you may freely discuss your 
participation with the study investigators or doctors not involved in this research. 
 
 
 
 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 24 of 29 
• COST OF PARTICIPATION 
 
The study will pay for research-related items and/or services that are provided only because you are 
participating in the study. These research-related items and/or services are explained in other areas 
of this consent form. 
  
You or your health plan may be responsible to pay for all the types of items listed below: 
• Items and services that would have been provided to you even if you were not in the study 
• Health care given during the study as part of your regular care 
• Items and services needed to give you study drugs or devices  
• Monitoring for side effects or other problems  
• Deductibles or co-pays for these items and/or services 
 
You will not be paid for taking part in this study. 
 
For more information on clinical trials and insurance coverage, you can visit the National Cancer 
Institute’s Web site at http://cancer.gov/clinicaltrials/understanding/insurance-coverage. You can 
print a copy of the “Clinical Trials and Insurance Coverage” information from this Web site. 
 
Another way to get the information is to call 1-800-4-CANCER (1-800-422-6237) and ask them to 
send you a free copy. 
 
• 
EMERGENCY CARE AND COMPENSATION FOR INJURY 
 
It is important that you promptly tell the study doctors if you believe that you have been injured 
because of taking part in this study. You can tell the researcher in person or call him/her at the 
number(s) listed below. 
 
If you are injured as a result of being in this study, UCLA will provide necessary medical treatment. 
The costs of the treatment may be covered by the University of California or the study sponsor or 
billed to you or your insurer just like other medical costs, depending on a number of factors. The 
University does not normally provide any other form of compensation for injury.  For more 
information about this, you may call the UCLA Office of the Human Research Protection Program 
at 310-825-5344 or send an email to mirb@research.ucla.edu. 
 
• PRIVACY AND CONFIDENTIALITY 
 
The study doctors will do their best to make sure that your private information is kept confidential. 
Information about you will be handled as confidentially as possible, but participating in research 
may involve a loss of privacy and the potential for a breach in confidentiality. Study data will be 
physically and electronically secured.  As with any use of electronic means to store data, there is a 
risk of breach of data security 
 
You will be asked to sign a separate form called the University of California Permission to Use 
Personal Health Information for Research that will allow UCLA to share information from your 
medical records with the study doctor so they can use your information as part of this study.  
 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 25 of 29 
Coding of Specimens and Medical Information.  
 
Your tissues, cells from your blood and medical information will be labeled with a code. Only the 
researchers conducting this study will have the information that matches the code to traditional 
identifying information, such as your name, address, phone number, or social security number. The 
information that matches the code to your identifying information will be kept in a safeguarded 
database. Only very few, authorized people, who have specifically agreed to protect your identity, 
will have access to this database. All other researchers and personnel, including those who will be 
working with your samples and medical information, will not have access to any of this identifying 
information about you.  
 
The research team, authorized UCLA personnel and regulatory agencies such as the FDA, may have 
access to study data and records to monitor the study. Research records provided to authorized, non-
UCLA personnel will not contain identifiable information about you. Publications and/or 
presentations that result from this study will not identify you by name.  
 
If photographs of you are taken, they may be used for educational purposes. Your identity will be 
protected by showing the minimum amount of your body that includes the lesion or reaction being 
pictured, and by disguising your eyes, birth marks or anything else that may make it easy for others to 
recognize that it is a picture of you. 
 
How information about you will be stored. 
 
All research data and records will be stored electronically on a secure network with encryption and 
password protection. 
 
How long information from the study will be kept. 
 
The researchers intend to keep the research data and records indefinitely for future research. 
 
BIOPSY TISSUE SAMPLE OR BLOOD CELLS REMAINING AT THE END OF THE 
STUDY 
 
You will be asked to consider whether you would permit part of this sample to be shared with other 
researchers. You will be asked to sign an additional consent form for this.  If you agree to have your 
sample shared with other researchers and later decide to withdraw, we may not be able to retrieve 
any or all of your sample from other researchers. The researcher is not required to store your 
sample(s) indefinitely. 
 
Use of My Specimens. 
 
Any specimens (e.g., tissue, blood, urine) obtained for the purposes of this study will become the 
property of the University of California. Once you provide the specimens you will not have access 
to them. The University may share your specimens in the future with other researchers or outside 
institutions. Information that identifies you will not be shared with anyone outside of UCLA. The 
specimens will be used for research and such use may result in inventions or discoveries that could 
become the basis for new products or diagnostic or therapeutic agents. In some instances, these 
inventions and discoveries may be of potential commercial value and may be patented and licensed 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2






MART-1 F5 TCR ICF 5/12/2016 
 
Page 28 of 29 
 
Public Information about this Study: 
• ClinicalTrials.gov is a website that provides information about federally and privately 
supported clinical trials. A description of this clinical trial will be available on 
http://www.ClinicalTrials.gov, as required by U.S. Law. This website will not include 
information that can identify you. At most, the website will include a summary of the 
results. You can search this website at any time.  
 
• The National Cancer Institute's (NCI) Clinical Trials Reporting Program (CTRP) is a 
comprehensive database of information about all NCI-supported clinical trials. The goal of 
this comprehensive database is to help NCI identify areas that need more clinical research 
and to help NCI decide which studies are most important to do first. The NCI requires that 
cancer clinical trials report information about how many subjects are enrolled in the trials 
and the outcome of the trials. Specific information about you as a subject will be included in 
the database.  This information will include information about your cancer, your study 
identification number, the month and year of your birth, as well as your gender, country of 
origin, race, ethnicity, and zip code.  This information will be maintained in a secure and 
confidential manner by the NCI CTRP in their electronic database.  The NCI CTRP has 
many safeguards in place for privacy, security, and limited authorized access. 
 
 
HOW DO I INDICATE MY AGREEMENT TO PARTICIPATE?  
 
If you want to participate in this study you should sign and date below. You have been given a copy 
of this consent form and the Research Participant’s Bill of Rights to keep.  You will be asked to 
sign a separate form authorizing access, use, creation, or disclosure of health information about you. 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2


MART-1 F5 TCR ICF 5/12/2016 
 
Page 29 of 29 
 
SIGNATURE OF RESEARCH SUBJECT 
 
I have read the information provided above.  I have been given an opportunity to ask questions and 
all of my questions have been answered to my satisfaction.  I have been given a copy of this form, 
as well as a copy of the Research Participant's Bill of Rights. 
 
BY SIGNING THIS FORM, I WILLINGLY AGREE TO PARTICIPATE IN THE RESEARCH IT 
DESCRIBES. 
 
 
__________________________________  
________________________ 
Signature of Subject  
 
 
 
date 
 
 
__________________________________ 
Name of Subject (Printed) 
 
 
SIGNATURE OF INVESTIGATOR 
 
I have explained the research to the subject, and answered all of his/her questions.  I believe that 
he/she understands the information described in this document and freely consents to participate. 
 
 
___________________________________  
___________________________ 
Signature of Investigator 
 
 
 
date (must be the same as subject's) 
 
 
__________________________________  
___________________________ 
Name of Investigator (Printed) 
 
 
Contact Number 
 
 
Protocol ID:IRB#10-001212    UCLA IRB Approved
Approval Date: 7/24/2017
Through: 7/18/2018   Committee: Medical IRB 2