1

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’

2

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

4

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

5

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

6

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

7

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)

9

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

10

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 +

11

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

13

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

14

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

15

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

17

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

19

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

20

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

10

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

21

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

22

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

23

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