systemic anti-tumor immunity and immune memory formation ......systemic anti-tumor immunity and...

Systemic anti-tumor immunity and immune memory formation by a novel TLR7/8 targeting agent NKTR-262 combined with CD122-biased immunostimulatory cytokine NKTR-214Saul Kivimäe, Rhoneil Pena, Marlene Hennessy, Phi Quach, Janet Cetz, Yolanda Kirksey, Wildaliz Nieves, Fiore Cattarruzza, Christie Fanton, Zhongxu Ren, Haiying Cai, BoLiang Deng, Wen Zhang, Neel K. Anand, Werner Rubas, Steve Doberstein and Jonathan Zalevsky

Nektar Therapeutics, San Francisco, CA

INTRODUCTION 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 has the potential to elicit clonal expansion of tumor antigen-speci�c cytotoxic T cells enabling ef�cacious anti-tumor immune therapies with durable and speci�c anti-tumor immune memory formation

• 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 NKTR-262 treated and abscopal lesions in multiple preclinical mouse tumor models

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

• NKTR-262 + NKTR-214 combination therapy is being evaluated in select

solid tumor indications in a phase 1/2 clinical study (NCT03435640)

Mice bearing bilateral CT26 tumors were treated in the right �ank tumors with 10 µg NKTR-262 (IT) and 0.8 mg/kg NKTR-214 (IV) administered as single agents or in combination. Immune cells in blood and both NKTR-262 treated (solid symbols) and untreated left �ank (open symbols) tumors were analyzed by �ow cytometry at indicated timepoints. Tumor antigen-speci�c CD8 T cells were labeled with AH1 speci�c MHC-peptide dextramers. (*, p<0.05, n=3-4, mean ± SEM)

Fraction of tumor T cell clones found in matched post-treatment blood was measured in bilateral CT26 tumor-bearing mice at indicated timepoints after NKTR-214 (0.8mg/kg, IV) single agent or combination treatment with NKTR-262 (10 µg, dosed IT into the right �ank tumors). TCR usage was determined utilizing ImmunoSEQ platform from Adaptive Biotechnologies

Tumor in�ltration and T cell clonality in tumors and blood and were assessed in bilateral CT26 tumor-bearing mice at indicated timepoints after NKTR-214 (0.8mg/kg, IV) single agent or combination treatment with NKTR-262 (10 µg, dosed IT in the right �ank tumors). TCR usage was determined utilizing ImmunoSEQ platform from Adaptive Biotechnologies

Mice bearing subcutaneous bilateral CT26 tumors were treated in the right �ank tumors with NKTR-262 (0.1 µg or 10 µg, IT) on Day 0. NKTR-214 (0.8 mg/kg, IV) was administered on Day 4. Immune cells in blood and both NKTR-262 treated and abscopal left �ank tumors (solid and open symbols, respectively) were analyzed by �ow cytometry on Day 1 (NKTR-262 single-agent effect) and Day 7 (combination treatment effect). (*, p<0.05 with bars indicating comparisons, n=4, mean ± SEM)

NKTR-262 and NKTR-214 combination treatment couples locally initiated tumor antigen release and presentation with systemic CD8 T cell expansion and tumor infiltration

Systemic T cell repertoire modulation in NKTR-262 and NKTR-214 treated tumor-bearing mice − combination treatment increases clonal T cell expansion in blood and tumors and enhances systemic distribution of tumor T cell clones

NKTR-262 and NKTR-214 combination selectively expands CD8 T cells and systemically sustains tumor antigen specific T cells

Poster #P364: Society for Immunotherapy of Cancer 2018 Annual Meeting

0

20000

40000

60000

80000

Tumor Dendritic CellsActivation (MHCII MFI)

Mea

n F

luo

resc

ence

Inte

nsi

ty

*

*

Day 1 Day 7

Vehicle treated

0.1 µg NKTR-262 + NKTR-214

10 µg NKTR-262 + NKTR-214

Tumor CD8 T Cells

0

5

10

15

20

25

% o

f L

ive

Cel

ls

*

*

*

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 7Day 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

*

*

CONCLUSIONS

• Intratumoral NKTR-262 treatment concurrently induces tumor antigen release and activation of antigen presenting cells enhancing CD8 T cell tumor in�ltration

• NKTR-262 and NKTR-214 combination results in sustained preferential expansion of CD8 T cells and a substantially larger fraction of tumor antigen-speci�c CD8 T cells than NKTR-214 monotherapy

• NKTR-262 and NKTR-214 combination treatment demonstrates curative ef�cacy in multiple syngeneic tumor models and durable anti-tumor immunity in complete responders

• NKTR-262 and NKTR-214 combination treatment increases clonality and enhances clonal expansion of T cell cells in blood and tumors

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

RESULTS

MECHANISM OF ACTION OF NKTR-262 AND NKTR-214 COMBINATION TREATMENT

Tumor re-challenge 10 weeks post tumor clearance after drug treatment

Intratumoral T Cell Clonality and Infiltration

T Cell Post-Treatment Clonality and Clonal Expansion in Blood

Blood Distribution of Tumor T Cell Clones

Mice bearing subcutaneous bilateral CT26 or EMT6 tumors were treated with a single NKTR-262 IT dose (20 µg) and NKTR-214 (0.8 mg/kg, IV, q9dx3) that led to 100% CRs in both CT26 and EMT6 tumor models. Ten weeks after tumor clearance mice were rechallenged with the same or alternate tumor cell line to evaluate spontaneous tumor rejection without drug treatment

Systemic dissemination of locally initiated antitumor immune response

NKTR-262 and NKTR-214 combination treatment leads to synergistic efficacy and durable anti-tumor immunity

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

NKTR-262Local i.t. dose

NKTR-214Systemic i.v.

TreatedtumorAbscopal

tumor

0 5 10 15 200

100

200

500

1000

1500CT26CR → CT26

Days After Rechallenge

Tu

mo

r V

olu

me

(mm

3 )

Day 4 Day 5 Day 9

Vehicle

NKTR-262

NKTR-214

NKTR-262 + NKTR-214

* * *

Day 4 Day 5 Day 90

20

40

60

80

100

CD8 T Cells in Blood CD4 T Cells in Blood%

of

T C

ells

(Mea

n ±

SE

M)

0

20

40

60

80

100

% o

f T

Cel

ls (M

ean

± S

EM

)* * *

0

2

4

6

8

10

Tumor Antigen SpecificAH1+ CD8 T Cells in Spleen

Day 5 After Treatment

*

0

10

20

30

Tumor Antigen SpecificAH1+ CD8 T Cells in Tumors

Day 5 After Treatment

% o

f Li

ve C

ells

(Mea

n ±

SE

M)

*

Day 5 Day 90

5

10

15

20

25

Tumor Antigen SpecificAH1+ CD8 T Cells in Blood

% o

f L

euko

cyte

s (M

ean

± S

EM

)

% o

f L

euko

cyte

s (M

ean

± S

EM

)

*

0.2 0.3 0.4 0.50.0

0.2

0.4

0.6

0.8

Day 4 (Tumors)

T C

ell I

nfi

ltra

tio

n(F

ract

ion;

Mea

n ±

SE

M)

0.2 0.3 0.4 0.50.0

0.2

0.4

0.6

0.8

Day 7 (Tumors)

0.2 0.3 0.4 0.50.0

0.2

0.4

0.6

0.8

Day 13 (Tumors)

Rig

ht Flank Tum

ors

0.2 0.3 0.4 0.50.0

0.2

0.4

0.6

0.8

T Cell Clonality(Mean ± SEM) (Mean ± SEM) (Mean ± SEM)

T C

ell I

nfi

ltra

tio

n(F

ract

ion;

Mea

n ±

SE

M)

0.2 0.3 0.4 0.50.0

0.2

0.4

0.6

0.8

T Cell Clonality0.2 0.3 0.4 0.5

0.0

0.2

0.4

0.6

0.8

T Cell Clonality

Left Flank Tum

ors

(Mean ± SEM)T Cell Clonality

0.00 0.05 0.10 0.15 0.200

20

40

60

80

100Day 7 (Blood)

Vehicle NKTR-214 NKTR-262 + NKTR-214

(Mean ± SEM)T Cell Clonality

0.00 0.05 0.10 0.15 0.200

20

40

60

80

100Day 13 (Blood)

T Cell Clonality(Mean ± SEM)

0.00 0.05 0.10 0.15 0.200

20

40

60

80

100Day 4 (Blood)

Exp

and

ed C

lone

s(M

ean

± S

EM

)

Day 4 Day 7 Day 13

5

10

15

20

25

% o

f Tu

mo

r T

Cel

l Clo

nes

in B

loo

d ( M

ean

± S

EM

)

Days After Treatment Start

Left Flank Tumors

Day 4 Day 7 Day 13

Vehicle NKTR-214 NKTR-262 + NKTR-214

Right Flank Tumors

Tu

mo

r V

olu

me

(mm

3 )

0

100

200

500

1000

1500

0 5 10 15 20

EMT6CR → EMT6

Days After Rechallenge0 5 10 15 20 25

0

100

200

500

1000

1500EMT6CR → CT26

Days After Rechallenge

Tu

mo

r V

olu

me

(mm

3 )

0 5 10 15 200

100

200

500

1000

1500CT26CR → EMT6

Days After Rechallenge

Tu

mo

r V

olu

me

(mm

3 )