NKTR-214, an engineered IL-2, selectively depletes intratumoral Tregs and expands immunotherapy-induced effector T cell responsesMeenu Sharma1, Hiep Khong1,2, Faisal Fa’ak1, Brent C Chesson1, Barbara M Pazdrak1, Laura Maria S Kahn1, Louise Janssen1, Uddalak Bharadwaj4, Binisha Karki1, Zhilan Xiao1, Yared Hailemichael1,

Manisha Singh1, David Tweardy4, Salah Eddine Bentebibel1, Cara Haymaker1,Chantale Bernatchez1, Adi Diab1, Jonathan Zalevsky3, Ute Hoch3, Willem W. Overwijk1,2

1Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 770302The University of Texas MD Anderson Cancer Center UThealth Graduate School of Biomedical Sciences, Houston, TX 77034

3Nektar Therapeutics, 455 Mission Bay Blvd South, San Francisco, CA 94158 

4Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, TX, 77054, USA.

High dose IL-2 has been used in treatment of metastatic melanoma and renal cell carcinoma. However, physiologic toxicities associated with IL-2 treatment, limited its use in anti-cancer therapies. 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 Tregs.

•  To assess the therapeutic synergy of NKTR-214 with CTLA-4 and PD-1-based checkpoint blockade therapy or with peptide-vaccination in CT26 colon carcinoma and B16 melanoma models. •  To investigate impact of treatment on proliferation and apoptosis of effector CD8+ T cells and immunosuppressive CD4+Foxp3+ Tregs, as well as effector cytokines and

chemokines in tumor and peripheral tissues. •  To identify mechanisms by which NKTR-214 mediates depletion of intratumoral Tregs while preserving them in periphery.

Introduction

Objectives

Results

Untreatedα-PD-1 α-CTLA-4NKTR-214

NKTR-214 + α-PD-1 + α-CTLA-4

α-PD-1 + α-CTLA-4

****α-CTLA-4

Untreated

α-PD-1

NKTR-214 + α-PD-1

NKTR-214 + α-CTLA-4

NKTR-214

***

**

*ns

D-4

D0

D4

D7

D11

D13 D17

D18

D5 α-PD-1 or α-CTLA-4

NKTR-214

CT26

D-7

D0

D4

D7 D11

D13 D17 D18

D5 α-PD-1 + α-CTLA-4

NKTR-214

CT26

0 35 700

50

100

Days after tumor induction

Per

cent

sur

viva

l

α-CTLA-4

Untreated

α-PD-1

NKTR-214 + α-PD-1

NKTR-214 + α-CTLA-4

NKTR-214

***

**

*ns

0 35 700

50

100

Days after tumor induction

Per

cent

sur

viva

l

NotreatmentNKTR-214Aldesleukin

Vaccine+AldesleukinVaccine+NKTR-214

Vaccine **

0 20 40 600

20

40

60

80

100

Days post tumor challenge

Per

cent

sur

viva

l

UntreatedNKTR-214AH1 VaccineAH1 Vaccine + NKTR

UntreatedNKTR-214AH1 VaccineAH1 Vaccine + NKTR

D-7 D0 D8 D16 D32

NKTR-214 Aldesleukin

Vaccination B16.F10

0 50 1000

20

40

60

80

100

Daysa&ervaccina-on

Perc

ent s

urvi

val

NotreatmentNKTR-214Aldesleukin

Vaccine+AldesleukinVaccine+NKTR-214

Vaccine **

D-4 D4

Vaccine NKTR-214

CT26

NKTR-214 (given every 8 days until experimental endpoint)

D12

NKTR-214 synergizes with checkpoint blockade

NKTR-214 synergizes with anti-cancer vaccines

NKTR-214 enhances proliferation and survival of CD8+ Teff while depleting intratumoral Tregs

Tumor

Spleen

Thy1.1+ CD8+ Teff Tregs CD4+ Teff

2 4 6

** ***

Unt

Vacc

Vacc + Aldesleukin

Vacc + NKTR

Abs no. of cells X105

(per g of tumor )

80

5

* ***

Unt

Vacc

Vacc + Aldesleukin

Vacc + NKTR

Abs no. of cells X105

(per spleen )

0 10 15 20 25

****

*

Unt

Vacc

Vacc + Aldesleukin

Vacc + NKTR

Abs no. of cells X103

(per g of tumor )

6420

****

ns

Unt

Vacc

Vacc + Aldesleukin

Vacc + NKTR

Abs no. of cells X103

(per g of tumor )

201050 15

nsns

ns

Unt

Vacc

Vacc + Aldesleukin

Vacc + NKTR

Abs no. of cells X105

(per spleen )

6420 82

nsns

ns

Unt

Vacc

Vacc + Aldesleukin

Vacc + NKTR

Abs no. of cells X104

(per spleen )

0 4 6 8 10

% Ki67+ Cells % Annexin V+ Cells

68±9% 24±6%

IL-2

IL-2 +

IFN-γ + TNF-α

**

Ki6

7

Foxp3

IL-2IL-2 + IFN-γ + TNF-α

0

50

100

% A

nnex

in+

Aqu

a+ ce

lls

****

- +012345

Treg

s (%

of C

D3)

NKTR-214

**

- +02468

10

Treg

s (%

of C

D3)

NKTR-214

p= 0.18CT26 (Tumor)

- +0

25

50

Treg

s (%

of C

D3)

NKTR-214

**B16 (Tumor)

- +0

250

500

IFN

-γ(R

NA

coun

ts)

NKTR-214

*

- +0

250

500

TNF-α(R

NA

coun

ts )

NKTR-214

*

- +0

20000

40000

IFN-γ(ng/ml)

NKTR-214

****

- +0

400

800

TNF-α(ng/ml)

NKTR-214

****

IFN-γ20,000

TNF-α

300

600

CCL-19

CCL19

CCL22

CXCL2

CXCL5

CCL3 / MIP-1α

CCL4 / MIP-1β

CCL11 / Eotaxin

CCL12 / MCP5

CCL17 / TARC

CXCL10 / IP-10

CXCL13 / BCA-1

20,000

40,000

60,000

80,000

200,000

400,000CXCL9 / MIG

CCL2 / MCP1

CCL5 / RANTES

Granzyme 50,000100,000

Tumor Spleen

Unt

reat

ed

Vacc

ine

Unt

reat

ed

Vacc

ine

Vacc

ine

+ N

KTR

Vacc

ine

+ N

KTR

Similar trends observed in human and mice post NKTR-214 treatment

Conclusions

020406080

100

Thy1.1+ CD8 Teff cellsTregs

ND

Unt Vacc Vacc+

NKTR-214

Vacc+

Aldesleukin

ns

****

***

*

020406080

100

ND

Unt Vacc Vacc+

NKTR-214

Vacc+

Aldesleukin

ns

****

***

*ns

ns

Thy1.1+ CD8 Teff cellsTregs

020406080

100

Thy1.1+ CD8 T cellsTregs

ND

Unt Vacc Vacc+

NKTR-214

Vacc+

Aldesleukin

ns ns

****

nsns

ns

020406080

100

Thy1.1+ CD8 T cellsTregs

Unt Vacc Vacc+

NKTR-214

Vacc+

Aldesleukin

ns ns

****

*****

ns

**

NKTR-214 treatment induced cytokines and chemokines

B16 (Tumor) B16 (Tumor) Human (Tumor)

Human (Tumor)

NKTR-214 -induced CD8+Teff-derived IFN-γ and TNF-α mediate selective depletion of intratumoral Tregs

BALB/C mice bearing 4 or 7 days old, palpable s.c. CT26 tumors left untreated or received anti-PD-1 or anti-CTLA-4 or combination of checkpoint blockade antibodies (i.p. on indicated days) with or without NKTR-214 (i.p. on indiacted days) given until the experimental endpoint. Kaplan-Meier survival curves, each with 5 to 10 mice per group. *P < 0.05, **P < 0.01, log-rank test.

C57BL/6 mice bearing 7-days old, s.c. B16 tumors received gp100-specific Thy1.1+ pmel-1 Teff cells i.v. Mice were either left untreated or received vaccine (hgp100/L-tyr and anti-CD40 given on left and right flank, s.c. with TLR-7 agonist applied topically). Untreated or vaccinated mice received either aldesleukin or NKTR-214 on indicated days.Kaplan-Meier survival curves, each with 5 mice per group. *P < 0.05, **P < 0.01, log-rank test.

BALB/C mice bearing 4 days old CT26 colon carcinoma s.c. tumors received either no treatment or vaccine (Ah1 peptide and anti-CD40 given s.c. on both the flanks with TLR-7 agonist applied topically) with or without NKTR-214. Kaplan-Meier survival curves, each with 5 mice per group, **P < 0.01, log-rank test.

C57BL/6 mice bearing 7-days old, s.c. B16 tumors received gp100-specific Thy1.1+ pmel-1 Teffs i.v.. Mice were either left untreated or received vaccine (hgp100/L-tyr and anti-CD40 given on left and right flank, s.c. and TLR-7 agonist applied topically). Mice were either left vaccinated or further received aldesleukin or NKTR-214. Tumors and spleen harvested on day 7 post treatment and levels of Thy1.1+ CD8+ Teff cells, CD4+ Teff cells and Tregs were analyzed by flow cytometry. (A) Absolute numbers of Thy1.1+ CD8+ Teff cells (left panel), Tregs (middle panel) and CD4+ Teff (right panel) in tumor (upper panel) and (B) spleen (lower panel) were analyzed on day 7 post treatment. Histograms showing (C) Ki67, (D) annexin V and viability dye aqua expression on tumor and spleen-infiltrating Thy1.1+ CD8+ Teff and Tregs on day 7 after treatment. Data represented as mean± SEM, (n=4-5,*P<0.05, **P<0.01, ****P<0.0001 unpaired t-test).

(A)

(B)

(C) (D)

Heatmap depicting level of various cytokines and chemokines analyzed with multiplex luminex assay on day 7 post treatment in tumor and spleen of indicated treatment groups.

C57BL/6 mice bearing 7-days old, s.c. B16 tumors received gp100-specific Thy1.1+ pmel-1 Teffs i.v. Mice were either left untreated or received vaccine or received vaccine with NKTR-214 with or without in vivo neutralizing antibodies against IFN-g and TNF-a given on day -1, day 2, day 4 and day 6. Proportions of Tregs in tumor (upper panel) and spleen (lower panel)

Tumor Unt NKTR Vacc Vacc+NKTR Vacc+NKTR

(α-IFN-γ+ α-TNF-α) 72±8 77±16 43±15 5±1 61±14

Foxp

3

CD4

6±2 7±3 5±1 7±1 8±1

Spleen

In vivo In vitro

Tregs (CD4+ CD25hi) were obtained from splenocytes of wild type C57BL/6 mice and cultured for 8 days in presence of anti-CD3/anti-CD28 beads and IL-2 with or without IFN-γ and TNF-α. (A) Expression of Ki67 and (B) expression of annexin V and viability dye aqua are shown, were analyzed on cultured Tregs on day 9. Data (mean±SEM) is representative of at least three-four independent experiments (**<0.01, ****P<0.0001, ns= non significant, unpaired t-test).

**

(A) Level of IFN-γ and TNF-α analyzed by luminex and(B) percentage of Tregs analyzed by flow cytometry, in tumors of untreated or vaccine plus NKTR-214 treated mice (of indicated tumor model). Analysis was done on day 7 post treatment. Data represented as mean±SEM (****P>0.001, unpaired t-test).

(C) level of IFN-γ and TNF-α analyzed by nanostring and (D) Proportions of Tregs (n=5, mean±SEM) analyzed by flow cytometry, in tumor of melanoma and renal cell carcinoma patients, pre (-) and on week 3 post NKTR-214 treatment (+) (*P>0.05, ns= non-significant, paired t-test).

(A)

(B)

(A)

(B)

(C)

(D)

NKTR-214 synergizes with checkpoint blockade as well as with vaccination to improve the survival, proliferation and tumor infiltration of effector CD8+ T cells while promoting selective intratumoral depletion of Tregs to establish effective anti-tumor immunity.