untitled h1
untitled h2

UCLA•CALTECH• USC•UCONN
Translational Program in Engineered Tumor Immunity TITLE: Adoptive Transfer of MART-1 F5 TCR Engineered Peripheral Blood Mononuclear Cells
(PBMC) after a Nonmyeloablative Conditioning Regimen, with Administration of MART-12635-Pulsed Dendritic Cells and Interleukin-2, in Patients with Advanced Melanoma VERSION DATE:
Study Agent:
01/10/2018, Amendment 15 Retroviral Vector MART-1 F5 TCR (F5 TCR) Engineered PBMC
PRINCIPAL INVESTIGATOR: Antoni Ribas, M.D.  
UCLA  
  
  
| 10-001212              |
|------------------------|
| 10-001212              |
| 13859                  |
| 000000                 |
| 53.08.0-h/BUA-2016-005 |
| 1600                   |
| NCT00910650            |  
CO-PRINCIPAL INVESTIGATOR: James S. Economou, M.D., Ph.D.  
UCLA  
  
  
ASSOCIATE CLINICAL INVESTIGATORS: (in alphabetical order) (with human subject responsibilities)  
| UCLA IRB #:                    |
|--------------------------------|
| UCLA ISPRC/DSMB                |
| FDA IND #:                     |
| NH OBA/RAC #:                  |
| UCLA IBC #:                    |
| UCLA GCRC #                    |
| Clinical Trial Registration #: |  
| Bartosz Chmielowski, M.D., Ph.D.   |
|------------------------------------|
| Alistair J. Cochran, M.D.          |
| Johannes Czernin, M.D.             |
| John A. Glaspy, M.D., MPH          |
| Siwen Hu-Lieskovan, M.D., Ph.D.    |
| Akira Ishiyama, M.D.               |
| Tara A. McCannel, M.D.             |
| Daniel Shin, M.D.                  |
| Arun Singh, M.D.                   |
| Deborah Wong, M.D., Ph.D.          |  
| UCLA   |
|--------|
| UCLA   |
| UCLA   |
| UCLA   |
| UCLA   |
| UCLA   |
| UCLA   |
| UCLA   |
| UCLA   |
| UCLA   |  
  
  
ASSOCIATE NON-CLINICAL INVESTIGATORS: (in alphabetical order) (no human subject responsibilities)  
| David Baltimore, Ph.D.          | CALTECH   |
|---------------------------------|-----------|
| Beata Berent-Maoz, Ph.D.        | UCLA      |
| Thinle Chodon, M.D., Ph.D.      | RPCI      |
| Begonya Comin-Anduix, Ph.D.     | UCLA      |
| Zoran Galic, Ph.D.              | UCLA      |
| UCLA                            |           |
| David W. Gjertson, Ph.D.        | Caltech   |
| James R. Heath, Ph.D.           |           |
| Paula Kaplan-Lefko, Ph.D.       | UCLA      |
| Donald Kohn, M.D.               | UCLA      |
| Richard C. Koya, M.D. Ph.D.     | RPCI      |
| William H. McBride, Ph.D., Sc.D | UCLA      |
| Bijay Mukherji, M.D.            | UCONN     |
| Michael E. Phelps, Ph.D.        | UCLA      |
| Caius G. Radu, M.D.             | UCLA      |
| Pin Wang, Ph.D.                 | USC       |
| Xiaoyan Wang, Ph.D.             | UCLA      |
| Owen N. Witte M.D.              | UCLA      |
| Lili Yang, Ph.D.                | UCLA      |
| UCLA                            |           |
| Jerome A. Zack, Ph.D.           |           |
untitled h1
table of contents 3.0 table

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 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
2 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 3 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
untitled h1
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 nonmyeloablative 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.  
5
untitled h1
1.1.1. cell dose escalation within

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.
untitled h1
1.1.2. cell dose de-escalation and

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).
untitled h1
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).
untitled h1
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.
untitled h1
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-
7 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.
untitled h1
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.
untitled h1
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 retrovirallytransduced with a MART-1 F5 TCR, followed by 3) IL-2 and 4) MART-126-35 peptide–pulsed DC.
untitled h1
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.
untitled h1
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.  
  
Patients will receive two more MART-1 26 -35 peptide pulsed DC vaccines at two weeks intervals that will be generated from cryopreserved PBMC following the same protocol of one week ex vivo differentiation culture in GM-CSF and IL-4 and peptide pulsing. Patients will undergo repeated peripheral blood sampling, PET CT scanning and biopsies of tumor deposits (Figure 1). 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, 5 to 10 ml blood samples will be collected daily during the first week after adoptive cell transfer routinely and plasma derived from these samples will be cryopreserved and archived for cytokine analyses when necessary.
untitled h1
1.8 experimental study agents 1.8.1

The retroviral vector, MSGV1-F5AfT2AB, was generated at the Surgery Branch/NCI by Dr. Steven A. Rosenberg and colleagues. It 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, and murine stem cell virus 3'LTR.  
The alpha and beta TCR chains are linked by a T2A self-cleaving sequence.
untitled h1
1.8.2 mart-126-35 peptide pulsed dendritic

Autologous DC will be differentiated from adherent peripheral blood monocytes in a one-week in vitro culture in GM-CSF and IL-4 as we have previously described 4.5 . DC will be pulsed with the MART-126-35 anchor-modified immunodominant peptide in the HLA-A2*0201 haplotype 6 and administered i.d.
untitled h1
1.9 timing of events

To account for the pilot investigational nature of the procedures in this protocol and anticipated problems in patient scheduling, timing of events are approximate and the study team will attempt to perform them on the dates included in this protocol, but variations on these dates will not be considered protocol violations.  
2.0 **Schedule of Events**  
| 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 | X      |     |      |    |    |    |    |
| MART-1                         | Screening           | Day                   | Day |        |     |      |    |    |    |    |
| -6                             | –5  and  -4         | Day  –4 to  -1        | Day | Day    | Day |      |    |    |    |    |
| 0                              | 1                   | 1 to  7               | Day | Day    |     |      |    |    |    |    |
| 14                             | 20 to  40           | Day                   | Day | Day    | Day | Day  |    |    |    |    |
| 30                             | 45                  | 60                    | 75  | 75- 90 | Day | Q2-3 |    |    |    |    |
| 90                             | mo up  to  Year  2a | Q6 o  up to  Year  5f |     |        |     |      |    |    |    |    |
| 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:              | x                     |     |        |     |      |    |    |    |    |
| PT/INR, PTT Autoantibody Panel | X                   | X                     |     |        |     |      |    |    |    |    |
| Thyroid Hormones               | X                   | X                     |     |        |     |      |    |    |    |    |
| Pregnancy Testc                | X                   | X                     |     |        |     |      |    |    |    |    |
| Infectious                     | disease             | X                     |     |        |     |      |    |    |    |    |
| serologies CT or MRI Scan of   | X                   | X                     |     |        |     |      |    |    |    |    |
| the Brain Otologic Visit       | X                   | X                     |     |        |     |      |    |    |    |    |
| Ophthalmologic Visit           | X                   | X                     |     |        |     |      |    |    |    |    |
| Leukapheresis                  | X                   | X                     |     |        |     |      |    |    |    |    |
| Plasmapheresis                 | X                   | X                     |     |        |     |      |    |    |    |    |
| Blood                          | for                 | plasma                | X   | X      |     |      |    |    |    |    |
| (cytokines)d Blood for         | cell                | X                     | X   | X      | X   | X    | X  | X  | X  | X  |
| analyses                       | (immune             | X                     |     |        |     |      |    |    |    |    |
| monitoring)e Biopsy            | X                   | X                     |     |        |     |      |    |    |    |    |
| ECHO or MUGA                   | X                   |                       |     |        |     |      |    |    |    |    |
| ECG                            | X                   |                       |     |        |     |      |    |    |    |    |
| PFT                            | X                   |                       |     |        |     |      |    |    |    |    |  
| Q12  mo up  to  Year  15g                                                                                                                                  |                     |                       |     |        |     |      |
|------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------|-----------------------|-----|--------|-----|------|
| PET/CT Scans                                                                                                                                               | X                   | X                     | X   |        |     |      |
| Inpatient admission                                                                                                                                        | X                   |                       |     |        |     |      |
| Cyclophosphamide                                                                                                                                           | X                   |                       |     |        |     |      |
| Fludarabine                                                                                                                                                | X                   |                       |     |        |     |      |
| MART-1                                                                                                                                                     | F5                  | TCR                   | X   |        |     |      |
| PBMC                                                                                                                                                       | Screening           | Day                   | Day |        |     |      |
| -6                                                                                                                                                         | –5  and  -4         | Day  –4 to  -1        | Day | Day    | Day |      |
| 0                                                                                                                                                          | 1                   | 1 to  7               | Day | Day    |     |      |
| 14                                                                                                                                                         | 20 to  40           | Day                   | Day | Day    | Day | Day  |
| 30                                                                                                                                                         | 45                  | 60                    | 75  | 75- 90 | Day | Q2-3 |
| 90                                                                                                                                                         | mo up  to  Year  2a | Q6 o  up to  Year  5f |     |        |     |      |
| Low dose IL-2                                                                                                                                              | X                   |                       |     |        |     |      |
| MART-126-35/Dendritic                                                                                                                                      | X                   | X                     | X   |        |     |      |
| Cell Vaccine 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. |                     |                       |     |        |     |      |  
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.
untitled h1
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 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
untitled h1
4.0 study objectives 4.1 primary

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. 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 MART126-35 peptide–pulsed DC vaccines.  
ii) Suboptimal number of MART-1 F5 TCR engineered T cells recovered from patients at prespecified time points.  
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. 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 14 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.
untitled h1
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.
untitled h1
5.0 background 5.1 current management

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.
untitled h1
5.2 rationale for immune-based therapies

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.
untitled h1
5.3 mart-1 melanoma antigen as

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
15 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.
untitled h1
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.
untitled h1
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.
untitled h1
5.6 retroviral vector-based human gene

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.
untitled h1
5.7 safety and regulatory issues

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, 16 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.
untitled h1
5.8 rationale for the use

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.
untitled h1
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.
untitled h1
5.10 mart-1 tcr-engineered t cell

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 18 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.
untitled h1
6.0 preliminary data 6.1 origin

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 MART1 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 19 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.
untitled h1
6.2 data using the mart-1

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.
untitled h1
7.0 eligibility criteria 7.1 inclusion

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.  
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.
untitled h1
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  
(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.
untitled h1
7.3. definition of study enrollment

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.
untitled h1
8.0 study procedures

This is a phase 2 study to determine the safety, feasibility and antitumor activity of retrovirustransduced 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.
untitled h1
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.
untitled h1
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
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.
untitled h1
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
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.
untitled h1
8.3. leukapheresis cell cultures and

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.
untitled h1
8.4 days -5 to -1

The conditioning chemotherapy will be initiated the next day following leukapheresis.
untitled h1
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.
untitled h1
chemotherapy administration days -5 to

- 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.
untitled h1
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.
untitled h1
prophylactic supportive care throughout the

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.  
- 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 JMedicine protocol and the clinical judgment.  
- Norethindrone (Norlutate) 10mg PO daily will be used for menstruating women to prophylaxis against menorrhagia.
untitled h1
8.5 day 0 mart-1 f5

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.
untitled h1
transgenic mart-1 f5 tcr pbmc

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.
untitled h1
patient monitoring after mart-1 f5

- 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  
- 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.
untitled h1
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.
untitled h1
8.6 days 1 14 and

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.
untitled h1
8.7 days 1-7 low dose

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
untitled h1
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.
untitled h1
supportive care during low dose

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 27 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.
untitled h1
blood collection for immune monitoring

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.
untitled h1
blood collection for plasma cytokine

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.
untitled h1
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.
untitled h1
8.9 days 20 to 40

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.
untitled h1
8.10 days 30 45 60

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.
untitled h1
8.11 permissible systemic therapies during

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:  
- 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.
untitled h1
8.12 days 75 to 90

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.
untitled h1
8.13 day 90 office visit

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.
untitled h1
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).
untitled h1
9.0 study agents 9.1 pbmc

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 nonmyeloablative 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.
untitled h1
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-12735 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 MART126-35 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:** 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.
untitled h1
9.1.2 pbmc transgenic for mart-1

Product Description. PBMC obtained from leukapheresis and activated for 48 hours with OKT3 (antiCD3 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 interferongamma, 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 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.  
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 antithymocite globulin (ATG).
untitled h1
9.2 mart-126-35 peptide pulsed dendritic

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 HLAA*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 HLAA*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.  
33
untitled h1
9.2.2 mart-126-35 peptide pulsed dendritic

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 antiCTLA4 antibodies), we have not observed SAE attributable to the MART-1-expressing DC.
untitled h1
9.3 interleukin-2 (aldesleukin proleukin recombinant

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 IL2 include diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, or constitutional symptoms and laboratory changes.
untitled h1
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.  
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.
untitled h1
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 highdose 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 36 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.
untitled h1
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.
untitled h1
10.0 generation of experimental cell

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.
untitled h1
10.1 generation of mart-1 f5

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.
untitled h1
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.
untitled h1
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.
untitled h1
10.1.4 clinical grade retrovirus msgv1-f5aft2ab

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                                                                                         | No          | growth | within | 14 | Apr-10-2007 |
| days                                                       | days                                                                                                        |                                                                                                             |             |        |        |    |             |
| Mycoplasma culture                                         | Negative for the                                                                                            |                                                                                                             |             |        |        |    |             |
| and vero indicator cells                                   | presence of  mycoplasma                                                                                     | Negative                                                                                                    | Apr-04-2007 |        |        |    |             |
| In-vitro viral assay  utilizing 3T3, MRC-5  and Vero cells | No CPE or                                                                                                   | No CPE or                                                                                                   | Apr-10-2007 |        |        |    |             |
| hermadsorption                                             | hermadsorption                                                                                              |                                                                                                             |             |        |        |    |             |
| 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 |        |        |    |             |  
| 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                                                                                              | No evidence of viral                                                                                              | Dec-21-2006        |             |
| contamination                           | contamination                                                                                                     |                                                                                                                   |                    |             |
| 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                             | HTLV-1/11 (PCR)*                                                                                                  | Negative                                                                                                          | Negative           | Nov-16-2006 |
| Contaminants                            | 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 coculture) 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                                                                                             | Cells confirmed to | Apr-10-2007 |
| Of murine origin                        | of murine origin                                                                                                  |                                                                                                                   |                    |             |
| Vector insert stability                 | Vector size/sequence  consistent with  expectant fragment  size                                                   | Vector size/sequence  consistent with  expectant fragment size                                                    | Apr-10-2007        |             |
| Vector                                  | Sequencing of the                                                                                                 | > 98% homology with                                                                                               | > 99.9% homology   | Sep-26-2008 |
| Sequencing                              | insert in the MCB                                                                                                 | the expected sequence                                                                                             |                    |             |  
Table 1b. Certificate of Analysis of the MSGV1-F5AfT2AB Final Product.  
| Test                     | Methods           | Limits    | Results   | Date   |
|--------------------------|-------------------|-----------|-----------|--------|
| No growth 14 days        | No growth 14 days | March-18- |           |        |
| culture for Bacteria and |                   |           |           |        |  
| Fungus Mycoplasma culture                                  | Negative for the                                                      |                                                                                  |                                                                      |              |
|------------------------------------------------------------|-----------------------------------------------------------------------|----------------------------------------------------------------------------------|----------------------------------------------------------------------|--------------|
| and Vero indicator cells                                   | 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                                                             | No CPE or                                                                        | Mar-18-2008                                                          |              |
| hermadsortion                                              | hermadsortion                                                         |                                                                                  |                                                                      |              |
| Endotoxin                                                  | Limulus amebocyte                                                     | Less than 0.33 EU/mL                                                             | Harvest 1-7: less than                                               | Mar-18-2008  |
| lysate                                                     | 0.06 EU/mL                                                            |                                                                                  |                                                                      |              |
| 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 cocuture) 108 cells from  production run                                                            | No Evidence of RCR                                                    | No Evidence of RCR                                                               | Mar-18-2008                                                          |              |
| Transgene                                                  | MART-126-35 tetramer                                                  |                                                                                  |                                                                      |              |
| expression                                                 | 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 MART126-35 peptide  stimulation using  MART-126-35 peptidepulsed K562/A2.1 cells                                                                                  | > 100,000 pg/ml of IFNγ production upon  MART-126-35 peptide  stimulation using  MART-126-35 peptidepulsed K562/A2.1 cells                                                                      | Sept-05-2008 |
untitled h1
10.1.5 clinical grade retrovirus msgv1-f5aft2ab

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                     | MART-126-35                                             | tetramer             |                       |             |      |
| (Transgene expression)                            | or dextramer staining in  activated PBMC                | >                    | 10%                   | MART-126-35 |      |
| tetramer                                          | or                                                      | dextramer            |                       |             |      |
| positive cells among CD3+  T lymphocytes          |                                                         |                      |                       |             |      |
| Sterility                                         | Aerobic and anaerobic  culture for bacteria and  fungus | 0.03%                | MART-126-35           |             |      |
| tetramer                                          | or                                                      | dextramer            |                       |             |      |
| positive                                          | cells                                                   | among                |                       |             |      |
| CD3+ T lymphocytes 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.                      |                                                         |                      |                       |             |      |  
| A CCEPTABILITY               |                      |                     |                   |                     |                         |
|------------------------------|----------------------|---------------------|-------------------|---------------------|-------------------------|
| TEST                         | METHOD               | RESULTS             | DATE              |                     |                         |
| CRITERIA                     |                      |                     |                   |                     |                         |
| Physical                     | gRT-PCR              | 10                  | viral             | 2.06 x  10 5 viral  | 1/30/2017               |
| viral                        | 5                    | x                   |                   |                     |                         |
| vector titer                 | particles/ ml        | particles/ml        |                   |                     |                         |
| Infectious                   | MART-126-35 tetramen | >10% MART-126-35    | 54.7% MART-126-35 | 1/15/2017           |                         |
| viral                        |                      |                     |                   |                     |                         |
| vector titer                 | staining             | tetramer            | positive          | clls                | tetramer positive cells |
| in                           |                      |                     |                   |                     |                         |
| (Transgene                   | transduced   PBMC    | among               | CD3+              | among               | CD3+                    |
| lymphocytes                  |                      |                     |                   |                     |                         |
| expression)                  | lymphocytes          |                     |                   |                     |                         |
| Sterility                    | 1/29/2017            |                     |                   |                     |                         |
| Aerobic                      | and                  | No growth           | within  14        | No growth within 14 |                         |
| anaerobic culture for        | dys                  | days                |                   |                     |                         |
| bacteria and fungus          |                      |                     |                   |                     |                         |
| Mycoplasma                   | MycoAlert test       | Negative (<1 ratio) | Negative (<1)     | 10/27/2017          |                         |
| <0.33 EU/ml                  | <0.25 EU/ml          | 10/27/2017          |                   |                     |                         |
| Endotoxin                    | Limulus              | amebocyte           |                   |                     |                         |
| lysate or Endosafe           |                      |                     |                   |                     |                         |
| PTS System                   |                      |                     |                   |                     |                         |
| Legend: EU: endotoxin units. |                      |                     |                   |                     |                         |  
Table 2b. MSGV1-F5AfT2AB Retrovirus Annual Recertification 2017:
untitled h1
10.1.6 retrovirus transduction method

The MSGV1-F5AfT2AB retrovirus vector lot will be stored in a monitored and locked < = -70ºC freezer in the UCLA HGMP/JCCC GMP Suite, 14 b floor, Factor Building (Viral Bank). Transductions will be performed in retronectin-coated 6-well plates.  
RetroNectin Coating. Wells will be pre-coated with retronectin (Takara Bio Inc., Japan), a recombinant chimeric fibronectin molecule. 1 ml of 10 µ α/ml retronectin in sterile PBS is placed per well in 6-well plates and incubated overnight at 4°C (aiternatively at room temperature for 2 hours). Wells are blocked with 1.5 ml of HBSS with 2.5% human serum albumin at room temperature for 30 minutes and washed with HBSS containing 2.5% HEPES.  
Retroviral Transduction. Two to four ml of thawed and 1:1 diluted MSGV1-F5AfT2AB retrovirus viral supernatant in TCR medium is applied to each retronectin-coated well of Transduction \#1 plates and centrifuged at 2000 for 2 hours at 32ºC. Retroviral supernatant is removed from transduction \#1 plates (leaving behind one ml), 2 x 10 6 activated PBMC per well in AIMV plus 5% heat inactivated human AB serum supplemented by 300 IU/ml of IL-2 are added and centrifuged at 1000g for 10 minutes at 32 °C. Plates are then incubated at 37°C overnight at 5% CO 2 . On the following day, after blocking and washing, two to four ml of thawed and 1:1 diluted MSGV1-F5AfT2AB retrovirus viral supernatant in TCR medium is applied to each retronectin-coated well of Transduction \#2 plates and centrifuged at 2000g for 2 hours at 32ºC. Then, viral supernatant is removed from each well. PBMC
are transferred from transduction \#1 plates to transduction \#2 plates and centrifuged at 1000g for 10 minutes at 32 °C. Plates are incubated at 37°C overnight at 5% CO2.  
Post-transduction Expansion. At the end of the transduction, cells are washed and maintained at 37ºC, 5% CO 2 in AIMV plus 5% heat inactivated human AB serum supplemented by 300 IU/ml of IL-2 at a density of 0.7 x 10 6 cells/ml for up to 96 hours from the initial transduction.
untitled h1
10.1.7 transgenic pbmc product washing

After completion of the cell transduction procedure and short term ex vivo expansion, the MART-1 F5 TCR transduced PBMC product is subjected to two washings, resuspended in saline containing 1%
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.
untitled h1
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).
untitled h1
10.1.9 in-process testing and final

In Process Testing. We will conduct the following in-process testings (Table 3a and 3b).  
| 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 3a. Activated PBMC (Day 2) In-process Testing: 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                                                                                                                                                                 | Negative by PCR                                                            |
| results are equivocal or positive 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 |                                                                            |  
Table 4a. MART-1 F5 TCR Transgenic PBMC Lot Release Testing with Results Available Before Administration to Patients:  
| Test Item                                                            | Acceptable Criteria                   |    |    |       |    |             |          |    |
|----------------------------------------------------------------------|---------------------------------------|----|----|-------|----|-------------|----------|----|
| Viability                                                            | > 70%                                 |    |    |       |    |             |          |    |
| Gram and fungal stainsa                                              | Negative                              |    |    |       |    |             |          |    |
| Endotoxin Assay b                                                    | ≤ 5 EU/ kg body weight per dose       |    |    |       |    |             |          |    |
| MycoAlert testc                                                      | Negative ;ratio<1 (readingB/readingA) |    |    |       |    |             |          |    |
| PCR/ELISA mycoplasma assay if the                                    | Negative by PCR                       |    |    |       |    |             |          |    |
| MycoAlert results are equivocal or positive 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 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).  
| Characterization Assays                    | Comments                                                                                                  |
|--------------------------------------------|-----------------------------------------------------------------------------------------------------------|
| Vector proviral copy per                   | Quantification of the copy number of provirus by qRT-PCR                                                  |
| transduced cell  Final product phenotyping | CD3, CD4, CD8, CD27, CD28, CD45RA/RO, CD62L, CCR7, PD-1                                                   |
| Transgene integration site                 | Determination of integration site(s)                                                                      |
| analysis  Structure of integrated          | Stability of integrated transgene                                                                         |
| 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 |  
Table 5. MART-1 F5 TCR Transgenic PBMC Optional Additional Testing:
untitled h1
10.2 generation of 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.
untitled h1
10.2.2 peptide manufacturing mart-1 f5

MART-1 peptide was purchased from Biosynthesis (Lewisville, TX) and was provided with a COA.  
Additional tests were performed to make it clinical grade. The aliquoted peptide for clinical use was lot release tested as defined in Table 6.
untitled h1
10.2.3 peptide aliquoting and storage

Peptides are solubilized in GMP-grade DMSO and stored aliquoted in single-use vials at -80°C.  
Storage site is the UCLA Gene and Cell Therapy Core Facility, 14 th floor Factor Building, in a monitored freezer with backup energy system containing material for experimental clinical trial use only.
untitled h1
10.2.4 mart-126-35 peptide working stock

This peptide working stock will undergo the lot release testing described in Table 6.  
Table 6. MART-1 26-35 Peptide Working Stock Lot Release Testing:  
| Test Item           | Acceptability Criteria      | Results             | Date            |    |         |       |    |            |
|---------------------|-----------------------------|---------------------|-----------------|----|---------|-------|----|------------|
| Gram Stain          | Negative                    | Negative            | 8/25/2008       |    |         |       |    |            |
| Fungal Stain        | Negative                    | Negative            | 8/25/2008       |    |         |       |    |            |
| Bacterial Culture   | No growth                   | No growth x 14 days | 09/10/2008      |    |         |       |    |            |
| Fungal Culture      | No growth                   | No growth x 14 days | 09/10/2008      |    |         |       |    |            |
| Endotoxin level     | < 0.5 EU/ml                 | 0.4 EU/ml           | 09/24/2008      |    |         |       |    |            |
| Potency:            | IFN- γ                      | 30,000 pg/ml of     | MART-1          | >  | 100,000 | pg/ml | of | 09/05/2008 |
| IFN-y production by | MART-1                      |                     |                 |    |         |       |    |            |
| ELISA               | upon                        | specific            | specific IFN- γ |    |         |       |    |            |
| MART-126-35         | ELISA using 1 x 10 6 MART-1 | production          |                 |    |         |       |    |            |
| peptide stimulation | TCR transduced cells        |                     |                 |    |         |       |    |            |
untitled h1
10.2.5 mart-126-35 peptide annual recertification

The stored peptide working stock aliquots will undergo the annual recertification testing described in Table 7.  
Table 7. MART-1 25 -35 Peptide Working Stock Annual Recertification:  
| A CCEPTABILITY               |                     |            |      |
|------------------------------|---------------------|------------|------|
| TEST                         | METHOD              | RESULTS    | DATE |
| CRITERIA                     | Negative            |            |      |
| Bacterial culture            | No growth           | 1/29/2017  |      |
| Microbiological              | Negative            |            |      |
| studies                      | 1/29/2017           |            |      |
| Fungal culture               | No growth           |            |      |
| Endosafe - MCS or            | <0.005 EU/µg        |            |      |
| Endotoxin Assay              | <0.1 EU/ug peptide  | 10/31/2017 |      |
| PTS System                   | peptide             |            |      |
| Mycoalert/ PCR               | Negative (0.12)     |            |      |
| Mycoplasma test              | Negative (<1 ratio) | 10/31/2017 |      |
| based assay                  |                     |            |      |
| Legend: EU: endotoxin units. |                     |            |      |  
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 6 C f or 1/ 2 h o ur a n d k e pt at 2 0 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 m) pri or t o u s e.
untitled h1
1 0. 2. 7 l

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 |                                   |
untitled h1
1 0. 2. 8 pl

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 |                                   |
untitled h1
1 0. 2. 9 d

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 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 C, 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 4 6 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 C i n 5 %  
C 0 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).
untitled h1
1 0. 2. 1 0

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.
untitled h1
1 0. 2. 1 1

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 i n 1 ml s er u m fr e e R P MI 1 6 4 0 a n d 1 0 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 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 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.
untitled h1
1 0. 2. 1 2

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 t o 1. 2 x 1 0 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 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.  
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.
untitled h1
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.  
| Culture Day        | Testing                                                                                                 | Lot Release Criteria                                                                                                                                        |
|--------------------|---------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Leukapheresis      | Gram stain                                                                                              | Negative                                                                                                                                                    |
| Sterility cultures | Negative to date*                                                                                       |                                                                                                                                                             |
| Plasma             | Gram stain                                                                                              | Negative                                                                                                                                                    |
| Sterility cultures | Negative to date*                                                                                       |                                                                                                                                                             |
| 0                  | Fresh/ Thawed PBMC                                                                                      | > 70% Viability                                                                                                                                             |
| +4                 | Gram stain                                                                                              | Negative                                                                                                                                                    |
| Sterility cultures | 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" |  
Table 10. MART-126-35/DC Lot Release Criteria:
*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.
untitled h1
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).
untitled h1
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.
untitled h1
10.3 samples for transgenic tcr

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.
untitled h1
11.0 evaluation of study endpoints

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.
untitled h1
11.1.1.1 definition of dose limiting

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:
- 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.
untitled h1
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 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.
untitled h1
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.
untitled h1
11.1.2 feasibility

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).  
| Parameter                                     | Criteria for Unfeasibility                         |
|-----------------------------------------------|----------------------------------------------------|
| Feasibility of generating MART-1 transgenic   | 3 or more preparations not meeting the lot release |
| cells that meet the lot release criteria.     | criteria in stage 1.                               |
| Feasibility of generating MART-126-35/DC that | 3 or more preparations not meeting the lot release |
| meet the lot release criteria.                | criteria in stage 1.                               |  
Table 11. Criteria to Declare the Study Unfeasible.
untitled h1
11.1.3 antitumor activity 11.1.3.1 malignancy

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.
untitled h1
11.1.3.2 definition of measurable and

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.
untitled h1
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.  
- 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.
untitled h1
11.1.3.3 definition of target and

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.
untitled h1
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.