Evaluation of Seneca Valley Virus Levels Mouse to Man · Evaluation of Seneca Valley Virus Levels Mouse to Man EMEA/IHC Meeting on Virus Shedding Rotterdam, The Netherlands ... October

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

Paul HallenbeckNeotropix, Inc.351 Phoenixville PikeMalvern, PA 19380www.neotropix.com

Evaluation of Seneca Valley Virus Levels Mouse to Man

EMEA/IHC Meeting on Virus SheddingRotterdam, The Netherlands

October 30, 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 trialOther assays used to detect nature of viral signal in vivoLessons learned


Discovery of Seneca Valley Virus (SVV-001)

Discovered serendipitously as a contaminant in an adenoviral vector preparation

Fetal Bovine Serum/Swine TrypsinBroad species tropism; A Natural host is swine In picornaviridae family; possibly new genusSelective tropism for tumors with neuroendocrine propertiesNot linked to any specific disease


Life Cycle


Phylogeny of SVV-001

0.1

HAV

AEV

AiV

BKV

PTV-1

ERBV-1

FMDV-O

ERAV

-1

LV

SV2PEV-8

HRV-14

HRV-2CV-A16BEV-1

PEV-9PV-1

CV-A21CV-B5

EV-70SEV-A1

998

1000

280

526

979

728914

545

490

758

1000

650

771

Hepatovirus

Parechovirus

TeschovirusAphthovirus

Erbovirus

Enterovirus + Rhinovirus

New genus?

0.1

HAV

AEV

AiV

BKV

EMCVTMEV

ERBV-1

FMDV-O

ERAV

HPeV-1

LV

SV2PEV-8

HRV-14

HRV-2CV-A16BEV-1

PEV-9PV-1

CV-A21CV-B5

EV-70SEV-A1

998

1000

280

526

979

728914

545

490

758

1000

650

771

Hepatovirus

Parechovirus

TeschovirusAphthovirus

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


H44

6H

187

H82

H20

9H

69AR

HC

C33

H11

84H

1963

DM

S153

H37

8H

2107

H52

6H

289

DM

S114

DM

S53

H13

39H

69H

2195

DM

S79

H14

6H

1618

H34

5H

1514

H17

70H

1299

H46

0H

OP-

62EK

VXH

OP-

92H

522

H23

H32

2MH

226

A549

H72

7H

835

UM

C-1

1

Y79

SK-N

EP-1

IMR

-32

D28

3Med

SK-N

-MC

WER

I-RB1

SK-N

-AS

SK-N

-SH

DAO

YSW

-13

H29

5RH

295

BON

AsPC

-1Bx

PC-3

-4

-3

-2

-1

0

1

2

3

4

SCLC NSCLC Pediatric Endocrine

Log

[1/E

C50

]

PHH

HAE

CH

AoSM

CH

CAE

CH

CAS

MC

HR

CE

HR

EH

PASM

CN

HA

HU

VEC

HM

VEC

Mon

ocyt

esPB

MC

-4

-3

-2

-1

0

1

2

3

4

Log

[1/E

C50

]

Human Tumor Cell Lines Normal Human Cells

Potent and highly selective cell killing in human tumor cells50% 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 observed

Selectivity of NTX-010 for Human Cancers


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 ModelUp to 5 x 1011 vp/kg

No dose-limiting toxicity


NTX-010 Efficacy in Tumor Models

Anti-tumor activity in 13/13 models testedXenograftSyngeneic (immunocompetent)OrthotopicMetastatic

NTX and 3 collaboratorsSt. Jude/NCI (Pediatric Preclinical Testing Program)

Pediatric models shown to predict clinical activityDurable complete responses in 3/3 models tested

Baylor College of MedicineEradication of metastases in orthotopic retinoblastoma

Johns HopkinsPrimary SCLC model


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


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


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


0 24 48 72 96 120 144 1680

1

2

3

4

5

6

7

SVV - Normal

SVV - TB

0

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


Biodistribution of SVV-001 RNA in A/J Mice

0 1 2 3 4 5 6 7 8 9 10 11 120123456789

Time (weeks)

SVV-

001

(Log

[R

NA

cop

ies]

)

0 1 2 3 4 5 6 7 8 9 10 11 120123456789

Time (weeks)

SVV-

001

(Log

[R

NA

cop

ies]

)

0 1 2 3 4 5 6 7 8 9 10 11 120123456789

Time (weeks)

SVV-

001

(Log

[R

NA

cop

ies]

)

BMBrainHeartKidneyLiverLung

LNOvarySpinal CordSpleenTailTestis

Subsequent analysis demonstrates that no infectious virus or full length genome exists post neutralizing

antibody titer presenceSlow degradation apparent


BrainTumor Heart

KidneyLiverLung

OvarySpleen

0 2 4 6 8 10 12 140123456789

10

Time (days)

SVV-

001

Con

cent

ratio

nLo

g [T

CID

50/m

g]

0 2 4 6 8 10 12 140123456789

10

Time (days)SV

V-00

1 C

once

ntra

tion

Log

[TC

ID50

/mg]

109 vp/kg IV bolus

Tumor-bearing Normal

Tumor-selective Replication in Mouse Tissues


Phase I Clinical Trial DesignTumors with one or more neuroendocrine markers

Small Cell (Lung, Elsewhere), Carcinoid, Others

Arm 1-Carcinoid Patients Single i.v. infusion in outpatient setting3 + 3 Dose Escalation in log incrementsDose level 1 = 10E7 viral particles/kg to 10E11 vp/kg Low permissive tumors

Arm 2-Small cell PatientsSingle i.v. infusion in outpatient setting3 + 3 Dose Escalation in log incrementsDose level 1 = 10E7 viral particles/kg/2 logs behind carcinoid armHigh permissive tumors-Elicits efficacy in high permissive tumors in mice

Intensive Monitoring of Viral kinetics/ClearanceSputum, stool, urine, serum, nasal swabInfectious Virus, qRT-PCR, neutralizing antibody titer

Clearance for two consecutive timepoints separated by 48 hrsNo less then two weeks of full monitoringDaily (days 1-5) week one; M, W, F (weeks 2-3), M, Th (weeks 4-6), M (weeks 7+)

Other assays as appropriate

SitesJohns Hopkins – Charlie RudinUS Oncology – 9 sites


Phase I Clinical Trial-Biosafety Considerations

Isolation of patientsChildren, pregnant women, limited travel, private bedroom and bathroom

Disinfect excretaMonitor viral sheddingEpidemiology amongst family members and direct caregiversContraception in men/Sterility in women


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


0 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

RN

A(c

opie

s/m

L)

0 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 2Pat 1 Pat 3

Time(study day)

SVV-

001

(TC

ID50

/mL)

0 10 20 30 40 50 60

10 4

10 5

10 6

10 7

10 8

10 9

10 10

BLQ

Feces RT-PCR


Time(study day)

SVV-

001

RN

A(c

opie

s/gr

am)

0 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


Time(study day)

SVV-

001

RN

A(c

opie

s/m

L)

0 10 20 30 40 50 60

10 3

10 4

10 5

10 6

10 7

10 8

BLQ

Feces IFT


Time(study day)

SVV-

001

(TC

ID50

/gra

m)

0 10 20 30 40 50 60

10 2

10 3

10 4

10 5

10 6

10 7

10 8

BLQ

Sputum IFT


Time(study day)

SVV-

001

(TC

ID50

/mL)

Viral Load – Cohort 1 (107 vp/kg)


0 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


Time(study day)

SVV-

001

RN

A(c

opie

s/m

L)

0 10 20 30 40 50 60

10 2

10 3

10 4

10 5

10 6

10 7

10 8

BLQ

Urine IFT


Time(study day)

SVV-

001

(TC

ID50

/mL)

0 10 20 30 40 50 60

10 3

10 4

10 5

10 6

10 7

10 8

10 9

10 10

BLQ


Time(study day)

SVV-

001

RN

A(c

opie

s/sw

ab)

Nasal Swabs RT-PCR

0 10 20 30 40 50 60

10 2

10 3

10 4

10 5

10 6

10 7

10 8

BLQ

Nasal Swabs IFT


Time(study day)

SVV-

001

(TC

ID50

/sw

ab)

Viral Load – Cohort 1 (107 vp/kg)


1e7 1e9 1e10

103

104

105

106

107

108

109

Infusion dose(vp/kg)

Seru

m S

VV-0

01 R

NA

(cop

ies/

mL)

1e7 1e9 1e10

102

103

104

105

106

107


Seru

m S

VV-0

01(T

CID

50/m

L)

RT-PCR TCID50

Monitoring Input Dose


Correlation of TCID50 vs. RT-PCR

2 3 4 5 6 7 84

5

6

7

8

9

10

r2 = 0.91Slope = 0.91

Log [TCID50]

Log

[RN

A c

opie

s]


0 10 20 30 40 50 60

10 4

10 5

10 6

10 7

10 8

10 9

10 10

BLQ

Feces RT-PCR


Time(study day)

SVV-

001

RN

A(c

opie

s/gr

am)

0 10 20 30 40 50 60

10 4

10 5

10 6

10 7

10 8

10 9

10 10

BLQ

Feces RT-PCR


Time(study day)

SVV-

001

RN

A(c

opie

s/gr

am)

0 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 11Pat 8 Pat 12Pat 10

Time(study day)

SVV-

001

RN

A(c

opie

s/gr

am)

0 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

RN

A(c

opie

s/gr

am)

107 vp/kg 108 vp/kg

109 vp/kg 1010 vp/kg

Shedding in Feces Decreases with Dose


107 108 109 10100

10

20

30

40

50


Tim

e to

Cle

aran

ce(d

ays)

Trend of Shorter Clearance Time with Dose


Day 8 Day 15 Peak

2468

10121416

107 108 109 1010

BLQLog 2

[Neu

tral

izat

ion

Tite

r]

Neutralization Response with Dose


0 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

RN

A(c

opie

s/m

L)

0 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

(TC

ID50

/mL)

0 5 10 15 20 25 302 0

2 5

2 10

2 15

2 20

Pat 13Pat 9Pat 1

SerumNeutralization

Pat 16 Pat 17 Pat 18

Time(study day)

αSV

V N

eutr

aliz

ing

Tite

r(fo

ld-d

ilutio

n)

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


Low-Rep 1e7 Hi-Rep 1e70

5

10

15


Tim

e to

Pea

k in

Ser

um(d

ays)

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


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 inpatients with higher doses

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

• 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


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 HopkinsCharles Rudin, PI

US Oncology


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


Adrenal Gland

Liver Metastasis Normal LiverKidney

PancreasSpleen

Patient 001 Tumor-selective Replication


Reaction Primer set Position Product size

R1 F2/ R5 348 / 1309 (5’end of the genome) 961 bpR4 F11 / R16 3052 / 4609 (Middle of the genome) 1557 bpR6 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


5e8 5e7 5e6 5e5 5e4 5e3 5e21Kb+ H+H-5e8 5e7 5e6 5e5 5e4 5e3 5e21Kb+ H+H- 5e1 5 .5 .251kb+

5e1 5 .5 .251kb+

• 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


Pat #1Day 4 H- H+1kb+ Pat #1

Day 17Pat #1Day 25

Normal human Seum RNA

SVV5e8 1kb+

Pat #1Day 4 H- H+1kb+ Pat #1

Day 17Pat #1Day 25

Normal human Seum RNA

SVV5e8 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 positiveassay 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

Evaluation of Seneca Valley Virus Levels Mouse to Man · Evaluation of Seneca Valley Virus Levels Mouse to Man EMEA/IHC Meeting on Virus Shedding Rotterdam, The Netherlands ... October

Documents