sarcoma immunotherapy: the future is near!

Sarcoma Immunotherapy:

The Future Is Near!

Seth M. Pollack, MD

Assistant Member

Outline

1. Introduction

2. Immune Response to Sarcoma Subtypes

3. Targeting NY-ESO-1

4. Looking into tumors

© Fred Hutchinson Cancer Research Center 2

Outline

1. Introduction





Sarcoma is a heterogeneous group of diseases


Sarcoma (1% of all cancer - percentages include children and adults)

Bone Sarcomas

(10%):

•Osteosarcoma

•Ewings Sarcoma

•Chondrosarcoma

•Giant Cell Tumor

•Other

Soft Tissue Sarcoma (STS)

GIST (18%)

RMS

Other “special” STS:

Kaposi’s

DFSP etc.

Non-GIST

Non-RMS

Not special

STS:

Or, in other

words, what I

usually call: STS

Ducimetriere et al 2011

Is STS really that rare?

Rare cancers are more than 25% of all adult cancers.

Approximately 12,000 Americans are diagnosed with STS annually (non-gist,

per American Cancer Society).

5000 people die annually.

Rare cancers lead to important scientific breakthroughs:

•Merkel Cell Carcinoma – 1500 patients/year

•ALL – 6590 patients/year

•Hodgkins Lymphoma – 8,500 patients/year

•Allogeneic transplant - 8000 patients/year

•Testicular cancer – 8720 patients/year

Greenlee et al. et al 2010

There are over 50 STS subtypes

Ducimetriere et al 2011

Total=438

Undifferentiated Pleomorphic Sarcoma (UPS)

Liposarcoma

Leiomyosarcoma

Other

Synovial

UPS – very highly mutated subtypes

LMS – less than UPS, but also highly mutated

SS – most common translocation associated sarcoma

Liposarcoma – genetically “simple”

“Liposarcoma” is actually at least 3 diseases


Well-

differentiated

(WD)

De-differentiated

(DD)

WD/DD

MRCL

Pleomorphic

Other/NOS

Basic Sarcoma Vocabulary

• Localized Disease

• Locally recurrent disease

• Metastatic Disease

• Neoadjuvant Therapy

• Adjuvant Therapy

• Palliative Therapy


What are the FDA approved drugs for sarcoma?

•First line therapy: Single Agent Doxorubicin (Average 4.6 month PFS)

•Recent approval: olaratumab (may improve OS combined with dox, phase III

results pending)

•Votrient (3 month PFS, no proven OS benefit – approved for STS other than

liposarcomas)

•Eribulin (2 month OS benefit, approved for liposarcoma only)

•Trabectedin (3 month PFS improvement liposarcoma and leiomyosarcoma)


Outline

1. Introduction





Different Types of Immunotherapy

• Cytokine

• Checkpoint inhibitor

• T cell therapy

• Vaccine

• Other


Checkpoint Inhibitors


Ott P A et al. Clin Cancer Res 2013;19:5300-5309

Checkpoint Inhibition in Melanoma


Topalian SL et al. N Engl J Med 2012;366:2443-2454.

TCR recognition of tumor requires MHC


T cell

TCR

tumor cell

peptide

MHC

Intracellular protein

activation

Retrospective Analysis


• Two highly mutated, genetically complex STS

types: UPS and LMS

• Two genetically “simple” STS types liposarcoma

(WD/DD and MRCL) and SS

Pollack et al., Cancer. In press

Focused Clustering Analysis


1" 2" 3" 4" 5" 6" 7"

Leiomyosarcoma"

Liposarcoma"

Pleomorphic/"

Undiff"Sarcoma"

Synovial"Sarcoma"

• Genes included if

p≤0.05 difference for

at least one subtype

• 367/760 genes

• Regions defined based

on dendrogram

clustering

• Most striking

separation for genes

related to antigen

presentation and T cell

infiltration


Class I MHC Molecules


UPS

SS

LMS

MRCL

WD D

D

0

500

1000

1500

2000

HLA-A

RN

A (

co

un

t)

*

**

UPS

SS

LMS

MRCL

WD D

D

0

500

1000

1500

2000

2500

HLA-B

RN

A (

co

un

t)

*

**

*

**

UPS

SS

LMS

MRCL

WD D

D

0

500

1000

1500

HLA-C

RN

A (

co

un

t)


Genes Related to T cell Infiltration


*

UPS

SS

LMS

MRCL

WD D

D

0

10

20

30

40

50

CD3D

RN

A (

co

un

t)*

**

UPS

SS

LMS

MRCL

WD D

D

0

50

100

150

200

250

IL7R

RN

A (

co

un

t)


TCR Vβ Sequencing


A

T-c

ell

fra

ctio

nM

ax. C

lon

al F

req

ue

ncy

B

*

C

Clo

na

lity

0.0

0.2

0.4

0.6

Lipo LMS UPS SS

0

10

20

30

40

Lipo LMS UPS SS

0.1

0.2

0.3

0.4

Lipo LMS UPS SS

**

**

*** *

*

A

T-c

ell

fra

ctio

nM

ax.

Clo

na

l F

req

uen

cy

B

*

CC

lon

alit

y

0.0

0.2

0.4

0.6

Lipo LMS UPS SS

0

10

20

30

40

Lipo LMS UPS SS

0.1

0.2

0.3

0.4

Lipo LMS UPS SS

**

**

*** *

*

A

T-c

ell

fra

ctio

nM

ax.

Clo

na

l F

req

ue

ncy

B

*

C

Clo

na

lity

0.0

0.2

0.4

0.6

Lipo LMS UPS SS

0

10

20

30

40

Lipo LMS UPS SS

0.1

0.2

0.3

0.4

Lipo LMS UPS SS

**

**

*** *

*

T-cell Fraction Clonality Max Frequency


Maybe those STS types rely more on PD-L1/L2?


UPS

SS

LMS

MRCL

WD D

D

0

20

40

60

80

100

CD274 (PD-L1)

RN

A (

co

un

t)

UPS

SS

LMS

MRCL

WD D

D

0

20

40

60

80

PDCD1LG2 (PD-L2)

RN

A (

co

un

t)


PD-1 Immunohistochemistry


4+ (above) and 5+ (below)

Staining showing infiltration with

PD-1+ cells

0 1 2 3 4 5

0

5

10

15

SS PD-1

# s

am

ple

s

0 1 2 3 4 5

0

2

4

6

8

10

UPS PD-1

# s

am

ple

s

0 1 2 3 4 5

0

2

4

6

8

LMS PD-1

# s

am

ple

s

0 1 2 3 4 5

0

5

10

15

Lipo PD-1

# s

am

ple

s


PD-L1 Immunohistochemistry


Very high PD-L1 Staining in

a UPS tumor

0 1 2 3 4 5

0

2

4

6

8

SS PD-L1

# s

am

ple

s

0 1 2 3 4 5

0

2

4

6

UPS PD-L1

# s

am

ple

s0 1 2 3 4 5

0

2

4

6

8

10

LMS PD-L1

# s

am

ple

s0 1 2 3 4 5

0

5

10

15

Lipo PD-L1

# s

am

ple

s


PD-1 and PD-L1 IHC Scores


SS

MRCL

WD/D

D L

ipo

LMS

UPS

0

1

2

3

4

5IH

C S

co

re

PD-1 Score

*

SS

MRCL

WD/D

D L

ipo

LMS

UPS

0

1

2

3

4

5

IHC

Sco

re

PD-L1 Score

*


PD-1 Inhibition in STS

Burgess/Tawbii ASCO 2016: SARC28 – 40 patients (4 of 10 UPS

responses, LMS – 0/10, SS – 1/10, Lipo 1/10)

Suzanne George ASCO 2016: 12 Uterine LMS - no responses

26

TAM impact for STS But are Killed by Trabectedin


Ganjoo et al., Am J Clin Oncol 2011 Germano et al. Cell 2013

TAM Markers in Uterine LMS

FHCRC 9717: Avelumab (anti-PD-L1) + trabectedin


PI: Pollack

For patients with liposarcoma and leiomyosarcoma.

FHCRC 9624: Doxorubicin + Pembrolizumab


Cycle 1: Pembrolizumab

200mgsingle agent

Cycle 2-7: Pembrolizumab

200mg+ Doxoribicin

Cycle ³8:

Pembrolizumab200mg

single agent

Phase I3+3 design:

Part 1: Doxorubicin 45 mg/m2

Part 2: Doxorubicin 75 mg/m2

Phase II2-stage design:

Part 1: 20 patients (requires ³ 2 responses)

Part 2: 15 patients PI: Pollack

Outline

1. Introduction





NY-ESO-1 is a CT Antigen Expressed by SS


Cancer Testis Antigens

(CTA) are well established

self-antigens - Not expressed at the protein level in most normal

tissues

- Unknown biologic function

- Epigenetically regulated

- NY-ESO-1, PRAME, MAGE family antigens included

-NY-ESO-1 frequently and

homogenously expressed by SS

-Positive 20/25; homogenous 14/20

Jungbluth et al., Int J Cancer 2001

Screening Tumors for Tissue Bank for CTA Expression


relative

row min row max

no

rmal te

stis

RL

B F

ibro

-M

el A

37

5U

266

HC

T11

6S

YO

-1M

RC

L 4

02

MR

CL 1

76

5S

YO

-1F

UJI

10

5K

CF

SS

W1

35

3S

W8

72

SK

-UT

-1o

ste

o 1

oste

o 2

oste

o 3

oste

o 4

oste

o 5

oste

o 6

oste

o 7

oste

o 8

ch

on

dro

1ch

on

dro

2ch

on

dro

3ch

on

dro

4ch

on

dro

5ch

on

dro

6lip

o 1

lipo

2lip

o 3

lipo

4lip

o 5

lipo

6le

io 1

leio

2le

io 3

leio

4le

io 5

leio

6le

io 7

leio

8le

io 9

leio

10

Ple

o 1

Ple

o 2

Ple

o 3

Ple

o 4

Ple

o 5

SS

1S

S 2

SS

3S

S 4

SS

5M

RC

L 1

MR

CL 2

MR

CL 3

MR

CL 4

MR

CL 5

MR

CL 6

MR

CL 7

MR

CL 8

MR

CL 9

MR

CL 1

0

Number Gene

BAGECTAG1BCTAG2CTCFLDDX53GAGE1MAGEA1MAGEA2MAGEA3/6MAGEA4MAGEA9MAGEA10MAGEA12MAGEC2RAGESSX1SSX2TAGPRAME

relative

row min row max

norm

al te

stis

RL

B F

ibro

-M

el A

37

5U

26

6H

CT

116

SY

O-1

MR

CL

40

2M

RC

L 1

765

SY

O-1

FU

JI

105

KC

FS

SW

13

53

SW

87

2S

K-U

T-1

oste

o 1

oste

o 2

oste

o 3

oste

o 4

oste

o 5

oste

o 6

oste

o 7

oste

o 8

ch

ond

ro 1

ch

ond

ro 2

ch

ond

ro 3

ch

ond

ro 4

ch

ond

ro 5

ch

ond

ro 6

lipo 1

lipo 2

lipo 3

lipo 4

lipo 5

lipo 6

leio

1le

io 2

leio

3le

io 4

leio

5le

io 6

leio

7le

io 8

leio

9le

io 1

0P

leo

1P

leo

2P

leo

3P

leo

4P

leo

5S

S 1

SS

2S

S 3

SS

4S

S 5

MR

CL

1M

RC

L 2

MR

CL

3M

RC

L 4

MR

CL

5M

RC

L 6

MR

CL

7M

RC

L 8

MR

CL

9M

RC

L 1

0

Number Gene


relative

row min row max

norm

al te

stis

RLB

Fib

ro-

Mel A

375

U26

6H

CT

11

6S

YO

-1M

RC

L 4

02

MR

CL 1

765

SY

O-1

FU

JI

105K

CF

SS

W135

3S

W872

SK

-UT

-1oste

o 1

oste

o 2

oste

o 3

oste

o 4

oste

o 5

oste

o 6

oste

o 7

oste

o 8

ch

ondro

1ch

ondro

2ch

ondro

3ch

ondro

4ch

ondro

5ch

ondro

6lip

o 1

lipo 2

lipo 3

lipo 4

lipo 5

lipo 6

leio

1le

io 2

leio

3le

io 4

leio

5le

io 6

leio

7le

io 8

leio

9le

io 1

0P

leo 1

Ple

o 2

Ple

o 3

Ple

o 4

Ple

o 5

SS

1S

S 2

SS

3S

S 4

SS

5M

RC

L 1

MR

CL 2

MR

CL 3

MR

CL 4

MR

CL 5

MR

CL 6

MR

CL 7

MR

CL 8

MR

CL 9

MR

CL 1

0

Number Gene


(NY-ESO-1)

relative

row min row max

no

rma

l te

stis

RL

B F

ibro

-M

el A

37

5U

26

6H

CT

11

6S

YO

-1M

RC

L 4

02

MR

CL

17

65

SY

O-1

FU

JI

10

5K

CF

SS

W1

35

3S

W8

72

SK

-UT

-1o

ste

o 1

oste

o 2

oste

o 3

oste

o 4

oste

o 5

oste

o 6

oste

o 7

oste

o 8

ch

on

dro

1ch

on

dro

2ch

on

dro

3ch

on

dro

4ch

on

dro

5ch

on

dro

6lip

o 1

lipo

2lip

o 3

lipo

4lip

o 5

lipo

6le

io 1

leio

2le

io 3

leio

4le

io 5

leio

6le

io 7

leio

8le

io 9

leio

10

Ple

o 1

Ple

o 2

Ple

o 3

Ple

o 4

Ple

o 5

SS

1S

S 2

SS

3S

S 4

SS

5M

RC

L 1

MR

CL

2M

RC

L 3

MR

CL

4M

RC

L 5

MR

CL

6M

RC

L 7

MR

CL

8M

RC

L 9

MR

CL

10

Number Gene


Pollack ASCO 2016

MRCL Expresses NY-ESO-1 Homogenously


• 25 cases tested, all positive

• >70% of cases, homogenous.

•MRCL cell lines can lysed by NY-ESO-1 specific effectors

• No other disease expresses NY-ESO-1 with this frequency

Pollack et al., Cancer 2012

Culturing NY-ESO-1 Specific Cells


tetramer

CD

8

<0.001% 8.51% 94.4%0.719% 1.6%

Stimulate Sort Expand

StimulateSort and Expand

SS and MRCL Leukapharesis

products lack clear tet+ populations.

After 2 stimulations, three wells were identified with clear tet+ populations.

Final product contained 57 x 109 cells and was over 94% CD8+,

tet+.

Pollack et al. JITC 2014

These Cells Kill Tumor Lines


Pollack et al. JITC 2014

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

80

100

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

80

100

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

80

100

E:T

% L

ysis

Mel A375

Mel 526

Patient 5

Patient 6

Patient 4

Patient 2

Patient 3

Patient 1

aLY TCR

High Affinity gp100 clone

High Affinity NY-ESO-1 Clone

(NY-ESO-1 +; gp100 -)

(NY-ESO-1 -; gp100 +)

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

80

100

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

80

100

E:T

% L

ysis

40 20 10 52.

5

0

20

40

60

80

100

E:T

% L

ysis

Mel A375

Mel 526

Patient 5

Patient 6

Patient 4

Patient 2

Patient 3

Patient 1

aLY TCR

High Affinity gp100 clone

High Affinity NY-ESO-1 Clone

(NY-ESO-1 +; gp100 -)

(NY-ESO-1 -; gp100 +)


Before

(from lung)

After

(from forehead) Before 10 Weeks After

T2 un

pulsed

T2 pu

lsed

Mel

A37

5

0

20

40

60

80

100

Pe

rce

nta

ge

Lysis

CD8+, Tet+ Cells Sorted and Expanded in vitro from Tumor Can Kill Targets

NY

-ES

O-1

C

lass I M

HC

0 102

103

104

105

0

5

10

15

20

LSR2-1_2537-S02 Post-Tx Blood D+71.fcs…CD8

<APC-A>: NY-ESO Dex

1.4298.6

0 102

103

104

105

0

1000

2000

3000

LSR2-1_2537-S02 Post-Tx TIL REP1.fcs…CD8

<APC-A>: NY-ESO Dex

0.1699.8

NY-ESO-1 tetramer (Gated on

CD8+ cells)

Peripheral

Blood Expanded TIL

0.16% >1.4%

Pt IFN1

However, because of safety issues with this trial, this

regimen was abandoned

LV305: A Novel NY-ESO-1 Vaccine


LV305 is a novel hybrid viral vector gene delivery system (ZVexTM) that expresses NY-ESO-1 RNA

designed to target DCs in vivo and stimulate CD8 T cell responses against this cancer testis antigen

ZVex

DC-SIGN

ZVex

DC

Envelope:

DC Targeting: •Sindbis virus envelope targets the Dendritic Cell receptor, CD209

(DC-SIGN)

Genome:

Safety: •3rd generation lentiviral vector backbone

•Modified to be replication incompetent and integration deficient

Specificity:

•Vpx prevents degradation within DCs

Slide courtesy of Immune Design

Increased NY-ESO-1 Tet+ Cells in A0201+ Patients


Hea

lthy SS

MRCL

Pt 1

-1 p

re

Pt 1

-1 p

ost

Pt 4

-3 p

re

Pt 4

-3 p

ost

Pt 1

-5 p

re

Pt 1

-5 p

ost

0.00

0.05

0.10

0.15

0.20

% tet+

Tetramer Staining in A2+ Patients(Percent of CD8+ Cells)

MAGE-A

3

MAG

E-A

4

MAGE-A

9

PRAM

E

NY-E

SO-1

Neg

Contr

ol

PHA

0

20

40

60

80

SF

U

1-1 PBMC Elispot

pre post

Pollack et al. Submitted

Pre and Post Tx TCR sequencing


1.68% of

Pre-tx T cells

2.51% of

Post-tx T cells

Clonality 0.11 Clonality 0.18

0.000001

0.00001

0.0001

0.001

0.01

0.000001 0.00001 0.0001 0.001 0.01

Abundance in P

BM

C-p

ost

Abundance in PBMC-pre

T cells absent in FFPE tumor (pre)

T cells present in FFPE tumor (pre)


Durable Tumor Regression Following LV305



Durable Tumor Regression Following LV305


5/5/2014 6/1/2016


Survival on the LV305 Vaccine Trial


0 5 10 15 20

0.0

0.2

0.4

0.6

0.8

1.0

Months

Pro

port

ion

Pro

gre

ssio

n F

ree

0 5 10 15 20

0.0

0.2

0.4

0.6

0.8

1.0

Months

Pro

po

rtio

n S

urv

ivin

g

All Sarcomas

N= 24 (progressed= 18)

95% CL: 2.6, 14.4

All Sarcomas

N= 24 (deaths= 4)

Pollack ASCO 2016

Outcomes on LV305 and Approved Drugs For Sarcoma


Treatment Indication PFS OS

LV305 STS 4.6

mo

Not

reached

Pazopanib

(Votrient)

STS (not

MRCL)

4.7

mo 12.5 mo

Trabectedin

(Yondelis)

MRCL after

Anthracycline

(all STS in EU)

4.2

mo 12.4 mo

Eribulin

(Halaven)

MRCL after

Anthracycline

2.6

mo 13.5 mo

Pollack ASCO 2016

CMB +/- Atezolizumab


≤ 80 pts with locally advanced,

relapsed, or metastatic

synovial sarcoma or MRCL

Combination Arm Sequential regimen of LV305 and G305 for 3

months

with atezolizumab q3w up to 2 yrs

Control Arm Atezolizumab q3w up to 2 yrs

Endpoints of PFS, safety, PFR

(at 6 mo), ORR, duration of

response, and OS Randomize

Screen,

biopsy, safety run-in Biopsy, then follow up to 2 years

Outline

1. Introduction






16

cm

Sarcomas Can

Be Quite Large

and Are

Relatively

Accessible

Presage’s Civo Device


2. Ultrasound

Measurements

M1

M2

3. Microinjection 1. Device Loading

1

2

3 4

7

6

8

5

4. Confirmation

and Skin Marking

Slide courtesy of Presage

2

3 4

6 1

8

7

5 PI: Pollack

FHCRC 9145: Intratumor TLR4 Agonist + Radiation


PI: Pollack

FHCRC 9145: Schema


Weekly GLA-SE injections through week 8.

1 2 43 765 8

rads

week

Pre-treatmentImaging

1st Post-treatmentImaging

Biopsy Biopsy

• Two, 6-patient cohorts treated: 5 mcg and 10 mcg.

• All patients were required to have unresectable of metastatic disease.

• GLA was injected directly into a tumor.

• Radiation was started to the injected tumor during the first week of treatment.

• Patients received high dose (generally 50 Gy) in few fractions (generally 5 or less).

• Current cohort using 20 mcg.

TLR4 Agonism Can Convert M2 to M1


0 102

103

104

105

0

20

40

60

80

100

CD163 high

0 102

103

104

105

0

20

40

60

80

100

0 102

103

104

105

0

20

40

60

80

100

CD206

CD115

% o

f m

ax

% o

f m

ax

% o

f m

ax

CD163

Before Treatment During Treatment

x

iNO

S

CD

16

3 x

CD

20

6

CD16

3

CD20

6

CD11

5

HLA

-DR

0

20

40

60

80

100

% p

os

. ga

ted

(C

D4

5+

, CD

11

B+

)Pre-treatment

Post-Treatment


Acknowledgments

Pollack Lab

Bailey Donahue

Sara Cooper (former)

Graeme Black

Taylor Hain

Olga Vitruk

FHCRC

Stan Riddell

Silvia Christien

Deb Banker

Chad He

Yuzheng Zhang

Mary Redman

Kelsey Baker

CPF

SCCA Sarcoma Program

Lee Cranmer

Venu Pillarisetty

Ed Kim

Chappie Conrad

Ben Hoch

Bob Ricciotti

Matt Spraker

Dave Seo

Other

Achim Jungbluth

Cassian Yee

Robin Jones

Immune Design

Jan H. ter Muelen

Frank Hsu

Hailing Liu

Adaptive Biotechnologies

Sharon Benzeno

Ryan Emerson

Marissa Vignali

Merck Research Labs

Terrill K. McClanahan

Jennifer Yearley

Erin Murphy

Wendy M. Blumenschein

Steven M. Townson

Special Thanks:

Gilman Family Sarcoma Fund

NCI 1K23CA175167-01

SARC

Sarcoma Foundation of America

sarcoma immunotherapy: the future is near!

Documents