Natural Killer Cells Plus IL-2 Following Chemotherapy to Treat Advanced Melanoma or Kidney Cancer

• Sponsor: National Cancer Institute (NCI) (NIH)
• Overall Status: Completed
• CT.gov ID: NCT00328861
• Collaborator: (none)
• Enrollment: 8
• Location: 1
• Arms: 2
• Duration (Months): 35
• Patients Per Site Per Month: 0.2

Study Details

Study Description

Brief Summary

Background:

• Natural killer (NK) cells are large lymphocytes (a type of white blood cell) that are important in the immune response to cancer.

• IL-2 (Aldesleukin) is a substance the body makes that controls the growth and function of many types of cells. The Food and Drug Administration has approved IL-3 for treating metastatic melanoma and kidney cancer. (Metastatic disease is cancer that has spread beyond the primary site.)

Objectives: To determine the safety and effectiveness of treating metastatic melanoma and kidney cancer with laboratory-treated NK cells and IL-2.

Eligibility: Patients 18 years of age or older with metastatic melanoma or kidney cancer who have previously been treated with high-dose IL-2.

Design:

• Leukapheresis. Patients under leukapheresis to obtain NK cells for the treatment regimen. Blood is collected through a needle in an arm vein and directed through a cell separator machine where white blood cells are extracted. The rest of the blood is returned to the patient through a needle in the other arm. NK cells are removed from the white blood cells and treated for re-infusion into the patient.

• Chemotherapy. Starting 8 days before infusion of the treated NK cells, patients receive intravenous (IV, through a vein) infusions of cyclophosphamide and fludarabine to suppress the immune system.

• NK cell infusion. Patients receive a 30-minute IV infusion of NK cells 2 days after the last dose of chemotherapy.

• IL-2 therapy. Within 24 hours of the NK cell infusion, patients receive high-dose IL-2 as a 15-minute IV infusion every 8 hours for up to 5 days. A second cycle of IL-2 is given about 14 days after the first.

• Blood tests and biopsy. Patients have frequent blood tests during the treatment period and may be asked to undergo a biopsy (surgical removal of a small piece of tumor or lymph node) at the end of treatment to look at the effects of the treatment on the tumor immune cells.

• Follow-up evaluation. Patients are evaluated 4-6 weeks after completing treatment. They have a physical examination, scans of tumor sites, blood tests and blood sampling (or leukapheresis) to examine the response to treatment. Patients who improve with treatment return for evaluations every month. Those whose tumor grows again after originally shrinking may receive one additional treatment course.

Condition or Disease	Intervention/Treatment	Phase
Metastatic Melanoma; Metastatic Kidney Cancer	Drug: Natural Killer (NK) Lymphocytes; Biological: IL-2; Drug: Cyclophosphamide; Drug: Fludarabine	Phase 2

Detailed Description

Background:

• Natural killer (NK) cells are large granular lymphocytes that are critical effector cells in the early innate immune response to pathogens and cancer.

• Previous and current clinical investigations have clearly demonstrated that T lymphocytes can mediate the regression of metastatic melanoma. However, not all patients with cancer are eligible for this type of immunotherapy either because resectable tumor is not available, the TIL do not expand sufficiently, or the tumor infiltrating lymphocytes (TIL) that do proliferate do not exhibit sufficient tumor specific reactivity.

• We have recently developed techniques for the in vitro isolation and expansion of anti-tumor NK cells to levels suitable for the treatment of cancer patients and are proposing in this protocol to evaluate therapy using these NK cells.

• In Surgery Branch pre-clinical experiments, we evaluated lysis of fresh melanoma cell digests, melanoma cell lines, renal cell carcinoma (RCC) lines, and normal peripheral blood mononuclear cells (PBMCs) by NK cells from several patients and demonstrated that NK cells could lyse some fresh melanoma digests, as well as melanoma cell lines and renal cell cancer (RCC) lines, while sparing normal allogeneic and autologous PBMCs.

Objectives:

• Determine the ability of the administration of autologous natural killer (NK) cells plus aldesleukin (IL-2) following a non-myeloablative lymphodepleting preparative regimen to mediate tumor regression in patients with metastatic melanoma or kidney cancer.

• Determine the rate of repopulation of the natural killer cells in treated patients.

• Determine the toxicity of this treatment regimen.

Eligibility:

• Patients, 18 years of age or older with metastatic melanoma or metastatic kidney cancer who have previously received high dose IL-2, with an Eastern Cooperative Oncology Group (ECOG) of 0 or 1.

• Patients may not have any active systemic infections, coagulations disorders, major medical illnesses of the cardiovascular, respiratory or immune systems or any form of autoimmune disease or immunodeficiency.

Patients must be eligible to receive high-dose IL-2.

Design:

• Patients will undergo apheresis on 03-C-0277 (Cell Harvest and Preparation for Surgery Branch Adoptive Cell Therapy Protocols) to obtain cells for generation of autologous natural killer cells.

• All patients will receive a non-myeloablative lymphocyte depleting preparative regimen of cyclophosphamide (60 mg/kg/day IV) on days -8 and -7 and fludarabine (25 mg/m^2/day

1. on days -6 through -2.

• On day 0 patients will receive the infusion of autologous natural killer lymphocytes and then begin the first cycle of high-dose IL-2 (720,000 IU/kg IV every 8 hours for up to 15 doses). A second cycle of IL-2 will be administered approximately 14 days later.

• Clinical and Immunologic response will be evaluated about 4 to 6 weeks

after the second cycle of IL-2.

-Using a small optimal Phase II design, two cohorts of patients, initially 16 in each cohort, will be enrolled, and if at least one of the first 16 patients has a clinical response (partial response (PR) or complete response (CR)), accrual will continue to 29 patients, targeting a 15% goal for objective response.

Study Design

• Study Type: Interventional
• Actual Enrollment: 8 participants
• Allocation: Randomized
• Intervention Model: Parallel Assignment
• Masking: None (Open Label)
• Primary Purpose: Treatment
• Official Title: Phase II Study in Metastatic Melanoma or Kidney Cancer Using Autologous Natural Killer Cells Plus Aldesleukin (IL-2) Following a Lymphodepleting Chemotherapy
• Study Start Date: May 1, 2006
• Actual Primary Completion Date: Jul 1, 2007
• Actual Study Completion Date: Apr 1, 2009

Arms and Interventions

Arm	Intervention/Treatment
Experimental: NK Cells + IL-2: Melanoma Melanoma (skin cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.	Drug: Natural Killer (NK) Lymphocytes; Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.; Other Names: NK cells ; Biological: IL-2; IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days.; Other Names: aldesleukin ; Drug: Cyclophosphamide; Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7.; Other Names: Cytoxan ; Drug: Fludarabine; Fludarabine 25 mg/m^2 day intravenous on days -6 through -2.; Other Names: Fludara
Experimental: NK Cells + IL-2: Renal Cell Renal cell (kidney cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.	Drug: Natural Killer (NK) Lymphocytes; Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.; Other Names: NK cells ; Biological: IL-2; IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days.; Other Names: aldesleukin ; Drug: Cyclophosphamide; Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7.; Other Names: Cytoxan ; Drug: Fludarabine; Fludarabine 25 mg/m^2 day intravenous on days -6 through -2.; Other Names: Fludara

Outcome Measures

Primary Outcome Measures

1. Objective Response [very 4-6 weeks for up to 1 year, and then every 6 months for up to 5 years.]

   Objective response (complete response (CR) or partial response (PR)) is measured by the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Complete response (CR) is the disappearance of all target lesions. Partial response (PR) is at least a 30% decrease in the sum of the longest diameter (LD) of target lesions taking as reference the baseline sum LD.

Secondary Outcome Measures

1. Safety [11/30/2006 - 7/31/2007]

   Here is the number of participants with adverse events. For the detailed list of adverse events see the adverse event module.

Eligibility Criteria

Criteria

• Ages Eligible for Study: 18 Years and Older
• Sexes Eligible for Study: All
• Accepts Healthy Volunteers: No

• INCLUSION CRITERIA:

1. Patients must have previously received high dose IL-2 (aldesleukin) and have been either non-responders (progressive disease) or have recurred.

2. Patients who are greater than or equal to 18 years of age, must have measurable metastatic melanoma or metastatic kidney cancer and no tumor reactive T cells available for cell transfer therapy.

3. Pathology for metastatic melanoma or metastatic kidney cancer to be confirmed by the National Cancer Institute (NCI) Laboratory of Pathology.

4. Patients of both genders must be willing to practice birth control for four months after receiving the preparative regimen.

5. Clinical performance status of Eastern Cooperative Oncology Group (ECOG) 0, 1.

6. Absolute neutrophil count greater than 1000/mm^3.

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

8. Hemoglobin greater than 8.0 g/dl.

9. Serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) less than three times the upper limit of normal.

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

11. Total bilirubin less than or equal to 2.0 mg/dl, except in patients with Gilbert's Syndrome who must have a total bilirubin less than 3.0 mg/dl.

12. Must be willing to sign a durable power of attorney.

EXCLUSION CRITERIA:

1. Less than four weeks has elapsed since any prior systemic therapy at the time the patient receives the preparative regimen, or less than six weeks since prior nitrosurea therapy.

2. 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.

3. Life expectancy of less than three months.

4. Systemic steroid therapy required.

5. Any active systemic infections, coagulation disorders or other major medical illnesses of the cardiovascular, respiratory or immune system, as evidenced by a positive stress thallium or comparable test, myocardial infarction, cardiac arrhythmias, obstructive or restrictive pulmonary disease.

6. Any form of autoimmune disease (such as autoimmune colitis or Crohn's Disease).

7. Seropositive 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.)

8. Seropositive for hepatitis B or C antigen.

9. Seronegative for Epstein-Barr virus (EBV).

10. Patients who are not eligible to receive high-dose Aldesleukin as evaluated by the following:

11. Patients who are 50 years old or greater who do not have a normal stress cardiac test (stress thallium, stress multi-gated acquisition scan (MUGA), dobutamine echocardiogram, or other stress test) will be excluded.

12. Patients who have history of electrocardiogram (EKG) abnormalities, symptoms of cardiac ischemia or arrhythmias who do not have a normal stress cardiac test (stress thallium, stress MUGA, dobutamine echocardiogram, or other stress test) will be excluded.

13. Patients with a prolonged history of cigarette smoking or symptoms of respiratory dysfunction who do not have a normal pulmonary function test as evidenced by a forced expiratory volume 1 (FEV1) less than 60% predicted will be excluded.

14. Patients who experienced toxicities during prior IL-2 administration that would preclude redosing with IL-2, i.e. myocardial infarction, mental status changes requiring intubation, bowel perforation or renal failure requiring dialysis.

Contacts and Locations

Locations

	Site	City	State	Country	Postal Code
1	National Cancer Institute (NCI)	Bethesda	Maryland	United States	20892

Sponsors and Collaborators

• National Cancer Institute (NCI)

Investigators

None specified.

Study Documents (Full-Text)

None provided.

More Information

Additional Information:

• NIH Clinical Center Detailed Web Page

Publications

• Berendt MJ, North RJ. T-cell-mediated suppression of anti-tumor immunity. An explanation for progressive growth of an immunogenic tumor. J Exp Med. 1980 Jan 1;151(1):69-80.

• ClinicalTrials.gov Identifier: NCT00328861
• Other Study ID Numbers: 060169; 06-C-0169; NCT00354055
• First Posted: May 22, 2006
• Last Update Posted: Nov 4, 2012
• Last Verified: Oct 1, 2012

• Keywords provided by , ,: Adoptive Cell Therapy; Cutaneous Melanoma; Clinical Response; Rate of Repopulation; Toxicity Profile; Metastatic Melanoma; Metastatic Kidney Cancer
• Additional relevant MeSH terms: Melanoma; Kidney Neoplasms; Carcinoma, Renal Cell; Aldesleukin; Cyclophosphamide; Fludarabine

Study Results

Participant Flow

Arm/Group Title	NK Cells + IL-2: Melanoma	NK Cells + IL-2: Renal Cell
Arm/Group Description	Melanoma (skin cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.	Renal cell (kidney cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.
Period Title: Overall Study
STARTED	7	1
COMPLETED	7	1
NOT COMPLETED	0	0

Baseline Characteristics

Arm/Group Title	NK Cells + IL-2: Melanoma	NK Cells + IL-2: Renal Cell	Total
Arm/Group Description	Melanoma (skin cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.	Renal cell (kidney cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.	Total of all reporting groups
Overall Participants	7	1	8
Age (Count of Participants)
<=18 years	0 0%	0 0%	0 0%
Between 18 and 65 years	7 100%	1 100%	8 100%
>=65 years	0 0%	0 0%	0 0%
Age (years) [Mean (Standard Deviation) ]
Mean (Standard Deviation) [years]	37.3 (11.0)	51.0 (0)	39.0 (11.3)
Sex: Female, Male (Count of Participants)
Female	4 57.1%	0 0%	4 50%
Male	3 42.9%	1 100%	4 50%
Ethnicity (NIH/OMB) (Count of Participants)
Hispanic or Latino	0 0%	0 0%	0 0%
Not Hispanic or Latino	7 100%	1 100%	8 100%
Unknown or Not Reported	0 0%	0 0%	0 0%
Race (NIH/OMB) (Count of Participants)
American Indian or Alaska Native	0 0%	0 0%	0 0%
Asian	0 0%	0 0%	0 0%
Native Hawaiian or Other Pacific Islander	0 0%	0 0%	0 0%
Black or African American	0 0%	0 0%	0 0%
White	7 100%	1 100%	8 100%
More than one race	0 0%	0 0%	0 0%
Unknown or Not Reported	0 0%	0 0%	0 0%
Region of Enrollment (participants) [Number]
United States	7 100%	1 100%	8 100%

Outcome Measures

1. Primary Outcome

Title	Objective Response
Description	Objective response (complete response (CR) or partial response (PR)) is measured by the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Complete response (CR) is the disappearance of all target lesions. Partial response (PR) is at least a 30% decrease in the sum of the longest diameter (LD) of target lesions taking as reference the baseline sum LD.
Time Frame	very 4-6 weeks for up to 1 year, and then every 6 months for up to 5 years.

Outcome Measure Data

Analysis Population Description

[Not Specified]

Arm/Group Title	NK Cells + IL-2: Melanoma	NK Cells + IL-2: Renal Cell
Arm/Group Description	Melanoma (skin cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.	Renal cell (kidney cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.
Measure Participants	7	1
Complete Response	0 0%	0 0%
Partial Response	0 0%	0 0%

2. Secondary Outcome

Title	Safety
Description	Here is the number of participants with adverse events. For the detailed list of adverse events see the adverse event module.
Time Frame	11/30/2006 - 7/31/2007

Outcome Measure Data

Analysis Population Description

[Not Specified]

Arm/Group Title	NK Cells + IL-2: Melanoma	NK Cells + IL-2: Renal Cell
Arm/Group Description	Melanoma (skin cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.	Renal cell (kidney cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.
Measure Participants	7	1
Number [Participants]	7 100%	1 100%

Adverse Events

Arm/Group Title	NK Cells + IL-2: Melanoma		NK Cells + IL-2: Renal Cell
Arm/Group Description	Melanoma (skin cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.		Renal cell (kidney cancer). Cyclophosphamide 60 mg/kg/day intravenous on days -8 and -7. Fludarabine 25 mg/m^2 day intravenous on days -6 through -2. IL-2 720,000 IU/kg/intravenous every 8 hours for up to 5 days. Thirty minutes infusion of natural killer (NK) cells 2 days after last dose of chemotherapy.
All Cause Mortality
	NK Cells + IL-2: Melanoma		NK Cells + IL-2: Renal Cell
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	/ (NaN)		/ (NaN)
Serious Adverse Events
	NK Cells + IL-2: Melanoma		NK Cells + IL-2: Renal Cell
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	0/7 (0%)		0/1 (0%)
Other (Not Including Serious) Adverse Events
	NK Cells + IL-2: Melanoma		NK Cells + IL-2: Renal Cell
	Affected / at Risk (%)	# Events	Affected / at Risk (%)	# Events
Total	7/7 (100%)		1/1 (100%)
Blood and lymphatic system disorders
Leukocyte count decreased	7/7 (100%)	7	1/1 (100%)	1
Lymphocyte count decreased	7/7 (100%)	12	1/1 (100%)	2
Neutrophil count decreased	7/7 (100%)	7	1/1 (100%)	1
Platelet count decreased	7/7 (100%)	7	1/1 (100%)	1
Activated partial thromboplastin time prolonged	1/7 (14.3%)	1	1/1 (100%)	1
Petechiae	4/7 (57.1%)	4	1/1 (100%)	1
Hemoglobin decreased	3/7 (42.9%)	3	0/1 (0%)	0
Cardiac disorders
Hypotension	1/7 (14.3%)	1	1/1 (100%)	1
Sinus tachycardia	1/7 (14.3%)	1	0/1 (0%)	0
Premature ventricular contractions	1/7 (14.3%)	1	0/1 (0%)	0
Gastrointestinal disorders
Nausea	6/7 (85.7%)	8	1/1 (100%)	1
Vomiting	2/7 (28.6%)	4	1/1 (100%)	1
Anorexia	1/7 (14.3%)	2	0/1 (0%)	0
Diarrhea	2/7 (28.6%)	3	0/1 (0%)	0
Dry mouth	1/7 (14.3%)	2	0/1 (0%)	0
Ear, nose and throat examination abnormal	2/7 (28.6%)	3	0/1 (0%)	0
Mucositis oral	1/7 (14.3%)	2	0/1 (0%)	0
General disorders
Fatigue	6/7 (85.7%)	8	1/1 (100%)	2
Fever	2/7 (28.6%)	2	0/1 (0%)	0
General symptom	2/7 (28.6%)	2	0/1 (0%)	0
Pain in extremity	2/7 (28.6%)	2	0/1 (0%)	0
Infections and infestations
Sepsis	1/7 (14.3%)	1	0/1 (0%)	0
Wound infection	1/7 (14.3%)	1	0/1 (0%)	0
Metabolism and nutrition disorders
Serum albumin decreased	4/7 (57.1%)	4	1/1 (100%)	1
Serum phosphate decreased	1/7 (14.3%)	1	1/1 (100%)	1
Aspartate aminotransferase increased	1/7 (14.3%)	1	0/1 (0%)	0
Serum calcium decreased	2/7 (28.6%)	2	0/1 (0%)	0
Serum potassium decreased	3/7 (42.9%)	3	0/1 (0%)	0
Serum sodium decreased	1/7 (14.3%)	1	0/1 (0%)	0
Blood uric acid increased	1/7 (14.3%)	1	0/1 (0%)	0
Bilirubin increased	1/7 (14.3%)	1	0/1 (0%)	0
Musculoskeletal and connective tissue disorders
Back pain	1/7 (14.3%)	1	0/1 (0%)	0
Nervous system disorders
Confusion	3/7 (42.9%)	4	1/1 (100%)	1
Peripheral sensory neuropathy	1/7 (14.3%)	1	0/1 (0%)	0
Headache	4/7 (57.1%)	7	0/1 (0%)	0
Neuralgia	1/7 (14.3%)	1	0/1 (0%)	0
Respiratory, thoracic and mediastinal disorders
Hemorrhage nasal	0/7 (0%)	0	1/1 (100%)	1
Hypoxia	0/7 (0%)	0	1/1 (100%)	3
Chest pain	1/7 (14.3%)	1	0/1 (0%)	0
Cough	2/7 (28.6%)	2	0/1 (0%)	0
Dyspnea	4/7 (57.1%)	5	0/1 (0%)	0
Skin and subcutaneous tissue disorders
Pruritus	1/7 (14.3%)	1	0/1 (0%)	0
Rash desquamating	2/7 (28.6%)	2	0/1 (0%)	0
Vascular disorders
Capillary leak syndrome	5/7 (71.4%)	6	1/1 (100%)	1

Limitations/Caveats

[Not Specified]

More Information

Certain Agreements

Principal Investigators are NOT employed by the organization sponsoring the study.

There is NOT an agreement between Principal Investigators and the Sponsor (or its agents) that restricts the PI's rights to discuss or publish trial results after the trial is completed.