A Phase I Study of NK Cell Infusion Following Allogeneic Peripheral Blood Stem Cell Transplantation From Related or Matched Unrelated Donors in Pediatric Patients With Solid Tumors and Leukemias

Background

- Despite progress in pediatric oncology, some patient subsets with hematologic
malignancies and pediatric solid tumors continue to experience extremely poor overall
survival. Allogeneic Hematopoietic Stem Cell Transplant (HSCT) is effective in some
high-risk hematologic malignancies.

- Allogeneic HSCT can be performed safely in these patient populations, but disease
recurrence is common and new approaches to enhance the antitumor effect of this therapy
are needed. NK mediated killing appears to confer improved outcomes after HSCT for
patients with AML and ALL, and NK cell infusions have induced complete remissions in
patients with AML.

- Preclinical data demonstrates that activated NK cells readily kill pediatric solid
tumors and leukemias, that large numbers of activated NK cells can be generated ex vivo
using artificial APCs and that the post-transplant period may be favorable for expansion
and survival of adoptively transferred NK cells.

Objectives

- To assess the feasibility and toxicity of infusing escalating doses of donor-derived
activated NK cell donor lymphocyte infusions (NK-DLI) on Days 7 plus or minus 2 days and
49 plus or minus 7 days following HLA-matched T cell depleted (TCD) PBSCT in patients
with metastatic or recurrent pediatric solid tumors and high risk leukemias who have
unrelated donors or related donors;

- To determine if patients treated in this manner experience rapid, sustained donor
engraftment and acceptable rates of aGVHD (less than 25% incidence of grade III or grade
IV).

Eligibility

-Patients 4-35 years with hematologic malignancies (e.g., ALL, AML, CML, HD, NHL), with a 5/6
or 6/6 HLA-matched related or 9/10 or 10/10 HLA matched unrelated donor.

Design

- Pre-transplant disease specific immune depleting chemotherapy and the preparative
regimen will be the same as that used previously on 02-C-0259 and 01-C-0125, for those
patients undergoing reduced intensity transplant.

- For patients with ALL or AML, a myeloblative regimen based on current COG standard-of-
care preparative regimen will also be included.

- Donors will undergo 1-3 apheresis sessions for filgrastim mobilized PBSC. This product
will be T cell and NK cell depleted prior to cryopreservation. NK cells selected from
the product will be used for ex vivo activation and expansion using KT64.4-BBL
artificial antigen presenting cells.

- A phase 1 cell dose escalation of donor derived NK-DLI will be performed using 3 dose
levels (1 x 105, 1 x 106 and 1 x 107 NK cells/kg) infused on days 21 more or less 3
post-PBSCT and a second infusion on day 49 more or less 7 post-PBSCT.

- Three patients will be enrolled at each dose level, with the cohort expanded to 6 if
dose-limiting toxicity occurs. An expanded group of 12 patients will be treated at the
highest dose level tolerated.

- INCLUSION CRITERIA: PATIENTS (RECIPIENT)

- Hematologic Malignancies Diagnoses:

1. Acute lymphoblastic leukemia (ALL) with a history of bone marrow relapse in
clinical remission (CR) #2 or greater, or in CR#1 if prior induction failure; or
with an M1 marrow if unable to achieve CR.

2. Philadelphia chromosome positive ALL patients who;

1. Have progressed through or relapsed following TKI therapy or conventional
myeloablative therapy

OR

2. Are ineligible to receive tyrosine kinase inhibitor (TKI) therapy AND
myeloablative HSCT

3. Acute Myelogenous Leukemia (AML) with a history of bone marrow relapse in
remission CR #2 or greater; or with an M1 marrow if unable to achieve CR; or in
CR#1 if prior induction failure; or any of the following High-Risk categories:

1. FLT3/ITD+ with high allelic ratio > 0.4 (HR FLT3/ITD+)

regardless of low risk features.

2. Presence of monosomy 7, monosomy 5, or del5q, without

inv(16)/t(16;16) or t(8;21) cytogenetics or NPM or CEBP(alpha)

mutations.

3. AML without inv(16)/t(16;16), t(8;21), NPM, CEPB(alpha) mutations, monosomy
7, monosomy 5, del5q, or HR FLT3/ITD+,

but with evidence of residual AML (greater than or equal to 0.1%) at end of
Induction I.

4. Hodgkin s and Non-Hodgkin s Lymphoma with refractory disease or relapse after at
least one salvage regimen, or after autologous stem cell transplant

5. Juvenile Myelocytic Leukemia (JMML) with less than 10% blasts in marrow and
blood, who are not eligible for effective standard therapies.

- Age: 4 to less than or equal to 35 years old at the time of enrollment for solid tumor
patients and 4 to less than or equal to 35 years old for hematologic malignancies.

- All previous cytotoxic chemotherapy must be completed at least 3 weeks prior to study
entry. Any prior non-hematologic vital organ toxicity (cardiac, pulmonary, hepatic,
renal) of any previous therapy must have resolved to grade 1 or less, unless specified
elsewhere in Inclusion Criteria for Patient (Recipient).

EXCEPTIONS:

There is no time restriction in regard to prior intrathecal chemotherapy provided there is
complete recovery from any acute toxic effects; or

Subjects receiving standard ALL maintenance chemotherapy will not require washout.

- All previous immunologic or molecularly targeted therapy must be completed at least 1
week prior to study entry. Any prior non-hematologic toxicity of any previous therapy
must have resolved to grade 1 or less, unless specified elsewhere in Inclusion
Criteria for Patient (Recipient).

- Prior investigational therapy must be completed at least 30 days prior to study entry

- Patients with prior autologous or allogeneic transplant are eligible. Patients must be
greater than 100 days post transplant and have no evidence of active GVHD.

- Performance status: ECOG 0, 1 or 2, or for children less than or equal to 10 years of
age, Lansky greater than or equal to 60. Life expectancy greater than 3 months.

- Availability of HLA-matched (5-6/6 antigen or 8/8 allele) related or unrelated donor.

- Cardiac function: Left ventricular ejection fraction greater than or equal to 45% by
MUGA or ECHO, fractional shortening greater than or equal to 28% by ECHO.

- Pulmonary function: DLCO >= 40% of the expected value corrected for alveolar volume
and hgb for reduced intensity transplant and DLCO >=55% for myeloablative regimen. For
children who are unable to cooperate for PFTs, the criterion is: No evidence of
dyspnea at rest, no exercise intolerance, and no requirement for supplemental oxygen
therapy.

- Liver function: Serum total bilirubin less than 2 mg/dl, serum AST and ALT less than
or equal to 2.5 times upper limit of normal. Patients with Gilbert syndrome are
excluded from the requirement of a normal bilirubin. (Gilbert syndrome is found in
3-10% of the general population, and is characterized by mild, chronic unconjugated
hyperbilirubinemia in the absence of liver disease or overt hemolysis).

- Renal function: Age-adjusted normal serum creatinine according to the following, or a
creatinine clearance greater than or equal to 60 ml/min/1.73 m(2):

- For age (years) of less than or equal to 5, a Maximum serum creatinine (mg/dl) of
0.8

- For age (years) of greater than 5 but less than or equal to 10, a Maximum serum
creatinine (mg/dl) of 1.0

- For age (years) of greater than 10 but less than or equal to 15, a Maximum serum
creatinine (mg/dl) of 1.2

- For age (years) of greater than 15, a Maximum serum creatinine (mg/dl) of 1.5

- Marrow function: ANC must be greater than 750/mm(3) (unless due to underlying disease
in which case there is no grade restriction), platelet count must be greater than or
equal to 75,000/mm(3) (not achieved by transfusion) unless due to underlying disease
in which case there is no grade restriction). Lymphopenia, CD4 lymphopenia,
leukopenia, and anemia will not render patients ineligible.

- Ability to give informed consent. For patients less than 18 years of age their legal
guardian must give informed consent. Pediatric patients will be included in
age-appropriate discussion in order to obtain verbal assent.

- Durable power of attorney form completed (patients greater than or equal to 18
years of age only).

- Female patients (and when relevant their male partners) must be willing to
practice birth control (including abstinence) during and for two months after
treatment, if of childbearing potential.

EXCLUSION CRITERIA: PATIENT (RECIPIENT)

- Uncontrolled infection.

- Active CNS malignancy as defined by:

1. Solid Tumors: History of untreated CNS tumor involvement. Extradural masses which
have not invaded the brain parenchyma or parameningeal tumors without evidence
for leptomeningeal spread will not render the patient ineligible. Patients with
previous CNS tumor involvement are eligible IF the CNS tumor(s) has been treated
and has been stable or resolving for at least 6 months; and if the patient does
not currently require steroids.

2. Lymphoma: tumor mass on CT scan or leptomeningeal disease

3. Leukemia: CNS 2 or CNS 3 classification.

- Lactating or pregnant females (due to risk to fetus or newborn).

- HIV positive (due to unacceptable risk associated with severe immune suppression).

- Hepatitis B surface antigen (HBsAg) positive or hepatitis C antibody positive with
elevated liver transaminases. All patients with chronic active hepatitis (including
those on treatment) are ineligible.

- Patients who require systemic corticosteroid or other immunosuppressive therapy.
Immunosuppressive therapy must be stopped at least 28 days prior to protocol C1D1.
Topical agents and/or inhaled corticosteroids are permitted.

- High risk of inability to comply with transplant protocol, or inability to give
appropriate informed consent in the estimation of the PI, social work, psychiatry, or
the stem cell transplant team.

- Fanconi Anemia

- Clinically significant systemic illness (e.g. serious active infections or significant
cardiac, pulmonary, hepatic or other organ dysfunction), that in the judgment of the
PI would likely compromise the patient s ability to tolerate protocol therapy or
significantly increase the risk of complications.

INCLUSION CRITERIA: DONOR

- Weight greater than or equal to 15 kilograms and for unrelated donors, greater than or
equal to 18 years.

- HLA-matched related or unrelated allogeneic donors. Genotypically identical twins may
serve as stem cell donors. Related donors must be 5 or 6/6 antigen matched. Unrelated
donors must be 8/8 allele matched.

- For donors less than 18 years of age, he/she must be the oldest suitable donor, their
legal guardian must give informed consent, the donor must give verbal assent, and
he/she must be cleared by social work and a mental health specialist to participate.

- For donors greater than or equal to 18 years of age, ability to give informed consent.

- Adequate peripheral venous access for apheresis or consent to use a temporary central
venous catheter for apheresis.

- Donor selection will be in accordance with NIH/CC Department of Transfusion Medicine
(DTM) criteria and, in the case of an unrelated donor, the National Marrow Donor
Program (NMDP) standards and FDA 21 CFR 1271.

EXCLUSION CRITERIA: DONOR

- History of medical illness that in the estimation of the PI or DTM/NMDP physician
poses prohibitive risk to donation including, but not limited to, stroke, hypertension
that is not controlled with medication, or heart disease. Individuals with symptomatic
angina or a history of coronary bypass grafting or angioplasty will not be eligible.

- Anemia (Hb less than 11 gm/dl) or thrombocytopenia (less than100,000/microliters).

- Identical twins will be excluded; the lack of MHC incompatibility will alter the
toxicity profile in such a way as to make the results uninterpretable.

- Breast feeding or pregnant females. Donors of childbearing potential must use an
effective method of contraception during the time they are receiving filgrastim. The
effects of cytokine administration on a fetus are unknown and may be potentially
harmful. The effects upon breast milk are also unknown and may potentially be harmful
to the infant.

- High risk of inability to comply with protocol requirements as determined by the
principal investigator and donor center team.

- Positive screening test for transfusion-transmissible infection in accordance with DTM
or NMDP donation standards, including HIV-positive, hepatitis B surface antigen
(HBsAg) positive or hepatitis C antibody positive.