Harnessing the innate and adaptive immune system to eradicate treated and distant untreated solid tumorsSaul Kivimäe, Werner Rubas, Rhoneil Pena, Joseph McLaughlin, Marlene Hennessy, Yolanda Kirksey, Wildaliz Nieves, Myong Lee, Clive Law, Kavitha Bhasi, Phi Quach, Janet Cetz, John L. Langowski, Christie Fanton, Jose Zandro Aquino, Zhongxu Ren, Haiying Cai, BoLiang Deng, Wen Zhang, Neel K. Anand, Jennifer A. Riggs-Sauthier, Steve Doberstein, Jonathan Zalevsky

Nektar Therapeutics, San Francisco, CA

INTRODUCTION

RESULTS

RESULTS

• Tumor antigen release and T cell priming by antigen presenting cells is a critical �rst step for tumor growth inhibition by the adaptive immune system

• Toll-like-receptor (TLR) stimulation can induce differentiation of functional antigen presenting cells in the tumor environment and reduce immune suppression in tumors facilitating T cell priming

• Pharmacological induction of tumor antigen presentation combined with sustained in vivo expansion of tumor speci�c CD8 T cells can potentially increase diversity and numbers of tumor killing cytotoxic T cells enabling ef�cacious anti-tumor immune therapies

• Combination treatment with a novel intratumoral TLR7/8 targeting agent NKTR-262 and a systemic CD122-biased agonist NKTR-214 leads to synergistic activation of innate and adaptive anti-tumor immune response resulting in highly ef�cacious growth inhibition of treated and abscopal lesions in multiple preclinical mouse tumor models

− Intratumorally delivered NKTR-262 provides local sustained release of TLR7/8 agonist with minimal systemic exposure

− Systemic sustained CD122-biased IL-2 pathway activation by NKTR-214 expands and maintains tumor in�ltrating CD8+ T cell clones

Bilateral subcutaneous tumors model NKTR-262 abscopal effect:

Single-sided subcutaneous tumors:

Right �ank tumors in mice bearing subcutaneous bilateral CT26 tumors were intratumorally treated with NKTR-262 (0.1 µg or 10 µg) on Day 0. NKTR-214 (0.8 mg/kg) was administered IV on Day 4. Immune cells in blood and in both treated and abscopal tumors were analyzed by �ow cytometry on Day 1 (NKTR-262 single agent activity) and Day 7 (NKTR-262 + NKTR-214 combination activity). (*p<0.05 with bars indicating comparisons)

Right �ank tumors in mice bearing subcutaneous bilateral CT26 tumors were treated intratumorally with NKTR-262 (10 µg). NKTR-262 and released TLR7/8 agonist concentrations were measured from both treated and abscopal left �ank tumors and from plasma at indicated timepoints after treatment.

CT26 tumor bearing mice were treated intratumorally with 10 µg of NKTR-262. Cytokine levels in treated tumors and in plasma were measured at Tmax of each cytokine (2 hr and 6 hr after treatment).

TLR7 and type I interferon pathway gene expression induction after NKTR-262 dose response measured by qRT-PCR relative to vehicle in CT26 subcutaneous tumors.(*p<0.05 relative to vehicle at Tmax)

A single NKTR-262 dose (20 µg) combined with NKTR-214 (0.8 mg/kg, i.v., q9d x 3 doses) inhibits tumor growth in several syngeneic mouse tumor models. Note: only the right �ank tumor was treated with NKTR-262 in bilateral models to assess abscopal ef�cacy in the untreated left �ank tumors (*p<0.05 relative to vehicle).

NKTR-262 and NKTR-214 engage non-overlapping immune mechanisms enhancing antigen presentation and anti-tumor T cell response

Complete anti-tumor immune response cascade induction by NKTR-262 and NKTR-214 − combination treatment optimally couples locally initiated intratumoral innate immune activation with systemic expansion and in�ltration of cytotoxic tumor reactive CD8 T cells

NKTR-262 + NKTR-214 combination treatment leads to relative decrease of immune suppressive cells in tumors

The injected tumors exhibit extended exposure to TLR7/8 agonist with minimal systemic exposure after local NKTR-262 administration

Rapid and transient activation of TLR7 pathway and type I interferon response genes in NKTR-262 treated tumors

Effective tumor growth inhibition by combining NKTR-262 with NKTR-214 in multiple preclinical syngeneic tumor models

Poster #P275: Society for Immunotherapy of Cancer 2017 Annual Meeting

Vehicle

NKTR-262 + NKTR-214

NKTR-262

NKTR-214

Vehicle

NKTR-262 + NKTR-214

NKTR-262NKTR-214

Treated tumors:

Abscopal tumors:

0 10 20 300

300

600

900

1200

EMT6 (Bilateral)Mammary Carcinoma

Days after treatment start

Tum

or

Vo

lum

e (m

m3

± S

EM

)

100% CR

CT26 (Bilateral)Colon Carcinoma

0 10 20 300

300

600

900

1200

Days after treatment start

Tum

or

Vo

lum

e (m

m3

± S

EM

)

100% CR

0

20000

40000

60000

80000

Tumor Dendritic CellsActivation (MHCII MFI)

Mea

n F

luo

resc

ence

Inte

nsi

ty

*

*

Day 1 Day 7

% o

f L

euko

cyte

s

0

20

40

60

Tumor Macrophages

**

Day 1 Day 70

10

20

30

40

Tumor CD4 Treg(CD25+ FoxP3+)

% o

f T

lym

ph

ocy

tes

*

Day 1 Day 70

1

2

3

4

5

Tumor Monocytes

% o

f L

ive

Cel

ls

*

Day 1 Day 7

Vehicle treated

Abscopal

0.1 µg NKTR-262

Abscopal

10 µg NKTR-262

Abscopal

+ NKTR-214

Day 1 Day 70

20

40

60

80

100

Lymph Node TargetedTumor Dendritic Cells

(CCR7+)

% o

f D

end

riti

c C

ells

*

0

20

40

60

Antigen ExperiencedTumor CD8 T Cells

(CD44hi)

% o

f C

yto

toxi

c T

Lym

ph

ocy

tes

*

Day 1 Day 7

Tumor CD8 T Cells

0

5

10

15

20

25

% o

f L

ive

Cel

ls

*

*

*

Day 1 Day 7

0

25

50

75

100

Tumor CD8/Treg

TIL

Rat

io

*

*

Day 1 Day 70

20

40

60

80

100

Checkpoint ReceptorsTumor CD8 T Cells

(PD-1+ CTLA-4+)

% o

f C

D8+

T L

ymp

ho

cyte

s *

Day 1 Day 7

Day 1 Day 70

20

40

60

80

100

Tumor Cell Viability

% o

f To

tal C

ells

* *

Day 1 Day 70

2

4

6

8

10

Tumor Neutrophils

% o

f L

ive

Cel

ls

*

*

Day 1 Day 70

20

40

60

80

100

Tumor Dendritic CellsActivation (CD86+)

% o

f D

end

riti

c C

ells

*

NKTR-262 and Released TLR7/8 Agonist Concentration in:

Treated Tumor

Time (hr)0 24 48 72 96 120 144

1

10

100

1000

10000

100000

Tum

or C

once

ntra

tion

(pm

ol/g

)

NKTR-262

TLR7/8 agonist

Plasma

1

10

100

1000

10000

100000

0 24 48 72 96 120 144

Time (hr)

Plas

ma

Conc

entr

atio

n (p

mol

/mL)

Abscopal Tumor

1

10

100

1000

10000

100000

0 24 48 72 96 120 144

Time (hr)

Tum

or C

once

ntra

tion

(pm

ol/g

)

2

0

20

40

60

80 Il12b

Fold

-ch

ang

e

6 24 48

20

10

20

30 Mx1

Hours post-dose

Fold

-ch

ang

e

6 24 482

0

2

4

6 Irf7

Hours post-dose6 24 48

Fold

-ch

ang

e

2

0

10

20

30

40

50Cxcl10

Hours post-dose Hours post-dose

Fold

-ch

ang

e

6 24 48

NKTR-262 (100 µg)NKTR-262 (10 µg)

Vehicle

NKTR-262 (1 µg)NKTR-262 (0.1 µg)NKTR-262 (0.01 µg)

*

Regulatory T cells are expanded selectively in peripheral blood

Intratumoral NKTR-262 administration leads to high proin�ammatory cytokines at treated tumor but reduced exposure in plasma

Day 1 Day 70.0

0.5

1.0

1.5

2.0

Blood CD4 Treg(CD25+ FoxP3+)

% o

f Leu

kocy

tes

*

Day 1 Day 70

5000

10000

15000

Blood CD4 Treg activation(CD25 MFI)

Mea

n Fl

uore

scen

ce In

tens

ity

*

Day 1 Day 70

2

4

6

8

Blood CD8/Treg

CD8/

Treg

Rat

io Vehicle

0.1 µg NKTR-262 + NKTR-214

10 µg NKTR-262 + NKTR-214

*

*

CONCLUSIONS• Intratumoral NKTR-262 treatment simultaneously induces tumor antigen release

and activation of antigen presenting cells

• NKTR-262 and NKTR-214 combination treatment relieves immune suppression selectively in tumor environment facilitating CD8 T cell activity

• NKTR-262 design enables TLR7/8 agonist release and induction of proin�ammatory cytokines at the injection site reducing systemic exposure

• NKTR-262 and NKTR-214 combination treatment optimally couples localized innate immune activation to systemic CD8 T cell expansion enhancing cytotoxic T cell in�ltration and activity in tumor lesions

NKTR-262Local i.t. dose

NKTR-214Systemic i.v.

TreatedtumorAbscopal

tumor

VehicleNKTR-262 + NKTR-214

0 10 20 300

250

500

750

1000

H22Hepatocarcinoma

*

Days after treatment start

Tum

or

Vo

lum

e (m

m3

± S

EM

)

0 5 10 15

WEHI-164Fibrosarcoma

*0

250

500

750

1000

Days after treatment start

Tum

or

Vo

lum

e (m

m3

± S

EM

)

0 5 10 150

500

1000

1500

2000

2500

3000

RM-1 − ProstateAdenocarcinoma

*

Days after treatment start

Tum

or

Vo

lum

e (m

m3

± S

EM

)

0 10 20 300

500

1000

1500

2000

2500

JC − MammaryAdenocarcinoma

*

Days after treatment start

Tum

or

Vo

lum

e (m

m3

± S

EM

)

Plasma and Tumor Cytokine Levels6 hr post dose

Co

nce

ntr

atio

n (p

g/m

L)

PlasmaTumor

IL-6

KC

/GR

O

TNFα

IL-1β

IFNγ

IL-5

IL-2

0

20

40

60

500

1000

1500

2000

100IL

-6

KC

/GR

O

TNFα

IL-1β

IL-5

IFNα

IFNγ

IL-2

IL-1

2p70

0

100

200

300

400

500

2000

4000

6000

Plasma and Tumor Cytokine Levels2 hr post dose

Co

nce

ntr

atio

n (p

g/m

L)