Treatment of B-Chronic Lymphocytic Leukemia (B-CLL) With Autologous CD40 Ligand and IL-2-Expressing Tumor Cells

B-CLL samples from patients will accession the GMP facility according to Standard Operating
Procedures (SOP). B-CLL cells will be isolated from peripheral blood or a leukopheresis
collection (depending on cell count) with a target of 10^10 (minimum 5 x 10^9) tumor cells.

B-CLL cells will be separated in two aliquots. One aliquot will be co-cultured for 18-24
hours with irradiated (3,000 cGy) wild-type MRC-5. Co-cultured B-CLL cells will then be
collected, washed and transduced by the human IL-2 adenoviral vector (AdhIL-2). After
transduction, B-CLL will be washed and frozen. The second aliquot will be co-cultured for
18-24 hours with irradiated (3,000 cGy) hCD40L-transduced MRC-5 co-cultured B-CLL cells will
then be collected, washed and frozen. CD40-ligand up-regulation will be checked immediately
after 24 hours co-culture gating on the CD5-CD19 double positive population: Jurkat human
leukemia T-cell line (constitutively expressing surface human CD40-ligand) will be used as
positive control for CD40-ligand up-regulation measurement. An aliquot of the
AdhIL-2-transduced B-CLL cells will be maintained in culture to check IL-2 secretion.
Adequate hCD40L-expression will be > 20% positive viable B-CLL cells by flow cytometry
measured immediately after co-culture on -transduced MRC-5. Adequate IL-2 production will be
50pg/10^6 cells/24hrs by ELISA, measured 72 hours after transduction. B-CLL cells will be
frozen and an aliquot sent for safety testing. The cells may be released as soon as all
safety testing is complete and adequate hCD40L expression and IL-2 production has been
verified, or stored while the patient is receiving other therapy as clinically indicated.

When vaccine administration is due, the cells will be thawed, mixed and irradiated
IL-2-B-CLL and hCD40L-B-CLL cells will be irradiated to 3,000 cGy, a level that permits
continued cytokine secretion and surface markers expression while preventing cell division
measured by cell number count and by Thymidine incorporation assay. Any contaminating MRC-5
cells will have received a total of 6,000 cGy, which entirely blocks their proliferation,
again measured by cell number count and by Thymidine incorporation assay.

Prior to infusion, an aliquot of the final vaccine product will be tested for sterility

Patients will be injected subcutaneously.

Schedule of vaccine administration Patients will receive a fixed dose (2 x 10^7) of IL-2
secreting B-cells together with (2 x 10^7) hCD40L expressing B-cells, representing a safe,
well tolerated and immunogenic dose from our previous study. The injection will be performed
subcutaneously in the deltoid region of the upper arm. Based on the pattern and duration of
anti-tumor immune response in our first study, patients will receive 8 doses of vaccine at
1-2 week intervals for 12 weeks (see table), followed by 4 doses of vaccine at 4 weekly
intervals. If an SP response is sustained during this second course, or a clinical response
obtained, 6 final doses of vaccine will be given, again at 4 weekly intervals. Time will be
measured as week post-first vaccine injection.

Administration IL-2-B-CLL and hCD40L-B-CLL cells will be given by subcutaneous injection in
Dulbecco's Phosphate Buffered Saline with 10% Human Serum Albumin according to the schedule
in a volume of up to 1mL.

Supportive Care Patients will receive supportive care for acute or chronic toxicity,
including blood components or antibiotics (other than for active infection at the time of
enrollment), and other intervention as appropriate.

PATIENT EVALUATION Pre-Treatment Evaluation A complete history and physical examination with
documentation of measurable lesions is necessary prior to admission.

Baseline serology will be performed at the time of blast collection. They will include
anti-HIV1, anti-HIV2, HIV1-Ag, anti-HTLV, HBs-Ag, anti-HBc, anti-HCV, anti-CMV and syphilis
serology. Results are to be obtained within 30-days of the tumor cell collection.

HLA typing will be obtained prior to vaccine administration, whether or not the patient is
eligible for bone marrow transplantation: it will serve to further analyze the immune
response after vaccination.

Evaluation During Vaccine Administration Vaccine administration. The injection will be
performed subcutaneously in the deltoid region of the upper arm. The following schedule will
be used

To be evaluable patients will receive at least 6 doses of vaccine. Time is measured as week
post-first vaccine injection: If an SP or non-SP tumor cell response is seen after 12 doses
of vaccine, the patients may receive an additional 6 doses according to the above schedule.

Patients shall be seen pre-study and at weekly intervals for 10 weeks, then on week 12 and
every four weeks for one year. Additional visits will be obtained as clinically indicated,
and annually thereafter for 2 years the patients will be followed.

NCI Working Group criteria for response are used:

Complete remission (CR): requires all of the following; disappearance of all palpable
disease; a lymphocyte count less than 4,000/uL; a neutrophil count greater than 1,500/uL; a
platelet count greater than 100,000/uL; a hemoglobin level greater than 11g/dL; and a bone
marrow aspirate lymphocyte percentage of less than 30% with no nodules.

Partial remission (PR): requires greater than or equal to 50% decrease in palpable disease
as well as greater than or equal to 50% improvement of all abnormal blood parameters or
fulfilling the criteria noted as CR.

Progressive disease (PD): is characterized by at least one of the following; greater than or
equal to 50% increase in palpable disease; greater than or equal to 50% increase in
lymphocyte count to at least 5,000/uL; transformation to a more aggressive histology.

Stable disease (SD): no change or a response less than PR is considered stable disease

Physical examination will be performed pre each injection. Injection sites, lymph nodes,
spleen and liver will be examined.

Biological studies: Peripheral blood: depending on the time-point (See Table 2 attached in
Section S), 30-40 ml of peripheral blood will be collected pre each injection and then every
four weeks for 6 months after the last injection and then annually thereafter for 5 years.
If a patient's hemoglobin is less than 8.0g/dl at any of the evaluation times, the amount of
blood drawn for the evaluation will be reduced and may be obtained over more than one
venipuncture, if necessary. If there is insufficient blood, these tests will be prioritized
in the order given. In the event that the patients clinical condition does not warrant
weekly blood draws, preference will be given to the following time points: week 0, week 4,
week 10, then 4 weekly for one year and then annually for 5 years. When any injection is
performed, the patient will be checked for CBC, differential, platelet count, BUN,
creatinine, SGOT and immune analysis. The following assays will be performed in order to
study the immune response: numeric and phenotypical characterization of circulating
leukocyte sub-populations, including analysis of CD4+, CD8+ and CD25+ T-lymphocytes and
CD16+ and CD56+ NK cells and tumor SP cells. When sufficient patient leukocytes are
available, we will also assess the number of precursors reacting against autologous B-CLL
cells using IFN-gamma ELISPOT assay. Plasma will be tested for the presence of
immunoglobulins against autologous B-CLL cells. A bone marrow aspirate sample will be
collected at the beginning of the vaccination period and at the end of the vaccination
period.

Disease Assessment: Patients with measurable disease at inclusion will have serial
measurement of B-CLL cell counts in peripheral blood and/or bone marrow, per the clinical
team recommendation. The B-CLL cell count will be considered for response in conjunction
with other criteria according to the definition given in Appendix B. Tumor response will be
evaluated at a minimum at four, ten and 28-32 weeks and 52 weeks, then 4 months after the
last injection and then annually for 5 years. Further follow-up will be per the referring
physician.