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Supplemental Methods

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									Chiocca et al: Phase I trial of Ad.hIFNβ glioma gene therapy

Supplemental Materials to: A Phase I, Dose-Escalation, Multi-Institutional Trial of Ad.hIFN- Gene Therapy for Recurrent Malignant Glioma: Safety and Correlative Molecular Results

E. Antonio Chiocca1, Katie M. Smith, B. McKinney, Cheryl A. Palmer, Steven Rosenfeld, Kevin Lillehei, Allan Hamilton, Betty K. DeMasters, Kevin Judy, David Kirn

Supplemental Methods

Adenoviral vector The vector was derived from a master seed stock of H5.010CMVhIFN- [1], which was generated through a collaboration between Biogen Idec and the University of Pennsylvania. The transgene product (natural hIFN-) is a single-chain, 166-amino-acid glycoprotein containing a single N-linked oligosaccharide. Ad.hIFN- was manufactured under GMP conditions by BioReliance Inc. (Rockville, MD). The vector was produced in HEK293 cell, vialed at a concentration of 3.4  1011 vp/mL and stored at -60C or below. The formulation contained 20 mM Tris (pH 8.0) and 10% glycerol. The vialed Ad.hIFN- was analyzed for sterility, activity, purity and for the presence of adventitious virus contaminants. The lots used in this study passed the specifications for transgene expression (> 4 ng/mL of hIFN-), plaque-forming ability ( 100 particles/plaque-

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Chiocca et al: Phase I trial of Ad.hIFNβ glioma gene therapy

forming unit), host cell DNA ( 100 ng/dose for DNA  200 base pairs), and endotoxin concentration ( 3.5 endotoxin units/mL). The lots used for this study contained  4 copies of replication-competent adenovirus (RCA) in 3  1010 vp.

Patient eligibility and study design Written, informed consent was obtained from all patients before initiation of any studyrelated procedures. Good Clinical Practice guidelines and the Declaration of Helsinki were followed. The centers underwent routine auditing of records and were visited by the corporate sponsor (Biogen Idec) before trial initiation to ensure compliance and expertise. Possible recurrent tumor was defined as the reappearance of gadolinium-enhanced tumor on MRI. Progressive tumor was defined as an increase in size on two successive gadolinium-enhanced MRI scans. Tumor had to be confirmed by biopsy of frozen sections to be consistent with malignant glioma at the time of initial stereotactic treatment with Ad.hIFN-. Tumor resection had to be clinically indicated, and at least one tumor mass to be resected had to be amenable to stereotactic injection according to the neurosurgeon. An Eastern Cooperative Oncology Group (ECOG) performance status of 0 – 2 was required. Patients were to be taking anticonvulsant therapy during the study and could not be intolerant to corticosteroids. Exclusion criteria included a diagnosis of anaplastic oligodendroglioma, brainstem or optic chiasm involvement of tumor, uncontrolled seizure disorder, a history or diagnosis of an invasive malignancy other than Grade 3 or 4 glioma within 5 years of enrollment, or anticipated communication between cerebral ventricles and tumor resection cavity. Patients with blood test results indicating abnormal organ function, prolonged prothrombin time, or low hematopoietic cell counts

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Chiocca et al: Phase I trial of Ad.hIFNβ glioma gene therapy

(platelets  100,000 plts/mm3 and/or absolute neutrophil count  1500 cells/mm3) were not eligible. All cancer treatments were to be completed at least 4 weeks prior to administration of Ad.hIFN-. Patients were not eligible if they had prior treatment with any gene delivery vector while previous treatment with either Gliadel wafers or stereotactic radiosurgery was allowed. Women were excluded if they were pregnant, nursing, or planning to become pregnant. Effective contraception was required during the study for both men and women. The doses for the clinical trial were selected based on preclinical toxicology data showing that the highest dose per kg. was approximately equal to the lowest dose per kg. that was well tolerated in macaques.. The MTD was defined as one dose below that at which two or more patients experienced a dose-limiting toxicity (DLT). DLTs were classified as all Grade 3 or 4 toxicities (CTC version 2.0) considered to have a definite or likely relationship to study treatment, in addition to Grade 3 hydrocephalus, meningitis, brain edema, intracranial bleeding, or seizures not controlled by appropriate medical therapy, (as defined in ref. [2]). The following events known to have a high frequency in this patient population, including deep vein thrombosis, pulmonary embolism, and hyponatremia were not to be considered as related to the therapy for DLT determination. An independent data safety monitoring committee reviewed patient safety data and was responsible for alerting the sponsor to possible safety concerns related to the conduct of the study. Details related to injection procedure

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Chiocca et al: Phase I trial of Ad.hIFNβ glioma gene therapy

Subjects were to receive corticosteroids (minimum dose 8 mg twice a day of dexamethasone) beginning 2 days prior to the first administration of Ad.hIFN-, and ending 1 week after the second administration. Before and after this period,

corticosteroid therapy was to be given at the discretion of the investigator. CT-guided or MRI-guided frame-based stereotaxis was employed for the first injection and intratumoral injection sites were planned to deliver the agent into contrastor gadolinium-enhancing regions. The rationale for the two sequential injections between day 1 and day 4 or 8 was to allow for the recovery of tumor tissue after the first stereotactic intratumoral injection to allow for gene marking and other correlative analyses to be performed, followed by the second intracavitary injection that possessed more of a “therapeutic” intent[3-5]. Injections into the tumor bed were uniformly distributed throughout the resection cavity wall but had to spaced by at least 1 cm; each injection was performed using a tuberculin syringe to the depth of the needle hub (about 10 mm.). Each injection was carried out over 1 minute with slow retraction of the needle to minimize reflux. Injections were not performed into ventricular cavities and were done to minimize reflux into cerebrospinal fluid spaces. Surgical excision of tumor and opening/closure of craniotomy was performed employing routine neurosurgical techniques. Patients were monitored for concomitant medications and adverse events until the time of study withdrawal (due to disease progression, patient refusal to continue or death). The change in schedule of second injection/ craniotomy from day 8 to day 4 was prompted by necessities in surgical scheduling of cases encountered by the sites which required a shortening of the interval between the two operations. 4

Chiocca et al: Phase I trial of Ad.hIFNβ glioma gene therapy

Brain histopathology Biopsy samples were placed in 10% neutral-buffered formalin for processing into paraffin blocks and slide preparation at the University of Alabama Neuropathology Division. All slides were read by a board-certified neuropathologist with expertise in tumor histology blinded to cohort. Glass slides were stained with hematoxylin and eosin and were analyzed for protocol-defined histological characteristics, including cellularity, nuclear pleomorphism, mitotic index, inflammatory cell infiltration, endothelial proliferation, and necrosis. Quantitative PCR for vector biodistribution The PCR primers and TaqMan probe (Applied Biosystems, Foster City, CA) were designed to amplify and detect a 72-base-pair segment corresponding to the E1 or E3 deletion. The sequences of the primers were as follows: E3 forward primer: 5’-TTG.CCA.GAG.AAC.ACC.AAG.ATT.G-3’: E3 reverse primer : 5’-TGG.TCA.TTT.TTC.CGT.CTG.TGA.A-3’

E3 TaqMan probe : 5’-6FAM-CAA.ATT.CGC.AAC.TGG.CGC.CCT.G-TAMRA-3. Samples from all patients were analyzed for the E3 deletion; samples from a subset of patients were analyzed for E1. The lower limit of quantitation for the assay was  50 copies/g DNA. Positive values 50 copies/g DNA were considered detectable but were not quantifiable. CPE Assay

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Chiocca et al: Phase I trial of Ad.hIFNβ glioma gene therapy

This assay was added late in the trial and specimens were only available for patients enrolled in the two high-dose cohorts. All cultures were terminated after 28

days, and spot slides were prepared from all remaining negative flasks and examined for adenovirus immunofluorescent staining.

Antibody assays

Fot detection of antibodies to hIFN serum samples were screened in an ELISA assay. A murine monoclonal anti-hIFN- antibody was coated on a 96-well plate.

Human IFN- was added to half of the wells for capture by the antibody on the plate; diluent was added to the remaining wells to measure background binding of the test samples. Diluted study samples and controls were added to both halves of the plate. Antibodies were detected by a peroxidase-conjugated polyclonal antihuman IgG antibody. Samples testing positive in the ELISA assay ( 20 international units [IU]) were then tested for the presence of neutralizing antibodies in a biologic screening assay. A549 cells were grown in microtiter wells and then incubated with hIFN- in the presence of serum samples or controls. The cells were then challenged with EMC virus, crystal violet stain was added to all plates, and CPE was read visually. If the serum sample was able to neutralize the protective effect of hIFN- against viral lysis of the cells to any degree, the sample was considered positive. Samples testing positive in the screening assay were retested at higher dilutions to determine neutralizing titer. The plates were scored

visually against the hIFN- standard; the titer is the inverse of the dilution of sample required to neutralize 10 IU/mL of hIFN-.

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Chiocca et al: Phase I trial of Ad.hIFNβ glioma gene therapy

For detection of antibodies to adenovirus, the assay control results were required to fall within a predetermined range to meet assay acceptance criteria. Samples that exhibited a net average optical density (coated side value minus sham-coated side value) of less than 0.100 were considered negative and reported as below the limit of quantification (BLQ). Samples testing positive were serially diluted and tested in a titration assay in which the titer was defined as the reciprocal of the highest dilution that yielded a positive result.

Supplemental results

Patient demographics and pathology The primary diagnosis was confirmed by an independent neuropathologist for patients in the 6  1010 vp and 2  1011 vp treatment groups (this procedure was not in place for lowest cohort but was instituted, by amendment, for cohorts 2 and 3). On central histopathologic assessment, one patient enrolled in the highest dose-cohort was determined to have grade III anaplastic oligoastrocytoma instead of GBM, as locally classified. As per protocol, all patients had received some form of previous treatment, in addition to primary surgical resection, prior to study treatment and had

relapsed/refractory disease.

Details of patient with DLT 7

Chiocca et al: Phase I trial of Ad.hIFNβ glioma gene therapy

The patient had a history of generalized seizure disorder secondary to his brain tumor. The patient tolerated intratumoral treatment on Day 1 without toxicity. The second dose was injected into the wall of the resection cavity on Day 4 as described in the Methods section. Of note, the tumor resection cavity was located along the inferior surface of the left temporal lobe, allowing for gravity dependent extravasation of the drug out of the tissues and into the subarachnoid space of the basilar cisterns. On Day 8, four days after the second dose of Ad.hIFN- into normal brain adjacent to the resection cavity, the patient began experiencing confusion and became progressively lethargic and dehydrated. On Day 9 he was hospitalized for Grade 3 confusion. On Day 11, following a generalized seizure, lumbar puncture analysis was unremarkable, with no evidence of meningitis or opportunistic viral infections. On Day 20, the patient experienced another generalized seizure. Continuous EEG monitoring revealed recurrent temporal lobe seizures. On Day 21, Grade 4 confusion was noted.

Hematologic studies Nine patients had elevated baseline levels of GGT (a marker of brain capillary osmotic activity). Elevated and fluctuating GGT levels were observed in all patients during the study, no patient experienced an increase in GGT of more than one grade from their baseline value, and the observed changes were transitory. There were no Grade 4 GGT elevations, and no changes in GGT were considered related to treatment. One patient (low-dose group) had transient increases in serum alanine transaminase (ALT), aspartate transaminase (AST), and GGT, which were considered unrelated to study

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Chiocca et al: Phase I trial of Ad.hIFNβ glioma gene therapy

treatment and attributable to the surgical procedure. One patient (2  1011 vp treatment group) had Grade 4 hyperglycemia on Day 30 that was considered unrelated to study treatment. This patient was not diabetic. Perhaps the Grade 4 hyperglycemia can be attributed to concomitant medication with steroids.

Brain Histopathology Baseline samples were evaluated in all patients, injections of different doses were performed in identical fashion and all slides were read by an experienced neuropathologist who was blinded to treatment group. From baseline to the time of surgical resection, no consistent changes were noted in tumor samples in overall cellularity, nuclear pleomorphism, mitotic index, inflammatory cell infiltrates, endothelial proliferation, necrosis and evidence of radiation-induced injury. Proliferation rate (MIB 1(+) cell percentage) ranged from 0.54% to 54.5% on Day 1 and from 0.9% to 31.8% on the day of tumor resection (Day 4 or 8); no consistent changes were demonstrated in paired samples.

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