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

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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

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

Paul Hallenbeck Neotropix, Inc.

351 Phoenixville Pike Malvern, PA 19380 www.neotropix.com

Evaluation of Seneca Valley Virus Levels Mouse to Man

EMEA/IHC Meeting on Virus Shedding Rotterdam, The Netherlands October 30, 2007

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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

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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

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

Life Cycle

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

Phylogeny of SVV-001

0.1

HAV AEV AiV BKV PTV-1 ERBV-1 FMDV-O ERAV

  • 1

LV SV2 PEV-8 HRV-14 HRV-2 CV-A16 BEV-1 PEV-9PV-1 CV-A21 CV-B5 EV-70 SEV-A1

998 1000 280 526 979 728 914 545 490 758 1000 650 771

Hepatovirus Parechovirus Teschovirus Aphthovirus Erbovirus Enterovirus + Rhinovirus New genus?

0.1

HAV AEV AiV BKV EMCV TMEV ERBV-1 FMDV-O ERAV HPeV-1 LV SV2 PEV-8 HRV-14 HRV-2 CV-A16 BEV-1 PEV-9PV-1 CV-A21 CV-B5 EV-70 SEV-A1

998 1000 280 526 979 728 914 545 490 758 1000 650 771

Hepatovirus Parechovirus Teschovirus Aphthovirus Erbovirus Senecovirus Enterovirus + Rhinovirus New genus?

  • Sequence comparisons of P1, 2C, 3C & 3D and unique

features indicate a likely new genus called Senecovirus

Cardiovirus

SVV-001 & 12 isolates

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

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

  • 4
  • 3
  • 2
  • 1

1 2 3 4

SCLC NSCLC Pediatric Endocrine

Log [1/EC50]

PHH HAEC HAoSMC HCAEC HCASMC HRCE HRE HPASMC NHA HUVEC HMVEC Monocytes PBMC

  • 4
  • 3
  • 2
  • 1

1 2 3 4

Log [1/EC50]

Human Tumor Cell Lines Normal Human Cells

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

No cell killing

  • bserved

Selectivity of NTX-010 for Human Cancers

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

Minimal Toxicity in Pre-Clinical Models

Tumor Brain Heart Kidney Liver Lung Ovary Spleen

Murine Model

No dose-limiting toxicity up to 1014 vp/kg No microscopic pathology at 1014 vp/kg Tumor-specific replication observed Up to 3 x 1011 vp/kg No dose-limiting toxicity No infectious virus detected in tissues 7 days after dosing

Porcine Model Non-human Primate Model

Up to 5 x 1011 vp/kg No dose-limiting toxicity

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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
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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
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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
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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

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

24 48 72 96 120 144 168 1 2 3 4 5 6 7

SVV - Normal SVV - TB

1 2 3 4 5

SVV Neut - Normal

Day 21 - 2/3 1/3 2/3

SVV Neut - TB

Time (hr) SVV-001

(Log [pfu/ml])

SVV Neutralization

(Log [serum dilution])

SVV Kinetics in A/J tissues

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

Biodistribution of SVV-001 RNA in A/J Mice

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9

Time (weeks) SVV-001 (Log [RNA copies])

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9

Time (weeks) SVV-001 (Log [RNA copies])

1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9

Time (weeks) SVV-001 (Log [RNA copies])

BM Brain Heart Kidney Liver Lung LN Ovary Spinal Cord Spleen Tail Testis

Subsequent analysis demonstrates that no infectious virus or full length genome exists post neutralizing antibody titer presence Slow degradation apparent

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

Brain Tumor Heart Kidney Liver Lung Ovary Spleen

2 4 6 8 10 12 14 1 2 3 4 5 6 7 8 9 10

Time (days) SVV-001 Concentration Log [TCID50/mg]

2 4 6 8 10 12 14 1 2 3 4 5 6 7 8 9 10

Time (days) SVV-001 Concentration Log [TCID50/mg]

109 vp/kg IV bolus

Tumor-bearing Normal

Tumor-selective Replication in Mouse Tissues

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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
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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

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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

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

10 20 30 40 50 60 Pat 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 BLQ Pat 1 Pat 3

Serum RT-PCR

107 vp/kg Time (study day) SVV-001 RNA (copies/mL) 10 20 30 40 50 60 10 2 10 3 10 4 10 5 10 6 10 7 10 8 BLQ

Serum IFT

107 vp/kg Pat 2 Pat 1 Pat 3 Time (study day) SVV-001 (TCID50/mL) 10 20 30 40 50 60 10 4 10 5 10 6 10 7 10 8 10 9 10 10 BLQ

Feces RT-PCR

107 vp/kg Pat 2 Pat 1 Pat 3 Time (study day) SVV-001 RNA (copies/gram) 10 20 30 40 50 60 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 BLQ

Sputum RT-PCR

107 vp/kg Pat 2 Pat 1 Pat 3 Time (study day) SVV-001 RNA (copies/mL) 10 20 30 40 50 60 10 3 10 4 10 5 10 6 10 7 10 8 BLQ

Feces IFT

107 vp/kg Pat 2 Pat 1 Pat 3 Time (study day) SVV-001 (TCID50/gram) 10 20 30 40 50 60 10 2 10 3 10 4 10 5 10 6 10 7 10 8 BLQ

Sputum IFT

107 vp/kg Pat 2 Pat 1 Pat 3 Time (study day) SVV-001 (TCID50/mL)

Viral Load – Cohort 1 (107 vp/kg)

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

10 20 30 40 50 60 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10

BLQ

Urine RT-PCR

107 vp/kg Pat 2 Pat 1 Pat 3 Time (study day) SVV-001 RNA (copies/mL) 10 20 30 40 50 60 10 2 10 3 10 4 10 5 10 6 10 7 10 8

BLQ

Urine IFT

107 vp/kg Pat 2 Pat 1 Pat 3

Time (study day) SVV-001

(TCID50/mL) 10 20 30 40 50 60 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 BLQ 107 vp/kg Pat 2 Pat 1 Pat 3

Time (study day) SVV-001 RNA (copies/swab)

Nasal Swabs RT-PCR

10 20 30 40 50 60 10 2 10 3 10 4 10 5 10 6 10 7 10 8 BLQ

Nasal Swabs IFT

107 vp/kg Pat 2 Pat 1 Pat 3

Time (study day) SVV-001

(TCID50/swab)

Viral Load – Cohort 1 (107 vp/kg)

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

1e7 1e9 1e10 103 104 105 106 107 108 109

Infusion dose

(vp/kg)

Serum SVV-001 RNA

(copies/mL) 1e7 1e9 1e10 102 103 104 105 106 107

Infusion dose

(vp/kg)

Serum SVV-001

(TCID50/mL)

RT-PCR TCID50

Monitoring Input Dose

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

Correlation of TCID50 vs. RT-PCR

2 3 4 5 6 7 8 4 5 6 7 8 9 10

r2 = 0.91 Slope = 0.91

Log [TCID50] Log [RNA copies]

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

10 20 30 40 50 60 10 4 10 5 10 6 10 7 10 8 10 9 10 10

BLQ

Feces RT-PCR

107 vp/kg Pat 2 Pat 1 Pat 3 Time (study day) SVV-001 RNA (copies/gram) 10 20 30 40 50 60 10 4 10 5 10 6 10 7 10 8 10 9 10 10

BLQ

Feces RT-PCR

108 vp/kg Pat 6 Pat 4 Pat 7

Time (study day) SVV-001 RNA

(copies/gram) 10 20 30 40 50 60 10 4 10 5 10 6 10 7 10 8 10 9 10 10

BLQ

Feces RT-PCR

109 vp/kg Pat 11 Pat 8 Pat 12 Pat 10 Time (study day) SVV-001 RNA (copies/gram) 10 20 30 40 50 60 10 4 10 5 10 6 10 7 10 8 10 9 10 10

BLQ

Feces RT-PCR

1010 vp/kg Pat 14 Pat 15 Pat 19

Time (study day) SVV-001 RNA

(copies/gram)

107 vp/kg 108 vp/kg 109 vp/kg 1010 vp/kg

Shedding in Feces Decreases with Dose

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

107 108 109 1010 10 20 30 40 50

Infusion dose

(vp/kg)

Time to Clearance (days)

Trend of Shorter Clearance Time with Dose

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

Day 8 Day 15 Peak

2 4 6 8 10 12 14 16 107 108 109 1010

BLQ

Log2 [Neutralization Titer]

Neutralization Response with Dose

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

5 10 15 20 25 30 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 BLQ Pat 13

Serum RT-PCR

Pat 9 Pat 16 Pat 17 Pat 18 Pat 1

Time (study day) SVV-001 RNA

(copies/mL) 5 10 15 20 25 30 10 2 10 3 10 4 10 5 10 6 10 7 10 8

BLQ

Serum IFT

Pat 9 Pat 13 Pat 16 Pat 17 Pat 18 Pat 1 Time (study day) SVV-001 (TCID50/mL) 5 10 15 20 25 30 2 0 2 5 2 10 2 15 2 20 Pat 13 Pat 9 Pat 1

Serum Neutralization

Pat 16 Pat 17 Pat 18 Time (study day)

αSVV Neutralizing Titer

(fold-dilution)

Viral Load – Small Cell Carcinoma (107 vp/kg)

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

Low-Rep 1e7 Hi-Rep 1e7

5 10 15

Infusion dose

(vp/kg)

Time to Peak in Serum (days)

Peak in Serum – Small Cell Carcinoma (107 vp/kg)

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

Viral Shedding Conclusions

  • Tumor selective replication in small cell as predicted by preclinical

– Low titers in 14 carcinoid patients despite high doses (max 10e10 vp/kg) – High titers in 3/6 small cell patients despite low doses (max 10e 7 vp/kg)

  • Shorter times to peak virus, shorter times to clearance, lower levels

secreted in excreta samples, and higher neutralization titers in patients with higher doses

  • RT-PCR is a reliable and sensitive indicator of maximum theoretical

signal

  • Excellent correlation between RT-PCR and Infectious virus assay in

most samples

  • No issues of transmission
  • Viremia appears before shedding
  • Discordance between RT-PCR signal and TCID50 in rare patients.

– In two patients RT-PCR signal persists but infectious virus signal does not – Other assays (Long PCR, Northerns, IHC) indicate that RT-PCR signal in discordant cases in mice and man is from degraded genomic RNA

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

Acknowledgements Neotropix

  • John Neefe: Senior VP Clinical and Regulatory
  • Kevin Burroughs: Director of Non-Clinical Studies
  • Seshidhar Reddy: Director of Technology and New

Products

Johns Hopkins

Charles Rudin, PI

US Oncology

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

Other Methods

  • Is virus infectious assay not detecting infectious

virus in tissues

– Sensitivity – Neutralizing ab response – Production equals output – Slow degradation – Does RNA signal indicate infectious virus?

  • Long PCR
  • Northerns
  • In Situ Hybridization
  • IHC
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EMEA/ICH Workshop on Viral/Vector Shedding | October 2007

Adrenal Gland Liver Metastasis Normal Liver Kidney Pancreas Spleen

Patient 001 Tumor-selective Replication

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

Reaction Primer set Position Product size R1 F2/ R5 348 / 1309 (5’end of the genome) 961 bp R4 F11 / R16 3052 / 4609 (Middle of the genome) 1557 bp R6 F19 / R24 5452 / 7009 (3’end of the genome) 1557 bp

Intactness of Genomic RNA in serum - RT-PCR Assay

Primers for long RT-PCR reactions:

R1 R4 R6

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

5e8 5e7 5e6 5e5 5e4 5e3 5e2 1Kb+ H+ H- 5e8 5e7 5e6 5e5 5e4 5e3 5e2 1Kb+ H+ H-

5e1 5 .5 .25 1kb+ 5e1 5 .5 .25 1kb+

  • SVV-001 RNA ranging from 0.25 to 5e8 copies per reaction amplified by three primer pairs
  • 353-bp product of human beta-actin gene from HeLa cells served as positive control
  • HeLa RNA without primers served as negative control
  • Two out of three fragments amplified at 50 copies/reaction, thus sensitivity of assay is

50 copies of SVV-001 genome per reaction.

Determination of SVV-001 RT-PCR assay sensitivity

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EMEA/ICH Workshop on Viral/Vector Shedding | October 2007 Pat #1 Day 4 H- H+ 1kb+ Pat #1 Day 17 Pat #1 Day 25 Normal human Seum RNA SVV 5e8 1kb+ Pat #1 Day 4 H- H+ 1kb+ Pat #1 Day 17 Pat #1 Day 25 Normal human Seum RNA SVV 5e8 1kb+

  • RT-PCR reactions performed with RNA isolated from equal volumes of Patient 0001 serum

at study days 4, 17 & 25)

  • Genomic RNA (5e8 copies) from SVV-001 served as primary positive control
  • 353-bp product of human b-actin gene from HeLa cellular RNA served as a secondary positive

assay control (H+)

  • HeLa RNA containing no primer serves as negative control (H -)
  • Only study day 4 serum sample gave positive signal

Analysis of RNA in Patient 0001 Serum