Modified Tumor Infiltrating Lymphocytes for Metastatic Melanoma

Background:

- Tumor Infiltrating Lymphocyte (TIL) administration and high dose interleukin (IL)-2
following lymphodepletion can mediate durable complete responses in patients with
refractory melanoma. Obstacles to administration of this therapy include failure to
establish TIL in vitro for about 20% of patients, long delays between tumor resection
and TIL establishment resulting in poor TIL attributes for therapy and patient
ineligibility due to progression, and requirements for large numbers of feeder cells
for TIL expansion to therapeutic numbers.

- The K562 cell line was engineered to express the 4-1BBL costimulatory molecule and CD64
(the high affinity Fc receptor for loading with antibodies such as OKT3). The
K562.CD64.4-1BBL Engineered Cells with Costimulation Enhancement (ECCE) replaced up to
75% of feeder cells in large scale TIL expansions. ECCE added to tumor cell suspensions
provided costimulation in trans resulting in rapid and reliable lymphocyte growth even
from tumors with no TIL growth under standard conditions.

- A cloned K562.CD64.4-1BBL-7F11 ECCE line was produced under Good Manufacturing Practice
(GMP) conditions and a master cell bank has been generated. An optimized protocol was
established to rapidly expand (REP) young TIL using minimum feeders and 7F11. These
ECCE REPed TIL retained tumor recognition and some other attributes of standard TIL,
but differed from standard TIL by containing fewer CD4+ cells and more natural killer
cells.

When 7F11ECCE were added directly to single cell tumor suspensions, young TIL cultures were
reliably generated even from patients who otherwise would not have a standard young TIL
culture for treatment.

Objectives:

Primary objectives:

- In cohort 1, to evaluate whether young TIL that are rapidly expanded using 7F11 ECCE to
replace some feeder cells and administered with IL-2 in patients following a non
-myeloablative conditioning regimen will result in clinical tumor regression in
patients with refractory metastatic melanoma.

- In Cohort 2, to evaluate whether young TIL generated using in trans costimulation with
7F11 ECCE in patients for whom standard young TIL did not grow can mediate tumor
regression after a nonmyeloablative conditioning with high dose IL-2 in patients with
refractory metastatic melanoma.

- Determine the toxicity of ECCE young TIL in these treatment regimens

Eligibility:

Patients who are 18 years of age or older must have:

- metastatic melanoma;

- Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1

- One or more lesions 2 cm or greater suitable for resection for TIL culture

Patients may not have:

- Concurrent major medical illnesses;

- Any form of immunodeficiency;

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

- Contraindications for high dose IL-2 administration.

Design:

- Patients will undergo resection to obtain tumor for generation of autologous young TIL
cultures.

- Parallel TIL cultures will be established using a) the standard technique with IL-2
only and b) the Engineered Cells with Costimulation Enhancement (ECCE) protocol using
irradiated K562.CD64.4-1BBL-7F11 (7F11) cells.

- After 10 to 20 days cultures will undergo evaluation for TIL establishment. Standard
TIL will be used preferentially and patients who have TIL established by standard
methods will be assigned to Cohort 1

- Cohort 1:

- TIL will undergo ECCE REP by exposure to OKT-3, IL-2, feeder cells and irradiated
7F11.

- Patients will receive a non-myeloablative lymphocyte depleting preparative regimen
of cyclophosphamide (60 mg/kg/day intravenous (IV)) on days -7 and -6 and
fludarabine (25 mg/m^2/day IV) on days -5 through -1.

- On day 0 patients will receive the infusion of autologous young TIL and then begin
highdose aldesleukin (720,000 IU/kg IV every 8 hours for up to 15 doses).

- Clinical and immunologic response will be evaluated about 4-6 weeks after
treatment.

- Using an optimal two-stage Phase II design, initially 18 patients will be
enrolled, and if three or more of the first 18 patients have a clinical response
(partial response (PR) or complete response (CR)), accrual will continue to 35
patients, targeting a 30% goal for objective response.

- If standard young TIL fail to grow then ECCE young TIL will be evaluated and patients
who have ECCE TIL available will be assigned to Cohort 2 .

- Cohort 2:

- Cultures from patients in Cohort 2 will be evaluated for ECCE TIL establishment.
If adequate ECCE TIL are available, TIL will undergo ECCE REP by exposure to
OKT-3, IL-2, feeder cells and irradiated 7F11.

- Patients will receive a non-myeloablative lymphocyte depleting preparative regimen
of cyclophosphamide (60 mg/kg/day IV) on days -7 and -6 and fludarabine (25
mg/m^2/day IV) on days -5 through -1.

- On day 0 patients will receive the infusion of autologous young TIL and then begin
highdose aldesleukin (720,000 IU/kg IV every 8 hours for up to 15 doses).

- Clinical and immunologic response will be evaluated about 4-6 weeks after TIL
infusion.

- Using a small optimal two-stage Phase II design, initially 9 patients will be
enrolled, and if one or more of the first 9 patients has a clinical response (PR
or CR), accrual will continue to 24 patients, targeting a 25% goal for objective
response.

- If TIL cultures were not established by either standard methods or ECCE young TIL
protocols, patients will be eligible for re-resection and evaluation of TIL from a
different site.

- INCLUSION CRITERIA:

1. Measurable metastatic melanoma with at least one lesion that is resectable for
tumor infiltrating lymphocytes (TIL) generation.

2. Patients with 3 or less brain metastases are eligible. Note: If lesions are
symptomatic or greater than or equal to 1 cm each, these lesions must have been
treated and stable for 3 months for the patient to be eligible.

3. Greater than or equal to 18 years of age.

4. Willing to sign a durable power of attorney

5. Able to understand and sign the Informed Consent Document

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

7. Life expectancy of greater than three months.

8. Patients of both genders must be willing to practice birth control from the time
of enrollment on this study and for up to four months after receiving the
preparative regimen.

9. Serology:

1. Seronegative for human immunodeficiency virus (HIV) antibody. (The
experimental treatment being evaluated in this protocol depends on an
intact immune system. Patients engineered cells with costimulation
enhancement (ECCE) TIL 14 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 seronegative for hepatitis C
antibody. If hepatitis C antibody test is positive, then patient must be
tested for the presence of antigen by reverse transcription polymerase
chain reaction (RT-PCR) and be hepatitis C virus ribonucleic acid (HCV RNA)
negative.

3. Women of child-bearing potential must have a negative pregnancy test
because of the potentially dangerous effects of the preparative
chemotherapy on the fetus.

10. Hematology:

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

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

3. Platelet count greater than 100,000/mm^3.

4. Hemoglobin greater than 8.0 g/dl.

11. 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.

12. More than four weeks must have elapsed since any prior systemic therapy at the
time the patient receives the preparative regimen, and patients' toxicities must
have recovered to a grade 1 or less (except for toxicities such as alopecia or
vitiligo). Patients may have undergone minor surgical procedures within the past
3 weeks, as long as all toxicities have recovered to grade 1 or less.

13. Six weeks must have elapsed since any prior anti-cytotoxic T-lymphocyte antigen
4 (CTLA4) antibody therapy to allow antibody levels to decline.

14. Patients who have previously received any anti-CTLA4 antibody and have
documented gastrointestinal (GI) toxicity must have a normal colonoscopy with
normal colonic biopsies.

EXCLUSION CRITERIA:

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

2. Systemic steroid therapy required.

3. Active systemic infections, coagulation disorders or other major medical illnesses of
the cardiovascular, respiratory or immune system, myocardial infarction, cardiac
arrhythmias, obstructive or restrictive pulmonary disease.

4. Any form of primary immunodeficiency (such as Severe Combined Immunodeficiency
Disease).

5. Concurrent opportunistic infections (The experimental treatment being evaluated in
this protocol depends on an intact immune system. Patients who ECCE TIL 15 have
decreased immune competence may be less responsive to the experimental treatment and
more susceptible to its toxicities).

6. History of severe immediate hypersensitivity reaction to any of the agents used in
this study.

7. History of coronary revascularization or ischemic symptoms

8. Any patient known to have an left ventricular ejection fraction (LVEF) less than or
equal to 45%.

9. Documented LVEF of less than or equal to 45% tested in patients with:

- Clinically significant atrial and/or ventricular arrhythmias including but not
limited to: atrial fibrillation, ventricular tachycardia, second or third degree
heart block

- Age greater than or equal to 60 years old

10. Documented forced expiratory volume 1 (FEV1) less than or equal to 60% predicted
tested in patients with:

- A prolonged history of cigarette smoking

- Symptoms of respiratory dysfunction