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11.1.3.4 objective response classifications

The following RECIST criteria will be the primary method utilized in this study for the assessment and reporting of tumor response data:  
- Complete Response (CR): Disappearance of all evidence of melanoma in target and nontarget lesions. CR must be confirmed by repeat assessments performed no less than 4 weeks after the criteria for response are first met to qualify as CR. This definition includes a pathological CR when hyperpigmented lesions visible by exam are biopsied and demonstrate no viable measurable melanoma and pigment accumulated in phagocytic cells.  
- Partial Response (PR): At least a 30% decrease in the sum of the longest diameter (LD) of target lesions taking as reference the baseline sum LD. Non-target lesions may persist provided there is no unequivocal progression in these lesions. PR must be confirmed by repeat assessments performed no less than 4 weeks after the criteria for response are first met to qualify as PR;
- Progressive Disease (PD): At least a 20% increase in the sum LD of the target lesions from the smallest sum LD recorded since the beginning of therapy or the appearance of one or more new lesions or unequivocal progression of existing non-target lesions; and
- Stable Disease (SD): Measurements demonstrating neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify as PD after the start of treatment taking as reference the smallest sum LD since the treatment started. During this time, non-target lesions may persist provided there is no unequivocal progression in these lesions.
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11.1.3.5 frequency of tumor measurements

Tumor assessments for response will be performed starting at the end of 3 months (Day 90) and every 2-3 months subsequently. Tumor assessment will also be performed at discontinuation from the study, unless these have been performed within the last 4 weeks prior to discontinuation. CR and PR must be confirmed by repeat assessments performed no less than 4 weeks after the criteria for response are first met to qualify as such response. Additional tumor measurements may be completed as needed.
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11.1.3.6 assessment of response for

In patients with in-transit metastasis only, at least one in-transit metastatic lesion should not be excised. If there is only one measurable lesion, biopsies should not interfere with the measurement of the largest diameter of the lesion. Response will be assessed as above, taking the measurement from pictures with a built-in ruler.
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11.1.3.7 duration of response

The duration of overall complete response will be measured from the time measurement criteria has been first met for CR until the first date that recurrent or progressive disease is objectively documented. The duration of overall response will be measured from the time measurement criteria is met for CR/PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started).
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11.1.3.8 time to disease progression

Defined as the length of time from the date of enrollment to the date of progressive disease first documented, death, or the start of secondary antitumor therapy, whichever occurs first. Subjects lost to follow-up will be censored at their last available follow-up date.
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11.1.4 definition of study failure

Study failure according to safety and feasibility considerations is defined as follows:  
1) Unexpected SAEs that are related to the transduction process or research reagent and required that the study be stopped.  
2) 3 or more DLT in the first stage with 8 patients. 3) Considered to be an unfeasible approach as defined above. 4) 1 or less patients with CR or PR among 8 patients in stage 1. 5) 4 or less patients with CR or PR among 22 patients in the completed study combining patients in stage 1 and 2.
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11.2 secondary study endpoints 11.2.1

TCR and vector presence will be quantitated in PBMC samples, obtained before adoptive transfer and at 7, 14, 30, 45, 60, 75 and 90 days after transgenic cell adoptive transfer. Thereafter, sampling will be every 3 months during the first 2 years, and then every 6 to 12 months. Analysis will be performed both using immune monitoring and molecular techniques. Detection of surface expression of the MART-1 TCR transgenic protein will be analyzed both by MHC tetramer or dextramer analysis and staining for the specific region. Molecular analysis of the persistence of cells bearing the MART-1 F5 TCR cDNA will be done by real time PCR techniques using primers specific for the transgenes and retroviral vector sequences. This testing will provide data to estimate the *in vivo* survival of lymphocytes derived from the infused cells.
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11.2.1.2 long term monitoring for

Analysis for detection of RCR by PCR will be performed at the National Gene Vector Biorepository
(NGVB) at the Indiana University Viral Production Facility (IU VPF) under the supervision of Dr.  
Kenneth G. Cornetta. Samples taken from cells prior to infusion will be archived and blood samples obtained from study patients at 3 and 6 months, and at one year post cell administration will be tested.  
Blood samples will be archived annually thereafter if all previous testing has been negative.  
If a patient dies or develops a neoplasm (other than melanoma recurrence) during this clinical trial, efforts will be made to assay a biopsy sample for RCR. If any post-treatment samples are positive, then the baseline final product will be tested for RCR. Further analysis of the RCR and more extensive patient follow-up will be undertaken, in consultation with the UCLA IRB, RAC and FDA.
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11.2.1.3 analysis of retroviral insertion

It is possible that expansion of specific T-cell clones will be observed as tumor reactive T cells proliferate in response to tumor antigens. Therefore, care will be taken to track T cell persistence both immunologically and molecularly. Blood samples for persistence of TCR gene transduced cells will be obtained at 3, 6, 12 months, and then annually thereafter. If any patient shows a high level of persistence of TCR gene transduced cells at month 6 (by semi quantitative DNA-PCR using primers specific for vector sequences) the previously archived samples will be subjected to techniques that would allow the identification of clonality of persisting TCR gene transduced cells. Such techniques may include T cell cloning or LAM-PCR. Clonality analysis is available through NGVL 69.  
If a predominant or monoclonal T cell clone derived from TCR gene transduced cells is identified during the follow-up, the integration site and sequence will be identified and subsequently analyzed against the human genome database to determine whether the sequences are associated with any known human cancers. If a predominant integration site is observed, the T cell cloning or LAM-PCR test will be used at an interval of no more than three months after the first observation to see if the clone persists or is transient. In all instances where monoclonality is persistent and particularly in instances where there is expansion of the clone, regardless of whether or not the sequence is known to be associated with a known human cancer, the subject should be monitored closely for signs of malignancy.
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11.2.1.4 immunological monitoring.

Immunological monitoring will consist of quantifying T cells bearing surface MART-1 F5 TCR by MART-126-35 tetramer or dextramer analysis. The functionality of these cells will be tested in ELISA
55 and/or multicytokine array assays, intracellular cytokine staining (ICS). Immunological assays will be compared between 1) pre-infusion PBMC, 2) an aliquot of the engineered PBL at the time of infusion, 3) cells recovered from patients' peripheral blood after adoptive transfer. Our published definitions for a positive or negative immunological response using standardized MHC
tetramer assays will be used for these two assays 70. These definitions are based on the assay performance specifications by defining changes that are beyond the assay variability with a 95% confidence level. For the other assays, differences of 1 fold will be considered indicative of true biologic differences. Additional testing may be performed with other assays, for example intracellular phosphoprotein staining for signaling networks, multiplexed microfluidic and nanotechnology-based immune monitoring assays 71,72.
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11.2.2 evaluation of mart-1 tcr

The ability to non-invasively image T cell responses to cancer hinges upon the detection of metabolic changes related to TCR transgenic cell proliferation *in vivo* detectable with PET probes.  
Patients will be injected with 200 MBq [18F]FDG at baseline and approximately 20-40 days after ACT,
with allowed variability of scheduling for 2-4 weeks of the intended dates. One hour post injection, a PET/CT scan covering the area from the base of the skull to the upper abdomen will be acquired. Scans of the extremities will be performed in patients with metastatic lesions in the arms or legs. The CT scan will be performed in a "low dose mode" (110 kVp, 30 mAs anode current) resulting in an effective dose of less than 1.3 mSv per scan 73. These doses are comparable with typical diagnostic CT or PET scans and below the limits set by federal regulations for research studies involving radioactive imaging agents (http://www.access.gpo.gov/nara/cfr/waisidx_01/21cfr361_01.html).  
Regional uptake of [18F]FDG within metastatic tumor sites and secondary lymphoid organs will be quantified by standardized uptake values (SUV) normalized to the body weight of the patient. As an internal quality control, SUVs will also be determined for several normal organs, such as muscle, liver and lungs. These measurements will allow us to identify technical problems in the SUV calculations, such as partially paravenous tracer administration 74. An attempt will be made to have all imaging studies will be performed on the same PET/CT system in order to eliminate confounding effects of differences in scanner sensitivity, spatial resolution or image processing.
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11.2.2.2 tumor biopsies

To correlate the [18F]FDG signal with the numbers and functional phenotype of MART-1 F5 TCR
transgenic T cells accumulating at the tumor site, percutaneous or image-guided biopsies of tumor lesions between study days 20-40, with allowed variability in this range, will be performed. The biopsies will be analyzed by H&E, IHC to quantify the numbers of T lymphocytes. If sufficient quantity of tissue is available, the study investigators will attempt to monitor the phenotype of the TIL obtained from tumor biopsy samples by MHC tetramer or dextramer, multicolor flow cytometry (FACS), and other immune monitoring assays.
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12.0 adverse event reporting 12.1

A serious adverse event or serious adverse drug reaction is defined as any untoward medical occurrence at any dose that:  
- Results in death. - Is life-threatening (immediate risk of death). - Requires inpatient hospitalization or prolongation of existing hospitalization.  
- Results in persistent or significant disability/incapacity. - Results in congenital anomaly/birth defect.
Progression of the malignancy under trial should not be reported as an adverse event. However, if the malignancy has a fatal outcome during the trial or within the safety-reporting period, then disease progression must be recorded as a Grade 5 serious adverse event. Medical and scientific judgment should be exercised in determining whether an event is an important medical event. An important medical event may not be immediately life threatening and/or result in death or hospitalization. However, if it is determined that the event may jeopardize the patient and may require intervention to prevent one of the other outcomes listed in the definition above, the important medical event should be reported as serious.
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sae reporting period

Events that meet the SAE definition will be reported within the following timelines based on the time of first knowledge of the SAE by a study investigator:
- SAE other than resulting in unexpected death:
o P.I.: Within 24 hours. o IRB, ISPRC, DSMB, IBC, NIH/RAC: Within 7 days. o FDA: Within 15 days.  
- Unexpected death:
o P.I.: Within 24 hours.  
o IRB, ISPRC, DSMB, IBC, NIH/RAC: Within 24 hours.  
o FDA: Within 7 days.  
In general, any SAE will be reported as soon as it is known by the study investigators and enough data is gathered for report filing. All SAE reports will be managed through the UCLA SAE Compliance Officer, working under the JCCC Regulatory Affairs Office. Initial reports may be completed or amended with any new relevant additional information. Copies of all reports will be maintained in the study regulatory file. Copies of any report sent to the FDA will also be sent to the study sponsor.
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12.2 definition of an adverse

An adverse event is any untoward medical occurrence in a clinical investigation patient administered a product or medical device; the event need not necessarily have a causal relationship with the treatment or usage.
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abnormal test findings

The criteria for determining whether an abnormal objective test finding should be reported as an adverse event are as follows:  
- Test result is associated with accompanying symptoms, and/or
- Test result requires additional diagnostic testing or medical/surgical intervention, and/or
- Test result leads to a change in trial schedule or discontinuation from the trial, significant additional concomitant drug treatment, or other therapy, and/or
- Test result is considered to be an adverse event by the investigator or sponsor.
Merely repeating an abnormal test, in the absence of any of the above conditions, does not constitute an adverse event. Any abnormal test result that is determined to be an error does not require reporting as an adverse event.
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12.3 causality assessment

The investigator's assessment of causality must be provided for all adverse events (serious and nonserious). An investigator's causality assessment is the determination of whether there exists a reasonable possibility that the investigational product caused or contributed to an adverse event. If the investigator's final determination of causality is unknown and the investigator does not know whether or not investigational product caused the event, then the event will be handled as "related to investigational product" for reporting purposes. If the investigator's causality assessment is "unknown but not related to investigational product", this should be clearly documented on the study records. In addition, if the investigator determines a serious adverse event is associated with trial procedures, the investigator must record this causal relationship in the source documents and CRF, as appropriate, and report such an assessment in accordance with the serious adverse event reporting requirements, if applicable.
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12.4 withdrawal due to adverse

Withdrawal due to adverse event should be distinguished from withdrawal due to insufficient response, according to the definition of adverse event noted earlier, and recorded on the appropriate adverse event CRF page. When a patient withdraws due to a serious adverse event, the serious adverse event must be reported in accordance with the reporting requirements defined below.
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13.0 compliance with good clinical

This study must be conducted in compliance with all local and federal regulatory requirements, in particular those which afford greater protection to the safety of the trial participants. This study will be conducted according to the current revision of the Declaration of Helsinki and with local and federal laws and regulations relevant to the use of new therapeutic agents. The ICF must contain a language readily understood by the potential study subjects.
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13.2 regulatory approvals

The Protocol and ICF must be approved by the following committees before initiation of patient enrollment: Local Committees:
o Institutional Review Board (IRB). o Institutional Biosafety Committee (IBC).  
o Institutional Scientific Peer Review Committee (ISPRC).  
Federal Committees:  
o Recombinant DNA Advisory Committee (RAC).  
o Food and Drug Administration (FDA).
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13.3 subject recruitment

Study subjects will be invited to participate by the study investigators as part of the discussion of standard and experimental therapy for locally advanced or metastatic melanoma. Patients may access the study investigators through:  
a) Subjects may be referred from UCLA or outside clinics. No patient will be approached without a referral or agreement from the patient's physician in order to uphold patient confidentiality.  
b) Subjects may be identified by the study investigators from their own patient pools while discussing standard and experimental options for advanced or metastatic melanoma.  
c) Study flyers will be posted in public places, including the oncology clinic waiting room, for subject self-referral.  
d) Subjects may self-refer to this study when inquiring about clinical trial options at UCLA.  
e) This study will be listed at the NIH web site listing active clinical trials, which also assures trial registration at a public site.
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13.4 gender and minorities

There will be no discrimination based upon sex or race. Subjects of any gender and ethnicity with melanoma have been and will be considered in the proposed clinical trials. There has been and will certainly be no discrimination due to gender or ethnicity, but the epidemiology of melanoma and HLA haplotypes may result in uneven minority population distribution. Since melanoma is slightly more prevalent in males (2/3), we expect that approximately 30-35% of the study population will be females. Most subjects referred to the UCLA Melanoma Program come from the UCLA Oncology Network, with over 50 offices throughout the Los Angeles County and Western US, including areas where there is a majority of subjects of Asian, Hispanic and African American descent. Cutaneous malignant melanoma occurs less frequently among non-white populations than among whites. Data from the California Cancer Registry reveal that, between 1988 and 1993, 95.5% of subjects with melanoma in California were non-Hispanic whites, 3.5% were Hispanic, 0.5% Asian, and 0.5% non-Hispanic blacks 75. The patients treated in previous melanoma clinical trials conducted over the past 5 years at the UCLA Melanoma Program mirror the gender and race characteristics of the malignant melanoma patient population in the Southern California area. Table 12 reflects the gender and minority distribution of subjects accrued to clinical trials in the UCLA Melanoma Program over the past 5 years. 93% were white and 33% were female, which is in accordance with the incidence of melanoma in our population.  
Table 12. Gender and Minorities Accrued to Prior Melanoma Clinical Trials at the UCLA Melanoma Program (2002-2007):  
| American Indian or Alaskan Native   | Asian or Pacific Islander   | Black, not of Hispanic Origin   | Hispanic   | White, not of Hispanic Origin   | Other or   | Total   |     |
|-------------------------------------|-----------------------------|---------------------------------|------------|---------------------------------|------------|---------|-----|
| Unknown                             |                             |                                 |            |                                 |            |         |     |
| Female                              | -                           | 1                               | 1          | 5                               | 52         | 1       | 60  |
| Male                                | -                           | 2                               | -          | 2                               | 117        | 1       | 122 |
| Unknown                             | -                           | -                               | -          | -                               | -          | -       | -   |
| Total                               | -                           | 3                               | 1          | 7                               | 164        | 2       | 182 |
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13.5 children

Metastatic melanoma is very rare in subjects younger than 18 years old. The use of the nonmyeloablative regimen in this protocol is a major procedure that entails serious discomforts and hazards for the patient, such that fatal complications are possible. It is therefore only appropriate to carry out this experimental procedure in the context of life threatening metastatic cancer. Since the efficacy of this experimental procedure is unknown, it does not seem reasonable to expose children to this risk without further evidence of benefit. Should results of this study indicate efficacy in treating metastatic melanoma, which is not responsive to other standard forms of therapy, future research will be conducted in the pediatric population to evaluate potential benefit in that patient population. However, patients with age less than 18 will be considered on a case-by-case basis, and a specific pediatric consent will be developed if necessary.
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13.6 informed consent procedure

13.6.1 Informed Consent Form (ICF). This study must be conducted in compliance with UCLA IRB,
IBC, ISPRC, the RAC and the FDA, and relevant informed consent regulations. The ICF must contain a language readily understood by the potential study subjects. The original consent form, signed and dated by the patient and by the person who conducted the informed consent discussion, will be included in the patient's UCLA medical chart. A copy of the signed ICF will be provided to the patient. There will be one consent form describing all of the study procedures, including the administration of genetically modified cells and the procedures for gene transfer. The rationale for including gene transfer and non-gene transfer procedures in the same consent form is that their separation would result in unnecessary duplication of text and non-linear explanation of the study procedures.  
13.6.2 Procedure of Consent. Subjects will be seen by one of the study investigators who will explain the study in detail including procedures, potential risks and side effects. Subjects will be encouraged to take additional time to consider the study and will be given the opportunity to take home the ICF for review and consideration. They will then be invited to meet with one of the investigators at another time. Subjects will be encouraged to have significant others or relatives with them at the time of the interviews. Questions from subjects, significant others and relatives will be encouraged. If the subject is non-English speaking, the study investigators will have the informed consent translated in the subject's native language. Ample time will be allowed for discussions and clarification of the goals of the research project with the trial investigators. Potential subjects will be asked if they wish to participate and told that it is voluntary and refusal will not affect their relationship with their physician or the care given to them at UCLA. No coercion will be used.
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13.7 subject enrollment

Subjects will be enrolled at the time the ICF is signed. Fulfillment of the eligibility criteria for each new subject enrolled will be prospectively and independently monitored by the UCLA Human Gene Medicine Program (HGMP) Compliance Officer.
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14.0 qualifications of personnel and

The UCLA Human Gene Medicine and Melanoma Programs: Antoni Ribas, James S.  
Economou, John A. Glaspy, William H. McBride, Bartosz Chmielowski, Arun Singh, Deborah Wong, Begonya Comin-Anduix, Richard C. Koya, Thinle Chodon, Donald Kohn. The first human gene transfer clinical trial conducted by Drs. Economou, Glaspy and McBride was initiated in 1992.  
This was a double retroviral vector gene marking of tumor infiltrating lymphocytes (TIL)76. Two adenovirus vector gene transfer-based clinical trials were conducted by Drs. Economou, Glaspy, 60 McBride and Ribas at UCLA. These clinical trials involved the generation of genetically manipulated DC-based cellular vaccines using adenoviral vectors for melanoma and hepatocellular carcinoma
(INDs 9908 and 11299).  
- **Antoni Ribas, M.D.**, Professor of Medicine and Surgery, Director of the Tumor Immunology Program Area at the JCCC,. Dr. Ribas is a translational medical oncologist and tumor immunologist, conducting experimental clinical trials in patients with melanoma.  
- **James S. Economou, M.D. Ph.D.**, Professor of Surgery and Microbiology, Immunology, and Molecular Genetics, Chief of the Division of Surgical Oncology, Deputy Director of the JCCC and the Director of the Human Gene Medicine Program (HGMP), and Vice Chancellor for Research at UCLA. Dr. Economou is an accomplished translational tumor immunologist and pioneer in clinical trials with retrovirus-modified tumor infiltrating lymphocytes (TIL).  
- **John A. Glaspy, M.D., M.P.H.**, Professor of Medicine, Vice-Chair of the Division of HematologyOncology, Director of the JCCC Clinical Research Unit Shared Resource, and the Associate Chief, UCLA 100 Medical Plaza Community Oncology Practice. Dr. Glaspy is a leading expert in clinical research, with a focus on melanoma and the development of cytokines and growth factors for human use.  
- **William H. McBride, D.Sc**. Professor and Vice Chair for Research, Director, Experimental Radiation Oncology. Dr. McBride is a recognized expert in animal models of tumor immunotherapy and radiation therapy.  
- **Bartosz Chmielowski, M.D., Ph.D.** Assistant Professor of Medicine in the Division of Hematology-Oncology. An expert in thymic development 77-79 and in the care of patients with malignant melanoma.  
- **Arun Singh, M.D**., Clinical Instructor in Medicine at the Division of Hematology-Oncology. Dr.  
Singh is a trained hematology-oncologist with expertise in high dose chemotherapy and IL-2 administration, and an expert in the use of molecular imaging to study immune responses to cancer.  
- **Deborah Wong, M.D., Ph.D.** Clinical Instructor, Division of Hematology and Oncology. Dr. Wong is a translational medical oncologist whose research and clinical interest is in improving the understanding and the treatment of patients with melanoma.  
- **Siwen Hu-Lieskovan, M.D, Ph.D.** Clinical Instructor, Division of Hematology and Oncology at UCLA. Dr. Hu-Lieskovan is a translational medical oncologist whose research and clinical expertise is in development of immune and targeted therapy for melanoma, lung cancer and other solid malignancies.  
- **Daniel Shin, M.D.** Fellow, Division of Hematology and Oncology at UCLA. Dr. Shin is a translational medical oncologist.  
- **Begoña Comin-Anduix, Ph.D.** Adjunct Associate Professor, Division of Surgical Oncology. An expert in immune monitoring and culture of human lymphocytes 70. Will lead the laboratory analysis of patient-derived samples.  
- **Beata Berent-Maoz, Ph.D.** Assistant Project Scientist, Division of Hematology and Oncology at UCLA. An expert in retroviral and lentiviral vector manufacture and transduction of target cells. Will be responsible for the GMP manufacture of TCR genetically modified cells and DC.  
- **Paula Kaplan-Lefko, Ph.D.** Project Scientist, Division of Hematology-oncology at UCLA.  
Experienced in writing industry-sponsored and investigator-initiated regulatory documents. Will coordinate the regulatory activities during the conduct of this clinical trial.  
- **Richard C. Koya, M.D., Ph.D**. Associate Professor of Oncology, Vector Development and Production Facility and Associate Director, Center for Immunotherapy at the Roswell Park Cancer Institute.,. An experienced molecular biologist and expert in gene therapy vectors, including retrovirus and lentiviral vectors.  
- **Thinle Chodon, M.D., Ph.D.** Assistant Professor of Oncology, Center for Immunotherapy and Facility Director of CFI Translational Research Operations at the Roswell Park Cancer Institute, an expert in retroviral and lentiviral vector manufacture and transduction of target cells. Will be responsible for the GMP manufacture of TCR genetically modified cells and peptide pulsed DC.  
- **Donald Kohn, M.D.**, Professor of Pediatrics and Molecular Microbiology and Immunology at UCLA. Dr. Kohn is an international leader in clinical trials of HSC genetically modified using retroviral vectors.  
The UCLA AIDS Institute Gene Therapy Program: Jerome A. Zack and Zoran Galic. Two clinical trials of HSC gene therapy in patients with HIV infection and AIDS, have been led by Dr. Zack (INDs 7328 and 10183). These trials involve introduction of an anti-HIV ribozyme into autologous CD34+
hematopoietic progenitor cells from HIV+ adults.  
- **Jerome A. Zack, Ph.D.** Professor of Medicine and Vice Chair of Microbiology, Immunology, and Molecular Genetics. Dr. Zack is a thymocyte development, embryonic stem cells and HIV
immunity expert.  
- **Zoran Galic, Ph.D.** Assistant Professor of Medicine. Dr. Galic is an embryonic stem cell and thymocyte development expert.
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the caltech/usc engineering immunology program

- **David Baltimore Ph.D.**, President of Caltech and Nobel Prize Laureate for the description of reverse transcriptase. Dr. Baltimore has a longstanding interest in the development of immunotherapy for cancer and infectious diseases, and has unique expertise in retroviral vectors for gene transfer.  
- **Pin Wang, Ph.D.** Associate Professor in Chemical Engineering and Material Sciences at USC. Dr.  
Wang is an expert in the genetic engineering of retroviral and lentiviral vectors.  
- **Lili Yang, Ph.D.**, Project Leader at the Engineering Immunology Program at Caltech. Dr. Yang is an expert in TCR genetic cloning and the genetic engineering of HSC.
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the university of connecticut immunotherapy

- **Bijay Mukherji, M.D.** Professor of Medicine at the University of Connecticut. Dr. Mukherji is a pioneer in cloning human T cells specific for tumor antigens and in the study of their immunobiology. Dr. Mukherji conducted the first dendritic cell-based clinical trial in human subjects.  
The UCLA *In Vivo* Cellular and Molecular Imaging Center: Caius Radu, Johannes Czernin,
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michael phelps.

- **Caius G. Radu, M.D.**, Associate Professor of Molecular and Medical Pharmacology. Dr. Radu is an expert in molecular immunology and *in vivo* imaging of immune responses in murine models.  
- **Owen N. Witte, M.D.** Howard Hughes Medical Institute Investigator at UCLA, Professor of Microbiology, Immunology, and Molecular Genetics, President's Chair in Developmental Immunology, Professor of Molecular and Medical Pharmacology, and Director of the Institute of Stem Cell Biology and Medicine at UCLA. Dr. Witte is a recognized molecular biologist who has pioneered an approach to non-invasively image antitumor T cell responses in alive animals.  
- **Johannes Czernin, M.D.**, Professor of Molecular and Medical Pharmacology. Dr. Czernin is a leading expert in the use of metabolic imaging for assessment of tumor response in patients.  
- **Michael E. Phelps, Ph.D.**, Norton Simon Professor and Chairman of Molecular and Medical Pharmacology, Chief, Division of Nuclear Medicine. Dr. Phelps pioneered PET imaging to examine molecular and cellular functions in humans.
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nanosystems biology cancer center (nsbcc)

- **James R. Heath, Ph.D.**, Elizabeth W. Gilloon Professor of Chemistry at Caltech and Director of the Caltech-UCLA-Institute for Systems Biology (ISB) NSB Cancer Center. Dr. Heath is a nanotechnology and microfluidics expert who has developed novel and highly multiplexed immune monitoring assays able to analyze limiting samples from tumor biopsies.
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biostatistics analytical support & evaluation

- **David Gjertson, Ph.D.** Professor of Biomathematics at UCLA. Dr. Gjertson is an experienced biostatistician who will provide overall statistical oversight.  
- **Xiaoyan Wang, Ph.D.** Assistant Professor of Medicine at UCLA. Dr. Wang is an experienced biostatistician who will work with Dr. Gjertson.  
The Jules Stein Eye Institute Ophthalmology Program: Bradley Straatsma, Tara McCannel. - **Tara McCannel, M.D., Ph.D.**, Assistant Professor of Ophthalmology. Dr. McCannel is an expert in ocular melanoma who has been involved in the monitoring of potential ocular toxicities of experimental immunotherapies tested in clinical trials at UCLA.
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otological exams akira ishiyama.

- **Akira Ishiyama, M.D.** Professor of Head and Neck Surgery at UCLA. He is an expert in otological analysis, in particular in the study of autoimmune phenomena in the middle and inner ear.  
In conclusion, the study investigators are very familiar with the use of retroviral vectors to genetically modify primary lymphocytes, the requirements of GMP standards for *ex vivo* cell culture and genetic manipulation, the requirements for GCP standards for conducting human experimental clinical trials, and have fully equipped clinical facilities that have been successfully reviewed by regulatory agencies for the conduct of cellular therapy and gene transfer studies.
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14.2 personnel training

a. **Clinical Personnel**: The following will be required courses, training sessions or certificates required for clinical study personnel:
- UCLA IRB Human subjects certificate. - UCLA IRB HIPPA certificate. - Certificate in Good Clinical Practice. - UCLA Human Gene Medicine Program Educational Course.  
b. **Laboratory Personnel**: The following courses, training sessions or certificates will be required for study laboratory personnel involved in TCR transgenic cell manufacturing:
- UCLA IRB Human subjects certificate. - UCLA IRB HIPPA certificate. - UCLA JCCC current Good Manufacturing Practice training.  
- UCLA Human Gene Medicine Program Educational Course.  
- UCLA IBC training on Biosafety Level 2. - UCLA IBC certificate in the use of biosafety cabinets.  
- UCLA IBC shipping of Biological Material - UCLA IBC training in biological waste management. - UCLA IBC training on Bloodborne Pathogens.
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14.3 hgmp/jccc cell and gene

UCLA has a dedicated, fully equipped and functional GMP suite specifically designed for cellular and gene transfer clinical trials. This facility is located on the 14th floor of the JCCC Factor Building and is jointly managed by the JCCC and the HGMP. It consists of a 2395 square feet BioSafety level 2 (BSL2) suite with 7 HEPA-filtered rooms. This secured area has centralized monitoring of air and equipment linked to the study investigators' pagers. Access is restricted to investigators with certified training and is supervised by the JCCC Regulatory Affairs Office. The 4 positive pressure laboratories are designed for *ex vivo* cell manipulation of cells, since it minimizes the possibility of outside pathogen contamination. The 3 negative pressure laboratories are designed for gene transfer procedures, since it ensures that the recombinant vectors will not contaminate the surrounding environment. There have been 18 investigator-initiated clinical trials in which the cell or gene therapy product has been manufactured by the investigator team in the JCCC GMP suite within RAC and FDA compliance.
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14.4. gene therapy viral bank

The clinical grade MSGV1-F5AfT2AB will be stored in a dedicated –80oC freezer with central, computerized monitoring system, with alarm and recording of all GMP storage systems (refrigerators, freezers and incubators). This freezer is located at the JCCC GMP Suite. This freezer is designed for exclusive use as a viral bank for ongoing gene therapy trials at UCLA, and is located in a secure area for containment to ensure proper storage. Access to the facility is guarded by two security doors with restricted access only to adequately trained and authorized individuals. Completion of a five-course training program is required to gain access to this GMP suite. This training program is provided by the JCCC Regulatory Affairs Office and the UCLA IBC.  
Under these conditions, retroviral vectors retain activity for a period exceeding 5 years if not subjected to freeze-thawing. Each aliquot of the final retroviral vector is calculated to be for a single use, therefore avoiding freeze-thawing cycles. All activities related to the GMP retroviral vector stored in the viral bank will be recorded in appropriate SOPs. This includes initial lot shipment, viral vector storage, viral vector dispensing, remaining samples after use to generate retrovirus gene-modified cell therapies, and yearly recertification.
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14.5. clinical facilities

Chemotherapy conditioning and MART-1 F5 TCR cell adoptive transfer will be delivered in an isolation hospital bed in the Hematology/Oncology Hematopoietic Stem Cell (HSC) transplantation ward. This is a fully staffed inpatient ward with experienced nurses, house officers, clinical consultants and attending physicians. This ensures that, in the event of an adverse reaction or any other adverse event, treatment will be promptly available. Patients will be placed in individual use rooms equipped with a high-efficiency particulate air (HEPA) filters that meet the precautions normally used for patients undergoing hematopoietic stem cell transplantation. While receiving the TCR transgenic PBMC adoptive transfer, patients will be continuously monitored. This inpatient stay will be under the administrative responsibility of the UCLA General Clinical Research Center (Isidro B. Salusky, M.D., G-CRC Director, telephone: 310-206-6987). This will provide an additional level of administrative and scientific oversight. The G-CRC staff provides expert care for research subjects and collaborates with investigators for the coordination and performance of clinical research. The G-CRC includes a Computer Center under the supervision of the G-CRC
Biostatistician Dr. Robert M. Elashoff, Ph.D. (telephone: 310-825-9421) to assist in data analysis.  
The UCLA Melanoma Program medical oncology outpatient clinic is located at the UCLA 100 Medical Plaza, Suite 550. This facility is staffed with an office manager, 3 front desk assistants, 6 nurses, 2 phlebotomists, 2 insurance specialists and one nurse practitioner.  
The UCLA Hemapheresis Unit is located at the 6th floor of the 200 Medical Plaza building, Suite 660. This unit has 6 staff nurses on a one-to-one basis, a supervisor, a coordinator, and a Hematology/Oncology fellow and attending doctors.
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14.6 compliance with protocol procedures

Human Gene Medicine Program (HGMP) Compliance Officer. Kit Shaw (contact phone: 310-2670584), Ph.D. Dr. Shaw worked on engineering and testing retroviral gene-transfer vectors in primary cells and small animal models. She has been the study director for IND study of gene transfer to hematopoietic stem cells (HSCs) for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID), where she had to ensure trial compliance with the regulatory bodies (FDA,
DSMB, RAC, IRB) overseeing the trial. She also has experience with IND submissions and cGMP for cell-based products. She is familiar with clinical laboratory operations in addition to research laboratory operations. Dr. Shaw will oversee activities involving gene transfer in the UCLA GMP suite and will prospectively review adherence to the protocol, RAC and FDA procedures. Dr. Shaw also serves on the Jonsson Comprehensive Cancer Center (JCCC) DSMB. For our investigator-initiated gene therapy trial, Dr. Shaw will perform real time monitoring and auditing of the study.  
JCCC Office of Regulatory Compliance. The manager of this office is Terra Hughes. Ms. Terra Hughes recently joined the JCCC from the UCLA IRB where she managed the Medical IRB committee 2. She has a bachelor's degree from the University of Southern California (USC) in Biology and a Masters degree from CSULA in Criminalistics. Ms. Hughes will oversee the adequate conduct of this clinical trial.  
Clinical Research Compliance Officer. Sujna Raval-Fernandes, Ph.D. (contact phone 310-7948693). Dr. Raval-Fernandes has a strong background in cancer research with more than 10 years of basic research experience. She has been in the Jonsson Comprehensive Cancer Center (JCCC)'s Office of regulatory Compliance for the past four and half years serving as the compliance officer and as a resource to investigators and clinical study staff in resolving quality assurance issues related to research procedures and documentation. She is responsible for monitoring and auditing of studies to ensure regulatory compliance. She also serves on the JCCC Data and Safety Monitoring Board
(DSMB). Her responsibilities also include timely review of all serious adverse events submitted to the DSMB. She ensures that the investigators provide the JCCC DSMB with periodic summary reports that include accrual, safety and efficacy data. For our investigator-initiated gene medicine study, she will serve to ensure compliance to the JCCC DSMB.  
Office of Research Participant Advocacy (ORPA). The ORPA was established to assure the safety and well-being of all participants in clinical research studies conducted at the UCLA G- CRC while maximally facilitating the research process. Stanley Korenman, M.D. (telephone: 310-794-1818), Associate Dean of Ethics and Professor of Endocrinology, serves as the Clinical Director of the office. Laurie Shaker-Irwin, Ph.D. (telephone: 310-794-7504) serves as the Research Subject Advocate and manager of the office. ORPA fosters policies for the ethical conduct of clinical research, assuring autonomy and respect in the informed consent process, justice in the selection of participants, and beneficence in the initial and ongoing review and evaluation of risks and benefits. The office enhances patient safety by providing guidance and education to investigators and research personnel in the areas of ethics, research integrity, data and safety monitoring, adverse event reporting, and Good Clinical Practices (GCP).
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15.0 data safety monitoring board

The Jonsson Comprehensive Cancer Center (JCCC) Data and Safety Monitoring Board (DSMB) was constituted in January 2001. The DSMB membership consists of representatives of the Cancer Center clinical research community. The Director of the Cancer Center appoints members to a term of 2 years and each member is selected for their professional expertise in oncology practice and research. The JCCC DSMB meets monthly to review all serious adverse event reports for JCCC institutional clinical trials at UCLA and those encountered in the UCLA TORI network where the JCCC DSMB has oversight. All serious adverse event reports, which have been filed since the previous meeting, are presented to the committee for review. For all JCCC and TORI studies, where the JCCC DSMB has primarily oversight for AE review, all AEs occurring within these studies shall be reported to the JCCC DSMB in a timely manner consistent with the UCLA IRB time requirements [ten days, two days for a death] regardless of relationship or seriousness. The JCCC Office of Regulatory Compliance (ORC) will review all submissions and staff will enter the information into the JCCC Clinical Trials database. Reports are generated for full JCCC DSMB review of those SAEs that have some component of relatedness to the study drug and may, at the discretion of the JCCC compliance officers, include SAE reports that may be incorrectly assessed by the PI. For institutional trials, where the JCCC DSMB has primary DSMB review responsibility, the office will request that the PI generate cumulative adverse event reports for biannual or annual review.  
The DSMB reviews each Serious Adverse Event reported and determines whether or not protocol modifications are warranted to ensure patient safety. In this review, prior occurrences of similar toxicity with the therapy under study are taken into consideration, as well as the seriousness of the event and the likelihood that it was related to a study drug. The DSMB may recommend no changes to the study if the event is expected or related to other causes such as the patient's underlying condition. The DSMB may request an expert's advice of other non-UCLA Principal Investigator with national experience to support their deliberations and decisions. The JCCC DSMB has the authority to recommend to the UCLA IRB the immediate halt to the study (i.e., discontinuation of any further treatment of enrolled patients and discontinuation of enrollment of new patients) should there be any serious unexpected toxicity that warrants further investigation. JCCC DSMB correspondences are 66 addressed to the Principal Investigator and copied to the UCLA IRB. Minutes of the DSMB meetings are recorded and processed into the computer file.  
15.2 JCCC DSMB Internal Monitoring Plan. The NIH and NCI policy statements allow for variable monitoring and reporting plans, commensurate with the potential risks and with the size and complexity of the trial. The monitoring plan must be sufficiently rigorous and effective to ensure subject safety and to ensure protocol compliance and data validity and integrity.  
15.2.1 Level of Risk of a Study. For trials overseen by the JCCC DSMB, the JCCC DSMB will determine the degree of risk of the study and will ensure that there are procedures in place to ensure the safety of the subjects that are enrolled in the trial. The intensity level of the monitoring is determined by the risk category. Some of the factors that must be considered when assigning the Level of Risk category include: 1. A biostatistical design and appropriate procedures for proper data management so that the information collected can be properly validated.  
2. Appropriate Serious Adverse Event reporting procedures must be in place.  
3. The study duration must be appropriate and must be based on a realistic rate of enrollment.  
4. Data collection and data management must be adequate to verify and ensure subject safety.  
15.2.2 Assignment of risk. Assigning risk ensures that the data and safety monitoring is based on the level of risk (low, medium, or high) to ensure that the data and safety monitoring activities are appropriate. Below are some of the criteria used to make a decision regarding the assignment of risk:  
- Expected duration of the study based upon the estimated rate of one. - Type of study population (e.g., children, geriatric)
- The procedures used in the trial are commensurate with the degree of risk.  
- Adequate data management systems in place and appropriate case report forms - Proper serious adverse event reporting procedures in place - Proper biostatistical design and data analysis procedures in place.
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15.3 monitoring/auditing activities.

The compliance officer of the JCCC Office of Regulatory Compliance [ORC] will monitor the clinical records for all human subjects enrolled onto JCCC institutional trials overseen by the JCCC DSMB. The JCCC compliance officer will perform real time review of informed consent form processes and the meeting of all inclusion and exclusion criteria at study entry. Active monitoring will offer the JCCC study teams prospective information that can be used to enhance the quality of research being performed contemporaneously. Auditing is a review of historic performance of the research effort and is performed on case report forms, regulatory files and source documents to measure the quality of the research effort in a retrospective manner.
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15.4 detailed reporting mechanism for

Each protocol is required to have a detailed description of the Adverse Event Reporting method. The DSMB expects each protocol to abide by the reporting time line and definitions consistent with the NCI and FDA guidelines. An adverse event is any undesirable experience associated with the use of a medical product in a patient (any unfavorable and unintended sign, symptom or disease temporally associated with the use of a medical treatment or procedure regardless of whether it is considered related to the medical treatment or procedure). Any clinical adverse event must be recorded on the case report form during the course of the study. The investigator must evaluate and document the adverse event for severity, grade it according to the NCI Common Toxicity Guideline, causal relationship to the study drug under study, take appropriate action to care for the patient and to document the outcome. All information recorded in the case report form must be verifiably documented in the source (i.e., medical record or physician office chart).  
When an adverse event occurs, it is the responsibility of the investigator to evaluate and record into the source documents, the nature of the symptom, prescribe the appropriate remedy and to report the event. The adverse event must be reported to the sponsor, the IRB, FDA and any other appropriate agency (e.g., IBC and NIH for gene medicine trials.) The assessment and reporting must occur in writing within 10 days, 48 hours for death.  
If the event is **Serious and Unexpected**, the event should be reported immediately (in writing within 10 days, 48 hours for death) by the Principal Investigator to the IND sponsor, UCLA IRB, JCCC DSMB and to any appropriate agency; the NIH and the UCLA IBC for gene medicine trial; directly to the FDA if it is an institutional IND trial; and to the NCI if it is an NCI sponsored trial. For institutional trials and for gene medicine trials, the JCCC ORC and Gene Medicine Compliance Officers will assist the investigator to ensure that all serious adverse events are properly documented and reported in accordance to federal and institutional requirements.  
All adverse events that occur in a research study overseen by the JCCC DSMB must be submitted, regardless of relationship, expectedness, or seriousness.
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15.5 data recording and retention

Data recording will be done in compliance with Good Clinical Practice. Medical records and notes should be clearly marked and permit easy identification of participation by an individual in the specified clinical trial. Medical records and source data should be completed within a reasonable amount of time for purposes of timely data collection and entry. The investigators are to record all data with respect to protocol procedures, drug administration, laboratory data, safety data and efficacy ratings on the case report forms (CRF). The CRF may be a printed, optical or electronic document. All corrections on a CRF and on source documents must be made in a way that does not obscure the original entry. The correct data must be inserted, dated and initialed/authorized by study site personnel. If the change is not obvious provide a reason.
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16.0 statistical methods

This is a two-stage phase II clinical trial aimed at determining the safety, feasibility and antitumor activity of gene transfer using retrovirus MSGV1-F5AfT2AB transduced PBMC. UCLA will be responsible for registering patients and for maintaining a complete database of the study information excluding laboratory assay data. The clinical laboratory assay data will be obtained from source documents from the UCLA medical records (including both paper and electronic files and laboratory results) and transcribed into CRFs. A shadow chart may be kept with copies of the source documents in a locked room at the UCLA Clinical Research Unit or under direct control of the study P.I. Failure to complete the study due to the stopping rules being invoked will be the main basis for determining safety and feasibility of this study. This study is intended to provide data that might allow the investigators to conduct a preliminary assessment of safety and feasibility.
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16.1 sample size determination

The Simon optimal two-stage design1 is used to determine the sample size, using the co-primary endpoint of response rate as the criterion for the clinical trial statistical design.
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mart-1 f5 tcr protocol 01/10/2018

This clinical trial is set up to rule out the null hypothesis that po ≤ 0.10 (i.e. to rule out that this combined therapy has a response beyond 10%, since several current treatment approaches achieve response rates of 10% in patients with advanced melanoma) versus the alternative that the effect-size = p t − p o > 0.25 has an expected sample size ( Ave n ) of 10.6 and a probability of early termination (PET) of 0.81. The alternative hypothesis of a response rate of 35% (and thus a difference between the null and alternative hypothesis of 25% or effect-size = ρ 1 − ρ 0 > 0.25) is chosen since it is felt to represent a clinically-meaningful difference and results in a study sample size that is feasible to be conducted within a pilot single-institution study. If the drug is actually not effective, there is α ≤ 0.05 probability of concluding that it is. If the drug is actually effective, there is a β ≤ 0.20 probability of concluding that it is not (Table 13). After testing the drug on 8 patients in the first stage, the trial terminates if only 1 patient responds. If the trial goes on to the second stage, a total of 22 patients will be studied. If the total number responding is less than or equal to 4, the combined immunotherapy approach of lymphodepletion plus MART-1 F5 TCR transgenic cell adoptive transfer, together with high dose IL-2 (up to Amendment 10)
or low dose IL-2 (starting in Amendment 11) and MART-1/DC vaccines, is rejected.  
Table 13. Calculations of Simon's Optimal Two-Stage Designs for Phase II Clinical Trials Clinical trials with p 1 - p 0 varying from 0.15 to 0.50 (α=0.05, β=0.20), calculated using the PASS 2005 software (Kaysville, Utah).  
| Effect size p 1 - p o                                                                                           |        |      |      |      |      |      |    |
|-----------------------------------------------------------------------------------------------------------------|--------|------|------|------|------|------|----|
| Po                                                                                                              | Key    | 0.20 | 0.25 | 0.50 |      |      |    |
| 0.15                                                                                                            | 0.30   | 0.40 |      |      |      |      |    |
| n =                                                                                                             | 10     | 9    | 5    | 4    | 3    | 3    |    |
| 0                                                                                                               | 0      | 0    | 0    | 0    |      |      |    |
| r =                                                                                                             | 0      |      |      |      |      |      |    |
| n =                                                                                                             | 29     | 17   | 18   | 16   | 8    | 7    |    |
| 0.05                                                                                                            | r =    | 3    | 2    | 2    | 2    | 1    | 1  |
| PET                                                                                                             | 0.60   | 0.63 | 0.77 | 0.82 | 0.86 | 0.86 |    |
| Ave n                                                                                                           | 17.6 3 | 12.0 | 7.9  | 6.2  | 3.7  | 3.6  |    |
| 18                                                                                                              | 10     |      |      |      |      |      |    |
| n =                                                                                                             | 8      | 4    | 3    | 2    |      |      |    |
| r =                                                                                                             | 2      | 1    | 1    | 0    | 0    | 0    |    |
| n =                                                                                                             | 43     | 29   | 22   | 15   | 9    | 8    |    |
| 0.10                                                                                                            | r =    | 5    | 2    | 2    |      |      |    |
| 7                                                                                                               | 4      | 3    |      |      |      |      |    |
| PET                                                                                                             | 0.73   | 0.74 | 0.81 | 0.66 | 0.73 | 0.81 |    |
| Ave n                                                                                                           | 24.7   | 15.0 | 10.6 | 7.8  | 4.6  | 3.1  |    |
| n =                                                                                                             | 19     | 9    | 7    | 6    | 3    | 2    |    |
| r, =                                                                                                            | 3      | 1    | 1    | 1    | 0    | 0    |    |
| n =                                                                                                             | 55     | 34   | 25   | 19   | 10   | 6    |    |
| 0.15                                                                                                            | r =    | 12   | 8    | 6    | 5    | 3    | 2  |
| PET                                                                                                             | 0.68   | 0.60 | 0.72 | 0.78 | 0.61 | 0.72 |    |
| Ave n                                                                                                           | 30.4   | 19.0 | 12.1 | 8.9  | 5.7  | 3.1  |    |
| Legend: n1: required number of patients in stage 1; r1: number of patients with an objective response that need |        |      |      |      |      |      |    |
| to be exceeded to proceed with the study; n: total number of required patients combining stage 1 and 2; r:      |        |      |      |      |      |      |    |  
number of patients with an objective response that are needed to be exceeded to conclude that the clinical trial had successfully ruled out the null hypothesis; Ave n: expected sample size; PET: probability of early termination.
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16.2 definition of evaluable patients

Patients evaluable for the primary analysis are those fully enrolled in the study after meeting the inclusion/exclusion criteria. Other patients will be considered un-evaluable and excluded from the primary analysis.
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16.3 efficacy analysis

The toxicities observed after each TCR transgenic cell infusion will be summarized in terms of type
(organ affected or laboratory determination such as absolute neutrophil count), severity (by Toxicity Table) and nadir or maximum values for the laboratory measures, time of onset (i.e. course number), duration, and reversibility or outcome. Tables will be created to summarize these toxicities and side effects by dose and by course. Baseline information (e.g. the extent of prior therapy) and demographic information will be presented, as well, to describe the patients treated in this pilot study. All responses will be reported.
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16.4 study stopping and non-stopping

It is recognized that AEs can occur frequently in this population based on the underlying metastatic melanoma and these can be SAEs. The review of SAEs will form the basis for potential early stopping of the study. Only unexpected SAEs that are related to the transduction process/research reagent would define a stopping rule. The review of these adverse events, and any decision to prematurely stop subject enrollment, will be determined by the UCLA DSMB and reviewed by the IRB. Absolute stopping rules for this clinical trial will be:  
1. Any death that is possibly related to the investigational agents namely MART-1 F5 TCR PBMC or MART-126-35 peptide pulsed DC.  
2. Two or more grade 4 events that are possibly related to MART-1 F5 TCR PBMC or MART-126-35 peptide pulsed DC.  
3. Any event of hematological malignancy must be considered study stopping criteria until oncogenesis related to retroviral mutagenesis, EBV lymphoma and Post Transplant Lymphoproliferative Disorder (PTLD) can be excluded.
Premature termination of the clinical trial may occur because of a regulatory authority decision, change in opinion of the FDA, RAC, IRB, the DSMB, or determination that there are problems in the cell product generation or the safety of their administration as descried in the assessment of primary study endpoints. Additionally, recruitment may be stopped for reasons of particularly low recruitment, protocol violations, or inadequate data recording.
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17.0 references

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Fleischhauer K, Tanzarella S, Wallny HJ, et al: Multiple HLA-A alleles can present an immunodominant peptide of the human melanoma antigen Melan-A/MART-1 to a peptide-specific HLA-A*0201+ cytotoxic T cell line. J Immunol 157:787-97, 1996 53. Yee C, Thompson JA, Byrd D, et al: Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci U S A 99:16168-73, 2002 54. Mackensen A, Meidenbauer N, Vogl S, et al: Phase I study of adoptive T-cell therapy using antigen-specific CD8+ T cells for the treatment of patients with metastatic melanoma. J Clin Oncol 24:5060-9, 2006 55. Wu X, Li Y, Crise B, et al: Transcription start regions in the human genome are favored targets for MLV integration. Science 300:1749-51, 2003 56. Gordon LK, Ribas A, Nusinowitz S, et al: Surveillance of the eye and vision in a clinical trial of MART1-transformed dendritic cells for metastatic melanoma. Control Clin Trials 25:400-7, 2004 57. Straatsma BR, Nusinowitz S, Young TA, et al: Surveillance of the eye and vision in clinical trials of CP-675,206 for metastatic melanoma. Am J Ophthalmol 143:958-969, 2007 58. Jaeger E, Bernhard H, Romero P, et al: Generation of cytotoxic T-cell responses with synthetic melanoma-associated peptides in vivo: implications for tumor vaccines with melanomaassociated antigens. Int J Cancer 66:162-9, 1996 59. Men Y, Miconnet I, Valmori D, et al: Assessment of immunogenicity of human MelanA peptide analogues in HLA-A*0201/Kb transgenic mice. Journal of Immunology 162:3566-73, 1999 60. Valmori D, Dutoit V, Schnuriger V, et al: Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity. J Immunol 168:4231-40., 2002 61. Schuler-Thurner B, Schultz ES, Berger TG, et al: Rapid Induction of Tumor-specific Type 1 T Helper Cells in Metastatic Melanoma Patients by Vaccination with Mature, Cryopreserved, Peptide-loaded Monocyte-derived Dendritic Cells. J Exp Med 195:1279-88., 2002 62. Timmerman JM, Levy R: Dendritic cell vaccines for cancer immunotherapy. Annu Rev Med 50:507-29, 1999 63. Morse MA, Coleman RE, Akabani G, et al: Migration of human dendritic cells after injection in patients with metastatic malignancies. Cancer Res 59:56-8, 1999 64. Fong L, Brockstedt D, Benike C, et al: Dendritic cells injected via different routes induce immunity in cancer patients. J Immunol 166:4254-9, 2001 65. Rosenberg SA: Progress in human tumour immunology and immunotherapy. Nature 411:380-4, 2001 66. Roth MD, Gitlitz BJ, Kiertscher SM, et al: Granulocyte macrophage colony-stimulating factor and interleukin 4 enhance the number and antigen-presenting activity of circulating CD14+ and CD83+ cells in cancer patients. Cancer Research 60:1934-41, 2000 67. Bertinetti C, Zirlik K, Heining-Mikesch K, et al: Phase I trial of a novel intradermal idiotype vaccine in patients with advanced B-cell lymphoma: specific immune responses despite profound immunosuppression. Cancer Res 66:4496-502, 2006 68. Therasse P, Arbuck SG, Eisenhauer EA, et al: New guidelines to evaluate the response to treatment in solid tumors [see comments]. J Natl Cancer Inst 92:205-16, 2000 69. Patterson AP: Minutes of the Recombinant DNA Advisory Committee - 9/21/05. http://www4.od.nih.gov/oba/rac/minutes/RAC_minutes_09-05.pdf, 2005 70. Comin-Anduix B, Gualberto A, Glaspy JA, et al: Definition of an immunologic response using the major histocompatibility complex tetramer and enzyme-linked immunospot assays. Clin Cancer Res 12:107-16, 2006 71. Kwong GA, Radu CG, Hwang K, et al: Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells. J Am Chem Soc 131:9695-703, 2009 72. Ma C, Fan R, Ahmad H, et al: A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells. Nature medicine 17:73843, 2011 73. Yaghoubi S, Barrio JR, Dahlbom M, et al: Human pharmacokinetic and dosimetry studies of [(18)F]FHBG: a reporter probe for imaging herpes simplex virus type-1 thymidine kinase reporter gene expression. J Nucl Med 42:1225-34, 2001 74. Weber WA: Use of PET for monitoring cancer therapy and for predicting outcome. J Nucl Med 46:983-95, 2005 75. Cress RD, Holly EA: Incidence of cutaneous melanoma among non-Hispanic whites, Hispanics, Asians, and blacks: an analysis of california cancer registry data, 1988-93. Cancer Causes Control 8:246-52., 1997 76. Economou JS, Belldegrun AS, Glaspy J, et al: In vivo trafficking of adoptively transferred interleukin-2 expanded tumor-infiltrating lymphocytes and peripheral blood lymphocytes. Results of a double gene marking trial. J Clin Invest 97:515-21, 1996 77. Chmielowski B, Muranski P, Ignatowicz L: In the normal repertoire of CD4+ T cells, a single class II MHC/peptide complex positively selects TCRs with various antigen specificities. J Immunol 162:95-105, 1999 78. Chmielowski B, Muranski P, Kisielow P, et al: On the role of high- and low-abundance class II MHC-peptide complexes in the thymic positive selection of CD4(+) T cells. Int Immunol 12:67-72, 2000 79. Chmielowski B, Pacholczyk R, Kraj P, et al: Presentation of antagonist peptides to naive CD4+ T cells abrogates spatial reorganization of class II MHC peptide complexes on the surface of dendritic cells. Proc Natl Acad Sci U S A 99:15012-7, 2002
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main protocol and consent modifications

While generating the forms to fill in the product lot release criteria we noted that the approved version of the study protocol did not specify which final product lot release tests should be received before administration of the cells to patients. The lot release criteria were detailed in Table 4 of the protocol, but both tests with immediate results and with delayed results were listed together. The key issues were the low likelihood of obtaining results of the copy number of viral insertions in the target cells, and the testing for replication competent retrovirus (RCR), which Dr. Reiser, the FDA product reviewer for this IND, had asked to be done at the Indiana University Viral Production Facility (IU VPF) under the direction of Dr. Ken Cornetta.  
Dr. Reiser at phone number 301-827-3420 who agreed that final results of the copy number and RCR were not required to be in hand before administering the TCR transgenic cells to patients.  
Dr. Reiser said that a plan should be in place in case the results were positive, including the contacting and treatment of study subjects and the reporting of the event to the FDA.  
With this clarification, we discussed that we would amend the protocol to clearly state which results would be in hand before TCR transgenic cell administration to patients, and which results would be available at a later time point. We also discussed that this protocol amendment would include a plan for patient and FDA notification. The lot release criteria have been broken down into two tables in the amended version of the protocol dated 03/16/09. Table 4a (page 37) details the results that need to be in hand before the release of the TCR transgenic cells for patient administration. Table 4b (page 37-38) details the results that may be available afterwards. After this table there is a specification of the plans to contact and treat the patients and how the local and federal regulatory agencies should be notified.  
b) Consent Form:
No changes.  
c) HS-1 Form:
No changes.  
Date: April 13, 2009 Main protocol and consent modifications; a) Protocol:  
- After discussion with the FDA reviewers in a series of teleconferences and email exchanges on April 7 and 8 (see attached minutes of these information exchanges), we deleted the requirement for quantitation of the number of copy inserts of retroviral vector in the final product. The FDA reviewers agreed with us that the quantitation of surface TCR expression in transduced cells was a good quantitative assessment of transduction efficiency. In fact, the previously IRB approved protocol had this test listed twice, as lot release criteria and as optional additional testing in the TCR transduced cells.  
- We clarified the intent of the schedule of events as the intent to perform the protocol procedures on those days. However, since this is a pilot study and needs to account for variation on patient outcome and scheduling issues, we clarify in this second amendment that the dates are approximate.  
- The use of antiementics and prophylactic antifungals was updated to match the standard practice in the stem cell transplantation ward at UCLA.  
- The administration of MESNA was corrected to continuous intravenous infusion for 24 hours.
b) Consent Form:
- We received the NIH Certificate of Confidentiality (CoC). Therefore, we added back the CoC
language to the consent form.  
- We corrected a typographical error in page 7.  
c) HS-1 Form:
No changes.  
Amendment 3 Date: May 26, 2009
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main protocol and consent modifications

a) Protocol:
- After new discussion with the FDA reviewers on April 27 (see attached minutes), we slightly modified our *ex vivo* culture plan after transduction to limit it to 4 days, which following the Guidance for Industry for Replication Competent Retrovirus (RCR) testing (November 2006) allows us to bank but not prospectively test for RCR in the final product. The FDA reviewers agreed with us that a reasonable plan would be to test samples from peripheral blood obtained at 3, 6 and 12 months after TCR transgenic adoptive transfer and only test the final product sample if one of the patient-derived samples is positive for RCR. The proposed S+/L- assay is a one-month assay with high costs that would not be able to provide results in time for patient safety. Therefore, we discussed with the FDA and went onto amend our protocol following the Guidance for Industry plan for studies where gene-modified cell therapies are cultured for less than 4 days. This was included in Table 4b and Sections 10.1.9 and 11.2.1.2.  
- The timing of sampling of blood of patients for RCR was modified to follow the Guidance for Industry document to samples at 3, 6 and 12 months, and stored samples annually thereafter (Section 11.2.1.2).  
- The reporting of the results of the mycoplasma PCR-based rapid test was changed, and we changed it in the protocol accordingly (Tables 4a and 10, and Section 10.2.10).  
- We deleted a 100 ml blood draw for immune monitoring analysis that was planned on day +1 since in the first two patients this blood draw provided very few lymphocytes since the blood draw happens at a time when the patient is pancytopenic. Analysis of these lymphocytes by immune monitoring assays did not provide much information.  
- We added a note in the schedule of events (Section 2) and in the protocol description in Section 8.6 text to note that 100 ml blood draws for immune monitoring analysis being planned for days 7 and 14 after TCR transgenic adoptive transfer can be done with a wider window since in our first 2 patients we have noted the recovering pancytopenia and hemodynamic state after high dose IL-2 may not make it clinically wise to obtain these blood draws on these particular days.  
- We added statements to the supportive care plan for patients to follow the clinical judgment and the standard practices in J-Medicine (Section 8.3).  
- Minor typographical errors were corrected throughout and marked in tracking mode in one of the protocol copies.
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b) informed consent form

- Upon review of the ICF we noted that there was no good explanation of the amount and timing of blood draws for immune monitoring analysis. Therefore, we added a paragraph in Section 4 of the Study Procedures to explain to patients that repeated blood draws are planned to obtain cells for immune monitoring analysis.  
- After a correspondence with the IRB, an additional change was made to the consent form to take out the explanation and checkbox for the request for autopsy.
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c) hs-1 form

- A new grant from our collaborator Dr. Bijay Mukherji from the University of Connecticut, with a UCLA subcontract, was added as funding source for this research project.  
- The timing of immune monitoring assays was added to the experimental procedures. - The tables of cell therapy lot release criteria were updated to match the tables included in the protocol after amendments 1, 2 and 3.  
Date: July 16, 2009
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main protocol and consent modifications

a) Protocol:
- A new section (8.12.) was added to allow the administration of booster MART-1 peptide pulsed DC vaccinations when the levels of MART-1-specific T cells would fall lower than 5% of total T
lymphocytes. The schedule of events was included in the new section 2.1.  
- The requirement for paired tumor biopsies as inclusion criteria was changed to performing biopsies when felt feasible by the study investigators.  
- An appendix at the end of the protocol detailing the sequential protocol amendments was added.  
- Minor typographical errors were corrected throughout and marked in tracking mode in one of the protocol copies.  
b) Informed Consent Form
- The ICF was updated to include the administration of 3 delayed DC booster vaccinations. - The details on how the study was funded were updated in the ICF. - The change in the requirement for tumor biopsies was added, where patients without easily accessible lesions for surgical biopsy would still be eligible for this protocol either with no biopsy, or with an image-guided biopsy.  
- The contact information for study investigators was updated.  
c) HS-1 Form:  
Date: October 13, 2009  
a) Protocol:
- Correction of the schedule of events to reflect the follow up of patients who are in response or without progression after 3 months.  
- A new grant from our collaborator Dr. David Baltimore from the California Institute of Technology, with a UCLA subcontract, was added as funding source for this research project.  
- The changes in the eligibility not strictly requiring paired tumor biopsies were included. - The addition of booster DC vaccinations was included.  
- Added subjects who are HLA-A*0205 (HLA-A2.5) positive as potential participants in this research, provided that we demonstrate that MART-1 F5 TCR transgenic cells produce >
30,000 pg/ml/million cells of IFN-γ production upon MART-126-35 peptide stimulation using MART-126-35 peptide-pulsed PBMC from the potential HLA-A*0205 (HLA-A2.5) subject candidate.  
- Added the Endosafe - MCS System as an alternative method for testing for endotoxin levels in TCR transgenic cells and dendritic cells.  
- Added to the pre-study procedures the placement of a double lumen central venous access catheter.  
- Added the possibility of using clinical grade human AB serum for the DC manufacture.
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b) informed consent form

- The dendritic cell vaccine booster part of the dendritic cell addendum consent was corrected to monthly vaccines.  
- The table at the end of the main consent form was updated to add the follow up visits, blood work and scans every 3 months for at least 2 years in patients who have no progression after the first 90 days of study participation.  
c) HS-1 Form:
- The changes in eligibility and procedures included in the protocol were made in the HS-1 form.  
Date: April 6, 2010
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main protocol and consent modifications

a) Protocol:
- The major modification in Amendment 6 was the increasing of the F5 TCR transgenic cell dose to 1010 and restarting the stage 1 of this protocol since with the prior version administering a cell dose up to 109 we did not meet the minimal hurdle of antitumor activity to proceed to stage 2 of this research after having entered the first 8 patients.  
- The description and references to the NCI Surgery Branch F5 TCR adoptive cell transfer protocol have been updated based on the published data included in the manuscript by Johnson *et al*. in Blood 2009.  
- C reactive protein (CRP) testing was added to the baseline exams since there is emerging evidence that it may provide information about the immune permissively of the tumor microenviroment.  
- The timing of procedures related to prior therapy was clarified.  
- The eligibility/exclusion for patients with prior brain metastasis was clarified. - The sources of clinical grade IL-2 and GM-CSF were updated since they have recently changed due to pharmaceutical company agreements and cross-licensing.  
- We added a description of Retronectin to the detailed explanation of reagents used in this study.  
- We added the possibility of samples being tested with novel immune monitoring assays that have become available in the past several months.  
- We added Dr. James Heath from Caltech as a non-clinical investigator. - Several typographic errors and inconsistencies with the laboratory procedures were corrected and updated.  
b) Informed Consent Form
- No modifications were made to the ICF.  
c) HS-1 Form:
- The changes in eligibility and procedures included in the protocol were made in the HS-1 form.  
Date: October 5, 2010  
a) Protocol:
- Deletion of Dr. Erika von Euw from the protocol roster since she is no longer involved in this research project.  
- Clarification of the enrollment of patients with a prior diagnosis of brain metastasis. - Updated information on the source of OKT3 for this protocol. - Updated details on the procedure of retroviral transduction of activated lymphocytes..  
- The inclusion of the testing for C reactive protein (CRP) in the Schedule of Events.  
b) Informed Consent Form
- Several typographical errors have been corrected in the ICF, and wording has been slightly modified to improve it.  
c) HS-1 Form:
- The relevant changes in the main study protocol were made in the HS-1 form.  
Date: December 17, 2010
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main protocol and consent modifications

a) Protocol: The following is a description of the major changes in Amendment 8 and the reasoning behind them:
1. Four days of Fludarabine at 25 mg/m2/day instead of 5 days:
The available information based on the course, correlative studies and outcome of the SAE on patient F5-10 strongly suggest that it was due to direct marrow toxicity from the conditioning regimen as the most likely cause (see full description of the SAE). The most likely cause of a delayed marrow aplasia is fludarabine. Therefore, we have decreased the number of fludarabine doses at 25 mg/m2/day i.v. daily from 5 days to 4 days.  
2. Capping of the administration of high dose IL-2 to up to 9 doses instead of 14 doses:
A potential adverse contributor to the marrow aplasia in patient F5-10 is the 14 doses of IL-2 that this patient received, which is higher than other patients treated in this protocol. The prior versions of the protocol included dosing IL-2 at 600,000 IU/kg every 8 hours for up to 14 doses based on tolerance. This patient tolerated all doses within the expected parameters and received all of them. In conversations with our colleagues at the NCI Surgery Branch, who have administered the same conditioning regimen followed by high dose IL-2 to over 200 patients, they feel that the dosing of IL-2 may be an adverse contributor. In addition, review of the literature shows that higher doses of IL-2 up to 15 (720,000 IU/kg, 8 hourly for 5 days) lead to grade 3/4 toxicities whereas decreasing it to a maximum of 9 (720,000 IU/kg, 8 hourly for 3 days) results in fewer toxicities and still have the anti-tumor activities in conjunction with the adoptive cell transfer (ACT) 7,21. In fact, there was a trend wherein less doses of IL-2 correlated with higher clinical responses. Therefore, we plan to cap the number of IL-2 doses to a maximum of 9 doses.
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3. using freshly harvested f5

The major problem for patients treated within this protocol has been relapse of melanoma after an initial tumor response. Our ongoing experiments analyzing the functionality of the TCR transgenic cells recovered from patients demonstrate a progressive loss of ability to secrete multiple cytokines upon MART-1 antigen exposure. This is reminiscent of preclinical studies of T cell exhaustion and lack of secondary response to antigen. Therefore, improving the *in vivo* functionality of the TCR transgenic cells continues to be a major goal of our research plans.  
A recent experiment in a murine model of ACT performed by our collaborators Drs. Lili Yang and David Baltimore from the California Institute of Technology (Caltech) indicated that cryopreservation has a profound detrimental effect on the *in vivo* long-term survival of the adoptively transferred T cells and their ability to have a secondary response to antigen exposure. We noted a similar effect within our clinical trial, since in patient F5-3 received a round of three delayed MART-1 peptide pulsed dendritic cell vaccines and had no secondary expansion of MART-1-specific T cells. These results have prompted us to modify our protocol to avoid cryopreservation of the TCR transgenic cells administered to patients, which is included in Amendment 8.
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b) informed consent form

- The use of freshly harvested F5 MART-1 TCR transgenic T cells for infusion in place of cryopreserved cells as depicted in Amendment 8  
- The use of high dose IL-2 every 8 hours following transgenic T cell infusion for a maximum 9 doses over 3 days has been adopted to replace the previous regimen of maximum 14 doses in Amendment 8
c) HS-1 Form
- As IRB submission at UCLA was converted to webIRB which is entirely done online, HS-1 form was no longer required.  
Date: April 20, 2011
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main protocol and consent modifications

a) Protocol:
- Addition of a PCR/ELISA mycoplasma assay if the MycoAlert results are equivocal or positive both during in-process testing and before cells are administered to the patient.  
- Lowering of the lot release criteria to 10% of tetramer positive cells for MART-1(26-35) as long as all the other lot release criteria are met.  
- Indication of performing additional testing for the number of vector copies. If higher than 5 we will propose a new dilution of the vector supernatant before using it for cell transduction.  
- Cardiac stress test on Day 75-90 has been removed.  
- Addition of potential risks associated with autoimmune toxicity from mispaired TCRs
- The use of Trimethoprim-Sulfamethoxazole and Ganciclovir will start from Day -6
- Replacement of William Quan by Kit Shaw as the Human Gene Medicine Program (HGMP)
Compliance Officer
- More detailed description of Day -6 events
- Minor formatting and wording modification b) Informed Consent Form
- Addition of potential risks associated with autoimmune toxicitiy from mispairing TCRs - Cardiac stress test on Day 75-90 has been removed
- Addition of Day -6 events to the schedule table in the end of the consent
- Addition of risk to include life-threatening infection and/or respiratory failure requiring mechanical ventilation
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amendment 10 date august 22

a) Protocol
- Addition of Sujna Raval-Fernandes as the compliance officer for the DSMB. - Addition of Arun Singh as a study investigator with patient contact. - Addition of Phyllis Wu as study coordinator and regulatory coordinator.  
- Change of manufacturer of OKT3 to Miltenyi: As per FDA recommendation on July 28, 2011, we replaced eBioscience OKT3 with Miltenyi OKT3.  
- Change of GM-CSF manufacturer to Berlex and IL-2 manufacturer to Prometheus only in accordance to FDA product reviewer recommendation on July 22, 2011. The sources of clinical grade IL-2 and GM-CSF were changed due to pharmaceutical company agreements and cross-licensing.  
- Added dextramer as an alternative option for tetramer for the purpose of quantification of F5 TCR transgenic cells. We had tested by staining the same sample side-by-side at least three times and obtained comparable results repeatedly. Test results attached.  
- Corrected "Blood for Plasma/serum" procedure in the schedule of events to "Plasmapheresis".  
Plasmapheresis is carried out at the time of leukapheresis for every patient on Day 0 and Day 30.  
- Correction of minor typographic errors.  
b) Informed Consent Form
- Addition of Arun Singh as a study investigator.  
- Description of a day 20-40 ECHO/MUGA was deleted.
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amendment 11 date november 7

a) Protocol
- Decrease the maximum cell dose for ACT to 109 total cells.  
- Changing the IL-2 administration from high dose (HD) intravenously to low dose (LD)
subcutaneously for up to 14 days.  
- Require having pulmonary function tests in all patients and change the patient exclusion criteria to exclude patients with a FEV1/FCV< 70% of predicted for normality.  
- Electrocardiogram (ECG) is added as a heart test for patients. - List pancytopenia caused by bone marrow infiltration by melanoma cells as a risk factor. - Added to exclusion criteria the following:
o History of, or significant evidence of risk for, chronic inflammatory or autoimmune disease (eg, Addison's disease, multiple sclerosis, Graves disease, Hashimoto's thyroiditis, inflammatory bowel disease, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, hypophysitis, pituitary disorders, etc.). Patients will be eligible if prior autoimmune disease is not deemed to be active (e.x. fibrotic damage of the thyroid after thyroiditis or its treatment, with stable thyroid hormone replacement therapy). Vitiligo will not be a basis for exclusion.  
o History of inflammatory bowel disease, celiac disease, or other chronic gastrointestinal conditions associated with diarrhea or bleeding, or current acute colitis of any origin.  
- Added "Must be willing to perform two biopsies at least" to inclusion criteria.  
- Revised screening blood test details, reflected in the schedule table and section 8.1 - In section 8.4, added "Norethindrone (Norlutate) 10mg PO daily will be used for menstruating women for prophylaxis against menorrhagia" under "Prophylactic Supportive Care Throughout the Pancytopenic Period".  
- Added 8.11 Permissible Systemic Therapies During the Protocol On-study Period - Change GM-CSF source to Genzyme - Revisions in response to FDA inquires regarding cross-referenced IND 14760.  
- Correction of minor typographic errors.  
b) Informed Consent Form
- Description of the LD IL-2 administration - Description of the SAE of intubation in a second patient. - Description of pancytopenia caused by bone marrow infiltration by melanoma cells as a risk factor. - Requirement of having pulmonary function tests in all patients and change the patient exclusion criteria to exclude patients with a FEV1/FCV< 70% of predicted for normality.  
- Electrocardiogram (ECG) is added as a heart test for patients. - Added MART-1 testing in the screening procedures.  
Date: July 25, 2013 Main Protocol and Consent Modifications: a) Protocol
- No changes were made to the protocol b) Informed Consent Form
- Description of an SAE in which the patient died due to melanoma progression with massive intraabdominal bleeding.  
Date: September 9, 2014 Main Protocol and Consent Modifications:
a) Protocol
- Changed contact information for several co-investigators on page 1
- Added Paula Kaplan-Lefko, Beata Berent-Maoz, Daniel Shin, Siwen Hu-Lieskovan and Xiaoyan Wang
- Removed Bradley Straatsma and Phyllis Wu - Blood tests (Mg, Phos and uric acid) in table in Section 2.0 were changed from Day -5 through Day 0 to only on Day -6.  
- Modified days of blood collection and physical exams in table in Section 2.0 and 8.0. - Added to section 8.0 and section 2.0 that changes in the range of 7 days up or down from the scheduled event will not be considered deviations from the study timeline. Removed the word "significant" referring to deviations.  
- On page 25, ECOG has been deleted from the list of tests to be performed in the hospital. - On page 26, "mineral panel" has been deleted from the blood tests to be monitored.  
- Clarification that physical exam at baseline will include weight, height, and vital signs and that weight measurement will only be obtained at subsequent physical exams if clinically indicated
(Section 8.1)
- Deleted that physical exam would include weight, height and vital signs on Day -6 (Section 8.3) since weight measurement is not standard inpatient practice.  
- In section 8.1 and 8.2, added that a new biopsy may not be required if an acceptable biopsy was performed within 60 days of prior to screening per coverage analysis for other trials.  
- In section 8.2, modified cardiac function test details.  
- Reduced IL-2 administration from twice daily for 14 days to twice daily for 7 days per IRB due to SAEs in IRB \#12-000153 and 13-001624.  
- In section 8.4, changed dose of Ativan from 2 mg to 1 mg - Added holding parameters for IL-2 and fevers due to IL-2 to section 8.7 - Changed section 8.9 to indicate that CT will be attenuated. - In section 8.15, removed optional repeated dendritic cell vaccines. - In sections 9.6.4 and 9.6.5, updated information regarding IL-4 and retronectin. - In section 10.1.5, Table 2b Annual Recertification was updated.  
b) Informed Consent Form
- Reduced IL-2 administration from twice daily for 14 days to twice daily for 7 days.  
- Removed optional repeated dendritic cell vaccines
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amendment 14 date august 1

a) Protocol
- Modified annual recertification tests required (Section 10.1.5)
b) Informed Consent Form
- No changes  
a) Protocol
- In section 10.1.5, Table 2b Annual Recertification was updated
- In section 10.2.5 MART-126-35, Table 7 Peptide Annual Recertification was updated, and modified annual recertification tests required.