622 FANGâ„¢ cytokine expression analyses update

622 FANGâ„¢ Cytokine Expression Analyses Update Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S240 CANCER IMMUNOTHERAPY III 620 Summary of[.]

Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy

S240

CANCER-IMMUNOTHERAPY III

620 Summary of bi-shRNAfurin/GM-CSF

Augmented Autologous Tumor Cell Vaccine

(FANG™) in Advanced Cancer of the Liver

Minal Barve,1,3 Phillip B Maples,2 Douglas Orr,5 Joseph Kuhn,6

Mitchell Magee,7 Jeffrey Lamont,5 Cynthia Bedell,1 Gladice

Wallraven,2 Beena O Pappen,2 Alyssa Roth,1 Staci Horvath,1

Derek Nemunaitis,1 Padmasini Kumar,2 Neil Senzer,1,2 John

Nemunaitis.1,2,3,4

1 Mary Crowley Cancer Research Centers, Dallas, TX; 2 Gradalis,

Inc., Dallas, TX; 3 Texas Oncology, P.A., Dallas, TX; 4 Medical

City Dallas Hospital, Dallas, TX; 5 Baylor Medical Center, Dallas,

TX; 6 WLS Surgical Associates, P.A., Dallas, TX; 7 Cardiovascular

Specialty Associates of North Texas, P.A., Dallas, TX.

There are limited systemic options available for the management

of advanced hepatocellular cancer (HCC) In a Phase I trial of a novel

autologous whole-cell tumor cell vaccine (FANG™) incorporating

a plasmid with an expressive GM-CSF transgene and a bifunctional

shRNA interference moiety targeting furin, we demonstrated

safety; confi rmed mechanism; verifi ed immunoeffectiveness via

tumor-specifi c gIFN ELISPOT conversion and ELISPOT-survival

correlation; and suggested an overall survival benefi t [Senzer et al Mol

Ther 2012; Senzer et al Vaccines and Vaccination in press] FANG™

vaccine, combining 1) knockdown of the proconvertase furin which

consequently limits tumor expression of TGFβ1 and TGFβ2 (both

potent inhibitors of immune response), 2) expression of GM-CSF and

3) provision of the full patient-specifi c tumor antigenic matrix, enables

a broadened dimensional approach to stimulation of the afferent arm

of the immune response We now provide further follow up of a subset

of 8 hepatocellular carcinoma (HCC) patients Vaccine manufacturing

was successful in 7/8 attempts (one failure due to insuffi cient cell

yield) Median GM-CSF expression was 144 pg/106 cells, TGFβ1

knockdown was 100%, and TGFβ2 knockdown was 93% of the vector

transported cells Five patients were vaccinated (1 or 2.5 x 107 cells/

intradermal injection, 6-11 vaccinations) No vaccine toxicity or

unique HCC-related toxicities were observed Three of these 5 patients

demonstrated evidence of an immune response per gIFN ELISPOT

assay Long-term follow up demonstrates survivals of 319, 729, 784,

931+ and 1043+ days of the FANG treated patients In conclusion,

the data support Phase II trial assessment of FANG vaccine in HCC

621 Phase I Study of FANG™ Vaccine in

Advanced Ewing’s Sarcoma

M Ghisoli,1,2 C Lenarsky,1 N Senzer,2,4 M Magee,3 J Lanoue,5 E

Mendeloff,6 C Bedell,2 P Kumar,4 D D Rao,4 G Wallraven,4 B

O Pappen,4 P B Maples,4 J Nemunaitis.1,2,3,4

1 Texas Oncology, PA, Dallas; 2 Mary Crowley Cancer Research

Centers, Dallas; 3 Medical City Dallas Hospital, Dallas;

4 Gradalis, Inc, Dallas; 5 Pediatric Surgical Associates, Dallas,

TX; 6 Congenital Heart Surgery Center, Medical City Children’s

Hospital, Dallas.

Ewing’s Sarcoma, uniquely characterized by expression of

EWS-FLI1 fusion protein, is the second most common malignant bone

tumor among children and adolescents Patients and physicians

continue to be frustrated by the lack of therapeutic gains in

meaningful improvement in overall survival for patients after failure

of conventional chemotherapy

The FANG™ vaccine contains a plasmid encoding both GMCSF

and an RNA interference (RNAi) moiety, bifunctional shRNAfurin

(bi-shRNAfurin) that targets and down regulates the proprotein

convertase Furin resulting in knockdown of both TGFb1 and

b2 The bi-shRNA technology is designed to maximize target

inhibition via a process involving amalgamated mRNA cleavage,

degradation and p-body sequestration GMCSF enhances dendritic

cell functionality and expands the cytotoxic T-cell population This

“triad” concept (enhanced effector cell activation + inhibition of innate immunosuppression + antigenic matrix exposure) promotes immune de-tolerization and enhances the afferent arm of the immune response

Based on our initial encouraging results in adult cancer in which safety, confi rmation of component mechanism and apparent survival advantage was demonstrated [Senzer et al Mol Ther 2012], a Phase

I study was initiated in patients (≥12 y.o.) with advanced/relapsed Ewing’s sarcoma who failed standard treatment options

Briefl y, the vaccine is cGMP manufactured at Gradalis, Inc following autologous tumor tissue harvest Vaccine is produced at

a dose of 1xcells/injection It is administered intradermally once

a month at a minimum of 5 to a maximum of 12 doses ELISPOT analysis of cytotoxic T cell function to autologous tumor antigens

is monitored at baseline, Months 2, 4, 6, 9, 12, and 18 GMSCF production is also measured

To date, 11 pediatric patients have entered the screening process:

7 with relapsed disease and 4 with progressive disease Vaccine manufacturing was successful in 8 of the 11 harvested patients (2 patients had insuffi cient viable cells and 1 had contaminated tissue) Five patients have received vaccine; three of whom experienced progressive disease Two patients received 8 and 4 doses, respectively, and both have stable disease (494 and 99 days from enrollment) A total of 16 doses have been given There have been no related serious adverse effects, one patient had a grade 1 treatment-related AE limited to skin erythema at the injection site Three of 3 evaluable patients showed ELISPOT activation (see Table) High levels of knockdown were achieved for TGFb1 (88–100%, mean 98%), TGFb2 (84–100%, mean 95%) and Furin (74–98%, mean 89%) This limited initial data shows the safety and feasibility of this study and possible clinical benefi t Updated results will be presented

ELISPOT rxn’s (+ > 10)

Mo 12 206

Mo 14 186

Mo 17 136

622 FANG™ Cytokine Expression Analyses Update

Padmasini Kumar,1 Connor Phalon,1 Beena O Pappen,1

Gladice Wallraven,1 Yang Yu,1 Fabienne Norvell,1 Himabindu Menakuru,1 Chris M Jay,1 Zhaohui Wang,1 Donald D Rao,1 John Nemunaitis,1,2,3,4 Neil Senzer,1,2 Phillip B Maples.1

1 Gradalis, Inc., Dallas, TX; 2 Mary Crowley Cancer Research Centers, Dallas, TX; 3 Medical City Dallas Hospital, Dallas, TX;

4 Texas Oncology, P.A., Dallas, TX.

We have developed an autologous whole cell tumor vaccine, FANG™, incorporating a plasmid encoding GMCSF and a novel bifunctional short hairpin RNA (bi-shRNA) targeting FURIN FURIN, also known as furin/PACE/SPC1, is an enzyme, which belongs to the subtilisin-like proprotein convertase family The proprotein convertases processes latent precursor proteins into their biologically active products One of the furin substrates is the family

of transforming growth factor beta (TGFβ) precursors which have been shown to play a signifi cant role in tumor progression, metastasis, and tumor induced immunosuppression

Molecular Therapy Volume 22, Supplement 1, May 2014

Copyright © The American Society of Gene & Cell Therapy S241

CANCER-IMMUNOTHERAPY III

An aliquot of cells from before and after transfection with FANG™

plasmid were placed in culture and incubated at 37ºC Cells and

supernatants were collected on Days 0, 1, 2, 3, 4, 7, 10 and 14

GMCSF, TGFβ1, TGFβ2 (R&D Systems) and Furin (USCN Life

Sciences) proteins were quantifi ed by enzyme-linked immunosorbent

assay (ELISA) kits We demonstrated earlier an average increase

(n=85) in the expression of GMCSF from 7.3 to 1,108 pg/106cells/

ml at Day 4 Mean TGFβ1 and β2 effective target knockdown was

93.5 and 92.5% from baseline, respectively (Senzer et.al Mol Ther

2011) Furin knockdown was 85.09% The percentage knockdown

of TGFβ1 and TGFβ2 were signifi cant with p <0.001 and consistent

with the Furin knockdown also with p<0.001 results

The number of vaccines manufactured in Phase I is currently at 79

In addition we have manufactured 81 vaccines for Phase II Ovarian

Cancer, 10 vaccines for Phase II Melanoma and 3 vaccines for Phase

II Colorectal cancers with liver metastasis A total of 173 FANG

vaccines all together were manufactured todate

We have reduced the number of time points the assay samples were

collected from 8 to 5 time points with Day 0, 3, 4, 7 and 10 only for

the supernatants collected for cytokine expression We show that

this reduction in assay time points does not alter the outcome of the

QC release criteria

Updated cytokine evaluation for Phase I and Phase II studies will be

presented in ASGCT 2014 An increase in the expression of GMCSF

and knockdown of TGFβ1, TGFβ2 and FURIN are consistently

achieved in all tumor cells transfected with FANG plasmid

623 HLA Typing Identity Test for Verifi cation

of Patient to their FANGTM Autologous Cancer

Vaccine

Yang Yu,1 Nicolas Taquet,1 John Nemunaitis,1,2 Phillip B Maples.1

1 Gradalis, Inc., Dallas, TX; 2 Mary Crowley Cancer Research

Centers, Dallas, TX.

cancer vaccine, Gradalis receives tumor sample deliveries from

multiple clinics and hospitals Identity match of the tumor sample

and the vaccine manufactured with the patient is an essential QA

component We have taken a number of steps to ensure the integrity

of Patient – Vaccine tumor collection / cGMP manufacturing /

clinical treatment continuum A most reliable method for specimen

identifi cation is DNA identity testing, which will verify the origin

of two samples We adopted the HLA (Human Leukocyte Antigen)

molecular genotyping by SSP-PCR (sequence-specific primer

polymerase chain reaction) as our identity method HLA testing

is a widely accepted method of identity testing commonly used in

solid organ and bone marrow transplantation and paternity testing

In comparison to the comprehensive STR (Short Tandem Repeat)

DNA profi ling, HLA typing is reliable, cost effective, and has a short

turnaround time Genomic DNA isolated from the peripheral white

blood cells of the patient is considered “correct” or “known” This is

compared to the tumor cells which are obtained on day-1 of vaccine

manufacturing before plasmid transfection The DNA extraction was

performed by the DNeasy kit (Qiagen) and DNA quantity and quality

is subsequently determined by Nano-Drop The ABDRDQ SSP-PCR

HLA Typing Kit which include Class I A Locus, B Locus and the

Class II DR/DQ loci from One Lambda, and Taq polymerase from

Promega were utilized for the HLA typing The positive HLA antigens

are identifi ed by post-PCR gel electrophoresis The confi rmation of

the tumor tissue origin from the patient is determined by the complete

molecular HLA typing match between the tumor tissue and the blood

We previously reported an analysis of 58 vaccine-blood sample

pairs on Phase I and Phase II FANG vaccine trials Since that time

another 23 Phase-II FANG vaccine-blood sample pairs have been

tested and analyzed All vaccine-blood sample paired HLA typing results are complete matches These results support HLA typing as

an identity verifi cation test for FANG™ vaccine

624 Randomized Phase II Trial of Adjuvant Autologous Tumor Cell Vaccine (FANG™) for High Risk Stage III/IV Ovarian Cancer: Preliminary Results

J Oh,3 M Barve,1,3 C Bedell,1 J Kuhn,5 B Fine,3 T P Heffernan,6

C M Matthews,3 C A Stringer,3 E C Koon,3 H Goodman,7 E

L Fleming,8 L R DeMars,8 M K Bergman,9 B O Pappen,4 P Kumar,4 P B Maples,4 N Senzer,1,4 J Nemunaitis.1,2,3,4

1 Mary Crowley Cancer Research Centers, Dallas; 2 Medical City Dallas Hospital, Dallas; 3 Texas Oncology, PA, Dallas; 4 Gradalis, Inc, Dallas; 5 WLS Surgery Associates, PA, Dallas; 6 North Texas Gyn Onc, Dallas; 7 Florida Cancer Specialists, West Palm Beach;

8 Dartmouth-Hitchcock Medical Ctr, Lebanon; 9 Cancer Care Northwest, Spokane.

Introduction: Phase I evaluation of FANG™ vaccine (GMCSF/ bi-shRNAi furin vector transfected autologous tumor) demonstrated safety; confi rmed GMCSF transgene expression and knockdown

of furin and consequent TGFβ1 and β2 expression; showed T-cell activation per gamma IFN-ELISPOT and resulted in a longer than expected survival duration thereby justifying Phase II testing [Senzer

et al Mol Ther 2012; Senzer et al Vaccines and Vaccin in press] The majority of women with Stage III/IV ovarian cancer who achieve

a complete clinical response with standard of care management involving debulking surgery and consolidation chemotherapy will relapse within 2 years

Methods: This is a 2:1 randomized Phase II open-label trial of FANG using tumor harvested at the time of surgical debulking Sixty patients achieving clinical complete response following primary surgical debulking and chemotherapy will undergo vaccine construction Given the high recurrence rate and subsequent progression seen with frontline standard of care treatment of advanced stage ovarian cancer, we initiated a randomized Phase II study of adjuvant FANG vaccination following initial debulking/chemotherapy Forty patients will receive 1.0x107 cells/intradermal injection once a month for up

to 12 doses and 20 will be followed as per standard of care without maintenance therapy Key endpoints include safety, immune response and progression free survival

Preliminary results: Thus far, 23 patients have been randomized, 15

of whom have received at least 1 FANG injection (103 total injections) and 8 have been randomized to No FANG treatment This trial design allows for control group cross over at time of progression No toxic effect has been observed A higher rate of ELISPOT response has been elicited in this minimum disease population in comparison

to that achieved in Phase I trial (92% vs 50%) Mean PFS internal difference of the FANG vs No FANG patients was 491 vs 337 days ELISPOT positivity at baseline, although rare after debulking surgery, may be identifi ed as a positive prognostic factor for PFS independent

of FANG vaccine Preliminary PFS of patients receiving FANG with negative ELISPOT (n=20) prior to randomization is shown in fi gure

In conclusion, Phase II accrual continues Updated results will

be presented