administration of tumor-specific cytotoxic t lymphocytes ......bollard et al, jit 2004 making lmp1...

CM. Bollard, G Dotti, S Gottschalk, E Liu, A Sheehan, M Mims, H Liu, AP. Gee,

MK. Brenner, HE. Heslop, CM. Rooney

Administration of Tumor-Specific Cytotoxic T Lymphocytes Engineered to Resist TGF-ß to Patients with EBV-Associated Lymphomas

• Significant failure rate of therapy for advanced stage or recurrent disease

• Long-term side effects of chemotherapy and

radiation

• EBV antigens expressed by up to 40% of lymphomas are potential targets for T cell immunotherapy

Rationale of Immunotherapy for EBV-positive Lymphoma

EBV Infected B cells

EBV +ve Lymphoma Cell

TGFb

EBV Specific Cytotoxic T Lymphocytes (CTL) Control EBV Infection in vivo

LMP1

LMP2A

Lytic EBNA 3 LMP 2 LP LMP 1 EBNA 2 EBNA1

PBMC

CTL

• LMP1 and LMP2A are potential CTL targets

Hodgkin R-S Cell/NHL Cell

LMP1 and LMP2A-specific CTL For Hodgkin and non-Hodgkin Lymphoma

EBNA1

LMP1

LMP2A

Bollard et al, JIT 2004

Making LMP1 and LMP2 Immunodominant Antigens

Gottschalk et al, Blood 2004 and Leen et al, JIT 2007

LMP-specific Cytotoxic T

Lymphocytes (CTL)

Ad5f35 LMP1-I-LMP2

PBMC IL-2

Lymphoblastoid cell line (LCL) Dendritic

Cells

Ad5f35 LMP2

OR

-10%

0%

10%

20%

30%

40%

50%

60%

70%

80%

Ratio 40:1 Ratio 20:1 Ratio 10:1 Ratio 5:1

% S

peci

fic

Lysi

s

AutoLCLAlloLCLPHABlasts

FC109Y08.133

CD8

ILL

100

101

102

103

104

101

102

103

104

0.03% 1.03%

18.96% 79.98%

FC109Y08.130

CD8

CLG

100

101

102

103

104

101

102

103

104

0.02% 0.19%

16.83% 82.95%

FC109Y08.131

CD8

FLY

100

101

102

103

104

101

102

103

104

0.17% 15.32%

19.48% 65.03%

FC109Y08.132

CD8

YLL

100

101

102

103

104

101

102

103

104

0.01% 2.95%

16.53% 80.50%

15.32% 0.19% 1.03% 2.96%

CD8

FLY-A2 LMP2 tetramer

CLG-A2 LMP2 tetramer

ILL-A29 LMP2 tetramer

YLL-A2 LMP1 tetramer

LMP1 & LMP2–Specific Activity in LMP-CTL from a Hodgkin Disease Patient

Relapsed Disease Arm (n=21)

• No toxicity • 11 CR (1 also given Rituximab) (includes 1PRCR)

• 2 very good partial responses (up to 36 mths) • 8 progressive disease (2-8 wks) Median clinical response: 1.5y

(range: >6 to >40 mths)

CR PR

n =21

Patients with disease at CTL infusion

Clinical Responses post LMP-CTL

Bollard et al, JCO 2013 in press

CR PR

n =21

X X X X X X X X

CR PR

NR

50% Disease Free Survival at 2 years

Clinical Responses post LMP-CTL in Patients with Active Disease

Year

Pro

port

ion d

isease-f

ree

0 1 2 30

0.2

0.4

0.6

0.8

1

Alascer

ALCI

P=0.744

LMP1/2-CTL study

LMP2-CTL study

Figure 4. Immune Reconstitution

LMP1

SFC

per

2x105

LMP2

SFC

per

2x105

LMP1

SFC

per

2x105

LMP2

SFC

per

2x105

Responders Non-Responders

Immune Reconstitution of LMP1 and LMP2-specific T cells in Patients Treated with

LMP1/2-CTL

Responders Non-

responders

Figure 4C. Immune Reconstitution

0

20

40

60

pre-infusion post-infusion

Survivin

SF

C p

er

2x

10

5 c

ell

s

0

10

20

30

40

50

60

70

pre-infusion post-infusion

PRAME S

FC

pe

r 2

x1

05 c

ell

s

0

20

40

60

pre-infusion post-infusion

Survivin

0

20

40

60

80

100

120

pre-infusion post-infusion

MAGEA4

0

20

40

60

pre-infusion post-infusion

PRAME

0

20

40

60

80

100

120

pre-infusion post-infusion

MAGE A4

Evidence of Epitope spreading in Responding Patients Treated with LMP1/2-

CTL

Can we make LMP-CTL resistant to the inhibitory effects of TGF-ß

secreted by Lymphoma cells?

• Inhibits CTL proliferation

• Inhibits cytotoxicity

- perforin

• Inhibits cytokine production

- IFN

TGFb Effects on CTL

Creating a Mutant TGFb Receptor II

Stop codon 597

Wild type

Receptor

Truncated TGFß Receptor II

Dominant Negative Receptor (DNR)

Transmembrane domain

Bollard et al, Blood 2002

MoMLV MoMLV

U3 R U5 U3 R U5

NcoI/BamHI

SD PBSQ

SA

+

TM domain

SFG:DNR

Retroviral vector SFG

DNR

Bollard et al, JIT 2004, Bollard et al, Blood 2002, Foster et al, JIT 2008

Rendering LMP-specific T cells Resistant to TGFb

Ad5f35 LMP1-I-LMP2

PBMC IL-2

EBV-LCL

DC

DNR-transduced LMP CTLs

SFG:DNR

• EBV+ malignant cells EBER and/or LMP1

and/or LMP2 positive Lymphoma cells

• Patients with relapsed Hodgkin Disease or NHL including after allogeneic SCT

Study Eligibility

CD4 and CD8 T cells are DNR-transduced

CD8 FITC

TG

FB

RII

PE

100

101

102

103

104

100

101

102

103

104

81.32%16.26%

2.39%0.03%

NON Trans

CD8 FITC

TG

FB

RII

PE

100

101

102

103

104

100

101

102

103

104

76.84%10.68%

10.19%2.29%Trans

n=6 CTL lines

XX

0

20000

40000

60000

80000

100000

120000

140000

Pt 1 Pt 2 Pt 3 Pt 4 Pt 5 Pt 6

Tra

ns

ge

ne

co

py n

um

be

r in

10

0n

g D

NA

1%

25% 28%

0%

5%

10%

15%

20%

25%

30%

35%

CD45RA+CD62L+

CD45RA-CD62L+

CD45RA-CD62L-

17%

4%

11%

0%

2%

4%

6%

8%

10%

12%

14%

16%

18%

20%

TGFβ+ CD3+ TGFβ+ CD3+ CD4+

TGFβ+ CD3+ CD8+

n=6 CTL lines

DNR-transduced CD4 and CD8 T cells are Predominantly Effector Memory

DNR-Transduced CTL are

LMP-specific

EBV-LCL

• 5 females and 3 males

• EBV+ HL

7 – relapsed post autologous SCT

1 – relapsed post allogeneic SCT

• Two previously treated with LMP-CTL alone

• All refused additional chemotherapy

Patients Studied

0.1

1

10

100

1000 CTLs

0.1

1

10

100

1000

10000 CTLs

DNR-transduced T-cells Persist in vivo for 5-23 months

Patient 1

Co

py n

um

be

rs in

10

3 n

g D

NA

0

Patient 3

0

• No dose limiting toxicity

• TGFb-resistent LMP-CTL may beneficial in EBV+ Lymphoma

• DNR-trans LMP-CTL persist up to 3 years

• Now plan to explore the use of DNR-CTL in other TGFb-secreting cancers

Conclusions