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

+)(-)

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

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+)

(-)

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

0 5 10 15 20 250

5

10

15

20

Days

%Te

tram

er+

(CD

8+) (-)

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

Tetramer+ CD8 T CellsCD8 T Cells

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

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+)

(-)

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

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

+)

(-)

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

UntreatedIL-2CIL-15C

NK Cells Elevated Following Treatment With Either Complexes

0 5 10 15 20 250

2

4

6

8

10

Days

%N

K (C

D45

+)

(-)

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

NK Cells

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

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