NKTR-214, an engineered cytokine, synergizes and improves efficacy of anti-cancer vaccination in the treatment of established murine melanoma tumor

Meenu Sharma1, Faisal Fa’ak1, Louise Janssen1, Hiep Khong1,2, Zhilan Xiao1, Yared Hailemichael1, Manisha Singh1, Christina Vianden1, Adi Diab1, Jonathan Zalevsky3, Ute Hoch3, Willem W. Overwijk1,2 1Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030

2University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030 3Nektar Therapeutics, 455 Mission, Bay Blvd South, San Francisco, CA 94158

Pmel-1 CD8+ T cells CD25+ Foxp3+ Tregs Pmel-1/Tregs Ratio

•  NKTR-214 efficiently synergized with vaccination, potently suppressing tumor growth and improving

survival of mice compared to vaccination with IL-2.

•  NKTR-214 significantly enhanced pmel-1 CD8+ T cell numbers in tumor and spleen.

•  NKTR-214 specifically decreased numbers of immune-suppressive Tregs in the tumor while maintaining

their numbers in spleen.

•  Despite the induction of very strong CD8+ T cell responses and anti-tumor activity, no gross toxicity was

observed.

• Accumulating evidence suggests that low baseline tumor infiltrating lymphocytes (TILs) are predictive for poor response to checkpoint inhibitor immunotherapies,1,2 thus agents designed to specifically activate and expand TILs may improve the overall success and utility of checkpoint inhibitor therapies in patients with low TILs

• Interleukin-2 (IL-2) is a cytokine that activates and expands tumor killing lymphocytes, but also potently activates suppressive T regulatory cells (Tregs) by binding to the heterotrimeric IL-2Rαβγ

• NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol and designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and NK cells over Tregs3

In this pre-clinical study, we investigated whether NKTR-214

can promote expansion and function of vaccination-induced,

tumor specific effector CD8+ T cells using the murine B16

melanoma model. We also studied how NKTR-214 impacts

the localization of effector CD8+ T cells and Tregs to tumor

and spleen.

1.  No treatment 2.  Aldesleukin (100,000 IU X 5 doses - D0,D1,D2)_every 8 days 3.  NKTR-214 (0.2 mg/kg) _every 8 days 4.  Vaccine 5.  Vaccine + aldesleukin (100,000 IU X 5 doses D0,D1,D2)_every 8 days 6.  Vaccine+ NKTR-214 (0.2 mg/kg)_every 8 days

Mice: C57BL/6

D0

Gp100 peptide TLR-7 agonist Anti- CD40 IL-2 or NKTR-214

D1

IL-2

D2

IL-2 NKTR-214 Or IL-2

D8

NKTR-214 Or IL-2

D16

D-6

Tumor Induction 300,000 B16 wild type cells

NKTR-214 Or IL-2

D24

Treatment repeated till

experimental end point

o  Gp100 peptide (50 ug/mouse)-L-tyr (adjuvant) o  anti –CD40 (50 ug/mouse) o  TLR-7 agonist (Imiquimod- 5 mice/pack)

Vaccine formulation

0 20 40 60 80 1000

20

40

60

80

100

No#treatmentNKTR-214#Aldesleukin

Vaccine##+#AldesleukinVaccine#+#NKTR-214#

Vaccine

**p= 0.0031

Days#a=er#vaccina?on

Perc

ent s

urvi

val

Pmel-1 T cell response Tregs

Survival

0 20 40 60 800

50

100

150

Untreated

NKTR321444Aldesleukin4

4Vaccine4Vaccine4+4Aldesleukin

p=0.039Vaccine4+4NKTR32144*

p=0.018*

Days4a@er4vaccinaBon

Tumor4size4(m

m² )

0"

50"

100"

150"

0" 10" 20" 30" 40" 50" 60" 70" 80"

No"treatment"

0"

50"

100"

150"

0" 10" 20" 30" 40" 50" 60" 70" 80"

Vaccine"!

Days"a9er"vaccina;on"

0"

50"

100"

150"

0" 10" 20" 30" 40" 50" 60" 70" 80"

NKTR?214"

0"

50"

100"

150"

0" 10" 20" 30" 40" 50" 60" 70" 80"

Aldesleukin"

0"

50"

100"

150"

0" 10" 20" 30" 40" 50" 60" 70" 80"

Vaccine""+""

Aldesleukin"

0"

50"

100"

150"

0" 10" 20" 30" 40" 50" 60" 70" 80"

Vaccine""+"

"NKTR?214"Tumor"size"(mm

2 )"

Tumor size

0

20

40

60

80

100

No#treatment

Vaccine#+#Aldesleukin

Vaccine#+#NKTR7214

Vaccine

45±8.8&%& 12±5.2&%& 6±3.3&%& 1.2±0.8&%&

No&treatment& Vaccine& Vaccine&&+&

Aldesleukin&

Vaccine&&+&

NKTRB214&

Foxp

3&

CD4&

Introduction

Objectives

NKTR-214 improves anti-tumor efficacy of peptide vaccines

NKTR-214 impacts the localization of Pmel-1 cells and Tregs between tumor and spleen

Tumor Spleen

Conclusions

References

Pmel-1 CD8+ T cells CD25+ Foxp3+ Tregs Pmel-1/Tregs Ratio

Pmel%1/Treg+ra-o

+(tum

or)+

0

1000

2000

3000

4000 **

0

200000

400000

600000

****

**

**

Numbe

r'of'P

mel,1'CD8

+'T'cells/'g

ram'of'tum

or'

10.5±2.3%) 11.4±1.2)%) 12.2±3.3)%) 11±2.4)%)

No)treatment) Vaccine) Vaccine))+)

Aldesleukin)

Vaccine))+)

NKTR@214)

Foxp

3)

CD4)

0

500000

1000000

1500000

2000000

***

No#treatment

Vaccine#+#Aldesleukin

Vaccine#+#NKTR7214

Vaccine

Num

ber'o

f'Pmel,1/'Spleen

'

0

20000

40000

60000

80000

100000

*ns

Num

ber'o

f'Tregs'/'Spleen'

0

50

100

150

***

Pmel%1/Treg+ra-o

+(Spleen)+

Tregs (% of CD4+ T cells)

Pmel-1 (% of CD8+ T cells)

3.4±2.2%' 72.3±5.2'%' 91.1±3.5'%' 95.2±3.5'%'

No'treatment' Vaccine' Vaccine''+'

Aldesleukin'

Vaccine''+'

NKTRA214'

Thy1.1

'

CD8'

Thy1.1

&

0.1±0.8%& 34.3±4.2&%& 65.8±3.5&%& 75.2±3.5&%&

No&treatment& Vaccine& Vaccine&&+&

Aldesleukin&

Vaccine&&+&

NKTRE214&

CD8&

transfer of TCR transgenic pmel-1 CD8+ T cells

D0#

Gp100#pep(de#TLR-7#agonist#An(-#CD40#Pmel-1#NKTR-214#

T#cell#response#:#Tumor/spleen#

D-6#

Tumor#Induc(on##300,000#B16#wild#type#cells#

D7#

1) Daud AI, Wolchok JD, Robert C, et al. J Clin Oncol. [Epub ahead of print] DOI:10.1200/JCO.2016.67.2477 2) Daud AI, Loo K, Pauli ML, et al. J Clin Invest. 2016;126(9):3447-52 3) Charych DH, Hoch U, Langowski JL, et al. Clin Cancer Res. 2016;22(3):680-90. 0 10 20 30 40 50 60 70

0

20

40

60

80

100Untreated

NKTR,21400Aldesleukin0

0Vaccine0Vaccine0+0AldesleukinVaccine0+0NKTR,2140 ***

***< 0.0001

< 0.0001

Days.a/er.vaccina6on

Thy1

.1 +

Pm

el-1

T c

ells

(% o

f CD

8+ T

cel

ls)

0 10 20 30 40 50 60 700

20

40

60

80

100Untreated

NKTR,21400Aldesleukin0

0Vaccine0Vaccine0+0AldesleukinVaccine0+0NKTR,2140 *

*p=0.03

p=0.01

Days.a/er.vaccina6on

CD

25+

Foxp

3+ T

regs

(% o

f CD

4+ T

cel

ls)

0

1000

2000

3000

4000

*

*ns

**

*Num

ber'o

f'Tregs/'gram

'of'tum

or'