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.