Evaluation of Seneca Valley Virus Levels Mouse to Man · Evaluation of Seneca Valley Virus Levels...
Transcript of Evaluation of Seneca Valley Virus Levels Mouse to Man · Evaluation of Seneca Valley Virus Levels...
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
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 trialOther assays used to detect nature of viral signal in vivoLessons 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 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
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
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
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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
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 ModelUp to 5 x 1011 vp/kg
No dose-limiting toxicity
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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
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
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
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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
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
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
107 vp/kg Pat 2Pat 1 Pat 3
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
107 vp/kg Pat 2Pat 1 Pat 3
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
107 vp/kg Pat 2Pat 1 Pat 3
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
107 vp/kg Pat 2Pat 1 Pat 3
Time(study day)
SVV-
001
(TC
ID50
/mL)
Viral Load – Cohort 1 (107 vp/kg)
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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
107 vp/kg Pat 2Pat 1 Pat 3
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
107 vp/kg Pat 2Pat 1 Pat 3
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
107 vp/kg Pat 2Pat 1 Pat 3
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
107 vp/kg Pat 2Pat 1 Pat 3
Time(study day)
SVV-
001
(TC
ID50
/sw
ab)
Viral Load – Cohort 1 (107 vp/kg)
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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
Infusion dose(vp/kg)
Seru
m S
VV-0
01(T
CID
50/m
L)
RT-PCR TCID50
Monitoring Input Dose
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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]
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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
107 vp/kg Pat 2Pat 1 Pat 3
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
108 vp/kg Pat 6Pat 4 Pat 7
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
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
107 108 109 10100
10
20
30
40
50
Infusion dose(vp/kg)
Tim
e to
Cle
aran
ce(d
ays)
Trend of Shorter Clearance Time with Dose
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
Day 8 Day 15 Peak
2468
10121416
107 108 109 1010
BLQLog 2
[Neu
tral
izat
ion
Tite
r]
Neutralization Response with Dose
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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)
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
Low-Rep 1e7 Hi-Rep 1e70
5
10
15
Infusion dose(vp/kg)
Tim
e to
Pea
k in
Ser
um(d
ays)
Peak in Serum – Small Cell Carcinoma (107 vp/kg)
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 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
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 HopkinsCharles Rudin, PI
US Oncology
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
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
Adrenal Gland
Liver Metastasis Normal LiverKidney
PancreasSpleen
Patient 001 Tumor-selective Replication
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 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
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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
EMEA/ICH Workshop on Viral/Vector Shedding | October 2007
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