G-CSF and AMD3100 to Mobilize Stem Cells in Healthy Volunteers

Peripheral blood progenitor cells (PBPC) are the most popular source of hematopoetic stem
cells for allogeneic transplantation because of technical ease of collection and faster
engraftment. Traditionally, granulocyte-colony stimulating factor (G-CSF) has been used to
procure the peripheral blood stem cell graft. Although regimens using G-CSF usually succeed
in collecting adequate numbers of PBPC from healthy donors, 5%-10% of the donors will
mobilize stem cells poorly and may require multiple large volume apheresis or bone marrow
harvesting. AMD3100 reversibly inhibits CXCR4 binding to stromal cell derived factor (SDF) -
1 and was recently discovered to be an effective agent to mobilize CD34+ cells into the
peripheral blood. In normal volunteers, administering AMD3100 after 4-5 days of G-CSF
resulted in a 3-3.5 fold increase in circulating CD34 cells compared to G-CSF alone. Recent
data has suggested that the combination of G-CSF and AMD3100 is superior to G-CSF alone for
mobilizing hematopoietic progenitor cells in heavily pretreated patients with multiple
myeloma or non-Hodgkin's lymphoma undergoing autologous hematopoietic transplantation.
Combining AMD3100 with G-CSF could be an effective strategy to improve the yield of PBPC
collected from allogeneic donors who mobilize poorly with G-CSF alone. However, the
biological impact of AMD3100 in this context on T cells and other cellular populations
contained within the allograft that mediate GVHD and graft-versus-leukemia (GVL) effects are
unknown.

We propose to collect PBPC from healthy volunteers following 5 days of G-CSF (10 mcg/kg/day)
and a single dose of AMD3100 (240 mcg/kg subcutaneous given 12 hours before starting
apheresis) to study the impact of combining these two mobilizing agents on the immunological
properties of the mobilized cells. A single 15 liter apheresis will be conducted on day 5
following the 5th dose of G-CSF. The immunological studies conducted on these mobilized
cells will be the same as our parallel study which is investigating the immune properties of
PBPCs mobilized with G-CSF or AMD3100 alone. If combining AMD3100 with G-CSF has no negative
impact on the immune populations involved in GVHD and graft-vs-leukemia effects, this
regimen could be used for allogeneic donors who fail to mobilize sufficient PBSC using G-CSF
alone.

Primary objective: To determine the cytokine polarization status of CD4+ T-cells collected
by apheresis following combination of AMD3100 and G-CSF compared to G-CSF mobilization.

Primary endpoint: the ratio of Th1 (intracellular IFN-g +) versus Th2 (intracellular IL-4+)
T-cells in the apheresis products collected from individual donors undergoing mobilization
with combination of G-CSF and AMD3100 to the ratio in apheresis product collected with G-CSF
alone (ratio published in literature).

Secondary endpoints: To examine 1) the cellular content and other immune properties of
mobilized cells; 2) yields of hematopoietic progenitor cells, immune cells, and other
cellular subsets collected by apheresis; and the 3) safety profile of AMD3100.

- INCLUSION CRITERIA:

Healthy volunteers greater or equal to 18 years old, less than or equal to 60 years.

Weight greater than 60 kg (132 pounds)

Normal renal function: creatinine less than 1.5 mg/dl l

Normal liver function: bilirubin less than1.5mg/dl, transaminases within normal limit

Normal blood count: WBC 3000-10000/mm3, granulocytes greater than 1500/mm3, platelets
greater than 150,000/mm3, hemoglobin greater than 12.5g/dl

Subject must be eligible for normal blood donation and fit to undergo apheresis procedure
(antecubital veins must be adequate for peripheral access during apheresis)

Ability to comprehend the investigational nature of the study and provide informed consent

EXCLUSION CRITERIA: any of the following

Active infection or history of recurrent infection or positive test for syphilis (RPR),
hepatitis B and C (HBaSAg, Anti-HCV), HIV and HTLV-1

History of autoimmune disease such as rheumatoid arthritis, systemic lupus erythematous

History of cancer within the past 5 years excluding basal cell or squamous cell carcinoma
of the skin

History of any hematologic disorders including thromboembolic disease

History of cardiac disease such as uncontrolled hypertension, peripheral vascular disease,
myocardial infarction, cardiac arrhythmias or related symptoms such as tachycardia, chest
pain, shortness of breath which have required medical intervention or treatment or a
Framingham coronary disease risk prediction score of greater than 10% 10 year CHD risk

History of heavy smoking with underlying pulmonary disease

History of cerebrovascular disease, transient ischemic attack, or stroke

Diagnosis of sickle cell anemia or sickle cell trait (to be screened by Hbg
electrophoresis)

Pregnant or lactating

Severe psychiatric illness: mental deficiency sufficiently severe as to make informed
consent impossible.

Mobilization with G-CSF within 90 days of protocol enrollment.