Anti-MART-1 F5 Lymphocytes to Treat High-Risk Melanoma Patients

Background:

We have engineered human peripheral blood lymphocytes (PBLs) to express an anti-MART-1
T-cell receptor (TCR) that recognizes an HLA-A*0201 restricted epitope derived from the
tumor infiltrating lymphocytes (TIL) clone DMF5.

We constructed a single retroviral vector that encodes both alpha and beta chains and can
mediate genetic transfer of this T cell receptor (TCR) with high efficiency without the need
to perform any selection.

In co-cultures with HLA-A*0201 positive melanoma, anti-MART-1 F5 TCR transduced T cells
secreted significant amount of IFN- but no significant secretion was observed in control
co-cultures with cell lines.

The anti-MART-1 F5 TCR transduced PBL could efficiently kill HLA-A*0201 positive tumors.
There was little or no recognition of normal fibroblasts cells.

This TCR is over 10 times more reactive with melanoma cells than the MART-1 F4 TCR that
mediated tumor regression in two patients with metastatic melanoma.

Poxviruses encoding melanoma antigens, similar to the ALVAC MART-1 vaccine have been shown
to successfully immunize patients against these antigens.

Objectives:

Primary objectives:

To evaluate the ability of four different strategies to enhance the persistence of
anti-tumor T cells in the circulation at 5-10 days, and at 31-35 days after treatment
(defined as F5 cells in cohorts 1 and 2, and aldesleukin in cohorts 3 and 4) and potentially
select one strategy for further study.

With Amendment E, the primary objective is to evaluate the ability of three different
strategies to enhance the persistence of anti-tumor T cells in the circulation at 5-10 days
and at 31-35 days after treatment (defined as F5 cells in cohort 5, aldesleukin in cohort 6,
and ALVAC MART-1 vaccine in cohort 7) and potentially select one strategy for further study.

Eligibility:

Patients who are HLA-A*0201 positive and 18 years of age or older must have:

- primary melanomas with lesions that are ulcerated and greater than or equal to 2.0 mm,
or any lesions that are greater than or equal to 4.0 mm in thickness, or greater than
or equal to 1 positive lymph node, or local recurrence, or resected metastatic disease,
within 6 months of surgical resection.

- must be clinically disease free at the time of protocol entry as documented by
radiologic studies within 4 weeks of patient entry.

- may have had prior adjuvant treatment with immunotherapy, including interferon, as long
as 3 weeks have elapsed since prior systemic therapy.

- normal values for basic laboratory values.

Patients may not have:

- ocular or mucosal melanoma;

- been previously immunized with MART-1;

- concurrent major medical illnesses;

- any form of primary or secondary immunodeficiency;

- severe hypersensitivity to any of the agents used in this study;

Design:

Peripheral blood mononuclear cells (PBMC) obtained by leukapheresis (approximately 1 times
10^10 cells) will be cultured in the presence of anti-CD3 (OKT3) and aldesleukin in order to
stimulate T-cell growth.

Transduction is initiated by exposure of approximately 10^8 to 5 times 10^9 cells to
retroviral vector supernatant containing the anti-MART-1 F5 TCR genes. These transduced
cells (called F5 cells) will be expanded and tested for their anti-tumor activity.

F5 cells will be administered intravenously at a dose of 1 times 10^9 to 7 times 10^10
cells.

Patients will be randomized into one of the following four cohorts:

1. F5 cells on day 0 alone

2. F5 cells on day 0 followed by the subcutaneous injection of 1.0 mg MART-1:26-35(27L)
peptide in Montanide ISA-51 VG on day 0 and day 30.

3. F5 cells on day 0 followed by the subcutaneous injection of 125,000 IU/kg/day
aldesleukin on days 0-4.

4. F5 cells on day 0 plus MART-1:26-35(27L) peptide in Montanide ISA-51 VG on day 0 and
day 30, and 125,000 IU/kg aldesleukin on days 0-4.

Starting with amendment E, the four cohorts above will be closed to accrual and
patients will be randomized to the following cohorts:

5. F5 cells on day 0 following subcutaneous injection of ALVAC MART-1 vaccine. Second dose
of ALVAC MART-1 vaccine is given on day 14.

6. F5 cells on day 0 following subcutaneous injection of ALVAC MART-1 vaccine and then
subcutaneous injection of 125,000 IU/kg/day aldesleukin on days 0-4. Second dose of
ALVAC MART-1 vaccine is given on day 14.

7. ALVAC MART-1 vaccine on days 0 and 14.

Patients will undergo complete evaluation with physical examination, computed tomography
(CT) of the chest, abdomen and pelvis (3 months and thereafter only) and clinical laboratory
evaluation at day 35, and 3 months after treatment and then every six months or until off
study criteria are met.

Each of the cohorts will be conducted using a two-stage MiniMax design. This design will try
to determine whether each of the modalities of administration can produce persistence of the
transferred cells at a frequency of greater than or equal to 5 percent of circulating
cluster of differentiation 8 (CD8) plus cells in 35 percent of patients as opposed to
undesirably low (15 percent), with a 3 percent probability of accepting a poor schedule and
15 percent probability of rejecting a good schedule.

Initially 22 patients will be enrolled in each cohort. If four immunologic responses
(persistence) are noted in a given cohort, then accrual to 39 patients would take place. The
cohort with the highest number of patients exhibiting persistence will be considered
immunologically active and will be considered worthy of further development. If this arm has
fewer than 11 of 39 patients with persistence, it will not be considered worthy of further
consideration.

Starting with amendment E, 10 patients will be enrolled in each new cohort (cohorts 5-7). If
on any of the three arms, there are 2 or more of 10 patients with 5% CD8+ circulating cells,
then this cohort will be considered worthy of further consideration.

- INCLUSION CRITERIA:

1. Primary melanomas with lesions that are ulcerated and greater than or equal to
2.0 mm, or any lesions that are greater than or equal to 4.0 mm in thickness, or
greater than or equal to 1 positive lymph node, or local recurrence, or resected
metastatic disease, within 6 months of surgical resection will be considered.
Patients must be clinically disease free at the time of protocol entry as
documented by radiologic studies within 6 weeks of patient entry. Patients must
have pathologic confirmation of cutaneous melanoma, with slides reviewed at
National Institutes of Health (NIH) (Department of Anatomic Pathology), and if
the diagnosis is not confirmed, the patient will be excluded from the study.

2. Human leukocyte antigens (HLA-A) 0201 positive.

3. Age greater than or equal to18 years.

4. Clinical performance status of Eastern Cooperative Oncology Group (ECOG) 0 or 1.

5. Able to understand and sign the Informed Consent Document.

6. Patients of both genders must be willing to practice effective birth control
during this trial because the potential for teratogenic effects are unknown.
Effective birth control requires use of an effective method from the following
list: Abstinence, Intrauterine device (IUD); Hormonal (Birth control pills,
injections, implants); Tubal ligation; Cervical cap; or Partner's vasectomy

7. Patients may have had prior adjuvant treatment with immunotherapy, including
interferon, as long as 3 weeks have elapsed since prior systemic therapy.

8. Serology:

1. Seronegative for human immunodeficiency virus (HIV) antibody. (The
experimental treatment being evaluated in this protocol depends on an
intact immune system. Patients who are HIV seropositive can have decreased
immune -competence and thus be less responsive to the experimental
treatment and more susceptible to its toxicities.)

2. Seronegative for hepatitis B antigen and hepatitis C antibody unless
antigen negative (The experimental treatment being evaluated in this
protocol depends upon an intact immune system and these conditions may have
possible immune system effects).

9. Hematology:

1. Absolute neutrophil count greater than 1000/mm^3 without the support of
filgrastim.

2. White blood cell (WBC) (greater than 3000/mm^3).

3. Platelet count greater than 90,000/mm^3.

4. Hemoglobin greater than 8.0 g/dl.

10. Chemistry:

1. Serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) less
or equal to 2.5 times the upper limit of normal.

2. Serum creatinine less than or equal to 1.6 mg/dl.

3. Total bilirubin less than or equal to 1.5 mg/dl, except in patients with
Gilbert's Syndrome who must have a total bilirubin less than 3.0 mg/dl.

EXCLUSION CRITERIA:

1. Ocular or mucosal melanoma.

2. Undergoing or have undergone in the past 3 weeks any systemic therapy except surgery
for their cancer, and must have recovered to a grade 1 from any adverse effects of
treatment prior to entry, other than those that do not have clinical implications,
e.g. vitiligo, alopecia.

3. Have autoimmune disease (such as autoimmune colitis or Crohn's disease) or any known
immunodeficiency disease, as evidenced by abnormal white blood count (WBC) count.

4. Concurrent systemic steroid therapy.

5. Known systemic hypersensitivity to any of the vaccine components, including egg
products or Neomycin.

6. Women of child-bearing potential who are pregnant or breastfeeding because of the
potentially dangerous effects of the treatment on the fetus or infant.

7. Have active systemic infections including concurrent opportunistic infections (The
experimental treatment being evaluated in this protocol depends on an intact immune
system. Patients who have decreased immune competence may be less responsive to the
experimental treatment and more susceptible to its toxicities).

8. Previous immunization with melanoma antigen recognized by T cells (MART-1).

9. Known hypersensitivity to any of the agents used in this study.