Efficacy and limitations of cytokine complex therapy on ...

Efficacy and limitations of cytokine complex therapy on pancreatic ductal adenocarcinoma

Taylor MesojednikMS2, UMN Medical School

Stromnes Lab

Pancreatic Ductal Adenocarcinoma (PDA)

• Most common form of pancreatic cancer

• 3rd leading cause of cancer-related mortality

• Resistant to chemotherapy• Tumor microenvironment

interferes with lymphocyte infiltration and activity

• Resistant to immune monotherapies

• Combinatorial approaches may enhance efficacy

T cellsTumor cells

MacrophagesPD-L1

Nucleus

Stromnes et. al., CIR, 2017

Cytokine Complexes• Cytokine bound to natural

receptor or a specific antibody

• Enhanced half-life in vivo• Expand CD8 T Cells and NK

Cells• IL-2 complexed with S4B6

mAb does not bind CD25• Reduces Treg expansion

during cytokine complex therapy

• IL-2C and IL-15C have demonstrated anti-tumor activity in murine melanoma models

(CD122) (CD132)

(CD25)“IL-2C”

“IL-15C”

Image Credit: Kristina Burrack, PhD – Adapted from: Waldmann, 2006, Nat Rev Immunol., 6:595-601. Votavova et al., 2014, Immunol Letters, 159:1-10.

Specific Aim

Determine the effect of IL-2 Complex (IL-2C) or IL-15 Complex (IL-15C) treatment on survival in a murine model of pancreatic cancer.

• Hypothesis: Treatment with cytokine complexes will expand effector lymphocytes and decrease tumor size and prolong survival in treated mice.

Cytokine Complex Experimental Design

0 6 7 9 10 1421

SurvivalIVISIVISIVIS5 x 104

KPC2a cells into B6-IL10-eGFP

pancreas

IL-2CIL-15C

PBMC Isolation

• Treatment Groups: Untreated (n=3), IL-2C (n=3), IL-15C (n=3)• IL-2C = IL-2C: IL-2/IL-2Ab (S4B6, Bio X Cell)• IL-15C = IL-15:IL-15Ra-Fc (R&D Systems)

Cytokine Complex Treatment Reduces Tumor SizeUn

treat

edIL

-2/IL

-2R

IL-1

5/IL

-15R

7 14 21 28 35 42x107

6.0

4.0

2.0

Radiance (p/sec/cm3/sr) Color Scale Min= 5.5e6 Max= 6.6e7

0 7 14 21 28 35 42 49105

106

107

108

109

Days

Rad

ianc

e

UntreatedIL-2:IL-2RIL-15:IL-15R

0 5 10 15 20 250

2

4

6

8

10

Days

%N

K (C

D45

+)(-)

IL-2:IL-2RIL-15:IL-15R

UntreatedIL-2CIL-15C

• Significant difference in radiance between untreated mice and IL-2C (p<0.05) and IL-15C (p<0.01)

Cytokine Complex Treatment Prolongs Mouse Survival

0 7 14 21 28 35 42 490

20

40

60

80

100

Days

Survival

0 5 10 15 20 250

2

4

6

8

10

Days

%N

K (C

D45

+)(-)


Untreated (n=3)IL-2C (n=3)IL-15C (n=3)

• Endpoint: Radiance > 1x108

• Significant survival difference

between both cytokine

complexes and untreated

animals (p<0.05)

• Experiment is ongoing

• IL-2C n=1

• IL-15C n=2

PBMC Flow Gating Strategy

Lymphocytes Single Cells Live, CD45+

CD8 x NK1.1 CD8 x CD4CD44 x Tetramer CD25

CD8

CD8

CD44

NK1 CD4 CD25Tetramer(Through CD8+) (Through CD4+)

CD45

L/DFS

CHFSCW

SSCA

FSCA

Representative gating for PBMCs isolated from a day 10 cheek bleed

Circulating CD8 T Cells Elevated Following IL-2C Treatment

0 5 10 15 20 250

10

20

30

Days

%C

D8

(CD

45+)

(-)


0 5 10 15 20 250

5

10

15

20

Days

%Te

tram

er+

(CD

8+) (-)


Tetramer+ CD8 T CellsCD8 T Cells

0 5 10 15 20 250

2

4

6

8

10

Days

%N

K (C

D45

+)

(-)



Circulating CD4 T Cells Elevated Following IL-15C Treatment

0 5 10 15 20 250

5

10

15

20

25

Days

%C

D4

(CD

45+)

(-)


0 5 10 15 20 250

20

40

60

80

100

Days

%C

D25

+ (C

D4+

) (-)

IL-2:IL-2R

IL-15:IL-15R

The CD25 and PD-1 staining on T cells from day 10 from experiment 1is really surprising, almost like an artifact because in some samples 90% of cells are PD-1+….so did something happen on the FACs collection that day?

We should just interpret it with caution because saw this in untreated mice and have never seen that before

CD25+ CD4 T CellsCD4 T Cells

0 5 10 15 20 250

2

4

6

8

10

Days

%N

K (C

D45

+)

(-)



NK Cells Elevated Following Treatment With Either Complexes

0 5 10 15 20 250

2

4

6

8

10

Days

%N

K (C

D45

+)

(-)


NK Cells

0 5 10 15 20 250

2

4

6

8

10

Days

%N

K (C

D45

+)

(-)



Spleen TumorTetramer+ CD8 T Cells NK Cells

LAG3

IL-10-eGFP

PD-1

Spleen Tumor

LAG3

IL-10-eGFPPD

-1

PD1, LAG3, and IL-10 elevated on Tumor-Specific CD8+ T cells in Untreated Tumor vs Spleen at Endpoint

% of Tetramer+ CD8 T Cells % of NK Cells

PD1+

LAG3+

IL-1

0+

NKG2A+

0

20

40

60

80

% of NK Cells

Spleen

Tumor

PD1+

LAG3+

IL-1

0+

NKG2A+

0

20

40

60

80

100

% of CD8+, Tetramer+ T Cells

Spleen

Tumor

PD-1 LAG3 IL-10 NKG2A PD-1 LAG3 IL-10 NKG2A

PD1+

LAG3+

IL-1

0+

NKG2A+

0

20

40

60

80

100

% of CD8+, Tetramer+ T Cells

Spleen

Tumor

PD1, LAG3, and IL-10 elevated in Tumor Specific CD8s in Untreated Tumor vs Spleen at Endpoint

Conclusions and Future DirectionsConclusions:• IL-2C and IL-15C therapy both prolonged survival and reduced tumor size in a

murine model of PDA• IL-15C appears to have the most pronounced antitumor activity • Tumor recurrence despite therapy supports the need for a combinatorial

treatment strategy or for additional treatments• A higher proportion of intratumoral lymphocytes expressed PD-1, LAG3 and IL-

10 compared to spleen suggesting that the tumor microenvironment is inducing a suppressive phenotype in infiltrating effector lymphocytes as part of its immune evasion

Future:• Examine the phenotype and function of host immune cells immediately

following cytokine complex treatment (day 14)• Test safety and efficacy of cytokine complex therapy in combination with

engineered T Cell therapy and other immunotherapies• Test the impact of prolonged cytokine complex treatment

Acknowledgements

Stromnes LabIngunn Stromnes

Adam BurrackJackson RaynorMeagan Rollins

U of MSara Hamilton

Kristina BurrackJeffrey Miller

NIH T35 Research Program in Infection & Immunity

Daniel MuellerStephanie Krischuk

Efficacy and limitations of cytokine complex therapy on ...

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