nktr-214, an engineered il-2, selectively depletes ... · nktr-214, an engineered il-2, selectively...
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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.
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