Study of Gene Modified Immune Cells in Patients With Advanced Melanoma

Adoptive Transfer of MART-1 F5 TCR Engineered Peripheral Blood Mononuclear Cells (PBMC) After a Nonmyeloablative Conditioning Regimen, With Administration of MART-126•35-Pulsed Dendritic Cells and Interleukin-2, in Patients With Advanced Melanoma

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

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

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

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

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

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

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

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

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

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

Inclusion Criteria:

• Histologically confirmed melanoma that is considered surgically incurable with either:

  • Stage IIIc melanoma including locally relapsed, satellite, in-transit lesions or bulky draining node metastasis.
  • Stage IV melanoma (M1a, M1b or M1c). At least 1 lesion amenable for outpatient biopsies; this should be a cutaneous or palpable metastatic site or a deeper site accessible by image-guided biopsy that is deemed safe to access by the treating physicians and interventional radiologists. Patients without accessible lesions for biopsy but with prior tissue available from metastatic disease would be eligible at the investigator's discretion.

• MART-1 positive melanoma by RT-PCR or Immuno-histochemical (IHC).

• HLA-A*0201 (HLA-A2.1) positivity by molecular subtyping*.

• Age greater than or equal to 18 years old.

• Life expectancy greater than 3 months assessed by a study physician.

• A minimum of one measurable lesion defined as:

  • Meeting the criteria for measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST).
  • Skin lesion(s) selected as non-completely biopsied target lesion(s) that can be accurately measured and recorded by color photography with a ruler to document the size of the target lesion(s).

• No restriction based on prior treatments.

• Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0 or 1.

• Adequate bone marrow and hepatic function determined within 30-60 days prior to enrollment, defined as:

  • Absolute neutrophil count >= 1.5 x 109 cells/L.
  • Platelets >= 100 x 109/L.
  • Hemoglobin >= 10 g/dL.
  • Aspartate and alanine aminotransferases (AST, ALT) =< 2.5 x ULN (=< 5 x ULN, if documented liver metastases are present).
  • Total bilirubin =< 2 x Upper Limit of Normal (ULN) (except patients with documented Gilbert's syndrome).
  • Creatinine < 2 mg/dl (or a glomerular filtration rate > 60).
  • Must be willing and able to accept at least two leukapheresis procedures.
  • Must be willing and able to accept at least two tumor biopsies.
  • Must be willing and able to provide written informed consent.

• Patients with HLA-A*0205 (HLA-A2.5) positivity by molecular subtyping may be eligible if there is demonstration that they can correctly present the MART-126-35 epitope as stimulators for IFN-gamma production by MART-1 F5 TCR transgenic cells.

Exclusion Criteria

• Previously known hypersensitivity to any of the agents used in this study.

• Received systemic treatment for cancer, including immunotherapy, within one month prior to initiation of dosing within this protocol. However, cell harvesting by leukapheresis may be performed before one month from prior therapy if the study investigators consider that it will not have a detrimental impact on the generation of the two cell therapies in this protocol.

• History of, or significant evidence of risk for, chronic inflammatory or autoimmune disease (eg, Addison's disease, multiple sclerosis, Graves disease, Hashimoto's thyroiditis, inflammatory bowel disease, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, hypophysitis, pituitary disorders, etc.). Patients will be eligible if prior autoimmune disease is not deemed to be active (e.g. fibrotic damage of the thyroid after thyroiditis or its treatment, with stable thyroid hormone replacement therapy). Vitiligo will not be a basis for exclusion.

• History of inflammatory bowel disease, celiac disease, or other chronic gastrointestinal conditions associated with diarrhea or bleeding, or current acute colitis of any origin.

• Potential requirement for systemic corticosteroids or concurrent immunosuppressive drugs based on prior history or received systemic steroids within the last 4 weeks prior to enrollment (inhaled or topical steroids at standard doses are allowed).

• HIV seropositivity or other congenital or acquired immune deficiency state, which would increase the risk of opportunistic infections and other complications during chemotherapy-induced lymphodepletion. If there is a positive result in the infectious disease testing that was not previously known, the patient will be referred to their primary physician and/or infectious disease specialist.

• Hepatitis B or C seropositivity with evidence of ongoing liver damage, which would increase the likelihood of hepatic toxicities from the chemotherapy conditioning regimen and supportive treatments. If there is a positive result in the infectious disease testing that was not previously known, the patient will be referred to their primary physician and/or infectious disease specialist.

• Dementia or significantly altered mental status that would prohibit the understanding or rendering of informed consent and compliance with the requirements of this protocol.

• Clinically active brain metastases. Radiological documentation of absence of active brain metastases at screening is required for all patients. Prior evidence of brain metastasis successfully treated with surgery or radiation therapy will not be exclusion for participation as long as they are deemed under control at the time of study enrollment.

• Pregnancy or breast-feeding. Female patients must be surgically sterile or be postmenopausal for two years, or must agree to use effective contraception during the period of treatment and 6 months after. All female patients with reproductive potential must have a negative pregnancy test (serum/urine) within 14 days from starting the conditioning chemotherapy. The definition of effective contraception will be based on the judgment of the study investigators.

• Since IL-2 is administered following cell infusion:

  • Patients will be excluded if they have a history of clinically significant ECG abnormalities, symptoms of cardiac ischemia or arrhythmias and have a left ventricular ejection fraction (LVEF) < 45% on a cardiac stress test (stress thallium, stress MUGA, dobutamine echocardiogram, or other stress test).
  • Similarly, patients who are 50 years old with a baseline LVEF < 45% will be excluded.
  • Patients with ECG results of any conduction delays (PR interval >200ms, QTC > 480ms), sinus bradycardia (resting heart rate <50 beats per minute), sinus tachycardia (HR>120 beats per minute) will be evaluated by a cardiologist prior to starting the trial. Patients with any arrhythmias, including atrial fibrillation/atrial flutter, excessive ectopy (defined as >20 PVCs per minute), ventricular tachycardia, 3rd degree heart block will be excluded from the study unless cleared by a cardiologist.
  • Patients with pulmonary function test abnormalities as evidenced by a forced expiratory volume at one second/forced vital capacity (FEV1/FVC)< 70% of predicted for normality will be excluded.