Shp1 Loss Drives Robust Anti- Tumor Immunity and Enhances ...
Transcript of Shp1 Loss Drives Robust Anti- Tumor Immunity and Enhances ...
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
11
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
12
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