Evaluation of Seneca Valley Virus Levels Mouse to Man EMEA/IHC - PowerPoint PPT Presentation

Evaluation of Seneca Valley Virus Levels Mouse to Man EMEA/IHC Meeting on Virus Shedding Rotterdam, The Netherlands October 30, 2007 Paul Hallenbeck Neotropix, Inc. 351 Phoenixville Pike Malvern, PA 19380 www.neotropix.com EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Introduction � Brief background of Seneca Valley Virus (SVV) � Assays used to detect SVV in vivo. � Kinetics/Biodistribution in Mice � Kinetics/Shedding in Patients treated with SVV in Phase I Clinical trial � Other assays used to detect nature of viral signal in vivo � Lessons learned EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Discovery of Seneca Valley Virus (SVV-001) � Discovered serendipitously as a contaminant in an adenoviral vector preparation � Fetal Bovine Serum/Swine Trypsin � Broad species tropism; A Natural host is swine � In picornaviridae family; possibly new genus � Selective tropism for tumors with neuroendocrine properties � Not linked to any specific disease EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Life Cycle EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Phylogeny of SVV-001 • Sequence comparisons of P1, 2C, 3C & 3D and unique features indicate a likely new genus called Senecovirus Enterovirus + Rhinovirus Enterovirus + Rhinovirus PEV-9PV-1 PEV-9PV-1 CV-A21 CV-A21 BEV-1 BEV-1 CV-B5 CV-B5 CV-A16 CV-A16 EV-70 EV-70 HRV-2 HRV-2 HAV HAV SEV-A1 SEV-A1 Hepatovirus Hepatovirus HRV-14 HRV-14 AEV AEV PEV-8 PEV-8 650 650 SV2 SV2 New genus? 771 771 LV LV 998 998 1000 1000 AiV AiV 758 758 1000 1000 HPeV-1 280 280 490 490 Parechovirus Parechovirus -1 545 545 728 728 914 914 BKV BKV ERAV ERAV 979 979 PTV-1 Aphthovirus Aphthovirus 526 526 Teschovirus Teschovirus FMDV-O FMDV-O 0.1 0.1 SVV-001 & Erbovirus Erbovirus EMCV Senecovirus New genus? 12 isolates TMEV ERBV-1 ERBV-1 Cardiovirus EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Selectivity of NTX-010 for Human Cancers Human Tumor Cell Lines 4 Normal Human Cells 4 3 3 2 2 Log [1/EC 50 ] Log [1/EC 50 ] 1 1 No cell killing 0 0 observed -1 -1 -2 -2 -3 -3 -4 -4 H446 H187 H82 H209 H69AR HCC33 H1184 H1963 DMS153 H378 H2107 H526 H289 DMS114 DMS53 H1339 H69 H2195 DMS79 H146 H1618 H345 H1514 H1770 H1299 H460 HOP-62 EKVX HOP-92 H522 H23 H322M H226 A549 H727 H835 UMC-11 Y79 SK-NEP-1 IMR-32 D283Med SK-N-MC WERI-RB1 SK-N-AS SK-N-SH DAOY SW-13 H295R H295 BON AsPC-1 BxPC-3 HRCE HRE NHA PHH HAEC HAoSMC HCAEC HCASMC HPASMC HUVEC HMVEC Monocytes PBMC SCLC NSCLC Pediatric Endocrine � Potent and highly selective cell killing in human tumor cells � 50% of tested SCLC are permissive � 100% of tested pediatric cancers permissive � No cell killing or viral replication observed on normal human cells � ≥ 10,000 fold selectivity in cell killing of highly permissive tumor vs. normal cells EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Minimal Toxicity in Pre-Clinical Models Murine Model � No dose-limiting toxicity up to 10 14 vp/kg � No microscopic Tumor Brain Heart Kidney pathology at 10 14 vp/kg � Tumor-specific replication observed Liver Lung Ovary Spleen Porcine Model Non-human Primate Model � Up to 3 x 10 11 vp/kg � Up to 5 x 10 11 vp/kg � No dose-limiting toxicity � No dose-limiting toxicity � No infectious virus detected in tissues 7 days after dosing EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

NTX-010 Efficacy in Tumor Models � Anti-tumor activity in 13/13 models tested � Xenograft � Syngeneic (immunocompetent) � Orthotopic � Metastatic � NTX and 3 collaborators � St. Jude/NCI (Pediatric Preclinical Testing Program) � Pediatric models shown to predict clinical activity � Durable complete responses in 3/3 models tested � Baylor College of Medicine � Eradication of metastases in orthotopic retinoblastoma � Johns Hopkins � Primary SCLC model EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Measuring Virus Levels in Vivo • Broad based – Tissues, serum, plasma, whole blood – Excreta samples • Stool, urine, serum, sputum, nasal swabs • Sensitivity • Reproducibility • Linearity • Practicality • Collection • Storage • Shipping • Stability • What assay tells you EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Assays • Real Time PCR – Quantitatively measures small piece of viral genome • Sensitive assay-measures maximum signal • Detects virus in the presence of some functional inhibitors – Example-Antibodies • High throughput • High reproducibility • High specificity • Does not measure infectious virus-only measures small piece of genome • Subject to contamination and false positives • Subject to degradation – Specific considerations necessary depending upon sample type • Example-Stool – PCR inhibitors in stool requiring sample dilution • Infectious virus – TCID50 • Measures infectious virus • Two logs less sensitive then RT-PCR assay • May be inhibited or inactivated by numerous cmpds – Example-antibodies • Labor intensive and expensive • Low throughput • Lower specificity – Specific considerations still necessary depending upon sample type • All samples filtered due to sterility issues EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Kinetics/Biodistribution of SVV-001 RNA in A/J Mice • Kinetics in serum and tissues in mice +/- tumors • Single IV injection • Doses analyzed – 10E9, 3 x 10E11, 10E14 vp/kg • Multiple tissues collected – Over 2 weeks, 3, 6 and 12 weeks • Assays – Genomic RNA by quantitative real-time RT-PCR – Infectious virus by TCID50 – Neutralizing antibodies EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

SVV Kinetics in A/J tissues 7 5 6 SVV Neutralization (Log [serum dilution]) Day 21 - 4 5 (Log [pfu/ml]) SVV-001 3 2/3 4 2/3 3 2 1/3 2 1 1 0 0 0 24 48 72 96 120 144 168 Time (hr) SVV - Normal SVV Neut - Normal SVV - TB SVV Neut - TB EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Biodistribution of SVV-001 RNA in A/J Mice 9 BM LN 8 Brain Ovary (Log [RNA copies]) 7 Heart Spinal Cord 6 SVV-001 Kidney Spleen 5 Liver Tail 4 3 Lung Testis 2 1 9 0 8 (Log [RNA copies]) 0 1 2 3 4 5 6 7 8 9 10 11 12 7 Time (weeks) 6 SVV-001 5 9 4 8 (Log [RNA copies]) 3 7 2 6 SVV-001 1 5 0 4 0 1 2 3 4 5 6 7 8 9 10 11 12 3 Time (weeks) 2 1 0 Subsequent analysis demonstrates that no infectious 0 1 2 3 4 5 6 7 8 9 10 11 12 virus or full length genome exists post neutralizing Time (weeks) antibody titer presence Slow degradation apparent EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Tumor-selective Replication in Mouse Tissues 10 9 vp/kg IV bolus 10 10 Tumor-bearing Normal 9 9 SVV-001 Concentration SVV-001 Concentration 8 8 Log [TCID 50 /mg] Log [TCID 50 /mg] 7 7 6 6 5 5 4 4 3 3 2 2 1 1 0 0 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 Time (days) Time (days) Tumor Heart Liver Ovary Brain Kidney Lung Spleen EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Phase I Clinical Trial Design Tumors with one or more neuroendocrine markers � � Small Cell (Lung, Elsewhere), Carcinoid, Others � Arm 1-Carcinoid Patients Single i.v. infusion in outpatient setting � � 3 + 3 Dose Escalation in log increments � Dose level 1 = 10E7 viral particles/kg to 10E11 vp/kg � Low permissive tumors � Arm 2-Small cell Patients � Single i.v. infusion in outpatient setting 3 + 3 Dose Escalation in log increments � � Dose level 1 = 10E7 viral particles/kg/2 logs behind carcinoid arm � High permissive tumors-Elicits efficacy in high permissive tumors in mice � Intensive Monitoring of Viral kinetics/Clearance � Sputum, stool, urine, serum, nasal swab � Infectious Virus, qRT-PCR, neutralizing antibody titer � Clearance for two consecutive timepoints separated by 48 hrs � No less then two weeks of full monitoring � Daily (days 1-5) week one; M, W, F (weeks 2-3), M, Th (weeks 4-6), M (weeks 7+) � Other assays as appropriate � Sites � Johns Hopkins – Charlie Rudin � US Oncology – 9 sites EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Phase I Clinical Trial-Biosafety Considerations � Isolation of patients � Children, pregnant women, limited travel, private bedroom and bathroom � Disinfect excreta � Monitor viral shedding � Epidemiology amongst family members and direct caregivers � Contraception in men/Sterility in women EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

How we use assays • Monitor dosing – Verify input dose • Monitor replication to distinguish high and low replicators – Presence of permissive tissue (tumor) – Monitor for safety • Monitor route and kinetics of shedding – Clearance for safety • Monitor immune response to virus – Patient safety – Correlative with input virus and viral replication EMEA/ICH Workshop on Viral/Vector Shedding | October 2007