Adoptive T-cell Therapy for Melanoma:Trials and Tribulations in the

Quest for FDA ApprovalLaszlo G. Radvanyi

University of Texas, MD Anderson Cancer CenterSITC Annual Meeting

Bethesda, MDOctober 27, 2012

Disclosures

• Genesis BioPharma

-

SAB

Presence of tumor-reactive T-cells in metastatic melanomas (TIL)

G1: Low SSC cells

100 101 102 103 104

CD8

100

101

102

103

104

MA

RT-

1 te

tram

er

0.6

31

100 101 102 103 104

CD8

100

101

102

103

104

gp10

0 te

tram

er

0.39

31.2

gp100 tetramer

M. Ross, L. Radvanyi

MART-1 tetramer

Gall bladder-associated metastasis

Directly infuse high numbersof expanded Tumor-infiltrating Lymphocytes (TIL)

Adoptive T-cell therapy (ACT):Increasing the tumor-specific T-cell army

Blockade

Tumor

IL-2

TIL versus single target Agapproaches (TCR/CAR transduction)

Mutanome(mutatopes)

Differentiationand self Ags

Recognizedby T cells

OKT3 + IL-2 + feeders

IL-2

REP stage

HD IL-2

Current state-of-the-art “selected”

TILACT protocol for melanoma

Lymphodepletedpatient (Cy + Flud)

Pre-REP stage(30-100 x 106) (20-150 x 109)

3-5 wks 2 wks

Tumor line /cryo

tumor cells

Selection of tumor-specific (↑

IFN-γ) fragments for REP

Timing of preconditioning and TIL+IL-2 therapy: MDACC

Day

215 22 26215 22 26

-7 -5 -1

0 1

-7 -5 -1

1

infusionTIL

High-doseIL-2

High-doseIL-2

Lymphodepletion

cytoxan fludarabine

Patient #2150/2153

Beforetherapy

1 month

18 months

Patient #2054/2256

Responses to TIL therapy

Response to TIL therapy

P re‐treatment1‐2 months  

Pos t‐treatment

Response of brain metastasis to TIL

Waterfall plot of tumor regression in first 31 treated patients: MDACC

48% response rate(>50% decrease in tumor burden)

Clinical Response data from MDACC (as of July 10, 2012)

Best overall response:

*Some patients are still undergoing clinical response

Number ofpatients CR* PR* Total

51 2 (4%) 21(41%) 23 (45%)

12

Poster #1:

Bernatchez

et al.Adoptive cell therapy using expanded autologous

tumor-

infiltrating lymphocytes in metastatic

melanoma patients:Role of specific lymphocyte subsets

Kaplan-Meier curves of overall survival at MDACC (N=31)

Landmark analysis (from 3 month post-TIL)

Overall survival(from time of TIL infusion)

Growing Network of TIL Therapy Centers

NCI, MDMDACC, TX Sheba, IsraelMoffitt, FL

Uppsala, SwedenNKI, NL

Copenhagen, DKSeattle, WA

• Durable clinical responses in 40-50% of metastatic melanoma patients (NMA preparative regimen)

• Collectively, over 300

patients have been treatedover last 10 years with autologous

TIL+IL-2 (NMA)

Toronto, ON

Roadblocks to TIL Commercialization

Licensing /Commercialization

Roadblocks

• Labor-intensive • Long process (5-7 wks)

Selection of tumor-reactive TIL needed for best efficacy:-

Need tumor targets-

Unreliable in vitro assays

Open culture systems not amenable to automation

Practical “off-the-shelf”APCs

for TIL expansionNeeded (feeder problem)

No predictive biomarkersfor patient selection up-front:-

Tumor microenvironment-

Systemic/genetic elements

No reliable biomarkersof TIL product potency:-

Need biomarkers in TIL

Problem Solution• No reliable biomarkers of infused TIL potencypredictive of response

• Phenotypic biomarker analysis:-

T-cell EM subsets (CD8)-

Novel markers-

Function (Ag-specific / polyclonal)

• No predictive biomarkers forpatient selection for therapy:-

clinical response-

initial TIL outgrowth -

expanded TIL phenotypes

• Predictive tests before tumorresection for TIL outgrowth:-

IHC biomarkers in tumors-

gene expression in tumors-

systemic/genetic markers (blood)

• No method to isolatetumor-specific TIL up-front for expansion (save time)

• Selection from fresh TIL isolatesusing activation markers?

• PBMC feeder problem andavailability (costs)

• Off-the-shelf APCs

with definedstimulatory molecules

• Open systems too muchmanual handling (labor/cost)

• Automated closed systems forselection and expansion

Total TIL infused and CD8+ T cellsare critical parameters

CR/PR PD/SD0

20406080

100120140160

Clinical Response

Tota

l TIL

s in

fuse

d (x

109 ) p= 0.0002

CR/PR PD/SD0

20

40

60

80

100

p= 0.0009

Clinical Response

% C

D8+ T

-cel

l in

TIL

Total TIL and % CD8 TIL Infused andClinical Response: Sheba and MDACC Data

CR/PR PD/SD0

20

40

60

80

100 P= 0.0204

Clinical Response

% C

D8+ T

-cel

ls in

TIL

Sheba (N= 49) MDACC (N= 29)

CR/PR PD/SD0

20

40

60

80

100 P= 0.0011*

Clinical Response

% C

D8+ T

-cel

ls in

TIL

CR/PR PD/SD0

20406080

100120140160

P= 0.0003*

Clinical Response

Tota

l TIL

s in

fuse

d (x

109 )

Sheba + MDACC combined (N= 78)

CR/PR PD/SD0

20406080

100120140160

P< 0.0001*

Clinical Response

Tota

l TIL

s in

fuse

d (x

109 )

CR/PR PD/SD0

20

40

60

80

100P= 0.0002

Clinical Response

% C

D8+ T

-cel

ls in

TIL

CR/PR PD/SD0

20

40

60

80

100

120 P= 0.0077

Clinical Response

Tota

l TIL

s in

fuse

d (x

109 )

CR/PR PD/SD0

20

40

60

80

100

120

140P< 0.0001*

Clinical Response

Tota

l CD

8+ T

ILs

infu

sed

(x 1

09 )

Analysis of CD8+ T-cell differentiation status in infused TIL

TEM TEFF

CD45RA-CD62L+CD27+CD28+

CD45RA-CD62L-CD27+CD28+GB+Perf-

CD45RA-CD62L-CD27-CD28+GB++Perf+

PD-1BTLATIM-3

CD45RA-/+CD62L-CD27-CD28-CD57+GB++Perf++

TTDETCM/MSC

PD-1 and BTLA expression on CD8+ TIL

PD-1 BTLA TIM-30

20

40

60

80

100P< 0.05

CD8+ T-cell marker%

in C

D8+ p

opul

atio

n

Analysis of all patients (N=31)

FSC

SSC

Aqua

SSC

CD8

CD

4

PD-1

BTL

A

19 2

74 4

TIL #2144

Viable

FSC

SSC Viable

CD8

CD

4

PD-1

BTL

A

48 13

32 7

Aqua

SSC

TIL #2124

FSC

SSC Viable

CD8

CD

4

PD-1

BTL

A

48 13

32 7

Aqua

SSC

TIL #2124FSC

SSC

Aqua

SSC

CD8

CD

4

PD-1

BTL

A

17 50

13 20

TIL #2131

Post-REP TIL (treated)

BTLA+ TIL correlated with positive clinical response and not PD-1+ or TIM3+

CR/PR PD/SD0

20

40

60

80

100 P= 0.855

Clinical Response

% in

CD

8+ pop

ulat

ion

CR/PR PD/SD0

20

40

60

80

100 P= 0.104

Clinical Response

% in

CD

8+ pop

ulat

ion

P= 0.4987 P= 0.4943

CR/PR PD/SD0

20

40

60

80

100 P= 0.0023

Clinical Response

% in

CD

8+ pop

ulat

ion

P= 0.0034

CR/PR PD/SD0

20

40

60

80

100 P= 0.855

Clinical Response

% in

CD

8+ pop

ulat

ion

CR/PR PD/SD0

20

40

60

80

100 P= 0.104

Clinical Response

% in

CD

8+ pop

ulat

ion

P= 0.4987 P= 0.4943

CR/PR PD/SD0

20

40

60

80

100 P= 0.0023

Clinical Response

% in

CD

8+ pop

ulat

ion

P= 0.0034

PD-1 BTLA TIM-3

BTLA: “B-

and T- Lymphocyte Attenuator”

• Ig

family member (monomer like PD-1).• Negative costimulatory

molecule binding HVEM.

• May be a novel CD8+ T-cell differentiation marker.

Poster #8:

Haymaker et al.BTLA: New marker for a highly proliferative

CD8+ TIL subset

associated with melanoma regression during adoptivecell therapy

Persistence of CD8+BTLA+ TIL clones

Vβ gene family

Freq

uenc

y

CD8+BTLA-CD8+BTLA+

Infused TILDay 7

Day 21Day 40

CD8+BTLA+

Infused TIL

Day 7

Day 21

Day 40

CD8+BTLA-

Vβ gene family

Freq

uenc

y

CD8+BTLA-CD8+BTLA+

Infused TILDay 7

Day 21Day 40

CD8+BTLA+Infused TILDay 7

Day 21Day 40

Infused TILDay 7

Day 21Day 40

CD8+BTLA+

Infused TIL

Day 7

Day 21

Day 40

CD8+BTLA-Infused TIL

Day 7

Day 21

Day 40

Infused TIL

Day 7

Day 21

Day 40

CD8+BTLA-

BTLA+ BTLA-

Day 56

Infused TIL Vβ genes Persisting Vβ genes in bloodBTLA+ BTLA-

Day 0

Can biomarkers or signatures be identified as predictors of TIL efficacy?

Tumor

•T cell subsets• DCs•iNOS•NT•MDSCs•Chemokinesignatures

•IHC•Gene expression•RPPA

TIL

•Phenotype (subsets)•Function•Senescence issues•Gene expression•Ag specificity

•CD8 subsets•CTL activity•Gene expression•Telomere length•Methylome•Mutanome

andneo-epitopes?

Inbornhost factors

•“Immune phenotype”of patient•Tumor progressionfactors

•SNPs

(GWAS)•Inflammatorycytokines (serum)•Tumor markers

(serum)

SurgeryInitial TIL expansion

(Pre-REP phase)

4-5 weeks

FFPE / frozen tissue

PBMCSerum/plasma

Sample ofpre-REP TIL

TIL rapid expansion

(REP phase)

2 weeks

TILinfusion+ HD IL-2

Sample ofpost-REP TIL

10-12 weeks

PBMC and serum(day 14, 21, 40-42, 70

post TIL infusion)

PBMCSerum/plasma

Lymphodepletion

Predictive biomarkers in TIL therapy

IHC predictive markers?

Predictive biomarkers by IHC in original tumors used to grow TIL

1.

T cells:-

CD3, CD8, CD4 (peri-tumoral

/ intra-tumoral)

-

Foxp3-

PD-1 / BTLA -

TNF-R family (4-1BB / OX40)

2. Negative and positive markers of inflammation:-

protein nitrotyrosination

(NT) peroxynitrite

- iNOS-

CXCL10/IP-10

3. Macrophages/myeloid cells/DCs:-

CD163 -

S100A9

-

CD66b- DC-LAMP

4. Other immunosuppressive factors:- pSTAT3- IDO

PT / IM

IT

PT / IM

IT

PT / IM

IT

PT/IM

IT

CD8 in original tumor used to grow TIL(peritumoral

near invasive margin

versus intratumoral

Association between CD8 by IHC and CD8 % in expanded TIL (N= 42 pts)

% CD8 in TIL infused

% C

D8

by

IHC

P = 0.049, r2 = 0.305

Total CD8

P = 0.046, r2 = 0.310

% CD8 in TIL infused

% C

D8

by

IHC

Peri-tumoralPeritumoral

/ invasive margin

Association between CD8 expression by IHC in tumor and TIL clinical response

P = 0.08 P = 0.0072

P = 0.0273

PeritumoralP = 0.75 P = 0.17

P = 0.01

Intratumoral

P = 0.23 P = 0.0064

P = 0.0073

Total CD8

Peritumoral

/ invasive margin

Association between higher CD8 % atinvasive margin tumor and survival

Time (months)

≥35%<35%

Ove

rall

surv

ival

(%)

Log Rank P= 0.04

PT / IM CD8:

Relevance of TIL at the invasive margin (peri-tumoral)

Tumorin vivo

Relevance of TIL at the invasive margin (peri-tumoral)

Tumor fragmentex vivo

IL-2

Can we select up-front tumor-specificTIL for expansion?

OKT3 + IL-2 + feeders

IL-2

REP stage

HD IL-2

Lymphodepletedpatient (Cy + Flud)

Pre-REP stage

3-5 wks 2 wks

Selection of tumor-specific (↑

IFN-γ) fragments for REP

Can we select up-front tumor-specificTIL for expansion?

OKT3 + IL-2 + feeders

REP stage

HD IL-2

Lymphodepletedpatient (Cy + Flud)

Pre-REP stage

3-5 wks 2 wks

Selection

TCR activation induced costimulatorymolecules (Ig

and TNF-R families)IC

OS

CTLA4

PD-1

+ - +

4-1B

BOX40

Ig superfamily TNF-R family

59

288

5

6139

0.20

PD-1

4-1B

B4-

1BB

PD-1

No tumorcells

Plus tumorcells (24 h)

4-1BB and PD-1 induction

Presence of recently activated 4-1BB+and OX40+ T cells in tumor isolates

0

10

20

30

40

-5 25 55 85

CD8+

41BB+ Ox40+

% o

f T-c

ell s

ubse

t

A B

0

10

20

30

40

-5 15 35 5541BB+ Ox40+

CD4+

% o

f T-c

ell s

ubse

t

B

4-1BB staining by IHC

Selection of 4-1BB+ CD8+ T cellshighly enriches tumor Ag-specific cells

TIL isolationfrom tumors

Anti-41BB Ab beadselection

REP

ELISPOT

41BBselected Unselected

Control

Ag-specific

Handling before REP

IL-2

HD IL-2

30-100 x 106

20-150 billion

1000-2000 X

Lymphodepletedrecipient (NMA)

Anti-4-1BBAnti-4-1BB

Addition of agonistic anti-4-1BB Abs during TIL production

Anti-4-1BB Ab

increases CD8+ T-cell yield and preserves CD28 expression

IL-2 IL-2+4-1BB0

20

40

60

80

100

120

140

160

CD

8+ T

-cel

l yie

ld (x

106 c

ells

)TIL 2014

0 10 30 100

500

0

5

10

15

Anti-4-1BB Antibody [ng/ml]

CD8+

cel

l cou

nt (x

10^6

)TIL 2354

0 10 30 100

500

0

20

40

60

80

Anti-4-1BB [ng/ml]

CD8+

cell

coun

t (x1

0^6)

P< 0.05

Pre-REP

IL-2

IL-2+4-1BB

CD

28

CD

28C

D28

CD8

CD8

CD8

45

26

51

Pre-REP

IL-2

IL-2+4-1BB

CD

28

CD

28C

D28

CD8

CD8

CD8

45

26

51

Anti-41BB during TIL expansionincreases GB and Perforin

expression

Perforin changes

0

20

40

60

80P<0.05

P<0.05

CD

8+ P

erf+

(%)

Pre-REP REP REP+α41BB

3:1 1:1 1:30

5

10

15

20 Post-REPPost-REP+4-1BB

TIL 2292

Effector:Target

Cas

pase

3-c

leav

age

(%)

2473

+OKT3

2473

4-1B

B REP+OKT3

2478

+OKT3

2478

41BB R

EP+OKT324

81+OKT3

2481

41BB R

EP+OKT3

0

1000

2000

3000

4000

5000

IFN-γ

(pg/

ml)

“Off-the-shelf”

APCs

for TIL REP:Engineered K562 cells

K562 dAPC

CD64

CD86

4-1BBL

mb IL-15

eGFP

Generation #1 aAPC(master cell bank made)

Problem Solution• No reliable biomarkers of infused TIL potencypredictive of response

• Phenotypic biomarker analysis:-

T-cell EM subsets (CD8)-

Novel markers-

Function (Ag-specific / polyclonal)

• No predictive biomarkers forpatient selection for therapy:-

clinical response-

initial TIL outgrowth -

expanded TIL phenotypes

• Predictive tests before tumorresection for TIL outgrowth:-

IHC biomarkers in tumors-

gene expression in tumors-

systemic/genetic markers (blood)

• No method to isolatetumor-specific TIL up-front for expansion (save time)

• Selection from fresh TIL isolatesusing activation markers?

• PBMC feeder problem andavailability (costs)

• Off-the-shelf APCs

with definedstimulatory molecules

• Open systems too muchmanual handling (labor/cost)

• Automated closed systems forselection and expansion

What the future TIL expansion protocol will look like (21-day process)?

Cell suspension

Clinical-grade cell sorting

4-1BB+

Tumor

1 day

Pre-REP(7 days)

21 days

REP(14 days)

PD-1+4-1BB+ICOS+

Closed dual bioreactor

TIL+aAPC+

IL-2/15/21

IL-2/15/21

AcknowledgementsChantale

Jie

Qing

Minying

Richard Jessica Cara Geok

• NCI• Melanoma Research Alliance• Adelson

Medical Research Foundation• Mulva

Foundation• Gilson-Longenbough

Foundation

Steve Rosenberg (NCI)Mark Dudley (NCI)

Jim Yang (NCI)Michal Besser

(Sheba)Jacob Schachter

(Sheba)Ena

Wang (NCI)Franco Marincola

(NCI)Nick Restifo

(NCI)Carl Ware (La Jolla)

• Prometheus (Proleukin)• Bristol Myers Squibb (4-1BB)• Genentech

(BTLA)

Patrick HwuTIL Lab at MDACC