TABLE OF CONTENTS

Page

SCHEMA........................................................................................................................................ 2

1. OBJECTIVES........................................................................................................................... 6

2. BACKGROUND...................................................................................................................... 7

2.1 Role of Vascular Endothelial Growth Factor (VEGF) in Angiogenesis............................... 7

2.2 Malignant Mesothelioma

2.3 Preclinical Studies with Vascular Endothelial Growth Factor Antisense (VEGF-AS)........ 11

2.4 Animal Clinical Toxicology............................................................................................. 13

2.5 Pharmacokinetic studies 14

2.6 Different VEGF compounds 14

2.7 Phase I/II trial of VEGF-AS in humans with refractory malignancy 16

3. DRUG FORMULATION AND PROCUREMENT............................................................ 20

3.1 VEGF-Antisense Oligonucleotide (VEGF-AS).......................................................... 20

3.1.2 How Supplied......................................................................................................... 20

3.1.3 Reconstitution.......................................................................................................... 20

3.1.4 Route of Administration........................................................................................... 21

3.1.5 Storage................................................................................................................... 21

3.1.6 Stability................................................................................................................... 21

3.1.7 Compatibility........................................................................................................... 21

3.1.8 Availability 21

4. PATIENT SELECTION......................................................................................................... 22

4.1 Eligibility Criteria ...................................................................................................... 22

4.2 Exclusion Criteria........................................................................................................... 23

...............................................................................................................................................

5. TREATMENT PLAN............................................................................................................ 25

5.1 Drug Administration................................................................................................... 25

5.2 Common Toxicity Criteria.............................................................................................. 27

5.3 Supportive Care Guidelines....................................................................................... 27

5.4 Duration of Therapy................................................................................................... 27

6. EXPECTED TOXICITIES/DOSE MODIFICATIONS....................................................... 28

6.1 Expected Toxicities.................................................................................................... 28

6.2 Dosing Delays/Dose Modifications........................................................................... 28

6.3 Definition of Unacceptable toxicity 29

7. CORRELATIVE/SPECIAL STUDIES................................................................................. 29

7.1 Biologic studies of VEGF and VEGFR status 29

8. STUDY CALENDAR............................................................................................................. 31

9. CRITERIA FOR EVALUATION AND ENDPOINT DEFINITIONS............................... 33

9.1 Study Outcome 33

9.2 Definition of Response 33

9.3 Time to Tumor Progression (TTP) 33

9.4 Best Overall Response/Endpoint Definitions 33

9.5 Duration of Response 33

9.6. Overall Survival 34

10. REGULATORY AND REPORTING REQUIREMENTS.................................................. 34

10.1 Adverse Drug Experience Reporting........................................................................ 34

10.1.1 AEs to be reported to VGTI.................................................................................... 34

11. STATISTICAL CONSIDERATIONS.................................................................................. 36

11.1 Summary of Design 36

11.2 Analysis of Results 41

12. RECORDS TO BE KEPT AND DATA SUBMISSION SCHEDULE 46

13. REFERENCES....................................................................................................................... 47

APPENDICES

APPENDIX A, Performance Status Scale

APPENDIX B, IMIG Staging System

APPENDIX C. Blood Processing

APPENDIX D. Data and Safety Monitoring Plan

1.0 OBJECTIVES

Primary objective:

1.1.1 To determine the time to progression of patients with malignant mesothelioma who are treated with two different doses of VEGF-AS.

Secondary objectives:

1.2.1 To determine the objective response rate of patients with malignant mesothelioma who are treated with VEGF-AS

1.2.2 To determine the toxicity experienced by patients with malignant mesothelioma who are treated with VEGF-AS.

1.2.3 To determine median and overall survival of patients with malignant mesothelioma who are treated with VEGF-AS.

Laboratory objectives:

1.3.1 To measure plasma VEGF levels before, during, and after therapy as a predictor of outcome.

1.3.2 To collect and store serum samples for possible future assessment of other antiangiogenic inhibition markers.

2.0 BACKGROUND

2.1 Malignant Mesothelioma

The causative relationship between asbestos and malignant mesothelioma was first recognized in 1960, when an unusually high incidence of the disease was observed in South African asbestos miners (Wagner, 1960). About 2500-3000 cases are diagnosed in the United States each year (Kindler, 1999). Malignant mesothelioma usually arises from the pleura or peritoneum; less common sites of origin include the pericardium and the tunica vaginalis. Extensive local progression results in death either from respiratory failure or from bowel obstruction and inanition. Median survival ranges from 6 to 15 months (Kindler, 1999). Prognostic factors predictive of poor survival include nonepithelial histology, chest pain, thrombocytosis, leukocytosis, anemia, poor performance status, advanced age, elevated LDH, symptom onset within 6 months of diagnosis, weight loss, and male gender (Herndon, 1998; Curran, 1998).

Current therapeutic interventions have not appreciably affected the natural history of malignant mesothelioma (Alberts, 1988). Surgery is possible in only a small percentage of patients who present with early stage disease. Most mesothelioma patients have locally advanced disease, advanced age, or other comorbid conditions that preclude surgery (Kindler, 1999).

Radiotherapy may be used in selected patients for palliation of pain, but has no impact on survival (Ball, 1990). Therefore the use of a systemic anticancer agent is the only treatment option for the majority of patients with malignant mesothelioma. Unfortunately, reproducible response rates above 20% are infrequently achieved even with the most active cytotoxic agents, which include doxorubicin, methotrexate, cisplatin, mitomycin, and alpha-interferon, and complete responses are rare (Ong, 1996; Ryan, 1998). New agents with novel mechanisms of action are clearly needed for this disease.

2.2 Role of Vascular Endothelial Growth Factor (VEGF) in Angiogenesis

Angiogenesis is the process whereby new blood vessels sprout in response to local stimuli. The process of angiogenesis involves the release of angiogenic factors, activation of metalloproteases to break down the extracellular matrix, followed by remodeling. The switch to the angiogenic phenotype is crucial in both tumor progression and metastasis.¹ The key factor involved in signaling for angiogenesis in nearly all human tumors is vascular endothelial growth factor (VEGF).^2,3 VEGF is a critical growth factor necessary for blood vessel formation. Loss of only one allele in the VEGF gene in knockout mice causes embryonic death.^4,5 Likewise, the VEGF receptors are essential for blood vessel formation as shown in gene knockout experiments in mice. ^6,7 Heightened expression of VEGF receptors in the endothelial cells of the tumor vasculature further attests to the significance of VEGF in tumor angiogenesis.^8,9

VEGF binds to at least three known receptors: flt-1/VEGFR-1, flk-1/KDR/VEGFR-2 and neuropilin-1.^10-12 VEGFR-2 is responsible for mitogenic signaling¹³, while VEGFR-1 participates in cell migration.^14-16 VEGF is regulated by several factors including hypoxia, cytokines such as IL-1, activation of certain oncogenes (Ras, Raf, Src), and loss-of-function mutations of p53 and the Von Hippel-Lindau genes.^17-22 Elevated tumor or serum VEGF levels has been predictive of poor survival.^23-24 The prognostic value of VEGF has been proposed to be related to enhanced angiogenesis in the tumor^25-34.

Ectopic expression of VEGFR-2 in non-endothelial cell lines does not lead to a VEGF mediated mitogenic response³⁵, suggesting that only the endothelial cells are configured to carry mitogenic VEGF signal to the nucleus. During neoplastic transformation, non-endothelial cells may acquire aberrant expression of VEGFR-2 and the downstream signaling to VEGF. Autocrine growth factor activity in tumor cells may contribute to metastatic potential and poor outcome.

VEGF is an autocrine growth factor for Kaposi's sarcoma,³⁶ in which tumor cells express an endothelial cell phenotype.³⁷ VEGF inhibitors including VEGF-antisense (VEGF-AS) have been shown to synergize with other agents such as epidermal growth factor antibody against colon carcinoma.³⁸ VEGF-AS has been shown to inhibit VEGF expression as well as blocking the binding of VEGF to VEGFR resulting in growth inhibition of tumor cell lines that express VEGFRs. Furthermore, VEGF-AS has an additive anti-tumor effect when combined with cytotoxic chemotherapeutic agents. In addition to having anti-angiogenic properties, VEGF-AS may also directly inhibit tumor growth in tumors that express the VEGF receptors.

2.3 Role of angiogenesis and VEGF in mesothelioma

Patients with malignant mesothelioma have higher circulating levels of VEGF than patients with any other solid tumor (Vermuelen, 1996; Linder, 1998). In mesothelioma, VEGF expression correlates with microvessel density (Ohta, 1999). A high intratumoral

microvascular density is associated with a poor survival in patients with mesothelioma

(Kumar-Singh, 1997; Ohta, 1999).

2.4 Pre-Clinical Studies with Vascular Endothelial Growth Factor Antisense (VEGF-AS)

VEGF-AS and VEGFR neutralizing antibodies directly inhibit tumor cell proliferation in vitro:

A wide variety of tumor cell lines express both VEGF and VEGF receptors (VEGFRs). There is a range of response to VEGF inhibition when studied in vitro in our lab. Notably, tumor cell lines that showed the most inhibition of cell viability were those that expressed both VEGF and VEGF receptors. Melanoma and ovarian carcinoma cell lines showed the greatest response, similar to that of KS cell line (KS Y-1). In sharp contrast, cell lines that failed to show response were erythroleukemia (HL-60), T cell leukemia HuT78 and fibroblast (T1) cell lines that lack both VEGF and VEGF receptor expression.

Dose dependent inhibition of cell growth was observed in cell lines that express VEGFRs, the same lines that showed growth inhibition with VEGF-AS. Cell lines that do not express VEGFRs did not show toxicity after VEGF-AS exposure. Blocking VEGFR-1 by itself does not initiate mitogenic signaling; but rather, it potentiates VEGFR-2 signaling by forming heterodimers with it that have a higher VEGF binding affinity. VEGFR-1 antibody does inhibit cell proliferation, although not as strongly as the VEGFR-2 antibody. The differences however, may be related to the activity of the antibody, or less efficient competition of VEGF/receptor interaction. In combination the VEGFR-1 and -2 antibodies were more potent inhibitors of tumor cell proliferation than either alone.

Inhibition of tumor growth in vivo

VEGF-AS (also known as VEGF AS-3) has been tested in murine xenograft models of human ovarian carcinoma and melanoma. Treatment of mice bearing Hey ovarian carcinoma xenografts with 10 mg/kg of VEGF AS-3m resulted in > 90% tumor inhibition. VEGF AS-3m, which is a mixed backbone oligonucleotide containing four ribonucleosides instead of deoxyribonucleosides flanking each end, see figure below, retained anti-tumor activity in the Hey ovarian tumor xenograft model in the absence of either an innate or adaptive immune system. Similarly, VEGF AS-3 (without any mixed backbone) was active against human melanoma M21 xenografts in athymic mice. In addition, an additive effect was observed when VEGF AS-3m was combined with low dose paclitaxel in M21 tumor xenografts, illustrating that the combined treatment regimes were more potent than either agent used alone.

Figure #1: VEGF-Antisense

VEGF-AS-3 was also active in an orthotopic prostate cancer model. Expression of VEGF increases with advancing prostate carcinoma and even more so when the tumor becomes hormone independent. Palliative therapy is the only treatment for non-resectable tumors. Prostate gland stroma plays a critical role in tissue remodeling and tumor regulation. Direct tumor implantation of the mouse prostate gland with the human prostate tumor cell line (PC3) was performed to determine if inhibition of VEGF would have an anti-tumor effect. Treatment was delayed to ten days post implantation, and the treatment consisted of AS-3m daily at a dose of 10mg/kg. Mice were sacrificed three weeks after tumor implantation and the prostate gland was harvested for analysis. All control mice (n=6) developed tumor at the site of injection in the prostate. Small tumors were seen in only two of six treated mice. This data substantiates the activity of this compound in the orthotopic prostate cancer tumor model.

Growth curves were also examined for apoptotic cells by TUNEL assay in ovarian tumor xenografts. Tumors from control mice showed occasional apoptotic cells not adjacent to the vessel. In contrast apoptosis in the tumors of AS-3m treated mice was more extensive in both perivascular cells and cells distant from vessels were involved. Apoptosis of both tumor cells and endothelial cells is observed, consistent with the proposed dual mode of action of AS-3m in VEGFR-2 positive tumor cells.

Effect of AS-3m on VEGF levels in vivo:

Human (Hey) tumor xenografts were harvested 24 hours after the last dose of therapy and tumor lysates were prepared. VEGF levels were quantitated and adjusted for total protein. Both human (tumor derived) and mouse (host derived) VEGF was inhibited in a dose dependent manner by AS-3m. In a representative experiment approximately 60% reduction in the levels of both human and mouse was observed.

Preclinical development of phosphothiorated AS-3 complimentary to VEGF was undertaken based on the in vitro inhibition of VEGF expression and in vivo inhibition of tumor growth ^36,39. Mice studies revealed kidney toxicities at 40 and 50 mg/kg but no toxicity at doses lower than 40 mg/kg. Monkey studies revealed transient elevation of serum Bb complement fragments at doses of 7.5 mg/kg on days 5 and 19, but were comparable to control group values on day 28. The Bb concentration in the 7.5mg/kg/day was comparable both in males and female monkeys on the various days of observations. No other abnormalities were observed either in the complement activation or clotting time.

2.5 Animal Clinical Toxicology

Mouse Toxicology

The toxicity profile of VEGF-AS was evaluated in the murine model. Mice were given intravenous doses of VEGF-AS on 8 days (on days 0, 1, 2, 3, 4, 7, 8, and 9) and at four dose levels: 10, 20, 30, 40 and 50 mg/kg. Control animals were treated with equal volumes of normal saline.

Administration of intravenous doses of VEGF-AS at doses of 40 mg/kg and 50 mg/kg resulted in the development of lesions in the kidney. Lesions in the kidney were characterized by both hyperplasia and degeneration with apparent recovery by later time points.

Monkey Toxicology

The toxicity profile of VEGF-AS was investigated in cynomolgus monkeys using slow intravenous bolus injection on Wednesday through Sunday of study weeks 1, 3 and 4 (total of 15 injections). The monkeys were assigned to three groups: three male monkeys received doses of 2.5 mg/kg/day; three male and female monkeys received 7.5 mg/kg/day once daily on Days 1-5, 15-19, and 22-26; and three male control monkeys received 0.9% saline on the same schedule as test animals. The monkeys were sacrificed on Day 30, 4 days after the last dose.

Monkeys were observed daily for vital signs, body weight; and blood was obtained for coagulation and complement activation analyses at scheduled intervals. Urine and blood analyses, EKG and ophthalmic examination were performed at baseline and prior to sacrifice. Blood samples for plasma toxicokinetic analysis were collected relative to dosing on Days 1 and 5.

All monkeys survived to scheduled terminal sacrifice. There were no treatment-related findings in the clinical observation, ophthalmic, electrocardiographic, body weight change, or anatomic pathology (macroscopic, microscopic and organ weight) data.

Serum Bb complement fragments were elevated primarily in 7.5 mg/kg/day males at Days 5 and 19, but were comparable to control group values on Day 28. The Bb concentrations in the 7.5 mg/kg/day females were similar to those observed in male monkeys. These transient elevations of serum Bb complement fragments are not apparently adverse to the animals.

No treatment-related differences were noted in the hematology, coagulation, urinalysis, or other clinical chemistry data. No adverse effects were observed for VEGF-AS at a dose level of 7.5 mg/kg/day when administered to cynomolgus monkeys by a slow bolus intravenous injection once daily for 5 days during study weeks 1, 3 and 4.

2.6 Pharmacokinetics

The pharmacokinetics and tissue distribution of VEGF-AS in Balb/c nu/nu mice was determined after a single IV dose of 30 mg/kg. Following IV administration of VEGF-AS the peak plasma concentration at 5 minutes was 34.26 ± 12.89 mg/ml. Intact AS-ODN could be detected up to 24 h. At 48 h, VEGF-AS was not detectable. The decrease in plasma concentration of AS-ODN followed a biexponential pattern with an initial half-life (t_1/2_a) of 0.33 ± 0.031 h and a terminal half-life (t_1/2_b) of 8.23 ± 2.31 h. The area under the plasma concentration-time curve was 16.95 ± 4.53 mg.h/ml, with a total body clearance of 1875.23 ± 589.84 mL/h/kg and volume of distribution of 21.47 ± 4.80 L/kg.

Following IV administration of VEGF-AS in cynomolgus monkeys, the peak plasma concentration at 5 minutes was 22.60 ± 3.51 mg/ml at 2.5 mg/kg dose and 67.50 ± 17.50 mg/ml at 7.5 mg/kg respectively. Intact VEGF-AS could be detected up to 4 h. At 24 h, VEGF-AS was not detectable. The terminal half-lives (t_1/2) were 0.55 ± 0.05 h and 0.68 ± 0.11 h at low and high dose respectively. The area under the plasma concentration-time curve increased from 11.36 ± 2.39 mg.h/ml at 2.5 mg/kg dose to 51.84 ± 14.53 mg.h/ml at 7.5 mg/kg dose. The total body clearance was 227 ± 51.51 mL/h/kg at 2.5 mg/kg, while it was 152.31 ± 33.27 mL/h/kg at 7.5 mg/kg dose. The volume of distribution was 0.18 ± 0.04 L/kg at low dose, and 0.15 ± 0.03 L/kg at high dose respectively.

2.7 Comparison with other VEGF compounds

Several other VEGF inhibitors are currently in clinical development that target only VEGF. VEGF-AS however targets other VEGF family members to include VEGF, VEGF-C and VEGF-D. In addition, VEGF inhibitors in clinical development, such as SU5416 have had issues related to solubility, with diluents such as cremaphor which cause phlebitis and liver toxicity. The anti-VEGF antibody, bevacizumab, has been associated with thromboembolic complications, renal bleed, hypertension, stroke, and myocardial infarction.

In pre-clinical study, VEGF AS3 has shown remarkable safety profile in primates. Murine toxicity studies showed renal lesion only at very high dose levels that are not expected to be achieved in human trials. We, however, remain vigilant of any and all toxicities and acknowledge that safety comes first.

A comparison of the corresponding areas of the VEGF family members is shown below: the highlighted bases indicate identity between either VEGF-B, -C, -D or PlGF and VEGF. Homology between the genes is not high in this region. Comparison of the sequences in the human and mouse VEGF genes that are complementary to AS-3. Mouse sequence shown here is nucleotides 288-308 of the sequence reported by Claffey and coworkers.⁴⁰ Identity is indicated by highlighted blocks.

Figure #2: Base identity between VEGF –A, -B,-C,-D and PlGF

Figure #3: Receptor usage of VEGF family members

Figure #4: VEGF-AS targets VEGF-A, VEGF-C and VEGF-D

Western blot

2.8 Phase I study of VEGF-AS in humans with refractory malignancy

1. Study objectives: (a) To determine the dose limiting toxicity

(DLT) and maximum tolerated dose (MTD)of VEGF-AS given as an IV infusion daily X 5 days (initial regimen), or, given daily x 5 days every other week for 4 months (amended regimen), in patients with relapsed or refractory malignancy, with stable disease or better after initial 28 day study period; (b) To evaluate the pharmacokinetic profile of VEGF-AS; (c) Secondarily, to preliminarily identify any evidence of objective tumor response in patients treated with IV VEGF-AS; (d) To determine VEGF A levels in plasma, pre and post infusions, and to compare therapeutic results with VEGF-A levels.

2. Study design: Phase I, dose escalation study, accruing 3 patients to each level. In the absence of DLT at a given level, escalation to the next level occurred. If DLT occurred at any given level, a total of 6 patients were to be added to that dose level. No dose escalations were permitted within patients. The MTD was to be the dose level where 0 or 1 DLTs were observed.

3. Definition of dose limiting toxicity (DLT): Any grade 3

coagulopathy; any grade 2 thrombotic event; grade 2 neuropathy; any grade 3 or 4 non-hematologic toxicity; grade 3 or 4 drug related thrombocytopenia; grade 4 drug related other hematologic toxicity.

4. Treatment dose levels employed:

LEVEL 1	15 mg/m2
LEVEL 2	22.5 mg/m2
LEVEL 3	30 mg/m2
LEVEL 4	37.5 mg/m2
LEVEL 5*	47 mg/m2
LEVEL 6*	59 mg/m2
LEVEL 7*	74 mg/m2
LEVEL 8*	85 mg/m2
NEXT LEVELS*	Increase in 11 mg/m2 increments

*The study design changed at Level 5, to allow additional protocol treatment in patients with stable disease or better after the first 5 day infusion. In these subjects, additional VEGF-AS was given as an IV infusion, daily x 5 days, every other week for a total of 8 cycles (4 months).

5. Entry criteria: (a) Any malignancy for which standard therapeutic

measures are no longer effective; (b) All stages with measurable or evaluable disease; (c) Age 18 years or older; (d) KPS of 50% or better, and estimated survival of at least 3 months; (e) Bilirubin £ 1.5 upper limit of normal (ULN); AST/ALT £ 2.5 ULN; (f) Absolute neutrophil count ³ 1500/mm3; platelets ³75,000/mm3 (unless due to marrow involvement); (g) Creatinine £1.5 mg/dl; (h) Signed informed consent

6. Exclusion criteria: (a) involvement of brain or CSF; (b) History of

deep vein thrombosis or pulmonary embolism or stroke; (c) History of CHF, unstable angina, cardiac arrhythmia, or MI; (d) Chemotherapy or XRT within 4 weeks; (e) Major surgery within 2 weeks; (f) Pregnant or lactating females.

7. Patient characteristics: 26 patients have been entered, will full follow-up now available. The median age is 57 years (range 19-83). Males=19; females-7. Prior therapy included systemic chemotherapy in 24 (92%); biologics/immunotherapy in 13 (50%); radiation in 9 (35%) and stem cell transplant in 1 (4%). Tumor types accrued included lymphoma in 5 (21%); sarcoma in 4 (13%); AIDS-related Kaposi’s sarcoma in 3 (13%); renal cell carcinoma in 3 (13%); colon cancer in 2 (8%); lung cancer in 2 (8%), and miscellaneous cancers in 7 (25%), including multiple myeloma, pancreas, melanoma, myoepithelioma, prostate cancer, thyroid and adenoid cystic carcinoma in one patient each.

8. Toxicity

CTC GRADE

TYPE	GRADE 1	GRADE 2	GRADE 3	GRADE 4	% Toxicity
Neutropenia	--	--	0	0	0
Anemia	--	0	1	0	4
Low platelet	--	0	0	0	0
Fatigue	5	0	0	0	19%
Constipation	4	0	0	0	15%
Hypotension	3	0	0	0	12%
Diarrhea	1	2	0	0	12%
Perioral numbness	2	0	0	0	8%
Insomnia	2	0	0	0	8%

Four serious adverse events have been reported. These include 3 patients who have died due to progressive malignant disease. Additionally, one patient with lymphoma, with pre-existing neutropenia due to extensive marrow involvement, developed neutropenic fever.

Through dose level 9 (96 mg/m²), there has been no dose limiting toxicity; no significant hematologic toxicity, except one case of grade 3 anemia in a patient who was transfusion dependent prior to study initiation; no evidence of coagulopathy or thrombotic event; no significant alteration in complement profiles; no significant cardiac toxicity.

9. Plasma VEGF levels on study: Plasma VEGF levels were

measured by ELISA at baseline, 24 hours after completion of VEGF-AS infusion; 5 days after completion of infusion; and weekly in patients on the revised schedule. VEGF levels are available on 25 patients. The median baseline VEGF level was 140 pg/ml (range 16-686 pg/ml). The VEGF levels declined by 25% or more at 24 hours in 14/25 (56%); were unchanged in 6/25 (24%); and increased by more than 25% in 5/15 (20%).

10. Preliminary data on clinical response

In an ongoing Phase I trial of VEGF-AS in patients with relapsed or refractory malignancies, one objective complete remission has been observed in a patient with AIDS-related Kaposi’s sarcoma lasting for 4 months. One patient with cutaneous T-cell had a mixed response and several patients with various solid tumors to include malignant mesothelioma have had stable disease.

3. DRUG FORMULATION AND PROCUREMENT

3.1.1 VEGF-Antisense Oligonucleotide (VEGF-AS)

VEGF-AS is manufactured by Avecia, Milford, MA for VasGene Therapeutics. The clinical vials are formulated by PrimaPharm, Inc., San Diego, CA. Each lyophilized vial contains 55 mf of VEGF-AS.

3.1.2 How Supplied

VEGF-AS is supplied by VasGene Therapeutics Inc. as a lyophilized powder with vial containing 55 mg of VEGF-AS for injection

3.1.3 Reconstitution

To reconstitute, first add 5.5 mL 0.9% Sodium Chloride, USP for injection to the vial containing the VEGF-AS. Swirl gently to dissolve. The resulting concentration of VEGF-AS is 10 mg/ml. Withdraw the required amount of the VEGF-AS solution as needed through a syringe and mix it in the bag containing saline.

3.1.4 Route of administration

Intravenous infusion over at least 60 minutes

3.1.5 Storage

The intact vials should be stored frozen (-20⁰C).

3.1.6 Stability

The antisense oligonucleotides are demonstrated to be quite stable as lyophilized products at –20⁰C. This has been observed with antisense for c-Raf and for Bcl/2. However, we are conducting a thorough stability program with the lyophilized preparation of VEGF-AS under the guidelines of ICH. We believe that there are no issues with the stability of the product after reconstitution.

CAUTION: The single-use lyophilized dosage form contains no antibacterial preservatives. Therefore, it is advised that the reconstituted product be discarded 8 hours after initial entry.

Stability of VEGF-AS Solution at 37^oC for 7 days shown below b [pk3] y polyacrylamide gel electrophoresis

3.1.7 Compatibility

VEGF-AS is compatible with 0.9% sodium chloride injection, USP.

3.1.8 Availability

Drug Ordering: To obtain a supply of VEGF-AS, the investigational pharmacist should call Dr. Parkash Gill [pk4] at 323-865-3909 during normal business hours (9:00 am to 5:00PM, Pacific Standard Time). The agent will be shipped via express courier to arrive on a weekday.

Drug Accountability:

The Investigator, or a responsible party designated by the investigator, must maintain a careful record of the inventory and disposition of all drugs received from VasGene Therapeutics.

4. PATIENT SELECTION

4.1 Eligibility Criteria

4.1.1 Patients must have histologically or cytologically confirmed malignant pleural or peritoneal mesothelioma, epithelial, sarcomatoid, or mixed subtype.

4.1.2 Patients must have had undergone prior systemic treatment, radiotherapy, or surgical procedure of curative intent for malignant pleural or peritoneal mesothelioma. Prior intrapleural cytotoxic agents (including bleomycin) will be included as prior therapy.

4.1.3 Patients must have measurable disease, defined as at least one lesion that can be accurately measured in at least one dimension (longest diameter to be recorded) as >20 mm with conventional techniques or as >10 mm with spiral CT scan. Pleural effusions and ascites are not considered measurable lesions.

4.1.4 Patients with pleural mesothelioma must be IMIG stage >II (see appendix B for the IMIG staging system).

4.1.5 Age > 18 years.

4.1.6 ECOG performance status < 2 (Karnofsky > 50%, see Appendix A) and an estimated survival of at least 3 months

4.1.7 Patients must have adequate organ and marrow function as defined below:

Absolute neutrophil count >1,000/ml

Platelets >50,000/ml

Total bilirubin < 2.0x the upper limits of institutional normal

AST/ALT < 2.5x the upper limits of institutional normal

Serum creatinine < 2.0 mg/dl

4.1.8 The effects of VEGF-AS on the developing human fetus are unknown. For this reason, women of child-bearing potential and men must agree to use adequate contraception (hormonal or barrier method of birth control) prior to study entry and for the duration of study participation. Should a woman become pregnant or suspect she is pregnant while participating in this study, she should inform her treating physician immediately.

4.1.9 Ability to understand and the willingness to sign a written informed consent document.

4.1.10 Patients with history of prior malignancy may be accrued provided that other eligibility criteria are met.

4.1.11 Must have medical records available to document start date of most recent prior therapy and time to progression for most recent prior therapy.

4.2 Exclusion Criteria

4.2.1 Patients who have had chemotherapy, immunotherapy or radiotherapy within 4 weeks prior to entering the study and 6 weeks from nitroso-urea-containing chemotherapy. All patients should have recovered from all toxicities of prior therapy.

4.2.2 Patients receiving therapy with other investigational agents at the time of study enrollment.

4.2.3 Patients with cancer involving the brain, or brain metastases.

4.2.4 Uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements

4.2.5 Pregnant and nursing women are excluded from this study as VEGF-AS may be harmful to the developing fetus or nursing infant.

4.2.6 Patients who had any major surgery within 4 weeks

5.0 TREATMENT PLAN

5.1 Drug administration

Patients will receive VEGF-AS as an intravenous (IV) infusion over a minimum of 60 minutes given three days weekly (Monday, Wednesday, Friday, (d1, d3 d5) for a total of 8 weeks. In subjects with stable or improved disease at 8 weeks, additional VEGF-AS will be given twice weekly for a total of 24 weeks, or until tumor progression, or toxicity.

Patients will be randomized to the following treatment doses of VEGF-AS as follows:

VEGF-AS VEGF-AS

100 mg/m² 200 mg/m²

TIW weeks 1-8 TIW, weeks 1-8

Re-evaluate for response

Patients with Stable or better disease after 8 weeks can continue to receive BIW therapy (Monday, Thursday or Tuesday Friday) at their respective dose level until disease progression or unacceptable toxicity.

In the event of a holiday, treatment can be omitted or given one day later. Patients will be permitted to miss up to 2 weeks of therapy for reasons unrelated to toxicity. Patients who have treatment delays of greater than 2 weeks not due to toxicity will be removed from therapy.

There will be no increase in dose among individual subjects

5.2 Toxicity Criteria

Life-threatening reactions which may be due to drug administration and all fatal reactions occurring on this study (during or within 30 days of treatment) will be reported immediately by phone. A written report will be submitted within ten (10) working days. The first occurrence of any previously unknown clinical event, regardless of grade, will be reported by phone within 24 hours to the IRB and to the Clinical Director at VasGene Therapeutics Inc (VGTI). A written report is required.

5.3 Supportive Care Guidelines

Colony stimulating factors (CSFs) should not be used as prophylaxis during the first 5 days of therapy but can be given at the discretion of the investigator, if the subject develops febrile neutropenia.

Other appropriate supportive care medications may be administered at the investigator's discretion.

5.4 Duration of Therapy

Patients will initially be treated on a three times weekly schedule for 8 weeks, unless treatment is stopped earlier due to one of the reasons outlined below. In patients with stable or better disease after 8 weeks, they will be allowed to receive an additional twice weekly therapy until tumor progression or toxicity. Reasons for early termination of study include:

§ Disease progression

§ Intercurrent illness that prevents further administration of treatment

§ Unacceptable toxicity

§ Patient decides to withdraw from the study

§ General or specific changes in the patient's condition that render the patient unacceptable for further treatment in the judgment of the investigator.

6. EXPECTED TOXICITIES/DOSE MODIFICATIONS

6.1 Expected Toxicities

The study will utilize the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 3.0, for toxicity and Adverse Event Reporting. Additionally, a copy of the CTCAE version3.0 can also be downloaded from the CTEP home page (http://ctep.info.nih.gov/). All appropriate treatment areas should have access to a copy of the CTC version 3.0.

Side effects seen in animals that were given doses far exceeding that planned for this study include lesions in the kidney and heart and changes in immune function.

VEGF-AS has also been studied in 30 humans to date. Most side effects have been minor (grade 1 or 2). The most common side effects seen in these patients (all less than 20%) were as follows: fatigue, constipation, low blood pressure, diarrhea, numbness around the lips, and difficulty sleeping. One patient had a grade 3 decrease in red blood cells; this individual was red cell transfusion dependent at study entry. No other grade 3 toxicity has been seen

Proteins similar to VEGF-AS have been given to humans and are associated with the following side effects: fatigue, fevers, chills, hot flashes or a flushing sensation, back pain, muscle aches, tender or swollen lymph nodes, lowering of blood counts, abnormal liver and kidney tests, abnormal blood clotting tests, upset stomach, nausea, vomiting, decrease appetite, skin rash, high or low blood pressure, and fluid retention. Another side effect could be an increased risk of developing blood clots and/or an increased risk of bleeding or bruising. It is possible that an allergic reaction could develop resulting in symptoms such as rapid heart beat, flushing, difficulty breathing. If not treated effectively, a severe allergic reaction could result in death.

6.2 Dosing Instructions and Modifications

6.2.1 For patients that weigh > 130% of ideal body weight, use the following formula 0.5x (ABW + 1.3xIBW). To calculate ideal body weight (IBW) in adults:

Males = 50 kg + [(2.3)(HT-60)] kg

Females = 45.5 kg + [(2.3)(HT-60)] kg

where HT is a patient’s height in inches

6.2.2 Dose Modifications:

Hematologic Toxicity

All treatment should be held permanently for patients experiencing drug related grade 3 thrombocytopenia, or grade 3 febrile neutropenia, or grade 4 neutropenia, unless these cytopenias are consistent with the patients’ baseline status, and secondary to marrow infiltration by malignant disease. There will be no dose modification of anemia.

All other non-hematologic toxicities:

For > Grade 3 or greater drug related toxicity, all therapy will be held permanently.

Doses that are missed on any day of any cycle due to toxicity will not be administered at a later day. Doses that are missed for reasons other than toxicity may be administered at a later day at the discretion of the principal investigator.

6.3 Definition of Unacceptable Toxicity

Unacceptable toxicity for dose limiting toxicity (DLT) will be defined as:

6.3.1 Hematology: Grade 4 thrombocytopenia or febrile neutropenia that is not definitely attributed to marrow infiltration by malignant disease

6.3.2 Non-Hematologic: All Grade 3 or 4 non-hematologic toxicities possibly related to drug

7. CORRELATIVE/SPECIAL STUDIES

7.1 Biologic studies of VEGF and VEGFR status

Samples for biologic studies to include VEGF levels in plasma will be collected at baseline (time 0); and then weekly for the first 5 weeks. Plasma is to be drawn into Acid/Citrate Dextrose (ACD) tubes and processed as specified in Appendix C.

STUDY CALENDAR

Except where specified, baseline/pre-therapy evaluations will be performed within 14 days of first study drug administration. Diagnostic tests, x-rays, and diagnostic biopsies (i.e. bone marrow) may be done within 30 days prior to the start of therapy. In the event that the patient’s condition is deteriorating, laboratory evaluations should be repeated within 48 hours. If the day 1 occurs within 48 hours of baseline, the same day 1 evaluation do not need to be repeated.

Study week

Every 4 weeks

End of

Study

Day of Rx

3 , 5

10, 12

17, 19

24, 26

31,33

38,40

45,48

53, 55

Medical history

Physical exam

Vital signs^a

Height/Weight/PS

CBC w/diff,plts

Serum chemistry^b

PT/PTT/Fibrinogen

B-HCG^c

EKG

Radiologic evaluation/Tumor Measurements^d

X^d

Plasma for VEGF and correlative studies^e

Toxicity

VEGF-AS^f

a) Vital signs: Blood pressure and pulse rate within 1 hour prior to administration of each dose; during treatment, vital signs monitored every 60 minutes for up to 1 hour post drug infusion.

b) Including alkaline phosphatase, total bilirubin, SGOT[AST], electrolytes, protein, creatinine

c) Serum pregnancy test (women of childbearing potential)

d) Radiologic evaluation for tumor measurement will be performed every 8 weeks. For responding patients (SD, PR, or CR) response must be confirmed 4 weeks after first observation of response, using the same diagnostic technique.

e) Plasma for VEGF levels and correlative studies will be obtained using ACD tubes (Yellow) See Appendix C for processing instructions.

f) Subjects with stable or improved disease parameters at 8 weeks will be allowed to receive additional VEGF-AS given BIW or until tumor progression or toxicity

9.0 CRITERIA FOR EVALUATION AND ENDPOINT DEFINITIONS

For the purposes of this study, patients should be reevaluated for response every 8 weeks. In addition to a baseline scan, confirmatory scans should also be obtained 4 weeks following initial documentation of objective response.

9.1. Definitions

Response and progression will be evaluated in this study using the new international criteria proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) Committee [JNCI 92(3):205-216, 2000]. Changes in only the largest diameter (unidimensional measurement) of the tumor lesions are used in the RECIST criteria. Note: Lesions are either measurable or non-measurable using the criteria provided below. The term “evaluable” in reference to measurability will not be used because it does not provide additional meaning or accuracy.

9.1.1 Measurable disease

Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as >20 mm with conventional techniques (PET, CT, MRI, x-ray) or as >10 mm with spiral CT scan. All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters).

9.1.2 Non-measurable disease

All other lesions (or sites of disease), including small lesions (longest diameter <20 mm with conventional techniques or <10 mm using spiral CT scan), are considered non-measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonis, inflammatory breast disease, abdominal masses (not followed by CT or MRI), and cystic lesions are all non-measurable.

9.1.3 Target lesions

All measurable lesions up to a maximum of five lesions per organ and 10 lesions in total representative of all involved organs should be identified as target lesions and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter) and their suitability for accurate repeated measurements (either by imaging techniques or clinically). A sum of the longest diameter (LD) for all target lesions will be calculated and reported as the baseline sum LD. The baseline sum LD will be used as reference by which to characterize the objective tumor response.

9.1.4 on-target lesions

All other lesions (or sites of disease) should be identified as non-target lesions and should also be recorded at baseline. Non-target lesions include measurable lesions that exceed the maximum numbers per organ or total of all involved organs as well as non-measurable lesions. Measurements of these lesions are not required but the presence or absence of each should be noted throughout follow-up.

9.2 Guidelines for Evaluation of Measurable Disease

All measurements should be taken and recorded in metric notation using a ruler or calipers. All baseline evaluations should be performed as closely as possible to the beginning of treatment and never more than 4 weeks before the beginning of the treatment.

Note: Tumor lesions that are situated in a previously irradiated area are not considered measurable.

The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up.

Imaging-based evaluation is preferred to evaluation by clinical examination when both methods have been used to assess the antitumor effect of a treatment.

Clinical lesions. Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules and palpable lymph nodes). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended.

Conventional CT and MRI. These techniques should be performed with cuts of 10 mm or less in slice thickness contiguously. Spiral CT should be performed using a 5 mm contiguous reconstruction algorithm. This applies to tumors of the chest, abdomen, and pelvis

Ultrasound (US). US should not be used to measure tumor lesions.

Cytology, Histology. The cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease.

9.3 Response Criteria

9.3.1 Evaluation of target lesions

Complete Response (CR): Disappearance of all target lesions

Partial Response (PR): At least a 30% decrease in the sum of the longest diameter (LD) of target lesions, taking as reference the baseline sum LD

Progressive Disease (PD): At least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions

Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum LD since the treatment started

9.3.2 Evaluation of non-target lesions

Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level

Incomplete Response/

Stable Disease (SD): Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limits

Progressive Disease (PD): Appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions

Although a clear progression of “non-target” lesions only is exceptional, in

such circumstances the opinion of the treating physician should prevail, and

the progression status should be confirmed at a later time by the review panel

(or study chair).

Note: If tumor markers are initially above the upper normal limit, they must

normalize for a patient to be considered in complete clinical response.

9.3.3 Evaluation of best overall response

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria (see section 9.3.1).

Target Lesions

Note:

× Patients with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be classified as having “symptomatic deterioration.” Every effort should be made to document the objective progression, even after discontinuation of treatment.

× In some circumstances, it may be difficult to distinguish residual disease from normal tissue. When the evaluation of complete response depends on this determination, it is recommended that the residual lesion be investigated (fine needle aspirate/biopsy) before confirming the complete response status.

9.4 Confirmatory Measurement/Duration of Response

9.4.1 Confirmation

To be assigned a status of PR or CR, changes in tumor measurements must be confirmed by repeat assessments that should be performed 4 weeks after the criteria for response are first met. In the case of SD, follow-up measurements must have met the SD criteria at least once after study entry at a minimum interval of 8 weeks.

9.4.2 Duration of Overall response

The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started).

The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that recurrent disease is objectively documented.

9.4.3 Duration of stable disease

Stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started.

9.5 Progression-Free Survival

Time to progression (as defined above) or death is measured from the start of treatment.

9.6 Time to Tumor Progression (TTP)

Time to tumor progression (TTP) will be the primary clinical end-point of this study. TTP will be defined as the time from first day of treatment to the first observation of disease progression or death due to disease. If failure has not occurred, failure time is censored at the time of last follow-up. Tumor evaluation will bee performed monthly until such time that tumor progression has been identified.

Time to tumor progression will be compared to the TTP achieved with the last therapy used immediately prior to the onset of VEGF-AS therapy in each study subject.

9.7 Overall Survival

Defined as the time from first day of treatment to time of death due to any cause. If a patient is still alive, survival time is censored at the time of last follow-up.

10. REGULATORY AND REPORTING REQUIREMENTS[pk5]

10.1 Regulatory Reporting for investigational agents:

This study will utilize the CTC version 3.0 for toxicity.

10.1.1 All Adverse Events for VEGF-AS should be reported to VGTI as described earlier followed by a written report to IRB.

10.1.2 AEs to be reported to VGTI:

· all grade 4 and 5 AEs including unrelated or unlikely unexpected and expected events

· any unexpected grade 2-5 AE (an expedited report is required for unexpected grade 2 and 3 AEs with an attribution of possible, probable or definite)

· any death within 30 days of drug administration

· any hospitalization (or prolongation of existing hospitalization) for medical events equivalent to CTC grade 3, 4, 5

Attribution Categories:

Probable – the adverse event is likely related to the investigational agent(s)

Possible – the adverse event may be related to the investigational agent(s)

Unrelated [pk6] – the adverse event is doubtfully related to the investigational agent(s)

10.1.3 A copy of the submitted SAE report must be sent to the Study Coordinator by fax (323.865.0371) or by email (espina@usc.edu) for distribution. The toxicity form should be sent to Coordinator within 24 hours. Any supporting documentation (i.e., laboratory, pathology, progress notes, discharge summary, autopsy, etc.) explaining the AE should accompany the submitted report.

10.1.4 Submitted reports must be filed with each collaborating site’s IRB as follows:

USC: within 10 working days

If there is any question about whether a particular adverse reaction should be reported, you may call VGTI. VGTI encourages submission of reports even if there is only a suspicion of a drug effect.

Questions regarding ADR reporting should be directed to the Study Coordinator (telephone # 323-865-0371, beeper 213.287-1968, FAX # 323-865-0133, e-mail espina@hsc.usc.edu). All life threatening toxicities mandate a telephone contact within 24 hours of the event between the reporting institution’s principal investigator (PI) and the PI’s or their designates of the other participating institutions.

Table: Expedited Reporting for VEGF-AS

UNEXPECTED EVENT

EXPECTED EVENT

Grades 2 – 3

Attribution of Possible, Probable or Definite

Grades 4 non Hematologic and 5

Regardless of Attribution

Grades 1 – 3

Grades 4 and 5

Regardless of Attribution

Grade 2 – Expedited report within 10 working days.

Grade 3 – Report by phone to IDB within 24 hrs. Expedited report to follow within 10 working days.

Grade 4 Hematologic

Report by phone to IDB within 24 hrs. Expedited report to follow within 10 working days.

(Grade 1 – Adverse Event Expedited Reporting NOT required.)

Report by phone to VGTI within 24 hrs. Expedited report to follow within 10 working days.

This includes all deaths within 30 days of the last dose of treatment with an investigational agent regardless of attribution.

Any late death attributed to the agent (possible, probable, or definite) should be reported within 10 working days.

Adverse Event Expedited Reporting NOT required.

Report by phone to VGTI within 24 hrs. Expedited report to follow within 10 working days.

This includes all deaths within 30 days of the last dose of treatment with an investigational agent regardless of attribution.

Any late death attributed to the agent (possible, probable, or definite) should be reported within 10 working days.

· For Hospitalization only - Any medical event equivalent to CTCAE Grade 3, 4, 5 which precipitated hospitalization (or prolongation of existing hospitalization) must be reported regardless of expected or unexpected and attribution.

· Telephone reports to the VGTI (323) 865-3909.

11.0 STATISTICAL CONSIDERATIONS

11.1 Summary of Design

This will be a pilot randomized Phase II trial. Fifteen patients will be randomized to each of 2 treatment arms for a total of 30 patients planned for this trial.

11.2 Analysis of Results

11.2.1 Analysis of Clinical Results

All patients who are randomized will be accounted for in the analysis of the results; reasons for going off study will be documented for each patient. Baseline clinical and demographic information will be presented to describe each patient treated, as well as the time to progression observed on the prior regimen. In comparing the dose levels, an intent to treat analysis will be performed. Although we do not anticipate deviations from the assigned treatment arm, we may compare the results of the “as randomized analysis to those of the “as treated” group to establish the consistency of the findings.

The NCI CTCAE v. 3.0 will be used to assess the side effects. Toxicities observed at each of the three dose levels will be summarized in terms of type (organ affected or laboratory determination), severity, time of onset, duration, and reversibility or outcome. For each dose level, we will calculate the proportion of patients who experience a TTP “benefit” and construct exact 95% confidence intervals. The TTP ratio will also be summarized by constructing histograms and calculating medians, quartiles and ranges. All objective responses will also be documented and summarized. Kaplan-Meier curves will be drawn to display the overall survival and the log-rank test and estimates of relative risk based on the log-rank statistics will be performed. 95% confidence intervals will be constructed for the median overall survival. Results will be summarized overall and by the assigned dose. We will also stratify patients according to disease and VEGF-receptor status. Although within each substratum of patients, there will not be a sufficient number of patients to formally test for differences in dose levels, these analyses will be used descriptively to estimate the association between dose and TTP and overall survival.

12.0 RECORDS TO BE KEPT AND DATA SUBMISSION SCHEDULE

12.1 Confidentiality of Records

The original data collection forms will be stored at the originating institution. At USC, the forms will be kept in secure file cabinets in the CISO; the forms will be stored in secure cabinets in the Division of Oncology

12.2 Patient Consent Form

At the time of registration, three signed and dated copies of the patient Informed Consent form with the Human Rights will be available (for patient, patient's medical chart and one for the Clinical Investigations Support Office (at USC).

12.3 Registration Eligibility Worksheet

At the time of registration, the information requested on the On-Study/Eligibility Form will be submitted to the Study Coordinator at USC who can be reached by phone at 323-865-0371 or fax at 323-865-0133.

12.4 Randomization and Registration

At the time a patient is deemed eligible for the trial, the patient will be registered by completing the eligibility worksheets and contacting the CISO office (323-865-0451). Patients will then be registered into the Norris Cancer Hospital system, stratified and then randomized using a program created by the study statistician which uses the minimization method and permuted block design. This program will provide the treatment assignment, a print out of which will be provided to the research team so they will have the treatment assignment.

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APPENDIX A

PERFORMANCE STATUS SCALE

Karnofsky Scale

Normal, no complaints 100%

Able to carry on normal activity 90%

Minor signs or symptoms of disease

Normal activities for effort 80%

Cares for self, unable to carry on 70%

Normal activity or to do active work

Requires occasional assistance, but 60%

Able to care for most of own needs

Requires considerable assistance and 50%

Frequent medical care

Disabled, requires special care and assistance 40%

Severely disabled, hospitalization 30%

indicated though death not imminent

Very sick, hospitalization necessary 20%

Active supportive treatment necessary

Moribund 10%

Dead 0%

APPENDIX B: NEW INTERNATIONAL MESOTHELIOMA INTEREST GROUP

(IMIG) STAGING SYSTEM FOR MALIGNANT PLEURAL MESOTHELIOMA

TIa Tumor limited to the ipsilateral parietal pleura including mediastinal and diaphragmatic pleura. No involvement of the visceral pleura.

TIb Tumor involving the ipsilateral parietal pleura including mediastinal and diaphragmatic pleura. Scattered foci of tumor also involving the visceral pleura.

T2 Tumor involving each of the ipsilateral pleural surfaces (parietal, mediastinal, diaphragmatic, and visceral pleura) with at least one of the following features:

• .Involvement of the diaphragmatic muscle.

• .Confluent visceral pleural tumor (including the fissures), or extension of tumor from visceral pleura into the underlying pulmonary parenchyma.

T3 Describes locally advanced but potentially resectable tumor.

Tumor involving all of the ipsilateral pleural surfaces (parietal, mediastinal, diaphragmatic, and

visceral pleura) with at least one of the following features:

• Involvement of the endothoracic fascia.

• Extension into the mediastinal fat.

• Solitary, completely resectable focus of tumor extending into the soft tissues of the chest wall.

• Non-transmural involvement of the pericardium.

T4 Describes locally advanced, technically unresectable tumor.

Tumor involving all of the ipsilateral pleural surfaces (parietal, mediastinal, diaphragmatic, and

visceral pleura) with at least one of the following features:

• Diffuse extension or multifocal masses of tumor in the chest wall, with or without associated rib destruction.

• Direct transdiaphragmatic extension of tumor to the peritoneum.

• Direct extension of tumor to the contralateral pleura.

• Direct extension of tumor to one or more mediastinal organs.

• Direct extension of tumor into the spine.

• Tumor extending through to the internal surface of the pericardium with or without a pericardial effusion; or tumor involving the myocardium.

N: Lymph Nodes

NX Regional lymph nodes cannot be assessed.

NO No regional lymph node metastases.

NI Metastases in the ipsilateral bronchopulmonary or hilar lymph nodes.

N2 Metastases in the subcarinal or the ipsilateral mediastinal lymph nodes including the ipsilateral internal mammary lymph nodes.

N3 Metastases in the contralateral mediastinal, contralateral internal mammary, ipsilateral or contralateral scalene or supraclavicular lymph nodes.

M: Metastases

MX Presence of distant metastases cannot be assessed.

MO No (known) distant metastases.

MI Distant metastases present.

STAGE I Ia: T1aN0M0

Ib: T1bN0M0

STAGE II T2N0M0

STAGE III ANY T3M0

ANY N1M0

ANY N2M0

STAGE IV ANY T4

ANY N3

ANY M1

Appendix C

Peripheral Blood Mononuclear Cells (PBMCs) and Plasma Levels of VEGF Processing Instructions and Methodology

Sample Collection

Approximately 7 mL of whole blood is to be collected into a citrate containing vacutainer tubes (either ACD tube- Yellow top or CPT- speckled blue) at each sampling time, specified in the Schedule of Events. Immediately upon blood drawing, the sample should be placed in an ice bath to chill the sample prior to centrifugation.

Sample Processing

· Blood samples will be immediately centrifuged using a 30 minutes of blood collection at approximately 2500 rpm and 4^oC for 10 minutes

· Plasma will be removed, and aliquoted into 2 cryogenic vials (1.8 mL capacity vials)

· Vials will be labeled with the following information: patient identifier, date of collection, treatment day.

· Samples will be immediately frozen and stored at approximately -70^oC until time of analysis.

· Resuspend the cell pellet in phosphate-buffered saline (PBS), layer over a polymer gel density gradient and spin at 2500-3500 rpm for 20-30 minute. Collect the band containing mononuclear cells (It is important not to contaminate the cells with erythrocytes), place in a 50 mL centrifuge tube and wash in PBS by centrifugation at 2200 rpm for 5 minutes at room temperature. Resuspend the pellet in 1 mL PBS and transfer equal volumes to two microfuge tubes. Spin the tubes for 2 minutes at 2000-6000 rpm then decant the PBS. Freeze both tubes at -70^oC.

Labeling Tubes

Label each tube with information below using a durable label and ink

· Protocol Number

· Patient initials

· Patient ID number

· Date and time of blood collection

· Specimen type: ACD Plasma

· Specimen purpose: VEGF-Levels

All specimens will be sent to:

Rizwan Masood, Ph.D.

Norris Cancer Hospital and Research Institute

1441 Eastlake Avenue, Room 624

Los Angeles, CA 90033

Phone: 323-865-0624

VEGF levels in patient samples will be determined using a solid phase double-ligand enzyme-linked immunosorbent assay (ELISA) obtained from R&D Systems (Minneapolis, MN) according to the manufacturer’s instructions. Microtiter plates are provided coated with monoclonal antibody specific for VEGF. Standards, samples, and VEGF antibody conjugated to horseradish peroxidase will be pipetted into the wells and any VEGF present will be sandwiched by the immobilized antibody and the enzyme-linked polyclonal antibody specific for VEGF. After removal of excess of any unbound substances and/or antibody-enzyme reagent, a substrate solution (hydrogen peroxide and chromogen tetramethylbenzidine) will be added to the wells and color developed in proportion to the amount of VEGF bound. The color development will be stopped after 30 min at room temperature and the intensity of the color measured at 450 nm in a microplate reader (Molecular Devices, Sunnyvale, CA).

APPENDIX D

DATA AND SAFETY MONITORING PROCEDURES FOR USC/NORRIS COMPREHENSIVE CANCER CENTER

1.0 Institutional Oversight and Protocol Audits

The Quality Assurance Committee (QAC) of the USC/Norris Comprehensive Cancer Center (USC/NCCC) has responsibility for the oversight of clinical trials conducted as part of the USC/NCCC research activities. All clinical protocols involving cancer patients or asking a cancer-related question are required to undergo scientific review by the USC/NCCC Clinical Investigations Committee (CIC). No cancer-related clinical trial may be activated without CIC review and approval; the USC Institutional Review Board (IRB) will not review a cancer-related clinical protocol that has not been reviewed and approved by the CIC. At the time of the CIC review, those studies that are classified as a clinical trial (i.e. involving an intervention) - thereby requiring QAC monitoring – are identified.

It is the responsibility of the study principal investigator (PI) to monitor the progress of each of his or her trials on an ongoing basis. The QAC will then review, on a monthly basis, all reported serious adverse events as well as all major study violations. In addition, at set intervals – depending on the class of the trial – the QAC will review the overall progress of the trial, including the cumulative toxicity, response to treatment and outcome, and adherence to protocol. Occasionally, for a randomized Phase II trial or for a multi-center trial Phase II/Pilot Trial or a study with potentially higher than average risk to patients, a formal Data and Safety Monitoring Committee (DSMC) will be formed to oversee the progress of the study. All Phase III trials are required to have a DSMC comprised of members that are not involved in the conduct of the trial. It is the responsibility of the CIC to review the monitoring plans at the time of the initial review of each protocol. These plans must include criteria for suspending the trial in the event of excessive toxicity, as well decision rules for terminating based on efficacy endpoints.

1.1 Annual Review

All open, in-house trials are reviewed once a year by the USC/Norris Comprehensive Cancer Center (USC/NCCC) Quality Assurance Committee (QAC). This annual review includes the following: evaluation of the current accrual relative to the planned total accrual and planned interim analyses; examination of all reported violations; examination of all reported AE’s; review of past audits and correspondence with the trial principal investigator (PI); review of results of current audit (by an outside agency or by the USC/NCCC QAC); review of previous correspondence between the PI and the QAC. Based on these reviews, the QAC will decide one of the following: recommend that the study continue unchanged and review in 12 months; request an explanation or a plan for change – to address an identified problem - before a final decision is made by the QAC; recommend a specific amendment or change to address an identified problem; recommend termination of the study. If the PI does not respond within 30 days (by the next QAC meeting) a letter is sent to the USC/NCCC Clinical Investigations Committee (CIC) with a recommended action. The PI then has 30 days to appeal to the CIC or the CIC will decide on an appropriate action. The results of all annual reviews, as part of the QAC minutes, are submitted to the CIC at the next CIC meeting.

1.2 Protocol Audits

All open, in-house (i.e. USC initiated) trials that are not monitored by another agency (e.g. Theradex) are audited by the USC/Norris Cancer Center Quality Assurance Committee (QAC) once a year. In addition, selected in-house protocols that are monitored by Theradex, will also be audited by the QAC. The review is done on the anniversary month of the initial IRB review date. Members of the Cancer Center involved in clinical research are asked to serve as auditors; generally one M.D. and one other person are asked to audit each patient selected. 20% of patients accrued during the past 12 months – and a minimum of 2 patients – are selected. Criteria for selection include: major response observed, treatment at the MTD of a Phase I trial, or some inside information that there might have been a problem. If none of these criteria are met, then patients are selected randomly. The audit involves a review of the hospital chart (i.e. source documentation) and evaluates the following: documentation of eligibility (including failure to obtain appropriate informed consent, and deviations from assessment of eligibility that would either (i) render that patient ineligible or (ii) make it impossible to establish eligibility – as well as the actual status of the patient); documentation of adherence to protocol-specified treatment and follow-up; evaluation of toxicity; and evaluation of response or other outcome.

Failure to report violations or required SAE’s is viewed as very serious. Responsible data managers and research nurses are counseled (by the CISO Associate Director) and the PI is requested to propose a plan to avoid such breeches in the future. SAE’s are forwarded to the IRB and the CIC is informed.

For all audited protocols (even those with no or minor issues), a report is created summarizing the results of the audit and, if appropriate, suggestions are provided for improving the conduct of the trial. Copies of all audit reports are sent to the CIC for review.

1.3 Review of Serious Adverse Events

If an adverse event (AE) is judged to be a serious adverse event (SAE), then the procedures to report the SAE depend on the drugs or the intervention in the protocol. For example, if the trial involves an investigational agent, then the PI must report certain SAE’s to the study sponsor within 24 hours and to the USC IRB within 5 days. If the trial involves drugs that are commercially available and that have been extensively studied (e.g. the use of methotrexate, doxorubicin, vinblastine, and cisplatin as adjuvant therapy for bladder cancer), then only SAE’s that result in death or that are unexpected, life-threatening, and thought to be possibly, probably or definitely associated with one of the drugs would be reported to the FDA and the USC IRB. The details regarding the specification of reportable AE’s and the procedures for reporting the SAE’s are given in the protocol.

The QAC will review all toxicities and AE’s that are reported to the IRB. All toxicities that are reported outside to agencies (e.g. the NCI, FDA, Office of Biotechnology, or the sponsoring pharmaceutical company) must also be reported to the IRB. The forms that are used to report toxicities or adverse events to the IRB (which may include adverse events that are not reported to any outside agency) are computer generated and tied to the USC/NCCC clinical trials database. Therefore at each QAC meeting, all adverse events and toxicities reported to the IRB since the last QAC meeting are listed and reviewed. At the time of the QAC review, a list of all past reported AE’s for the open protocols is also available for reference.

This review of SAE’s and other reported AE’s is done by the QAC on a monthly basis. For investigational agents, this QAC review is used to verify that the appropriate agencies and sponsors have been notified in a timely fashion with the appropriate documents, as well as to examine the pattern of SAE’s to date. For commercially available drugs, this monthly QAC review will be able to identify patterns of unexpected or increased toxicity.

The QAC will flag for follow-up, patterns of severe or relatively frequent AE’s. Based on the pattern of reported AE’s, the QAC will make recommendations on whether the study should continue unchanged, should be amended or modified, or be suspended, based on unacceptable risk to the trial participants – as well as considering the criteria in the protocol for suspending the trial in the event of excessive toxicity. These recommendations are forwarded to the CIC for action. If the CIC recommends suspension (temporary or permanent), the CIC will be responsible for notifying the IRB, as well as the sponsor (the NCI or the pharmaceutical company).

Finally, one function of the audits (see below) is to verify that all required reporting of AE’s has occurred.

1.4 Monitoring protocol violations

Data managers and research nurses are required to report major protocol violations to the QAC. This is done by completing a computer form – thereby ensuring that the information is stored in the USC/NCCC clinical trials database. Major violations are defined as:

(a) deviation from protocol treatment by 10% of dose, delay in treatment of a month beyond the prescribed amount, failure to hold a drug for reasons of toxicity, or administration of an inappropriate drug

(b) departure from the follow-up schedule (a) leading to an inadequate assessment of toxicity or (b) compromising the assessment of the outcome measures.

These reported violations are then reviewed and discussed at the next QAC meeting. Repeat violations and patterns are flagged. A memo is sent to the PI, requesting a plan to avoid these violations in the future. Sometimes the QAC will suggest a specific modification of procedures or protocol amendment. If the PI does not respond or if the violations continue, the QAC will report this to the CIC with a recommendation to either close the study or suspend accrual until the problems can be resolved. In practice, because of the close co-operation between the QAC and the CISO leadership, problems can be addressed on a one-to-one basis, usually bringing in the data manager or research nurse – and formal action is not required.

1.5 Monitoring accrual

The USC/NCCC clinical trials database is used to document the registration of all patients who are enrolled onto CIC-approved clinical trials. Thus at any given time, the clinical trials database should be up-to-date (within 24 hours) of accrual to studies.

At the monthly QAC meetings, all studies that have not accrued any patients during the preceding 12 months are flagged. A memo is sent to the PI requesting an explanation for the lack of accrual. If the explanation is not satisfactory, or if no response is received, a second memo is sent informing the PI that the protocol will be presented to the CIC with the recommendation for closure. At that time, the PI may appeal the closure to the CIC, but in the past, the CIC has consistently supported the QAC recommendation.

1.6 Monitoring study progress and interim analyses

Each protocol is required to describe the plans for interim analyses and stopping rules (for Pilot, Phase II and Phase III trials) or the rules for dose expansion, escalation, and de-escalation (for Phase I trials). In addition all Phase II and III trials must have criteria for suspending the trial in the event of excessive toxicity. The proposed plans and rules are reviewed by the CIC; no trial is activated without appropriate procedures in place – as assessed by the CIC. For each protocol, at the time of activation, a very brief summary of the proposed design (generally the total target accrual and the planned times for analysis) is stored in the clinical trials database. Every month, the design and the current accrual is reviewed for every open in-house protocol (approximately 40-50 at any given time). For every study that has reached the time for the scheduled interim analysis, a copy of the report summarizing the analysis, with the rationale for continuing (or discontinuing) the study is requested and (once obtained) is stored in the official protocol folder in the CISO office. Studies that do not supply an adequate summary of the interim analysis will be closed. The procedure for closing is the same as that described above: a memo is first sent to the PI requesting either a copy of the interim analysis report or an explanation of an issue; if no response is received or the response is not adequate, a second memo is sent informing the PI that the QAC will request that the CIC close the protocol. The PI then has 30 days to correct the situation or present an acceptable plan to the CIC, or the CIC will close the study.

At the monthly review, if a protocol has exceeded its planned total accrual (this very, very rarely happens) the PI will be notified of this, and the QAC will request that the CIC close the protocol.

2.0 Adherence to Protocol – Responsibilities of the Principal Investigator Study Team

It is the responsibility of the Principal Investigator (PI) to ensure that patient recruitment and enrollment, treatment, follow-up for toxicities and response, and documentation and reporting are all performed as specified in the protocol. When a study is opened at two institutions, the PI at each institution will assume the responsibilities for the day-to-day monitoring of the trial, as described below.

2.1 Eligibility

The PI will review the patient eligibility (with assistance from the Research Nurse – who will assemble the required source documents, and do an initial review) prior to registering the patient on study.

2.2 Informed Consent

Prior to registering the patient on study, the study research nurse will review the informed consent, to ensure that the patient has signed and dated the form, and that the form has been signed and dated by the person obtaining the consent as well as appropriate witnesses. The dates of all persons signing the consent must agree and must be before or on the date of study entry. If the informed consent is signed and the patient is registered on the same day that treatment is started, then the time of signature and treatment start must also be included.

2.3 Treatment

The PI is responsible for ensuring that treatment is given per protocol. If treatment is administered by another physician, the study research nurse will review the treatment orders with the PI. Regardless of who the treating physician is, there will be only one research nurse for the study at the LAC+USC Medical Center and one research nurse for the study at the USC/Norris Cancer Center. The PI will review the status of each patient on-study, with the research nurses and treating physicians, on an on-going basis.

2.4 Evaluation of Safety

Generally it is the study research nurse who schedules the visits to evaluate toxicity to treatment, who initially interviews the patient to discuss the symptomatic side effects of the treatment, and who initially reviews the lab results. At the USC/Norris Comprehensive Cancer Center, the Informatics Core has developed toxicity forms that tailor the collection of side effects and toxicity information for each protocol and allow the comparison of past toxicities with the current side effects. These are presented to the treating physician who will decide on the appropriate course of action – follow-up with additional tests or treatment. The PI will review the toxicity assessment and changes in schedule on an on-going basis.

2.5 Evaluation of Treatment Response

The study research nurse will schedule the tests or scans that are necessary to evaluate response to treatment. Although the treating physician will evaluate the results to establish the response, it is the responsibility of the PI to review all the scans and tests of all the patients to confirm the initial assessment of the response.

2.6 Data Management

2.6.1 Patient Charts

All written source documents are maintained in the patient chart which is stored in the Department of Medical Records at the hospital. X-rays and other images are stored in the Department of Radiology. These are the permanent, official documents for each patient on-study. A copy of the signed informed consent, physician’s notes, orders, test results and pathology notes are maintained in the patient chart.

It is usually the responsibility of the data manager to ensure that the patient chart contains all the required documents.

2.6.2 Research Charts

To facilitate adherence to the protocol schedule and data management, research charts are often created to collect copies of the informed consent, relevant notes, orders and results, that are in the hospital chart. Protocol calendars, worksheets, checklists, and completed case report forms may also be kept in the research chart. While the research charts do not contain the official, original documents, they do contain copies of all documents that are required for the study. These are maintained in the Clinical Investigation Support Office until the study is completed and the results are published and no further need is anticipated. These are then stored off-site.

It is usually the responsibility of the data manager to ensure that the research chart contains all the required documents.

2.6.3 Case Report Forms

It is the responsibility of the data manager to complete the required case report forms. For in-house trials, paper versions are stored in the research chart; for certain protocols, the information is directly entered into the Cancer Center clinical trials database. If errors are identified and corrected, a copy of the source document is attached with an explanation of the change, the date of the correction and the person making the correction.

It is the responsibility of the PI to review the research chart once the patient has completed treatment. At this time, the PI will review the all the toxicities experienced by the patient, as well as the overall response to treatment. A final check-list/verification is provided for the PI to indicate that she or he has reviewed the eligibility, treatment, toxicities and overall response. The case report forms are kept in the Research Chart.

3.0 Agency Reporting and Regulatory/Administrative Issues

3.1 IRB Review and Review by NCI-CTEP or FDA

3.1.1 Initial Review and Approval

The trial will not be activated and no patients will be enrolled in the trial until the study has received final approval from all sponsoring agencies, as well as the USC IRB. It is the responsibility of the study PI to verify that all stipulations have been met and that the official approval documents have been received.

3.1.2 Protocol Amendments

All modifications to the study plan or protocol document must be submitted to the USC IRB, as well as to all the sponsoring agencies (FDA, NCI, etc.) for review and approval. It is the responsibility of the study PI to ensure that the appropriate agencies have been informed of the proposed amendments and that these have been reviewed and approved. Until final approval is received, no patients may be treated according to the modified plan.

3.1.3 Serious Adverse Event and Toxicity Reporting

Procedures and requirements for reporting toxicity are described explicitly in the protocol. In the protocol, the following are specified:

(1) the definition of a serious adverse event (SAE)

(2) the agencies, in addition to the USC IRB, who are to be notified

(3) the information required and the procedures for notification

(4) the time frames for reporting the SAE’s

3.1.4 SAE Reporting with Multi-Institutional Studies

For studies in which two or more institutions enroll patients and an investigator at the USC/Norris Cancer Center is the study PI, the following additional procedures are described in the protocol:

(1) requirements for other institutions to report SAE’s to USC – and the required time frame

(2) procedures for USC to notify other institutions of SAE’s experienced at any of the participating institutions

3.2 Drug Tracking and Accountability

The USC School of Pharmacy and the Norris Hospital Department of Pharmacy Services provide investigational drug support services, including those services that are FDA-mandated such as drug tracking and accountability. Investigational drugs for patients treated on protocol, at the L.A. County General Hospital or Women’s Hospital, are managed and dispensed from a central facility located in the Norris Hospital. The services provided include distribution and control of investigational drugs, as well as research support. Prior to the initiation of any protocol involving an investigational agent, it is the responsibility of the PI to meet with a Research Pharmacist to arrange all aspects of drug procurement, dispensing, tracking and accountability. A Research Pharmacist is present at all CIC reviews and will flag those trials which have not adequately addressed the necessary issues. These protocols will not be approved (and forwarded to the IRB) unless the delinquent issues have been addressed.

3.2.1 Distribution and Control of Investigational Drugs

All aspects of drug tracking and accountability are coordinated with the central pharmacy facility at the Norris Hospital. For example, when a new prescription involving an investigational drug, is submitted to the pharmacy, a copy of the signed informed consent is also required to verify the status of the patient and the protocol. Services provided include:

3.2.1.1 procurement, storage, and inventory control of investigational drugs

3.2.1.2 preparation and dispensing of investigational drugs

3.2.1.3 maintenance of comprehensive inventory control and dispensing records for at least two years after an IND is terminated or its new drug application (NDA) is approved

3.2.1.4 maintain a computerized cross-reference system on investigational drugs that cross-references protocol number, drug name, patient name, principal investigator, and ordering investigator

3.2.1.5 insure compliance with FDA, NCI (or other sponsoring agency), JCAH, ASHP, Federal and State of California regulations and USC/Norris Hospital Policies and Procedures for inventory control and dispensing of investigational drugs

3.2.1.6 provide the Principal Investigator and research staff with investigational agent inventory and dispensing summaries as needed

3.2.1.7 maintain a Quality Assurance Monitoring program

3.2.1.8 provide necessary investigational drugs inventory control documentation and logs for both internal and external Quality Assurance audits by MCI, cooperative group, and other sponsoring agencies.

3.2.2 Research Support

In addition, the central research pharmacy facility will provide the following services to support the research aspects of the protocols:

3.2.2.1 review all proposed intramural Cancer Center protocols and provide input regarding all aspects of use of investigational agents (completeness and accuracy of agent background section, procedures on preparation and administration of agents, impact of pharmacokinetics and preclinical animal toxicology data on agent, schedule of administration, trial design, etc.)

3.2.2.2 provide PI and Protocol File investigational drug inventory and dispensing summaries on a quarterly basis

3.2.2.3 participate in intramural Quality Assurance audits

3.2.2.4 assist center investigators in external Quality Assurance audits by sponsoring agencies

3.2.2.5 provide special services as needed (such as maintaining codes and preparing/dispensing placebo treatments for double-blind trials, etc.).

3.3 Final Reports

The study PI will comply with the reporting requirements of the sponsoring agency and will file the final report (with the FDA or NCI) as required. The protocol will describe when the final analyses will begin after accrual is completed. A copy of the dataset that was used to produce the final analyses will be stored for future reference.