Natural Killer Cells and Bortezomib to Treat Cancer

Natural killer (NK) cells are innate immune lymphocytes that are identified by the expression
of the CD56 surface antigen and the lack of CD3. Unlike antigen specific T cells, NK cells do
not require the presence of a specific tumor antigen for the recognition and killing of
cancer cells. Our in vitro studies have demonstrated that pretreatment of malignant cells
with bortezomib significantly enhances NK-mediated tumor cytotoxicity by sensitizing cells to
TNF-related apoptosis-inducing ligand (TRAIL). TRAIL is a member of tumor necrosis factor
family of cytokines that promotes apoptosis. Importantly, in our laboratory, in vitro
expanded NK cells isolated from patients with metastatic cancers or hematological
malignancies exhibited significantly more cytotoxicity against their tumor cells when tumors
were pre-treated with bortezomib compared with untreated tumor controls. These findings
suggest that drug-induced sensitization to TRAIL could be used as a novel strategy to
potentiate anticancer effects of autologous adoptively infused NK cells in patients with
cancer.

Murine studies conducted in our laboratory have also established that bortezomib treatment
sensitizes tumors in vivo to killing by adoptively infused syngeneic NK cells; murine renal
cell carcinoma line (RENCA) tumors in BALB/c mice grew significantly slower and survival was
prolonged when syngeneic NK cell infusions were given following bortezomib treatment compared
to mice receiving NK cell infusions alone or bortezomib alone. This anti-tumor effect was
further potentiated by eradicating T-regulatory cells prior to adoptive NK cell infusion and
by administering interleukin-2 after adoptive NK cell infusion.

Recently, our laboratory has developed techniques for the in vitro isolation and expansion of
NK cells to levels suitable for the treatment of cancer patients. Furthermore, we have also
established good viability and sterility of these expanded NK cells which, compared to fresh
NK cells, have increased surface expression of TRAIL and have enhanced cytotoxicity against
tumor cells.

We therefore propose this non-randomized, Phase I, dose escalating study designed to evaluate
the safety and the anti- tumor effects of escalating doses of adoptively infused ex vivo
expanded autologous natural killer (NK) cells against metastatic cancers or hematological
malignancies sensitized to NK TRAIL cytotoxicity with Bortezomib.

The primary study objective is to determine the safety (maximum tolerated dose) of escalating
NK cell doses of adoptively infused ex vivo expanded autologous NK cells in subjects with
treatment refractory metastatic tumors or hematological malignancies that are sensitized to
NK cell cytotoxicity using bortezomib. Secondary objectives will include the anti-neoplastic
effects of this treatment regimen (assessed using standard disease specific response
criteria) and the toxicity profile associated with extended cycles of protocol therapy.

The primary endpoint will be assessed at day 21 (3 weeks after the Day 0 NK cell infusion).

- INCLUSION CRITERIA:

1. Diagnosed with histologically confirmed metastatic solid tumor - cancer of the
lung (small cell or non small cell), prostate (adenocarcinoma), colorectum,
kidney (renal cell carcinoma), pancreas (adenocarcinoma),or malignant melanoma,
metastatic Ewing's sarcoma, or metastatic epithelial neoplasms and adenocarcinoma
of unknown primary, and disease confirmed to be metastatic and unresectable for
which standard curative or beneficial treatments are no longer effective

OR

Diagnosed with a hematological malignancy (multiple myeloma, [MM] chronic
myelogenous leukemia [CML] or chronic lymphocytic leukemia [CLL] or small
lymphocytic lymphoma [SLL]) and disease resistant or refractory to standard
therapy and CLL/SLL patients are required to have failed prior treatment with at
least one nucleoside analogue. Myeloma patients are required to have disease
which has progressed following treatment with bortezomib.

2. At least 4 weeks since any prior systemic therapy (excluding corticosteroid
therapy) to treat the underlying malignancy (standard or investigational). NOTE:
subjects on FDA-approved tyrosine kinase inhibitors or other targeted therapies
for RCC such as mTOR inhibitors that have evidence of disease progression on
therapy may continue these medicines until the time of study enrollment (as
accelerated disease progression following discontinuation of these drugs has been
described).

3. At least 2 weeks since prior palliative radiotherapy.

4. Ages greater than or equal to 18 years and less than or equal to 70 years.

5. Evidence of progressive disease over a 3-month interval.

6. RBC transfusion independent (solid tumor patients only).

EXCLUSION CRITERIA:

1. Disease not evaluable radiographically (applies to solid tumor patients only).

2. Disease involving greater than 25% of the liver radiographically (estimated based on
review of liver lesions seen on CT scan).

3. History of an allogeneic hematopoietic stem cell transplant.

4. Brain metastases (with the exception of patients with a single brain metastasis less
than 1cm treated with either sterotactic or gamma knife radiotherapy) due to poor
prognosis and potential for neurological dysfunction that would confound evaluation of
neurological and other adverse events).

5. Peripheral neuropathy of grade greater than 1, which would require reduction of
bortezomib dose.

6. Acute diffuse infiltrative pulmonary disease.

7. Acute pericardial disease.

8. Life expectancy less than 3 months.

9. ECOG performance status 2, 3 or 4.

10. Uncontrolled concurrent illness including, but not limited to, symptomatic congestive
heart failure, unstable angina pectoris, life threatening cardiac arrhythmia. Patients
with symptoms of coronary artery disease, cardiac arrhythmias or an abnormal thallium
stress test must be evaluated and cleared by cardiology prior to enrollment.

11. Ongoing or active infection

12. Contraindication for administration of pentostatin, bortezomib, and/or interleukin-2.

13. Allergy or hypersensitivity to bortezomib, boron or mannitol by history.

14. Concurrent use of corticosteroids.

15. For all tumor types:

Marrow function characterized by

-Absolute neutrophil count less than 500/mcL (must be present off growth factors)

Organ function characterized by

- Total bilirubin greater than 3 times upper limit of normal

- AST (SGOT)/ALT (SGPT) greater than 4 times upper limit of normal

- Creatinine clearance less than 50 cc/min based on a 24 hour urine collection

- Left ventricular ejection fraction less than 40% by echocardiogram (ECHO)

- Hypercalcemia greater than 2.5 mmol/L

For all Hematologic malignancies:

Marrow function characterized by

- Neutrophil count less than or equal to 500/mcl

- Platelets less than or equal to 20,000/mcl

16. HIV-positive patients

17. Hepatitis C positive patients (Hep C PCR positive)

18. Active Hepatitis B infection (Hep B surface antigen positive)

19. Pregnant or nursing

20. Psychiatric illness/social situations that would limit compliance with study
requirements and ability to comprehend the investigational nature of the study and
provide informed consent.