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Genetic Modification for Immune Evasion
Stephen Gottschalk
T-cell Therapy
‘T-cell Therapy has shown promiseT-cell Therapy has shown promise in clinical studies’
BUT‘The majority of administered T-cell
products are sensitive to immune evasion mechanisms employed by tumors’
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Suboptimal T-cell products: sensitive to the immunosuppressive Tumor environment
Genetic Modification of T cells
• Render T cells resistantRender T cells resistant – to immune evasion mechanisms employed by tumors
• Improve T-cell function– cytokines/cytokine receptors– anti-apoptosis genes– silencing negative regulators– silencing negative regulators ……
• Antigen-specific T cells– Chimeric antigen receptors (CAR)– TCR
3
Expression of DN TGF- R
Tumor cells secrete factors to inhibit immune cells
TGFTGF--Inhibition of T-cell effector function
Bollard et al 2002
Induction of T-cell apoptosis
Expression of IL-12
si RNA FAS knock down
FASFAS--LL
Clinical Study opened in 2009
Immunoregulatory cytokines
IL-12
Wagner et al 2004
Dotti et al 2005
ILIL--1010
Expression of DN TGF- R
Tumor cells have MHC processing defects & antigen expression is heterogeneous
TGFTGF--Inhibition of T-cell effector function
Bollard et al 2002
Induction of T-cell apoptosis
Expression of IL-12
si RNA FAS knock down
FASFAS--LL
Clinical Study opened in 2009
Immunoregulatory cytokines
IL-12
Wagner et al 2004
Dotti et al 2005
ILIL--1010
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CAR vs TCR T cells
TCR T llCAR T ll TCR T cellsCAR T cells
T-cellT-cell
CAR vs TCR T cells
TGFTGF--FASFAS--LLNo
ILIL--1010
NoCostimulatory
Molecules
TCR T cells: incomplete T-cell activation
CAR T cells: Addition of costimulatory signaling domains into CAR endodomain
T-cell activation in the ABSENCE ofcostimulatory molecules
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CAR T cells to overcome immune evasion
• Targeting cell surface antigens
• Targeting multiple antigens
• Targeting inhibitory stromal cells
Rationale for immunotherapy for osteosarcoma
• Prognosis remains poor for patients withg p p• Metastatic disease (< 20% survival)
• Central tumors (< 35% survival)
• Relapsed disease
• Significant acute toxicities
• Long-term effects• Disability after surgery
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HER2 expression and prognosis
• Member of the EGFR family of receptors
• Expressed by up to 80% of primary osteosarcoma• Expressed by up to 80% of primary osteosarcoma
• Important for malignant phenotype
• Worse overall survival rates
HER2 negative
HER2 positive
(Gorlick et al. JCO 1999)
2nd Generation HER2-specific CAR
LTRLTR
GALV or RD114 pseudotypedretroviral particles
scFV
(Pule et al, Mol Thr 2005; Ahmed et al, Mol Thr 2009)
zeta
CD28
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Generation of CART cells by retroviral transduction
Retroviral Transduction + Retronectin + IL 2IL-2CD3/CD28 Retronectin + IL-2
IL-2
donor A
100 101 102 103 104
donor B
86.2% 83.0%
donor C
87.8%
100 101 102 103 104 100 101 102 103 104
donor A
100 101 102 103 104100 101 102 103 104
donor B
86.2% 83.0%
donor C
87.8%
100 101 102 103 104100 101 102 103 104 100 101 102 103 104100 101 102 103 104
T cells
Activated T cells
Day 0 Day 2 Day 3 Day 14-21 freeze
Control + NT T cells
HER2-CAR T cells inhibit 2ndary Sarcosphere formation+ HER2-T cells Sarcosphere
143B MNNG/HOS
Monolayer
143B MNNG/HOS
143B MNNG/HOS
(Rainusso et al; Cancer Gene Therapy 2012)
Monolayer
8
ControlWk
2
Antitumor activity in metastatic Lung Model
HER2-T cells
4
8
10
Wk
22
4
8
10
(Ahmed et al. Mol Ther 2009)
Safety concerns with HER2-CAR T cells
• 6 patients have received ‘conventional’6 patients have received conventional
HER2-specific T cells 1 - 4x1010 cells with no
side effects
• 2 patients have received HER2-CAR T cells
– 1st : 2x107 cells: no immediate toxicity
– 2nd: 1x1010 cells post nonmyeloablative
conditioning: developed ARDS and died
(Bernhard et al Cancer Immunol Imm 2008, Disis et al JCO 2009, Morgan et al Mol Thr 2010)
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T-cell Therapy for HER2-positiveOsteosarcoma
• To determine the safety and antitumor activity of HER2-CAR T cells
9 D l l 1 104 t 1 108 ll / 2• 9 Dose levels: 1x104 to 1x108 cells/m2
Characteristics of infused patients
• 9 females, 6 males
• Median age 17 3 (7 – 29)Median age 17.3 (7 29)
• 13 OS, 1 EWS, 1 DSCRT
• Disease status• 11 lung mets
• 1 lung and bone mets
• 1 lung and extraosseous mets
• 1 Liver mets
• All patients had failed multiple lines (>4) of salvage therapy
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Phenotype of GMP grade HER2-CAR T-cells
Frequency of CD3+CD45RO+ Frequency of Frequency of CD3 CD45RO Effector & Central memory T cells
q yCD4 & CD8 T-cell subsets
Characterization of GMP grade HER2-CAR T-cells from OS patients
Transduction CytotoxicityEfficiency
P<0.001
40
60
80
sis
(%)
NT T cellsHER2-CAR T cells
P<0.001
60
80
100
tive
(%)
P<0.001
n=9
0
20
K562 MDA NCI-H1299 LM7
Lys
0
20
40
NT T cells HER2-CAR T cells
Po
si
HER2 - HER2 +
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No increase in pro-inflammatory cytokines post infusion
DL1 DL1 DL1 DL2 DL2
DL3 DL3 DL5 DL5 DL5
20
40
60
80
100
conc
(pg
/mL
)
DL3 DL3 DL5 DL5 DL5
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100GMCSF IL-2IFN TNF
0
Pre 3h
Wk1
Wk2
Wk4
wk6
post Infusion
0
Pre 3h
…
Wk1
Wk2
Wk4
wk6
0
Pre 3h
…
Wk1
Wk2
Wk4
wk6
0
Pre 3h
…
Wk1
Wk2
Wk4
wk6
post Infusion post Infusion post Infusion
Dose Level 6(3x106 cells/m2)
In vivo persistence of adoptively transferred HER2-CAR T cells
Dose Level 7(1x107 cells/m2)
Dose Level 8(3x107 cells/m2)
0
5
10
15
20
25
25
0
510
1520
25
25
Tumor biopsy
0
5
10
15
20
25
20
25
post infusion
0
5
10
15
20
0
5
10
15
20
0
5
10
15
20
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Clinical Outcome
• 4 patients with stable disease• 1 patient for 6 weeks (then had tumor resected)• 1 patient for 6 weeks (then had tumor resected)• 1 patient for 12 weeks (then had tumor resected)• 1 patient for 4 months• 1 patient for 5½ months (ongoing)
• 11 patients with progressive disease
Conclusions: T-cell Therapy for sarcoma
• HER2-CAR T cells
– Have antitumor activity in preclinical models
• Adoptive transfer of HER2-CAR T cells in humans
– Evaluated cells doses safe (up to 3x107/m2)
– Limited T-cell persistenceLimited T cell persistence
• Plan
– Clinical: Escalate HER2-CAR T-cell dose to 1x108/m2
– Preclinical: Improve CAR; better preclinical models
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CAR T cells to overcome immune evasion
• Targeting cell surface antigens
• Targeting multiple antigens
• Targeting inhibitory stromal cells
HER2 as an Immunotherapy target for GBM
• ~ 70% of GBM positive HER2
2• HER2– promotes malignant phenotype
– expressed in CD133+ glioma initiating cell population
IHC CD133 HER2
CD133-PE HER2-PE
CD133 –ve
CD133 +ve
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HER2-specific T cells induce regression of autologous GBM
Tumor l
NT T cells
HER2 T cells
3
6
9
only T cells T cells
day
urvi
val P
roba
bilit
y
Tumor only
NT T cells
HER2-T cells
14
(Ahmed et al, Clin Can Res 2010)
Su
Survival Time (days)
• Majority of GBMs are positive for CMV IE1 and pp65
CMV as an Immunotherapy target for GBM
Ph II dj t 65/DC i t d
pp65IE1
• Phase II adjuvant pp65/DC vaccine study:– Safe
– Induction of pp65-specific T-cell responses
– Prolonged survival in comparison to historic controls
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GBM patients have decreased T-cell responses to pp65
p=0 002 p=0 31
CMV.pp65 CMV.IE1
400
600
800
1000
per
2x10
5P
BM
C
200
400
600
per
2x10
5P
BM
C
p=0.002 p=0.31
0
200
SF
C
0S
FC
GBM Healthy
DonorGBM Healthy
Donor
(Ghazi et al, JIT 2012)
Generation of CMV-specific CTL using Ad5f35 Vectors
Ad5f35
PBMC
Ad5f35CMVpp65
LCL
+ IL-2
O/NAdherence
(Leen et al, Nat Med 2006)
CMV-specific CTL
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Generation of CMV-specific T cells from GBM patients
6 )
1000 ExpansionCytotoxic Activity
T-ce
ll nu
mbe
r (x
106
0.1
1
10
100
1 2 3 4 5 6Week
n=6Cytotoxic Activity
20
30
40
20
30
40
50
CMV(+) auto GBM
CMV(+) mismatched GBM
CMV(-) auto GBM
CMV(-) mismatched GBM
sis
(%)
0
10
0
10
20
T cell : target ratio40:1 20:1 10:140:1 20:1 10:140:1 20:1 10:140:1 20:1 10:140:1 20:1 10:1
Lys
Elispot Assay
0200400600800
10001200
pp65 Control
SF
C p
er 1
x105
cells
Generation of HER2-CAR CMV-specific CTL
Ad5f35
PBMC
Ad5f35CMVpp65
LCL
+ IL-2
O/NAdherence HER2-CAR
CMV-CTLHER2-CARretrovirus
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Characterization of HER2-CAR CMV-specific T cells
A
Cytotoxic ActivityHER2-CAR expression
HER2(+) autologous GBM
HER2(-) MDA-MB-468
GBM #4GBM #3
s(%
)
40
60
40
60
40
60
40
60
CD8 HER2-CAR
NLV
(C
MV
)
51.9% 51.9%
Elispot Assay
Lysi
s
T cell : target ratio40:1 20:1 10:140:1 20:1 10:140:1 20:1 10:140:1 20:1 10:140:1 20:1 10:1
0
20
0
20
0
20
0
20
0
100
200
300
400
500
600
pp65 neg Co pos Co
CMV-CTL
HER2-CAR CMV-CTL
SF
C p
er 1
x105
cells
Characteristics & Outcome of infused patients
• 4 patients with recurrent GBM
• Cell dose: 1x106/m2
• Outcome:
• 2 progressive disease
• 1 stable disease for 4½ months
1 ti l• 1 partial response
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PREINFUSION
Pre CTL 6 weeks 4 months
Clinical response post T-cell infusion
Conclusions: T-cell Therapy for GBM
• HER2 and CMV antigens are expressed in GBM
• Generation of clinical grade
HER2/CMV-specific T cells is feasible
• Clinical Trial:
S f d t f i– Safe data so far encouraging
• Plan:
– Escalate HER2/CMV-specific T-cell dose to 1x108/m2
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CAR T cells to overcome immune evasion
• Targeting cell surface antigens
• Targeting multiple antigens
• Targeting inhibitory stromal cells
CAFs play critical role in tumorgenesis
Immunosuppression
TGFTGF--CXCL14
TGFTGF--ILIL--1010
FASFAS--LLTumor cells
CAFs FAP
MMPs
VEGFVEGF
VEGFVEGF
Promote Tumor growth
cells
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Characterization of FAP-CAR T cells
FAP-expression in cancer ll li
25000 NT-T cellsp<0.05
FAP-CAR T cells recognize FAP+ targets: cytokine secretion
0
5
Hs894
SENMA
C666.1
PL45
U87
A549t sco
re
cell lines
A549 A549.mFAP A549.hFAP0
5000
10000
15000
20000FAP-T cells
IFN
(pg
/ml)
p<0.05
FAP-CAR T cells recognize FAP+ targets: cytolytic activity
10
15
A549
LCL
A549.hFAP
A549.mFAP
C
A549 A549.mFAP A549.hFAP0
20
40
60NT-T cells
FAP-T cells
% L
ysis
(1
0:1
E:T
Ra
tio)
p<0.005p<0.005
FAP-CAR T cells have antitumor activity in vivo
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In vivo induction of murine FAP
FAP-CAR T cells have antitumor activity in vivo
4 11 180.1
1
Days post tumor injection
Mu
rin
e F
AP
fo
ld e
xpre
ssio
n
FAP-CAR T cells target FAP+ cells in vivo
Untreated FAP-T cellsNT-T cells
Day 5
Day 15
Day 25
Pre-treatment
Normal NT-T cells FAP-T cells0
5
10
15
Mu
rin
e F
AP
fo
ld e
xp
ress
ion
FAP+ cells in vivo
p<0.005
0 20 40 60 800
20
40
60
80
100NT (n=9)
FAP (n=8)
Untreated (n=9)
Days post tumor injection
Pe
rce
nt
surv
iva
l
p<0.0005
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Targeting CAFs induces expression of pro inflammatory genes
NTC
003
NTC
002
NTC
001
NTC
004
NTC
005
NTC
006
FAP
00
5FA
P0
04
FAP
00
1FA
P0
02
FAP
00
3FA
P0
06 Pathway analysis - enrichment of genes involved in
• chemokine & cytokine-cytokine receptor interaction
• toll-like receptor pathway
• natural killer cell mediated cytotoxicity
• Jak-STAT pathway
• B-cell receptor signaling p g g
• antigen processing and presentation pathway
• complement and coagulation cascades
Combining FAP-CAR T cells with tumor-specific T cells enhances antitumor effects
Pre-treatment
Day 11
Day 23
FAP-T cells EphA2-T cells FAP- T cells +EphA2-T cells
Untreated
y
Day 33
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Conclusions: Targeting tumor stroma
• CAFs contribute to immunosuppressive
tumor environment
• Targeting CAFs results in antitumor effects and
induces expression of pro-inflammatory genes
• Targeting CAFs and tumor cells results in enhanced
antitumor effects
Conclusions
Tumors pursue a gamut of immuneTumors pursue a gamut of immune evasion mechanisms
BUTG ti difi ti h ld thGenetic modifications holds the
promise to engineer T cells that are resistant to these ploys
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Acknowledgements
Go LabSunitha KakarlaClaudia Gerken
CollaboratorsHER2-CAR Winfried Wels
GMP FacilityAdrian GeeZhuyong MeiClaudia Gerken
Johanna YiMamta KalraMelinda D’SouzaSimone KrebsTania Rodriguez CruzKato IwahoriDavid TorresLaTerrica Williams
Winfried Wels
Osteosarcoma patientsPete Anderson, Lisa Wang
Osteosarcoma stem cellsNino Rainusso, Jeff Rosen
GBMRobert Grossman, MuraliChintagumpala, Jack Su
CAGTNabil AhmedGianpietro DottiMalcolm BrennerHelen Heslop
Zhuyong MeiOumar DioufHumin ZhangVita Brawley
Chris DeRenzoPaulina Velasquez
Clinical ResearchBambi GrilleyBridget MedinaCatherine PereraEnli LiuTara Gray
Chintagumpala, Jack Su
PathologyJohn Hicks, Suzanne Powell
Helen HeslopCliona Rooney