Shp1 Loss Drives Robust Anti-Tumor Immunity and Enhances Macrophage Effector Function

Darienne MyersRevolution MedicinesRedwood City, CA

© 2019 Revolution Medicines Poster 3022 on Wednesday

Disclosure

• I am a full-time employee of Revolution Medicines

2

Shp1 is an Exciting IO Target with Pleiotropic Effects in Innate and Adaptive Immune Cells

• Why target Shp1?– Phosphatase that transduces signals from inhibitory receptors in multiple immune cell types– Mice with mutation in Shp1 (“motheaten mice”) develop autoimmune/inflammatory disease– Literature suggests attenuation of Shp1 activity could promote anti-tumor immunity

3

Proinflammatory cytokines,M1 polarization

Macrophages

Shp1

DON’T EAT ME

XEAT ME

Dendritic Cells (DCs)

SHP1

MHCPeptide

Shp1X

Antigen uptake and presentation,T cell stimulation

T Cells

SHP1

TCR

Shp1X

CD8+ T cell proliferation, killing, perforin production

J Exp Med. 2013 June 24;210(7):1419-1431J Immunol. 2012 July 13;189:1812-1825

J Leuk Biol. 2017. 102(3)657-675Immunity. 2011 March 25;34:385-395

J Immunol. 2011;186(7):3934-3945

Generation of a Novel Mouse Model for Global Inducible Deletion of Shp1

• No known selective inhibitor of Shp1 available à requires a genetic tool• Evaluate tumor growth in an adult animal

– Motheaten mice (“me” mutation) die within 2-3 weeks of birth• Analyze the integrated effect of Shp1 loss in all immune cell types

4Figure adapted from: J Leuk Biol 2017. 102(3):657-675

Cross to Cre-ERT2: allows for inducible deletion of Shp1 upon tamoxifen administration

Shp1fl/fl

*Collaboration between Revolution Medicines and Clifford Lowell, Clare Abram at UCSF

Time-course of Shp1 Deletion in Mouse Model is Amenable to Modeling Effects on Tumor Growth

• Tamoxifen dose regimen achieves ~70-80% Shp1 deletion (peripheral blood)

• MC38 tumors implanted coincident with detection of Shp1 deletion

5

Days: (relative to tumor implant)

Shp

1/E

rk2

(% o

f con

trol)

-7 0 7 14 21 28 350

255075

100125

Shp1fl/fl

Shp1fl/fl Cre-ERT2

Implant tumors, monitor growth

Tamoxifen(to KO Shp1)

Shp1fl/fl

Shp1fl/fl Cre-ERT2

Shp1

Erk

Shp1fl/flShp1fl/fl

Cre-ERT2

Day 13 post-implant

Mean +/- SD; n=4

Global Shp1 Deletion Delays Tumor Growth in a Model Resistant to Checkpoint Blockade

• Marked tumor growth inhibition of MC38 in mice lacking Shp1– MC38 line is insensitive to checkpoint blockade (anti-PD1)

6n=10/group; mean +/- SEM, ** p<0.01, unpaired t test on each\ time point; / indicates a mouse that left study, not included in average TV

0 10 20 300

1000

2000

3000

Days post-implant

Tum

or V

olum

e (m

m3 ) Shp1 WT

Shp1 KO

**

Tum

or V

olum

e (m

m3 )

0 10 20 300

1000

2000

3000

4000

Days post-implant

Tum

or V

olum

e (m

m3 )

0 10 20 300

1000

2000

3000

4000

Days post-implantTu

mor

Vol

ume

(mm

3 )

Days post-implant

Shp1 WTShp1fl/fl

Shp1 KOShp1fl/fl; Cre-ERT2

0 10 20 30 400

1000

2000

3000

4000

Days post-implant

Tum

or V

olum

e (m

m3 ) Isotype control

0 10 20 30 400

1000

2000

3000

4000

Days post-implant

Tum

or V

olum

e (m

m3 ) Anti-PD1

n=15/group

Analysis of tumor immune microenvironment

Shp1 Loss Promotes a Proinflammatory Tumor Immune Microenvironment

• No major differences in immune cell infiltrate into MC38 tumors

– Trend towards increased frequency of myeloid cells and M1 mf in tumors from Shp1 KO mice

• Altered immune cell functionality as opposed to composition of tumor microenvironment?

– Increased proinflammatory cytokines in tumor lysates

7n=6-10; *p<0.05, **** p<0.0001

Tumor Immunophenotyping

WT (Shp1fl/fl) KO (Shp1fl/fl Cre-ERT2)

Total=100

NK cellsgd T cellsCD8+CD4+ TeffTreggMDSCsmMDSCsM1 macsM2 macstumor DCsother

Total=100

NK cellsgd T cellsCD8+CD4+ TeffTreggMDSCsmMDSCsM1 macsM2 macstumor DCsother

n=4-5/group

IFNg IL12p70 IL1bWT KO WT KO WT KO

0

25

50

75

Cyt

okin

e (p

g/m

g) *

****

*

Proinflammatory Cytokines

• Proposed to bind to and transduce signals downstream of SIRPa that inhibit phagocytosis– Biochemical assay: activation of Shp1 by tandem-phosphorylated SIRPa peptide– Hypothesis: Shp1 loss would enhance macrophage phagocytosis of cancer cells

1 10 100

1000

0

10

20

30

40

50

[peptide] (nM)

DiF

MU

P h

ydro

lysi

s(m

RFU

/s/n

M e

nzym

e)

[SIRPa1426-459 diP peptide] (nM)

Phos

phat

ase

activ

ity

(DiF

MU

Phy

drol

ysis

)

Shp1 is the Putative Transducer of the “Don’t Eat Me” Signal Downstream of CD47-SIRPa

Anti-CD47 / Anti-SIRPa

*8 agents targeting this axis currently in clinical trials

TAA=Tumor Associated Antigen

EAT ME

Macrophage

Cancer cell

DON’T EAT ME

Inhibits phagocytosis

Promotesphagocytosis

Shp1

Opsonization

Shp1P P

Active

Shp1

Auto-inhibited

8

Shp1-deficient Mouse and Human Macrophages Exhibit Increased Phagocytosis

• We generated Shp1-deficient mouse (Shp1fl/fl Cx3cr1-Cre) macrophages• Similar results observed for human peripheral blood monocyte-derived macrophages (siRNA)

• Shp1-deficient macrophages exhibited increased phagocytosis of target cells relative to WT

9

In vitro Phagocytosis Assay with Mouse Bone Marrow-Derived Macrophages

a-CD47Isotype Control

Macrophage

Targ

et

Method Validation

Shp1 KO = 70-75% reduction in Shp1 protein; n=2 mice/group; mean +/- SEM; unpaired t test, *p<0.05Phagocytic Index (PI) = % of total macrophages that phagocytosed target cells

Spleen Tumor0

50

100

150

Shp1

/Erk

2 (%

of c

ontro

l) WT

KO

Raji Target Cells(Isotype Control)

WT KO0

5

10

15

20

Phag

ocyt

ic In

dex *

DLD1 Target Cells(Isotype Control)

Phag

ocyt

ic In

dex

(PI)

WT KO0

5

10

15

20

Phag

ocyt

ic In

dex

*

Shp1 Knockout-mediated Increase in Phagocytosis is Comparable to Blockade of “Don’t Eat Me” via CD47/SIRPa

• Effect of Shp1 KO is comparable to blocking the “don’t eat me” signal with anti-CD47• Raji target cells; similar results observed with DLD1 target cells (data not shown)

• Combining Shp1 loss with an opsonizing agent drives higher level of phagocytosis

10

Isotyp

e Con

trol

Anti-C

D47

Anti-C

D47 +

Fc bloc

k

Rituxim

ab0

10

20

30

40

Phag

ocyt

ic In

dex WT

KO

*

*

ns

**

*

ns

Isotyp

e Con

trol

a-CD47a-

CD47+ F

c bloc

kRitu

ximab

Phag

ocyt

ic In

dex

(PI)

Mean +/- SEM, *p<0.05, **p<0.01

EAT ME

Macrophage

Cancer cell

Shp1

DON’T EAT ME

Opsonization(Fc portion of a-CD47)

CD47

CD47

Anti-CD47

Model: Shp1 Loss Promotes Anti-Tumor Immunity via Multiple Immune Cell Types

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SHP1X

Phagocytosis à Release of Tumor Associated Antigens (TAAs)

TAAs are Processed and Presented by DCs

Dendritic Cell (DC)

Production of IL

-12

Antigen presentation

Activation of T

cells

in the TME

IFNg production

Impact on additional T cell effector functions?

Macrophage

T CellShp1X

TCR

MHC TAA

Pro-inflammatory cytokine release(IL1b)

X

Poster 3022 on Wednesday

Acknowledgements

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Revolution Medicines

Elsa Quintana

Amira Belwafa

Chris Schulze

Tiffany Choy

Tram Nguyen

Pete Wildes

Mallika Singh

Rich Hansen

Mark Goldsmith

Jacqueline Smith

University of California, San Francisco

Clifford Lowell

Clare Abram

Alia Welsh

Poster 3022 on Wednesday