Improving Blood Stem Cell Collection and Transplant Procedures

Peripheral blood stem cell transplant research carried out by the NHLBI BMT Unit focus on
transplant techniques designed to decrease graft versus host disease (GVHD), increase the
graft-versus-leukemia (GVL) effect and reduce the risk of post-transplant graft rejection.

Through incremental transplant clinical trials we have shown that by controlling the stem
cell (CD34+ cell) and T lymphocyte (CD3+ cell) dose, severe GVHD can be reduced whilst
beneficial GVL effects can be preserved. We found that T cell depleted transplants using the
Nexell/Baxter Isolex 300i system and subsequently, the Miltenyi CD34+ CliniMACs system to
obtain high CD34+ doses depleted of lymphocytes were safe to administer and associated with
less severe acute GVHD and promising response rates and overall survival. Our previous trials
have helped us to create the transplant environment (significant lymphodepletion and minimal
post transplant immunosuppression) that make for an ideal platform for adoptive cellular
immunotherapy

This protocol is designed to evaluate the safety and efficacy of an improved ex vivo graft
manipulation procedure using the Miltenyi CliniMACs CD 34, 3 and 19 selection system in
HLA-matched sibling allogeneic peripheral blood stem cell transplant. The manipulation of the
graft is the primary research intervention and all other aspects of clinical management on
this protocol are the standard of care. The target CD34+ dose range will be 3 times 10(6)/kg
to 10 times 10(6)/kg and the target CD3+ dose range will be 5 x 10(4)/kg to 1 times 10(6)/kg.
The technique will preserve greater numbers of NK cells. Once we demonstrate adequacy of
engraftment in the first ten recipients, we plan an amendment to permit further use of this
protocol to serve as a platform for several planned adoptive cellular therapy initiatives.

The protocol will accrue up to 44 subjects aged 10-75 with a hematological malignancy and
their HLA-matched sibling donors, in whom allogeneic stem cell transplantation from an
HLA-matched sibling would be routinely indicated. Diagnostic categories will include acute
and chronic leukemia, myelodysplastic syndromes, lymphomas, multiple myeloma and
myeloproliferative syndromes.

Subjects will receive a myeloablative conditioning regimen of cyclophosphamide (120 mg/kg
total), fludarabine (125 mg/m(2) total) and total body irradiation (1200 cGy with lung
shielding to 600 cGy), followed by an infusion of a stem cell product prepared using the
Miltenyi CliniMACS system for CD34, 3 & 19 selection. Older subjects will receive a lower
dose of irradiation (600 cGy) without lung shielding to reduce the regimen intensity.

The overall objective is to assess the feasibility of using this system as a platform for
cellular immunotherapy initiatives. The primary study endpoint will be overall survival at
day plus 200. Non-relapse mortality at day plus 200 will be monitored for safety. Secondary
endpoints will be standard transplant outcome variables such as non-hematologic toxicity,
incidence and severity of acute and chronic GVHD and relapse of disease.

- INCLUSION CRITERIA:

Patients in remission as well as patients with primary induction failure or refractory
disease will be considered for inclusion. At the discretion of the PI, patients may
continue standard of care treatment options to control their baseline disease burden up to
the start of the protocol.

RECIPIENT

Ages 10-75 years inclusive

Chronic myelogenous leukemia (CML):

- Subjects under the age of 21 in chronic phase

- Subjects ages 10-75 in chronic phase who have failed treatment with imatinib, have
intolerance to imatinib, or who did not receive imatinib at therapeutic doses within
the first 12 months from diagnosis.

- Subjects ages 10-75 in accelerated phase or blast transformation.

Acute lymphoblastic leukemia (ALL): any of these categories: ALL in first remission with
high-risk features (presenting leukocyte count >100,000/cu mm, Karyotypes t9; 22, t4, t19,
t11, biphenotypic leukemia) All second or subsequent remissions, primary induction failure,
partially responding or untreated relapse.

Acute myelogenous leukemia (AML): AML in first remission - except AML with good risk
karyotypes: AML M3 (t15; 17), AML M4Eo (inv 16), AML t (8; 21). All AML in second or
subsequent remission, primary induction failure and resistant relapse.

Myelodysplastic syndromes(MDS): any of these categories - refractory anemia with
transfusion dependence, refractory anemia with excess of blasts, transformation to acute
leukemia, chronic myelomonocytic leukemia, atypical MDS/myeloproliferative syndromes.

Myeloproliferative disorders including atypical (Ph negative) chronic myeloid and
neutrophilic leukemias, progressing myelofibrosis, and polycythemia vera, essential
thrombocythemia in transformation to acute leukemia or with progressive transfusion
requirements or pancytopenia.

Chronic lymphocytic leukemia refractory to fludarabine treatment and with bulky progressive
disease or with thrombocytopenia (less than or equal to 100,000/ micro-l) or anemia (less
than or equal to 10g/dl) not due to recent chemotherapy.

Non-Hodgkin s lymphoma including Mantle cell lymphoma relapsing or refractory to standard
of care treatments.

Multiple myeloma, Waldenstroms macroglobulinemia, unresponsive or relapsed following
standard of care treatments.

HLA identical (6/6) related donor.

For adults: Ability to comprehend the investigational nature of the study and provide
informed consent. For minors: written informed consent from one parent or guardian.
Informed oral consent from minors: the process will be explained to the minor on a level of
complexity appropriate for their age and ability to comprehend.

DONOR

Related donor, HLA identical (6/6) with recipient

Weight greater than or equal to 18 kg

Age greater than or equal to 2 or less than or equal to 80 years old

For adults: Ability to comprehend the investigational nature of the study and provide
informed consent. For minors: Written informed consent from one parent or guardian and
informed assent: The process will be explained to the minor on a level of complexity
appropriate for their age and ability to comprehend.

EXCLUSION CRITERIA:

RECIPIENT (any of the following)

Estimated probability of surviving less than three months

Major anticipated illness or organ failure incompatible with survival from transplant

Severe psychiatric illness. Mental deficiency sufficiently severe as to make compliance
with the transplant treatment unlikely and making informed consent impossible.

Positive pregnancy test for women of childbearing age.

HIV positive

DLCO adjusted for Hb and ventilation < 50% predicted

Left ventricular ejection fraction < 40% (evaluated by ECHO) or < 30% (evaluated by MUGA)

AST/SGOT > 10 times ULN (>grade 3, CTCAE)

Bilirubin > 5 times ULN (>grade 3, CTCAE)

Creatinine > 4.5 times ULN (>grade 3, CTCAE)

Prior allogeneic stem cell transplantation

DONOR (any of the following)

Pregnant or breast-feeding. Lactating donors are permitted provided breastmilk is discarded
during the days filgatrim (G-CSF) is given.

Unfit to receive G-CSF and undergo apheresis (abnormal blood counts, history of stroke,
uncontrolled hypertension)

Sickling hemaglobinopathy including HbSS, HbAS, HbSC

Donors who are positive for HIV active hepatitis B (HBV), hepatitis C (HCV) or human T-cell
lymphotropic virus (HTLV-I/II)

Severe psychiatric illness. Mental deficiency sufficiently severe as to make compliance
with the BMT treatment unlikely, and making informed consent impossible.

Age greater than or equal to 80 years old

Children who weigh less than or equal to 18 kg and are < 2 years of age.