Oncolytic Vi
V rotherapy
i
for
for Multiple Myeloma
Stephen J Russell
Mayo Clinic
Scottsdale,
Scottsdale Arizona
Rochester,
Rochester Minnesota
Jacksonville,
Jacksonville Florida
Mayo Clinic College of Medicine
Mayo Clinic Comprehensive Cancer Center
Oncolytic viruses for cancer therapy
"Onco" = cancer
"lytic" = killing
· Harness the destructive power of viruses
· Tu
T mor
u
specific
· Efficient killing
killing of
o
of malignant
malignant cells
· Stimulate host's
host' anti
anti--tumor
tumor activity
· Recombinant "armed" oncolytic viruses
· Can be combined with conventional therapies
Adenovirus
HSV--1
1
NDV
Reovirus
Coxsackie
Vaccinia
V
Measles
Multiple Myeloma is an
appealing target for
intravenous virotherapy
Disseminated from outset
Low antiviral Ab titers
Attenuated measles for myeloma
HF
H
1
N
PVC
M
F
H
L
15894
Bluming and Ziegler (1971) Lancet ii, 105-
N
PM
F
HL
106
-Efficiently infects and kills human my
yyeloma cells
(via CD46), spares normal cells
-Has potent antitumor activity against xenograft
models of human multiple my
pyeloma
Peng et al. Blood, 2002; 98: 2002-2007
Dingli et al. Blood, 2004; 103: 1641-1646
-Can be engineered to express additional genes;
recombinants are extremely stable
SLAM only
SLAM + CD46
High CD46 expression in primary myeloma cells
Unsorted bone marrow aspirated from patients with multiple myeloma
CD46 expression in primary myeloma cells
correlates with measles susceptibility
NIS gene insertion for imagin
gg g virus spread
p
(M
( V-NIS)
MV-NIS iv
NIS
N
P
M
F
H
L
Wait 7 days for
virus to spread.
PET/CT imaging
Give radioiodine
NIS protein
Intravenous MV-NIS: Dose-response studies
SCID mice
KAS6/1 xenografts, 0.5 cm
IV MV-NIS, one dose
d3
3000
UV-inactivated
2500
104
10 MV NIS
-
Tu
105 MV-NIS
m
106 MV-NIS
o
2000
d9
r
107 MV-NIS
volume
1500
(m
1000
m
3)
500
d14
0-10 0 10 20 30 40 50 60 70 80 90 100 110 120
Time After Therapy (days)
Minimum effective dose 5 x 106/kg
MV-NIS for myeloma: translation
Phase III
Preclinical
Clinical
Phase II
Research
Trials
Funding
RO1 CA100634
ROC
Translation
O1 CA125614
T
Phase I
Requirements
Angela Dispenzieri PI
JARI foundation
IV administration
RAID contract
Clinical
CT-SPECT imaging
Regulatory
Protocol
Approvals
7 dose levels (10e6 to 10e9)
Approvals
()
RAC
Cyclophosphamide addition
FDA
9 patients treated (3 dose levels)
IRB
Vector
IBC
Manufacturing
Mark Federspiel
Toxicology
Pharmacology
Publications
Kah Whye Peng
Peng et al, Blood 2001; 98, 2002-07
Peng et al. Blood 2003: 101, 2557-62
Dingli et al. Blood 2003: 102, 489-96
Dingli et al. Blood 2004: 103, 1041-46
Ong et al. Exp Hematol 2006: 341, 713-20
Myers et al. Clin Pharmacol Ther: 2007: 82, 700-710
MV-NIS trial data:
Malignant plasma cells are preferentially infected by MV-GFP
CD138+
CD138-
60
50
40
s
30
cell
GFP+%
20
10
0
1
2
45
6
7
8
Patients
Anti-
Anti Measles antibody titers, CD46 receptor
levels, QRT-PCR and 123I uptake in nine treated
MM patients
Patient #
1
2
3
4
5
6
7
8
9
Anti-MV Ab pre /
1:10
<1:10
< 1:10
<1:10
< 1:10
<1:10
< 1:10
< 1:10
< 1:10
post- Rx
1:160
1:20
1:10
1:20
1:160
1:10
<1:10
1:10
1:10
CD46 copies/cell
79771/
140187/
28258/
75147
53048/
15136/
45332/
63391/
17356/
CD138+/CD138-
10322
10145
11228
/9012
12113
5070
8580
7492
6929
Q-RT-PCR
- / - /-+a/ - /-+b/ - /-- / - /-+c/ - /-- / - /-- / - /+d - / - /+e
- / - /-
gargle/blood/urine
123
I uptake
-+
-
-
-
-
-
-
-
a 16440 and 1405 MV-
MV N gene copies /mcg RNA on day 15 and 22,
respectively
b 1021 MV-N gene copies /mcg RNA on day 8, respectively
c 3403 and 8250 MV-N gene copies
gp
/mcg RNA on day 8 and 15, respectively
d15800 and 702666 MV-N copies /mcg RNA on day 15 and 22, respectively
e 2760 MV-N copies /mcg RNA on day 15, respectively
Patient 2 on day 8 after MV-NIS
106 TCID
were administered by intravenous infusion
50
MV N mRNA was detected in the buccal swab
(16440 copies per g RNA)
FDG PET/CT
I-123 SPECT/CT
A
B
Coxsackievirus A21 (CVA21)
· (+) Sense RNA i
v rus
· Enterovirus Closely related to Poliovirus
· ~7 5
. kb genome
· Entry via ICAM-1 (CD54) and DAF (CD55)
· Shows potent oncolytic activity against melanoma
melanoma
xenografts, myeloma cell lines (DR Shafren 2004,
2007)
· R
t
epor dt
ed to cause hi
hi dl
nd ili b
m
l
para
i
ys s
d
an
i
myos ti
itis in
suckling mice, human (Yoeli et al 2002)
VP4 VP3 VP2 VP1 2A 2B 2C 3A 3B 3C
3D
CVA21 potent antimyeloma oncolytic,
yy
fatal toxicity
CPE of mouse tissueoverlayson H1-HeLa cells
Mice
Tumor
Liver
Spleen
Brain
Skeletal
Control Muscle
CVA21 Treated
Muscle
IV virus #1
+++
-
-
-
++
IV
I viru
vir s
u #2
#
+++
-
-
-
++
IV virus #3
+++
-
-
-
++
IT virus #1
+++
-
-
-
++
Novirus #1
-
-
-
-
-
· Coxsackievirus A21
A21 (CVA21) injected into mice carrying SQ
SQ multiple
multiple
myeloma tumors at 1x106 TCID50
· Complete tumor regression by 14 days
· Severe myositis developed 7-10 days post inoculation
· CVA21 recovered from tumor tissue and skeletal muscle only
miRNA targeting concept
· MicroRNAs are small, 21-23 nt regulatory RNAs mediating translational
repression & RNA degradation
· Incorporating microRNA target elements (miRTs) into the genomes of RNA
viruses may impair their stability (only in cells expressing the cognate
microRNA)
· Incorporation of tissue specific-miRTs into an oncolytic virus genome may
provide a new modality for targeting tissue tropism
Muscle cell:
cell
Recombinant CVA21 with
miRNA degrades viral genome
muscle-specific miRT
VPG
AAAA
Tumor cell:
Virus propagates
miRT
VPG
AAAA
VPG
AAAA
VPG
AAAA
Muscle-Specific miRNAs
· 3 highly conserved miRNAs (miR-1, miR-133,
miR-206) highly enriched in skeletal muscle
· Screening, done using lentiviral vectors, identified
a combination to two miR-133 plus two miR-206
target sequences as the best insert
insert for
for muscle-
specific shutdown of gene expression
Isolation & Propagation of
iRT
m
-conti
tai i
n ng CVA21
CVA21
H1-HeLa
Mel 624
10
10
9
9
8
8
7
7
6
6
WT
WT
miRT
5
miRT
5
4
4
3
3
2
2
1
1
0
0
0
2
4
6
8
12
24
0
2
4
6
8
12
24
Time (hrs)
Time (hrs)
· Growth kinetics of WT and miRT
viruses similar in non-muscle cells
Mechanistic Validation
Viabili
ab ty of
o infected cells
Virus yi
y el
e d from
o
infected cells
108
Mock
100
WT
miRT 107
WT
miRT
75
106
105
50
104
25
103
0
102
·H1-HeLa cells transfected with control or muscle specific miRNA mimics
·miRNA 133 & 206 offer significant protection individually
·Combining miRNAs 133&206 completely blocks miRT virus toxicity and
propagation on HeLa cells
In vivo studies (WT vs
vs miRT
miRT vs
vs RevT
RevT control)
KAS6/1 or Mel624 tumors
(SCID mice)
mice)
CVA21 (106 IT)
WT, miRT
iR , RevT
Mi
Mon t
itor for tumor regression
and hind limb paralysis
WT, miRT, and RevT viruses cause KAS 6/1
tumor regression but only miRT prolongs
survival
AB
Control
WT
2.0
0.5
1.5
0.4
0.3
1.0
0.2
0.5
Survival
0.1
E
Control
100
WT
0.0
0.0
miRT
0
7
14
21
28
35
42
49
0
7
14
21
28
35
42
49
RevT
80
Day
Day
C
D
60
miRT
RevT
40
0.5
0.5
20
0.4
0.4
0
0.3
0.3
0
102030405060
70
Day
0.2
0.2
0.1
0.1
0.0
0.0
0
7
14
21
28
35
42
49
0
7
14
21
28
35
42
49
Day
Day
microRNA targeting
· CVA21 host range can be restricted by incorporation
of muscle specific microRNA target sequences
(miRTs) into the genome
· This modification incapacitates the virus in cells
expressing cognate miRNAs where the viral RNA is
recognized and degraded
· The approach has subsequently been applied to
poliovirus
p
(neurotoxicit
(
y) and oncolytic adenoviruses
(hepatotropism)
· But even targeted viruses remain immunogenic........
Kelly et al, Nature Med. 2008: 14, 1278-83
CVA21 - infectious RNA production
Mlu 1
In vitro transcription
RNA purification
pGEM-CVA21
Ambion
Ambion
T7
+
3'
MEGAscript®
t® kit
kit
5'
G
ME AclearTM
Infectious RNA in vivo
RNA validation
SCID mice with subcutaneous
48 hours post
RNA
KAS6/1 or Mel624 xenografts (.5x.5cm)
transfection
marker
CVA21
(kb)
transcript
Mock transfected
-9
-7.4kb
-5
-3
1µg CVA21 RNA
-15
1.5
-1
-.5
Dose dependence of infectious RNA oncolytic activity
Multiple My
pyeloma tumor model
· KAS6/1 SQ xenografts in irradiated SCID mice
· Treated with one IT dose of CVA21 RNA (or Opti-MEM for control)
RNA
control
1g2g4g8g16g32g
dose
Mice with
tumor
0
33.3%
50%
100%
100%
100%
100%
reduction
Circumventing anti-viral antibodies:
Cellular vehicles for measles delivery
Monocytes, Myeloma cells.
Infection by measles virus
Carrier cells fuse with
Tumor cells, delivering
virus
Infected carrier cells
Migrate into sites of myeloma growth
Resist antibodies and complement
Fuse selectively into tumor
Virus spreads in tumor
Plasmacytomas are infiltrated by CD68+ macrophages
N=10 HPF per sample
Conclusions
· Oncolytic virotherapy is a promising approach for myeloma
· Measles, CVA21, VSV, Vaccinia and Adenovirus have all shown
promise in preclinical models
· Measles (MV-NIS) targets CD46 and is currently tested in a
Phase I clinical trial
· microRNA target insertion is
is an
an effective
effective new strategy to modify
virus host range, abrogating toxicities
· Oncolytic viruses can be formulated as (nonimmunogenic)
infectious nucleic acid or delivered in cellular carriers
carriers.
Oncolytic virotherapy for myeloma
Rl
Russe l
ll Lab
David Dingli
Clinical practice
Betsy Hadac
Toxicology Core
Beth Kelly
Kah Whye Peng
y
Myeloma
Apollina Goel
Sue Greiner
Angela Dispenzieri
Shruthi Naik
Rae Myers
David Dingli
Audelia Munguia
Nathan Jenks
Martha Lacy
Hongtao Li
Andrew Greenslade
Li
Morie Gertz
Stephanie Carlson
(Mary Harvey)
Susan Hayman
Linh Pham
Francis Buadi
Kah Whye
Whye Peng
Peng
Vector
V
Core
Vi
V ncent Rajkumar
Core
Mark Federspiel
Phil Greipp
Angela Dispenzieri
Linda Gregory
John Lust
Caili Tong
Guy Griesmann
Shaji Kumar
Kirsten Langfield
Steve Zeldenrust
Roberto Cattaneo
Julie Sauer
Sharon Stephan
Imaging
John Morris III
Henry Walker
Mike O'
O Connor
John Morris III
Henry Walker
Connor
Troy Wegman
Steph Carlson