Evaluation of T Cell Products – LMP-specific T cell...

PACT Meeting, Houston Bollard

1

CM Bollard

Evaluation of T Cell Products –LMP-specific T cell therapies for Lymphoma

CM Bollard

The Long Winding Road to Successful T cell Therapy

Priming naïve T cells in vitro

Tumor specific T cells in the clinic (CAR modified)

Improved APC generation

Low volumeGMP grade culture

systems

Multi-virus specific T cells in

the clinic

2000

Improved growth conditions – rapid

manufacture

Target antigen identification

1995

Proof of principleVirus specific T cells

Leukemia-specific T cells

Incorporating CD4 and CD8 responder


2

Bench to BedsideBench to Bedside

T cells EBV+ Lymphomaspost BMT

EBV-specificT cells

Can CTLs be used to treat “real” malignancies that occur in normal individuals?

EBV+ Hodgkin’sDisease and NHL

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

Rationale of Immunotherapy for EBV-positive Lymphoma

advanced stage or recurrent disease

• Long-term side effects of chemotherapy and radiation

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


3

Types of EBV LatencyTypes of EBV Latency

LMP 1

LMP 2

EBNA-1

EBNA-2

LMP 1

LMP 2EBNA 1

EBNA 1EBNA-1

EBNA-3a

EBNA-3b

EBNA-3c

LPLP

Type 3EBV lymphoma post transplantLymphoblastoid cell lines (LCL)

Type 2Hodgkin’s diseaseNasopharyngeal carcinoma

Type 1Burkitt’s lymphoma

EBV Infected

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

LyticLyticB cells

LMP1

LyticLyticEBNA 3a, b, cEBNA 3a, b, cLMP 2LPLMP 1EBNA 2EBNA1

PBMC

CTL

EBV +ve Lymphoma Cell

Inhibitory factors

LMP1

LMP2A

PBMC


4

EBV-Specific CTL GenerationEBV-specific CTL Generation

Step 1: LCL generationEBV

EBV-infected B cells (LCL)

EBV specific T cell Generation

IL-2

PBMC

g4-6 weeks

EBVPBMC

Step 3: QA/QCSterility

LMP2-CTL

Step 2: CTL expansion4-7 weeks

HLA typePhenotypeCytotoxicity

EBV-CTL

Gene Marked T-cells persisted for 12 months max

Marked CTL by in situ

EBV Specific CTL in EBV+ve Hodgkin Lymphoma

max EBV-CTL lines showed

small populations of T cells reactive against LMP2

Some expansion of LMP2-specific T cells in PB post i f i

PCR at tumor site

infusion. Anti-tumor effects seen

(20% CR/PR)

Bollard et al, J Exp Med 2004Straathof et al, J Immunol 2005


5



EBV-specificT cells

EBV+ Hodgkin’sdiseaseHow can we

improve and expand tumor specific T cells?

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

LMP1

• LMP1 and LMP2A are potential CTL targets

EBNA1EBNA1

LMP2A

Hodgkin R-S Cell/NHL Cell


6

Adherent PBMC

Making LMP1 and LMP2Immunodominant Antigens

GM-CSFIL-4

PBMC

IL-1bIL-6

TNF-aPGE-2

mDC

LCL LCLLCL

IL-15 IL-2 IL-2

PBMC LMP-specificCytotoxic T

Lymphocytes (CTL)

More recentlySubstituting

pepmixes for ad vectors

Bollard et al, JIT 2004, Straathof et al, JI 2005

Phenotype of Autologous LMP-CTL Lines Expanded from Lymphoma Patients

120

• 45%+/-15% CD45RA- 62L-• 34%+/- 5% CD45RA- 62L+

40

60

80

100

%

0

20

40

CD19+ Total CD3 TCR(ab)+ TCR(gd)+ CD4+ CD8+ CD3+

CD56+

CD56+

CD3neg

CD16+

CD3negCD19 CD3 TCR TCR CD4 CD8 CD3+ CD56+ CD16+

CD56+ CD3neg CD3neg


7

FC109Y08.133

104

0.03% 1.03%

FC109Y08.130

104

0.02% 0.19%

FC109Y08.131

104

0.17% 15.32%

FC109Y08.132

104

0.01% 2.95%

15.32% 0.19% 1.03% 2.96%

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 Patient with Hodgkin Lymphoma

60%

80%

Auto LCL

CD8

ILL

100

101

102

103

104

101

102

103

18.96% 79.98%

CD8

CL

G

100

101

102

103

104

101

102

103

16.83% 82.95%

CD8

FLY

100

101

102

103

104

101

102

103

19.48% 65.03%

CD8

YL

L

100

101

102

103

104

101

102

103

16.53% 80.50%

CD8

-20%

0%

20%

40%

60%

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

% S

peci

fic L

ysis Allo LCL

PHA Blasts

LMP1 target

LMP2 target

LMP2-specific Cytotoxic T Lymphocytes (CTL) are CD4+ and CD8+

T cells+LMP2

CMV

LMP2

T cell alone

T cells+CMV

CD8+ T cellsCD4+ T cells

0 200 400 600 800 1000

media

SFC per 1x105 cells


8

150

200

250

Broad LMP2-specific Activity Present in CTL line

1x10

5ce

lls

0

50

100

1 2 3 4 5 6 7 8 9 10

11

12

13

14

15

16

17

18

19

20

21

22

/23

CT

L

CT

L+

LC

LOverlapping peptide pool number

SF

C p

er

81

101 HLA type: A2;29/B7;15(62)A2= FLYALALLL (4+5+20)A2 CLG s

880

-19

1

21

41

61

CTLalone

peptide28

peptide29

ILL PYL Peptide32

Peptide33

Peptide92

Peptide93

Peptide94

Peptide95

LLW CLG FLY

A29=ILLARLFLY (4+20)

SF

C p

er 1

x105

cells

Broad LMP1-specific Activity Present in CTL line

300400500600700800

1x

10

e5

ce

lls

0100200300

1 2 3 4 5 6 7 8 9 10 CTLalone

LMP1pepmix

SF

C p

er

600700800900

1000

e5 c

ells

Overlapping peptide pool number

GLWIYLLEILWRLGATIWQLLAF

HLA type: A2;29/B7;15(62)

0100200300400500600

#21

#22

#23

#24

#25

#26

#27

#28

#29

#30

#31

#32

#33

#34

#35

#36

#37

#38

#39

#40

#91

#92

#93

#94

#95

#96

#97

#98

#99

#100

CT

L a

lone

SF

C p

er

1x10e

15mer peptide number

YLLEMLWRKLLIALWNLHGQALFL


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Autologous LMP2-CTL product (n=17)

CTL Product

Autologous LMP2 CTL product (n 17)OR LMP1 and LMP2 CTL product (n=33)• Dose escalation

- Level 1: 4x107/m2, - Level 2: 1 2x108/m2- Level 2: 1.2x108/m2

- Level 3: 3x108/m2

Diagnosis LMP2 CTLs

LMP1/2 CTLs

Total

Types of Lymphoma Treated on LMP-CTL Studies

Hodgkin’s Lymphoma 9 14 22NK/T lymphoma 3 8 11Diffuse large B cell lymphoma

3 4 7lymphomaPTLD (lung, kidney, liver) 1 3 4Other 1 3 5Total 17 32 50

Age range 8-79years


10

100

150300

400

100

150

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

LMP1/2-CTL

Pt1 Pt2 Pt3 LMP2

0

50

100

pre

1wk

2wk

4wk

6wk

8wk

0

100

200

pre

2wk

6wk

3mth

0

50

pre

1wk

2wk

4wk

8wk

3mth

15

20

25

60

80

100

15

20

25

30

20253035

FC p

er 1

05ce

lls

Pt4 Pt5 Pt6 Pt7

LMP1

No difference between DL1 and DL3 patients

Time post infusion

0

5

10

pre

1wk

2wk

4wk

6wk

8wk

3mth

0

20

40

60

pre

1wk

2wk

4wk

6wk

8wk

3mth

0

5

10

15

pre

1wk

2wk

4wk

6wk

8wk

3mth

05

101520

pre

1wk

2wk

4wk

6wk

8wk

3mth

S

FC120L04.019 FC120L04.020

Supraclavicular Lymph Node

LMP2LMP2--tetramer Positive CTL tetramer Positive CTL Detected in LNDetected in LN

Pre Post

FC113L04.062 FC113L04.063

0 12% 0 00%

100 101 102 103 104

CD8 FITC100 101 102 103 104

CD8 FITC

0.19% 0.21%

mer

s

LMP2 LMP2

CTL CTL

Peripheral Blood

100 101 102 103 104

CD8 FITC100 101 102 103 104

CD8 FITC

0.12% 0.00%

CD8

LMP2

tet

ram

CD8 CD8


11

103

104

350

400LMP1 LMP2

PB

0.5%

Rise in LMP-specific T cells Post Infusion and Accumulation in Lymph Node

CD8

IIL

100

101

102

103

104

101

102

150

200

250

300

350

C p

er 2

X10

5 cel

ls

103

104

LYMPH NODE29

pen

tam

er I

LL-P

E

0

50

100

pre wk1 wk2 wk4 wk6

SFC

CD8IIL

100

101

102

103

104

101

102

10 NODE

1.31%

LMP2

_A2

CD8-FITC

PREPREPREPRE POSTPOST

Clinical Responses post LMP-CTL

POSTPOST


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Relapsed Disease Arm (n=21)Patients with disease

at CTL infusion

Clinical Responses post LMP-CTL

• 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)

CR

at CTL infusion

Median clinical response: 1.5y (range: >6 to >40 mths)

CRPR

n =21

60% Disease Free Survival at 2 years

Clinical Responses post LMP-CTL in Patients with Active Disease

XXXXX CR

NR

ion

dise

ase-

free

0.4

0.6

0.8

1 P=0.744

CRPR

n =21

XXX

CRPR

Year

Pro

port

0 1 2 30

0.2 AlascerALCILMP1/2-CTL study

LMP2-CTL study


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Adjuvant Therapyn=29

Clinical ResultsClinical Results

Patients high risk for relapseat CTL infusion

Clinical Responses post LMP-CTL- As Adjuvant Therapy

• No toxicity• 14 patients post BMT • 15 post chemo alone• 1 died of cardiac disease

(at <8wks)

Relapse

at CTL infusion

( )• 27 remain in remission

- 1 relapsed 8 wks post CTL - CR median of 2.5 years

Remain in remission

Patients who received CTL as Adjuvant Therapy

1

Progression Free Survival Probability in LMP2-CTL vs LMP1/2-CTL groups

rtio

n di

seas

e-fr

ee

0.4

0.6

0.8

1 P=0.741

Year

Pro

por

0 1 2 30

0.2 AlascerALCI

LMP2-CTL study

LMP1/2-CTL study


14

XXXXXX

XXXXXXXXXX

Patients who received T cells as Treatment

Patients who received T cells as Adjuvant Therapy

Cause of death specific probability: All subjects

XXXX

j py

ncid

ence

Pro

babi

lity

LymphomaOther

04

0.6

0.8

1

Year

Cum

ulat

ive

In

0 1 2 30

0.2

0.4

Deaths from Other causesDeaths from Other causes

In adjuvant Group 8/26 patients died

Deaths from Other Causes

1 relapsed, died in CR after allo SCT3 second cancers (2 MDS , 1 Sarcoma) 3 infection1 cardiac disease

Confirms need for targeted therapies


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• No toxicity – especially when used as adjuvant therapy

Conclusions-LMP1/2 data

therapy• Accumulation of LMP-CTL at disease sites• Anti-tumor effects seen (13/21 patients PR/CR)• And what about the patients who relapse??

R d S b ll

Immune Evasion Strategies in Hodgkin’s Lymphoma - Do CTL have a chance?

Reed-Sternberg cellAPC

ILIL--1010LMP-1 LMP-2

EBNA-1

BARF0TGF ß

CD8+CTLIL-13

CD4+ TH2 CellCD4+ TH2 CellILIL--44

TGF-ßTARC

CD4+CD25+ CD4+CD25+ Treg CellTreg Cell


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EBV-specificT cells

EBV+ Hodgkin’sDiseaseCan Tumor-specific CTL Be

Genetically Modified to Become Resistant to Inhibitory Effects of Lymphoma Cells?

• Inhibits CTL proliferation

TGF Effects on CTL

• Inhibits cytotoxicity- perforin

• Inhibits cytokine production- IFN IFN


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Creating a MutantTGF Receptor II

Wild type Receptor

Truncated TGFß Receptor IIDominant Negative Receptor

Stop codon 597

Receptor (DNR)

Transmembrane domain

Retroviral vector SFG

Bollard et al, Blood 2002

MoMLV MoMLVU3 R U5 U3 R U5

NcoI/BamHI

SDPBSQ

SA+

TM domain

SFG:DNR

DNR

Rendering LMP-specific T cells Resistant to TGF

Ad5f35 LMP1-I-LMP2

EBV-LCL

DC SFG:DNR

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

PBMC IL-2DNR-transduced

LMP CTLs


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CD4 and CD8 T cells are DNRCD4 and CD8 T cells are DNR--transducedtransduced

RII

PE 10

3

104

2.39%0.03%

NONTrans

RII

PE 10

3

104

10.19%2.29%Trans

CD8 FITC

TG

FB

R

100

101

102

103

104

100

101

102

81.32%16.26%

CD8 FITC

TG

FB

R

100

101

102

103

104

100

101

102

76.84%10.68%

XX100000

120000

140000

nu

mb

er

NA

n=6 CTL lines

0

20000

40000

60000

80000

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

Tran

sgen

e co

py

nin

100

ng

DN

35%18%

20% n=6 CTL lines

DNRDNR--transduced CD4 and CD8 T cells are transduced CD4 and CD8 T cells are Predominantly Effector MemoryPredominantly Effector Memory

25%28%

10%

15%

20%

25%

30%

17%

11%6%

8%

10%

12%

14%

16%

1%

0%

5%

0%

CD45RA+ CD62L+

CD45RA-CD62L+

CD45RA-CD62L-

4%0%

2%

4%

TGFβ+ CD3+TGFβ+ CD3+ CD4+

TGFβ+ CD3+ CD8+


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DNRDNR--Transduced CTL are Transduced CTL are LMPLMP--specific specific

EBV-LCL

DNR-Transduced LMP-CTL are Functional in vitro

70000

80000

30000

40000

50000

60000

Pro

lifer

atio

n

+TGF

+TGF

0

10000

20000

NT NT+TGFb Trans Trans+TGFb

CTL

Non trans CTL Transduced CTL

+TGF


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• 5 females and 1 male

Patients StudiedPatients Studied

• EBV+ HL 5 – relapsed post autologous SCT1 – relapsed post allogeneic SCT

• Two previously treated with LMP-CTL• Two previously treated with LMP CTL alone

• All refused additional chemotherapy

100

1000 CTLs

DNRDNR--transduced Ttransduced T--cellscellsPersist Persist in vivo in vivo for 5for 5--23 months23 months

Patient 1

0.1

1

10

10000 CTLs

mbe

rs in

103

ng D

NA

0

Patient 3

0.1

1

10

100

1000

Cop

y nu

m

0


21

100

1000

DNRDNR--transduced Ttransduced T--cellscellsPersist Persist in vivo in vivo for 5for 5--23 months23 months

Patient 4CTLs

0.1

1

10

mbe

rs in

103

ng D

NA

0

1000

10000

Cop

y nu

m

0.1

1

10

100

1000

Patient 6

Patient 5 Patient 5 –– Partial ResponsePartial Response

Pre0.1

1

10

100

1000

sio

n3

hr

ek1

ek

2e

k3

ek4

ek6

th 2

th 3

th 4

th 5

th 6

th 7

eek

umbe

rs in

103

ng D

NA

0

Post

Pre

-infu

s

We

Wee

Wee

We

We

Mo

nt

Mo

nt

Mo

nt

Mo

nt

Mo

nt

Mo

nt

Mo

nth

7+

1 W

Cop

y nu


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Patient 2 Patient 2 –– Complete ResponseComplete ResponsePRE CR 8 wk

Response durable for over 34mthss

in

A 350400450500

LMP2-CTL

DNR Specific Assay over 34mths

SFC

per

2x10

5ce

lls

Copy

num

bers

103

ng D

NA

050

100150200250300350

pre week 2 CTL #2 week 4 week 8 month 3

DNR Specific Assay

Patient 1 Patient 1 –– Mixed Response to CRMixed Response to CR

120

140

160 EBV T-cells

DNR-trans T-cells

Anti-CD25 RITCTL

ers

in

NA 05

cells

0

20

40

60

80

100

pre wk3 wk6 wk8 mth3 mth 6 mth 9 mth 12

CRMixed

Response

Copy

num

be10

3ng

DN

SFC

per

2x10

Residual Tumor Cells LMP2neg Post CTL

Tumor Cells LMP2pos PreCTL


23

PPatient 3 atient 3 –– Partial ResponsePartial Response

250

300 DNR-T-cellsLMP1-CTLin

0

50

100

150

200

pre 2wks 4wks 8wks

LMP2-CTL

Copy

num

bers

10

3ng

DN

A

SFC

per

2x10

5ce

lls

POSTPRE Partial

Response

(8mths)

PRE POST

LMPLMP--specific CTL Accumulate specific CTL Accumulate in Tumor Sitesin Tumor Sites

Patient 3

CD3 T-cells infiltrating tumor sites post CTL

80

100

1204000 Tumor infiltrating T-cells

are LMP-specific

er llsPatient 3

80

90

Infused CTL line

0

20

40

60

LMP1 LMP2 Adhexon EBV

SFC

pe

2x10

5ce

Residual LMP2+ Tumor CellsPresent post CTL

0

10

20

30

40

50

60

70

Pool1 Pool 2 Pool 3 Pool 4 Pool 5 Pool 6 Pool 7 Pool 8 Pool 9 Pool 10

Tumor infiltrating CTL

LMP1 peptide pools

SFC

per

2x

105

cells

SFC

per

2x

105

cells


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Clinical Outcomes after DNR-transduced LMP-CTL

Pt ID Age Dose # Doses

Response post CTL

Duration of Response

CTL Persistence

1 27 2x107/m2 2Mixed

responseCR

14 months 12months

2 47 2x107/m2 2 Complete response

34 months+ 2 months

3 43 6x107/m2 8Partial

response18 months 23 months

4 47 6x107/m2 6 Stable Disease

12 months 18 months

5 32 2x107/m2 6 Very good PR

10 months + 10months+

6 28 6x107/m2 4 Stable Disease

7 months 5months

• No dose limiting toxicityTGF i LMP CTL

ConclusionsConclusions

• TGF-resistant LMP-CTL may beneficial in EBV+ Lymphoma

• DNR-trans LMP-CTL persist up to 12 monthsmonths

• Enrolment continues…


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• Evaluation of T cell products important to perform detailed immune reconstitution

Conclusions-LMP1/2 data

panalysis and persistence

• Accumulation of LMP-specific T cells at disease sites even when peripheral blood persistence lowN i i i ll h d dj• No toxicity – especially when used as adjuvant therapy

• Anti-tumor effects seen (PFS 60% at 2 years)

CAGT LaboratoryMalcolm BrennerCliona RooneyHelen Heslop

Acknowledgments

Helen HeslopAnn Leen

Barbara SavoldoGianpietro DottiSteve Gottschalk

Juan VeraAdrian Geed a Gee

Nabil AhmedCarlos Ramos

Carl AllenCollaborators

EJ ShpallJohn Barrett

Cath LabStephanie Ku, Sharon Lam, Russell Cruz,

Patrick Hanley, Alana Kennedy-NasserYasmin Hazrat, Mo Baki, Javier El-Bietar,

JW Blaney, Gerrit Weber, Francesco Saglio

Evaluation of T Cell Products – LMP-specific T cell...

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