Primary Objective:

1. Evaluate safety, feasibility, persistence, and anti-tumor effect of infused haploidentical donor-derived natural killer (NK) cells and low-dose interleukin-2 (IL-2).

Secondary Objectives:

1. Quantification of cytokine levels.
2. Assessment of NK cell immunophenotype and function.

Recipient:

Experimental Therapy:

NK cells are part of the immune system (the cells in the body that naturally fight disease and infection). NK cells can sometimes destroy tumor cells, particularly when the NK cells are "mismatched" for certain proteins called human leukocyte antigens (HLA). Researchers believe this ability to destroy tumor cells can be predicted by learning your and the donor's HLA types to see if they are a mismatch, and by checking for other specialized proteins on the donor's NK cells (called killer immunoglobulin receptors [KIR]) that identify tumor cells or infected cells.

The NK cells will be collected from the donor's blood and then processed using an experimental device called a CliniMACS device. This device is designed to separate out the NK cells from the rest of the donor's collected white blood cells, using a special magnet. Before being infused into the recipient (you, if you choose to take part), the collected NK cells will be treated with a study drug called interleukin-2 (IL-2) in order to try to activate the NK cells' killing ability. You will also receive IL-2 injections to try to help the NK cells survive after infusion and possibly increase in number.

Screening Tests:

Within 28 days before you can start treatment on this study, you will have "screening tests" to help the study doctor decide if you are eligible to take part in this study. The following tests will be performed:

• To check the status of the disease, you will have bone marrow aspirations and biopsies performed. To collect a bone marrow biopsy and aspirate, up to 2 areas of the hip bone are numbed with anesthetic, and a small amount of bone marrow and bone is withdrawn through a needle.
• For another check of the status of the disease, you will most likely need to have computed tomography (CT) scans of the neck, sinuses, chest, stomach area, and hip area (and if needed, the brain).
• If the doctor thinks it is necessary, the status of the disease will also be checked with standard/routine tests such as positron emission tomography (PET) scans, iodine-131-meta- iodobenzylguanidine (MIBG -- a test that uses injected radioactive material and a special scanner to locate a tumor), and/or a bone scan.
• To measure your heart rate and level of oxygen in the blood, you will have a pulse oximetry test. This test uses a clothepin-shaped device that goes on the finger for about a minute.
• You will have a physical exam, including measurement of vital signs (blood pressure, heart rate, temperature, and breathing rate).
• Your medical history will be recorded.
• Blood (about 4 tablespoons) will be drawn for routine tests and to check for diseases (infections such as the human immunodeficiency virus [HIV]).

You will be told the results of this test for any diseases. If you have one of these infections, you will probably not be able to participate in this study.

• Urine or an additional 4 teaspoons of blood may also be drawn for other routine tests to check the disease status.
• Blood (up to 6 tablespoons) will be drawn and used to test for HLA and KIR typing, and you and the donor will be told the results of this test.
• Females who are able to have children must have a negative serum pregnancy test (as part of the blood sample described above, or possibly an additional 1 teaspoon).

These screening tests would also need to be repeated before an additional NK cell infusion for this study, in order to see if you continue to be eligible to receive one. This will be described further below.

Identifying an Eligible Donor:

Your relative, who must share half of your HLA genes to be eligible for the study, will be tested to see if his or her KIR molecule has the "mismatch" that researchers believe should help the donor's NK cells to target the tumor cells in your body.

The donor's blood will also be tested to guard against the possibility of transmitting an infection to you during the NK cell infusion.

Samples of the donor's blood will be used to help researchers develop future tests that will be used to track how long the infused NK cells survive and function in recipients' bodies. In order to help track the cells after infusion, researchers prefer (but do not require) that if you are a female recipient, your donor should be male and if you are a male recipient, your donor should be female.

Conditioning Phase:

If you are found to be eligible to take part in this study, you will start the "conditioning" phase of this study within 4 weeks after the screening tests. Over the course of 6 days, you will receive chemotherapy with cyclophosphamide and fludarabine to weaken your immune system in order to help the survival of the infused NK cells, and then mesna to protect your bladder from side effects that cyclophosphamide may cause.

Cyclophosphamide, fludarabine, and mesna will preferably be infused through an indwelling catheter (a tube that remains in a vein, such as tunneled in the arm or through the chest). If you already have an indwelling catheter in place, you will not need to have a new one placed. If a new catheter is needed, however, you will be asked to sign a separate informed consent form for its placement.

The Conditioning schedule is the following:

• Starting 6 days before the NK cell infusion (considered Day -6) and once a day through Day -2, you will receive fludarabine by vein, over about 30 minutes.
• On Days -5 and -4, you will receive cyclophosphamide by vein, over about 2 hours each time.
• Five times per day on Days -5 and -4, you will receive mesna by vein, over about 15 minutes each time.

Infusion of NK Cells:

On Day 0 you will receive the NK cells by vein, preferably through an indwelling catheter. The doctor will decide what amount of NK cells will be infused, which will affect how long the infusion lasts, but usually it lasts less than 1 hour.

To help prevent an allergic reaction to the infused cells (such as fever and chills), you will receive Benadryl (diphenhydramine) by vein, over 15-30 minutes, and Tylenol (acetaminophen) by mouth. You will also receive fluids by vein to help decrease the risk of kidney damage.

You or a caregiver will be trained in how to perform the IL-2 injections yourself. This drug will be injected under the skin for 9 doses over the course of 3 weeks.

If the doctor decides you are not eligible to receive the NK cell infusion on Day 0, you will be taken off study without receiving the donor's NK cells.

The collected NK cells will be thrown away.

Blood Test for Measuring NK Cell Survival:

Blood (up to 4 teaspoons each time) will be drawn and tested to see how long the NK cells survive in your body. This blood will be drawn on Day 0 (before the NK cell infusion and again 2 hours later) and on Days 2, 7, 14, 21, and 28. (It is possible that this blood draw schedule will stop earlier if the disease gets worse or the infused NK cells can no longer be seen.)

Possible Additional NK Cell Infusion:

If the neuroblastoma responds and you did not suffer a new intolerable side effect from the NK cells, then you may be eligible to receive 1 additional infusion of NK cells. If so, the rest of the study procedures would be the same as before (the screening tests to determine your eligibility, the requirement that the donor still be eligible, the Conditioning Phase with chemotherapy, the IL-2 injections, and the blood tests). You must use the same donor as before, if he or she is still eligible.

Hospitalization:

So that you can be monitored for side effects, you will need to stay in the hospital from Day -6 until after the NK cell infusion (or longer if medically necessary).

Follow-Up Visits:

After your final NK cell infusion (which may be either the first or second one as part of this study), you will return for follow-up visits at least 3 times a week during the first 3 weeks after the last NK cell infusion, and then once on about Day 28. You will return for follow-up visits within 2 months of infusion and again 3 months after the last NK cell infusion. If possible, you will be asked to return for follow-up visits every 3 months up until one year after the last NK cell infusion. At these visits, the following procedures will be performed:

• Any changes in your medical history will be recorded, and a physical exam will be performed.
• Blood (up to 2 tablespoons) will be drawn for routine tests.
• CT scans of the neck, sinus, chest, abdomen, and pelvis will be performed.
• Up to 2 bone marrow biopsies/aspirations will be performed (within the first 28 days after infusion).
• Sometime in Months 2 or 3, a PET scan will be performed.
• Sometime in Months 2 or 3, and up to 2 more times between Months 3 and 12, urine or an additional 4 teaspoons of blood may be drawn for other routine tests to check the disease status.
• If you had a bone scan and/or MIBG at screening, or if the study doctor feels they are needed, you will have these tests repeated at Month 3.

This is an investigational study. Cyclophosphamide, fludarabine, mesna, and IL-2 are commercially available but not FDA approved for use in neuroblastoma. Injecting IL-2 under the skin is not FDA approved for use in increasing the production of NK cells. The CliniMACS device is not commercially available or FDA approved. Infusing NK cells in patients with neuroblastoma is also considered experimental. At this time and for this purpose, NK cell infusions, IL-2, and the CliniMACS device are being used in research only.

Up to 10 recipients and 10 donors will take part in this study. All will be enrolled at M. D. Anderson.

Donor:

NK Cells:

NK cells are part of the immune system (the cells in the body that fight disease and infection). NK cells can sometimes destroy tumor cells, particularly when the NK cells are "mismatched" for certain proteins called human leukocyte antigens (HLA). Researchers believe this ability to destroy tumor cells can be predicted by learning the donor's and the recipient's HLA types to see if they are a mismatch, and by checking for other specialized proteins on the donor's NK cells (called killer immunoglobulin receptors [KIR]).

Screening Tests:

Before you can take part in this study, you will have screening tests to help the doctor decide if you are eligible to take part. The following tests will be performed:

• Blood (up to 6 tablespoons) will be drawn and tested for HLA and KIR. This blood will also be checked for any diseases (mostly infections such as the human immunodeficiency virus [HIV]) that could possibly be passed on to the recipient. You will be told the results of this test. If you have one of these infections, you will not be able to participate in this study.
• Blood (up to 6 tablespoons) will be drawn before the recipient starts preparative chemotherapy. This blood will be used for comparison so researchers can learn how long your infused NK cells survive in the recipient's body.
• If your sex is different than the recipient's, extra blood (up to 6 tablespoons) will be drawn for fluorescence in situ hybridization (FISH) testing.

This is a test that is used to study how long your infused NK cells survive in the recipient's body.

• Females who are able to have children must have a negative blood pregnancy test (as part of a blood draw described above).

These screening tests would also need to be repeated before an additional leukapheresis procedure for this study, in order to see if you continue to be eligible to have one performed. This will be described further below.

Leukapheresis:

If you are still eligible to take part in this study, a sample of your white blood cells will be collected using a procedure called leukapheresis. For this procedure, blood will be continuously drawn from a vein in your arm and will pass through a tube into a machine. This machine will remove a sample of your white blood cells (the type of cell that fights infections), including the NK cells, and will leave the rest. The other blood cells will be given back to you through a tube and needle inserted in your other arm. This procedure should take up to 6 hours.

During leukapheresis, you will receive a continuous infusion of ACD-A (sodium citrate solution) to decrease the risk of your blood clotting in the tubes.

The level of calcium in your blood may drop to a lower level. Because of this, you will be given a liquid (by vein) that has calcium.

If not enough white blood cells can be collected in the first leukapheresis procedure, a second procedure may be done on the following day.

Your collected white blood cells will be processed in the laboratory, where researchers will use a machine (called a CliniMACS device) to separate out the NK cells and treat them overnight with a drug called interleukin-2 (IL-2). This is in order to make the NK cells stronger ("activated") before they will be given to the recipient.

If the recipient is not eligible to receive the infusion of NK cells, your collected NK cells will be destroyed.

Possible Additional Leukapheresis Procedure:

If the recipient's neuroblastoma responds, and he or she did not suffer a new intolerable side effect from the NK cells, then he or she may be eligible to receive 1 additional infusion of NK cells. This requires that the recipient use the same donor as before. If you agree, and if you continue to be eligible based on another round of the same screening tests, you will repeat the study procedures a second time.

Length of Study:

Your participation in this study will be over after the last leukapheresis that you agree to have performed (which may be either one or two). If you agree to participate in the optional procedures, your participation may last up to 12 months after the recipient's last NK-cell infusion unless:

• the recipient's neuroblastoma gets worse,
• the study is ended early,
• or the study chair removes participant(s) from the study.

This is an investigational study. For the recipient, this study is experimental. However, leukapheresis is a standard procedure for separating white blood cells from the rest of the body.

Up to 10 recipients and 10 donors will take part in this study. All will be enrolled at M. D. Anderson.

• Drug: Fludarabine
• Drug: Cyclophosphamide
• Biological: Natural Killer Cell Infusion

Inclusion Criteria:

• Minimal eligibility: See the following (2 to 3).
• KIR typing will be undertaken after informed consent is signed.
• HLA typing will be undertaken independent of consent.
• Inclusion criteria for recipient (within 4 weeks prior to lymphodepleting conditioning regimen and after consent-signing). As following (5 to 18).
• HLA-haploidentical relative is predicted to be alloreactive based upon the presence of the relevant KIR genes and incompatibility with the recipient. Three conditions must be met simultaneously: a) The KIR-ligand (HLA molecule) for the KIR-receptor gene in question must be ABSENT in the recipient. b) The KIR-receptor gene in question must be PRESENT,that is, not deleted in the DONOR. c) The KIR-ligand (HLA molecule) for the KIR-receptor gene in question must be PRESENT in the donor.
• Progression of neuroblastoma after autologous peripheral blood stem-cell transplantation
• Measurable neuroblastoma disease as defined by at least one radiographic (using any/all of the following modalities CT, MRI, PET, MIBG, Bone scan), abnormality attributed to malignancy
• Platelets >/= 50,000 x 10^ 9/L and hemoglobin (Hgb) >/= 9 g/dL, unsupported by transfusions in last seven days.
• ANC >/= 1,000 x 10^ 9/L, unsupported by cytokines in last seven days.
• Off prednisone or other immunosuppressive medications for at least 3 days
• Zubrod performance scale </= 2 or Lansky >/= 60.
• Adequate renal function defined as: Serum creatinine (Cr), for adults </=2 mg/dL, for children </=2 mg/dL or </=2 times upper limit of normal (ULN) for age (whichever is less). If abnormal renal function then Cr clearance >60 mL/min/1.73m^2.
• Adequate liver function defined as: Total bilirubin </=2 mg/dL and SGPT (ALT) </= 2.5 x ULN for age (unless Gilbert's disease or abnormal liver function due to primary disease).
• Pulmonary symptoms controlled by medication and pulse oximetry >/= 92% room air.
• Negative serum test to rule out pregnancy within 2 weeks prior to registration in females post menarche of childbearing potential (non childbearing is defined as greater than one year post-menopausal or surgically sterilized).
• Requirement of males and females of childbearing potential to use any form of contraception considered effective and medically acceptable by the Investigator.
• Negative serology for (HIV)human immunodeficiency virus.
• Not a breast-feeding female.
• Inclusion criteria for recipient to receive NK infusion(s) as assessed on Day 0. As following (20 to 29).
• Not currently using a ventilator.
• Not currently undergoing dialysis.
• Not currently using a Phase I, II, or III investigational agent. These agents should be stopped within 21 days of NK infusion.
• No requirement for supplemental oxygen.
• No new detected cardiac arrhythmia not controlled with medical management within prior 72 hour period.
• No hypotension requiring pressor support within prior 72 hour period.
• No uncontrolled infection defined as daily fever >/= 38.2 degrees Celsius and no new positive culture for bacteria, fungus, or virus within 72 hours prior to NK-cell infusion, if clinically indicated.
• No ascites requiring paracentesis within prior 72 hour period.
• No seizure activity or clinically detectable encephalopathy or new focal neurologic deficits within prior 72 hour period.
• Not taking corticosteroids by mouth or intravenously within prior 72 hour period.
• Inclusion criteria for donor (within 7 days of apheresis). See following (31 to 40).
• Related to recipient.
• Able and willing to undergo apheresis.
• Willing (but not required) to donate blood for NK cell studies.
• Willing to donate blood for baseline chimerism studies.
• Willing to donate blood for baseline fluorescence in situ hybridization studies.
• Screening infectious disease panel that meets standard medical eligibility criteria for allogeneic blood stem cell donation.
• CBC, differential and platelet studies that meet standard medical eligibility criteria for allogeneic blood stem cell donation.
• Negative serum test to rule out pregnancy within two weeks prior to registration in females of childbearing potential (non-childbearing is defined as greater than one year post-menopause or surgically sterilized).
• Meet standard medical eligibility criteria for allogeneic blood stem cell donation.
• The first two patients enrolled in this protocol will need to have prior cryopreserved autologous peripheral blood stem cells collected within 6 months and/or they are at least 6 months after autologous peripheral blood stem cell rescue. This criterion safeguards against the possibility that the lymphodepletion regimen may lead to prolonged myelosuppression.

Exclusion Criteria:

• Exclusion criteria for donor:(within 7 days of apheresis). As following 2) to 3)

• Active infection.

  1. Febrile
  2. on antibiotics.
• Breast-feeding female.