Download Jeronimo Cello Research Assistant Professor Center for Infectious

Uso racional de los virus para el tratamiento del cancer:
Immunoterapia oncolitica utilizando poliovirus atenuado
Jeronimo Cello
Research Assistant Professor
Center for Infectious Diseases
School of Medicine
Stony Brook University
Tópicos de la charla
Viroterapia para el tratamiento del cancer:
Desde la genesis a la manipulacion genetica
Desarrollo de un modelo animal para el tratamiento de
Neuroblastoma mediante el uso del virus de la poliomielitis
Viroterapia, muerte celular inmunogenica
y su impacto en tratamiento del cancer
Viroterapia para el tratamiento del cancer:
Desde la genesis a la manipulacion genetica
Experimentos de la naturaleza
Remisión espontánea y generalmente transitoria
de tumores coincide infecciones agudas virales
Leucemias
Linfomas
Sarampion
Influenza
EBV
Varicela
Virus Hepatitis
Replicative cycle of virus and mechanisms of transformation
of normal to tumor cells elucidated at molecular level
Descriptive studies
Mechanistic studies
Rational use of
viral oncoloysis
Unrestricted growth and proliferation
Tumors cells
Deficient in controlling nucleic acid synthesis
Viruses grow preferentially
in this intracellular environment
Oncolytic virotheraphy
Infection and killing of tumour cells by an oncolytic virus
Oncolytic viral therapies: Advantages over conventional therapies
• Viruses can be rapidly modified by recombinant DNA technology
Deletion mutants
Incorporation of of tissue specific promoters
Receptor mediated re-targeting
• Viruses promote tumor-specific inflammation
• Self-replicate within the cancer cells→Unique pharmacokinetic properties
• Low levels of toxicity
Desarrollo de un modelo animal para el tratamiento del
neuroblastoma mediante el uso del virus de la poliomielitis
Background
Neuroblastoma es el tumor solido extracranial mas comun de la infancia (40 % adrenal, 25% paraspinal ganglia, 15% torax, 5%
pelvis, 3% tumor cervicales, 12% miscelaneos)
Neuroblastoma es un tumor embrional del sistema nervioso simpatico que se origina en los neuroblastos que migran a lo largo
del neuroaxis y populan los ganglios simpaticos, glandula suprarrenal, etc.
Amplificacion del oncogene MYCN y deleccion del brazo corto del cromosoma 1 esta asociado con mal pronostico
Neuroblastoma es resistente a los tratamientos convencionales antitumor (radio y quimio)
Cellular functions of CD155
• CD155 binds strongly to nectin-3: function in cell-to-cell adhesion
• CD155 interacts with vitronectin:mediates cell-to-matrix contacts
• CD155 binds CD96 and DNAM-1 on NK cells: stimulates cytotoxicity of activated NK
•
CD155 is over expressed in tumor cells
PVR expression in neuroblastoma cells
SJ-N-JF
KP-N-SIFA
SK-N-SD
100
1
100
60
2
3
4
5
6
PVR
Cell count
M1
100
101
102
103
104
100
101
N2a
102
103
104
100
101
SHEP
100
102
103
104
SUZUKI
100
60
M1
100
101
102
103
104
100
101
102
M1
103
104
100
101
102
103
104
Lane
1: N2a
2: SJ-N-JF
3: KP-N-SIFA
4: SK-N-SD
5: SHEP
6: SUZUKI
Relative Fluorescence Intensity
PVR Sequencing (SHEP) : No abnormality
RESISTANT
SENSITIVE
Experimental treatment of human neuroblastoma
using live-attenuated poliovirus
Toyoda et al, Int J Oncol 2004
Tumor Xenotransplantation and Intratumoral Liveattenuated Poliovirus Injection.
Xenograft
Poliovirus Injection
3 - 4 weeks
0.5 - 0.8 cm3
1 x 107 SJ-N-JF cells in
0.2 ml of Matrigel were
injected s.c. into both
flanks of nude mice.
3x106 TCID50
Live-attenuated
poliovirus was injected
into the right flank
tumor
Tumor size
was
monitored
every 2-3
days.
Day 0
Vehicle-treated
Mouse
Live-attenuated
poliovirustreated Mouse
Day 7
Day 14
Sabin strains are genetically unstable and they can revert to full neurovirulence
Poliovirus strain neuroattenuated and stable
Nude mouse is immunodeficient and does not express CD155
Modelo raton inmunocompetente that express CD155
Mouse neuroblastoma cells susceptible to poliovirus infection
Poliovirus Genome
5' NTR
Structural Region
P1
VPg
VP2
I
VP3
Non-structural Region
P2
VP1
2A2B
2B
2C
3A 3C
IIIIIIV VVI
VP4
CLOVER IRES
-LEAF
A103
3' NTR
P3
VPg
3D
A
AA
n
Mutacion de A103 localizado entre el “cloverleaf” y IRES atenua la
neurovirulencia de poliovirus en ratones CD155 10.000 veces y le
le confiere un fenotipo ts en celulas neuronal de origen humano
Nuevo sitio de atenuacion
Identification of an RNA Hairpin in Poliovirus RNA That Serves as the Primary
Template in the In Vitro Uridylylation of VPg
Aniko V. Paul,1,* Elizabeth Rieder,1 Dong Wook Kim,1 Jacques H. van Boom,2
and Eckard Wimmer1
Cis-acting replication element (cre)appear to be
essential for minus-strand RNA synthesis
Functional Dissection of a Poliovirus cis-Acting Replication Element [PV-cre(2C)]: Analysis of Single- and Dual-cre Viral
Genomes and Proteins That Bind Specifically to PV-cre RNA
Jiang Yin, Aniko V. Paul, Eckard Wimmer, and Elizabeth Rieder*
cre
VP2 VP3 VP1 2A 2B 2C 3A3B 3C
cre
3D
VP4
cre
VP2 VP3 VP1 2A 2B 2C 3A3B 3C
cre
Dual-cre PV
VP4
Mono-cre PV
3D
Neuropatogenicidad en ratones CD155
Virus
PV1 (M)
Mono-cre PV1
Dual-cre PV1
LD50 (PFU)
102
107.2
107
Alteracion de la region conservada localizada
entre el cloverleaf y IRES atenua la neurovirulencia
de poliovirus
PVR expression in neuroblastoma cells
SJ-N-JF
KP-N-SIFA
SK-N-SD
100
1
100
60
2
3
4
5
6
PVR
Cell count
M1
100
101
102
103
104
100
101
N2a
102
103
104
100
101
SHEP
100
102
103
104
SUZUKI
100
60
M1
100
101
102
103
104
100
101
102
M1
103
104
100
101
102
103
104
Lane
1: N2a
2: SJ-N-JF
3: KP-N-SIFA
4: SK-N-SD
5: SHEP
6: SUZUKI
Relative Fluorescence Intensity
PVR Sequencing (SHEP) : No abnormality
RESISTANT
SENSITIVE
viral titer (pfu/ml)
Growth curve of mono-cre PV
1.E+09
1.E+08
1.E+07
N2aCD155
1.E+06
SK
1.E+05
Hela
1.E+04
1.E+03
1.E+02
0
2
4
6
8
12
hours after infection
24
Celulas N2a originadas de un
neuroblastoma espontaneo raton
Ratones CD155, H-2a negativo
A/J, H-2a
CD155 tg
A/J
CD155/CD155
-/H-2a/H-2a
H-2?/H-2?
CD155 tg A/J
Inmunocompetente
CD155+, H-2a+
CD155/ H-2a/H-2?
Immunization
Tumor transplantation
Treatment
1x108 pfu
mono-cre PV
7-10 days
Once a week
Neutralizing
antibody to PV:
< 8 (n=8)
(200 mm3)
3 weeks
Neutralizing
antibody to PV:
256-2024 (3
mice)
1x107 cells
N2aCD155
1x108 pfu
mono-cre PV
4000
3500
3000
mono-cre PV
2500
2000
1500
1000
500
0
-1
0
1
2
3
Days after mono-cre PV inoculation
4
5
133 A to G mono-cre PV
133 A to G
VP2
VP3
VP1
2A
2B
2C
3A 3B
3C
3D
VP4
Virus
mono-cre PV1
A133G mono-cre PV1
PLD50 (i.c.)
107.2 pfu
105.33 pfu.
LDH release assay (24h after infection)
100
80
no infection
60
mono-cre
40
133AtoG mono
wild type
20
0
N2a
SK-N-
SK-N-
SK-SH-
MC
SH
5Y
cell line
1 cm
None of the rechallenged mice developed tumors at the site of tumors cells reinoculation or elsewhere
Oncolytic therapy by A133Gmono-crePV activates the immune system against Neuro-2aCD155 cells
leading to antitumor activity that is likely to be independent of A133Gmono-crePV
naive splenocytes:N2aCD155 cells
Figure 1
splenocytes from cured mice:N2aCD155 cells
Naive splenocytes:N2a cells
Splenocytes from cured mice:N2a cells
A
18
16
14
12
10
8
6
4
2
0
Target cells:Effector cells
120
B
100
% Lysis
80
60
40
20
0
non depleted
NK-depleted
CD4+-depleted CD8+-depleted
.
Figure 2
Tumor transplantation
1x107 cells
Neuro-2aCD155
A
Treatment
1x108 pfu
A133Gmono-crePV
cured mice
2 months
10 days
A/J mice
II
I
Adaptive transfer
of splenocytes
III
Tumor transplantation
1x107 cells
Neuro-2aCD155
10 days
A/J mice
B
IV
V
: splenocytes from naïve mice
: splenocytes from cured mice
6000
5000
4000
Average volume: 2183 mm3
3000
2000
Average volume: 583 mm3
1000
Days after adaptive transfer of splenocytes
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
0
Tumor volume (mm3)
7000
Fig. 3A
Vaccination with different preparations
Immunization
i) PBS
ii) PV1(M)-infected Neuro-2aCD155 cells lysate
iii) Non infected Neuro-2aCD155 lysate
iv) Neuro-2aCD155 lysate admixed with PV1 (M)
1x108 pfu
mono-crePV
Thrice a week
I
3 weeks
Thrice a week
II
Monitoring
of mice survival
3 weeks
III
Intravenous Tumor transplantation
2x106 cell Neuro-2aCD155
Figure 3B
vaccination with PBS
naïve mouse
liver
kidney
1 cm
Figure 3C
PBS
PV1(M)-infected Neuro-2aCD155 lysate
Noninfected Neuro-2aCD155 lysate
Noninfected Neuro-2aCD155 lysate admixed with PV1(M)
(i)
(ii)
(iii)
(iv)
100
Percent of survival
80
(ii)
60
(i)
(iv)
(iii)
40
20
0
0
20
40
60
80
Days after intravenous tumor seeding
100
120
Viroterapia, muerte celular inmunogenica
y su impacto en tratamiento del cancer
Main immunogenic determinants of dying tumor cells
Plasma membrane molecules
P53 activation
Inflammatory components
End-stage degradation products
Viral oncolysate
Oncolytic virus
“Restauración de equilibrio inmunológico a través de la vacunación y inmunoterapias .
será fundamental para el dominio final sobre el cáncer.” L. Old (co-discoverer P53)
DEPT OF MOLECULAR GENETICS AND MICROBIOLOGY
SCHOOL OF MEDICINE
SUNY AT STONY BROOK
NIDIA DEJESUS
JERONIMO CELLO
HIDEMI TOYODA
JIANG JING
ELIZABETH RIEDER-ROJAS
ANIKO PAUL
ECKARD WIMMER
Background
Neuroblastoma is the most common extracranial solid tumor and the most common tumor occurring during infancy
It is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts (pluripotent sympathetic cells).
These cells invaginate, migrate along the neuraxis, and populate the sympathetic ganglia, adrenal medulla, and other sites.
Patients present metastatic disease, usually to lymph nodes, liver, skin, bone, and bone marrow
Infants younger than 1 year (40%) have a good prognosis, even in the presence of metastatic disease, whereas older patients
with metastatic disease (60%) fare poorly, even when treated with aggressive therapy
Abdominal cavity (40 % adrenal, 25% paraspinal ganglia)
or involve other sites (15% thoracic, 5% pelvic, 3% cervical tumors, 12% miscellaneous)
G102G103 virus sintetico antes inoculacion intracerebral
G102A103 virus aislado del cerebro de ratones CD155
LD50%, pfu
102
>108
>108
Poliovirus recombinants for the treatment of malignant glioma
and intracerebral neoplasia
Induction of
tumor cell death
Induccion de una
respuesta inmune
antitumor
Calreticulin
HMGB1
HSP70
Tumor eradication
HSP90
Recognition of
tumor cell
death
Uptake of apoptotic bodies
Maturation of dendritic cells
Presentation of tumor derived antigens
DC
Innate
immune response
NK
NKT
Adaptative
immune response
DC
CTL
T helper
CTL
T helper
T Regs
Stimulation of
anti tumor immune response
Viral Oncolysate
Induction of
immunogenic
tumor cell death
Preservation of
the immune
system
Calreticulin/HSP70/90
exposure
HMGB1/Pro
inflammatory
Mediators Release
Anthracyclines
Bortezomib
γ-irradiation
p53 restoration
Enhanced
immunogenicity
of tumor cell death
Complete
eradication of
tumor cells
Stimulation of NK cells
Inhibition of PTX3
Maturation of DC
Secretion of chemo
attractants
Enhanced cross
presentation
of tumor derived antigen
Mecanismos propuesto para una eficiente
Immunoquimio terapia