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Breaking Immune Tolerance by Targeting Key Immunosuppressive Pathways in the Tumor
Microenvironment
Rolf A. Brekken, PhD
Key Messages
Phosphatidylserine (PS) is exposed on the external surface of cells and vesicles in the tumor microenvironment
PS exposure is immunosuppressive
PS signaling is an immune check point that is upstream of PD-1/L1, CTLA-4 and IL-2 signaling
Antibody-mediated inhibition of PS results in immune activation and durable anti-tumor responses
3RAB
Disclosures of Relevant Financial RelationshipsI have the following financial relationships to disclose:• Consultant: Peregrine Pharmaceuticals Inc., and Synergys Biotherapeutics• Grant/Research Support: BerGenBio AS, Imclone Systems/Eli Llly, Affitech AS,
and Peregrine Pharmaceuticals, Inc.• Stock/ Stock Options: Peregrine Pharmaceuticals, Inc. • Co-Founder: Tuevol Therapeutics, Inc
PC and SM
PS and PE
Exposure of Phosphatidylserine (PS) on the surface of cells
Tumor
Hypoxia / reoxygenationTumor cell metabolites
Cytokines
ROS
Normal
Tumor cell, endothelial cell,
exosome
Cell under normal
conditions
Normal dying cell
Program cell death
Brief history of PS targeting
Philip E. Thorpe, Ph.D.
VTA
circa late 90s
In the Thorpe lab we were interested in developing targeting agents that specifically homed to tumor vasculature. PS was a target we recognized.
3G4
Mouse IgG3
2aG4
Mouse IgG2a
Bavituximab
Chimerichuman IgG1
PGN635
Fullyhuman IgG1
PS targeting agentsDeveloped in the Thorpe lab and @ Peregrine
(1N11)
β2-Glycoprotein 1-dependent binding of PS targeting agents to exposed PSβ2-Glycoprotein 1 is an abundant serum protein; antibody induced dimerization of β2GP1 increases affinity for PS by >1000-fold
Monovalent Binding is Low Affinity (1 µM)
V
III
IV
I
II
III
IV
I
II
V
Complex Binding is High Affinity (1
nM)
β2-Glycoprotein 1
V
III
IV
I
II
I
II
III
IVV
bavitixumab
8
Tumor type Name Treatment Growth reduction
Prostate PC3 2aG4 + docetaxel 98%
LNCaP 2aG4 + docetaxel 95%
LNCaP 2aG4 + ADT 99%
Pancreas Pan02 3G4 + gemcitabine 95%
HCC PLC/PRF/5 2aG4 + sorafenib 98%
C3A 2aG4 + sorafenib 75%
Lung A549 2aG4 + RT Cure (50%)
H460 2aG4 + RT 99.9%
Rectal SW837 2aG4 + capecitabine + RT 98%
Glioma F98 2aG4 + RT 99.9% (15% cures)
Combination therapy in rodent tumor models
Antibody-mediated PS targeting mediates binding of macrophages and destruction of tumor endothelium
C1 day
A Control IgG
2 days
endothelium
monocytes
endothelial remnants
5 days
collagen IV
Macrophages
Tumor-associated macrophages (TAMs)
M1ImmunosuppressivePro-angiogenicMaintain T-regsDo not perform ADCC
ImmunostimulatoryPro-inflammatoryTumoricidalPerform ADCC
IL-10TGF-β
M2
Arg-1+
FcγR FcγR
FcγR
IL-1, IL-12, TNF-α
NOSNO
PREDOMINANT SCARCE
F4/80 Arg-1 iNOS Merge2a
G4
C44
M1
TAM
s (%
are
a)
2aG4C44
5
3
1
0
4
2
M2
TAM
s (%
are
a)
2aG4C44
5
3
1
0
4
2M
1:M
2 ra
tio
2aG4C44
10
6
2
0
8
4
PS blockade increases M1 to M2 ratio of TAMs
Yi Y, et al CIR 2013
Rela
tive
mRN
A
iNOS
TNF-α IL-12
Ccl5
Cxcl11
CD80CD86
CD40
MHC IIArg-
1
TGF-β IL-
10
CD206Ym
-1Fiz
z-1Ccl2
2Ccl1
7
VEGF-A
VEGF-B
0.1
1
10
M1 markers
M2 markers
2aG4 Drives TAMs towards M1 in vivo
Yi Y, et al CIR 2013
C44
2aG4
CD11b /Gr1 /DAPiMDSCs
0
2
4
6
C44 2aG4
MD
SC
s (%
are
a)
PS blockade reduces tumor-infiltrating myeloid-derived suppressor cells (MDSCs)
100 101 102 103 104100
101
102
103
104
72.9
CD
11b
2aG4
100 101 102 103 104100
101
102
103
104
22.2
Gr1
C44
5 tumors pooled
Note: Similar data from blood and spleen
Yi Y, et al CIR 2013
20
60
45
30
15
0
% to
tal c
ells
MDSCs Macrophages DCs
60
45
30
15
02aG4C44
40
30
10
02aG4C442aG4C44
MDSCs : Gr-1+, CDIIb+ Macrophages: F4/80+ DCs: CD11b+, CDIIchi
MDSCs treated with 2aG4 differentiate into macrophages and DCs in vitro
Yi Y, et al CIR 2013
…… and the dendritic cells acquire maturation markers
100 101 102 103 104
025
6
100 101 102 103 104
025
6
100 101 102 103 104
025
6
100 101 102 103 104
025
6Even
ts
Bavi2aG4C44
CD86 MHC II
CD80 CD40
Yi Y, et al CIR 2013
PS blockade facilitates induction of tumor-specific cytotoxic T-cells
1. He et al, Antiphosphatidylserine Antibody Combined with Irradiation Damages Tumor Blood Vessels and Induces Tumor Immunity in a Rat Model of Glioblastoma. Clin Cancer Res 2009;15:6871-6880
Generation of antitumor T-cell immunity in syngeneic F98 glioma in rat2
Cytotoxic T-cell Generation2
Cytotoxic T-cells
Tumor-specific cytotoxicicity
Antigen presentation
Mature DC
MHCII CD40
CD80 CD86
T
Tumor blood vessel cell /Tumor cell /Microvesicle
T
T
Cure of transgenic model of prostate cancer (TRAMP) mice
Died of old age
0 100 200 300 400 500 600 7000
20
40
60
80
100
Age (days)
Pe
rce
nt s
urvi
val
Cx + C44 *
Cx + mch1N11 **
C44
mch1N11 *
N=23-30
Rx start @ week 16
1. AACR 2012 Annual Meeting: Yin et al, & Keystone 2014 Huang et al
mch1N11 = PS targeting agent
M2 Macrophage,MDSCsTumor blood Vessel/
Tumor Cells
Exposed PS in the tumor environment provides a fundamental upstream immunosuppressive signal
PS
PS receptors
PS
PS
PS
PS
• TGF-β• IL-10
Immature DCT cell
• M2 macrophages engage PS
through PS receptors • Signaling initiated
• Increased immunosuppressive
cytokines• Innate immunity is suppressed• M1 macrophages fail to
develop
• DCs fail to mature • Lack of antigen presentation to T-
cells• Impaired cytotoxic T-cell response
M1 Macrophage
iNOS+
1. PS Signaling 2. Immune System Signaling 3. Immune Suppression
XFcγ receptor
Normal Cell
No PS Exposed
PS Signaling Suppresses Immune Surveillance
Why is this important?1. Prevents auto-immunity2. Hallmark of cancer
Antibody-mediated PS blockade stimulates development of M1 macrophages and T-cell mediated anti-tumor effects
Tumor blood vessel cell /Tumor cell
PS
PS
PS
PS
PS
• IL-12• TNF-α
CD40 CD80
CD86MHCII
Mature DC Cytotoxic T-cell
Tumor-specific cytotoxicicity
Antigen presentation
Tumor blood vessel cell /Tumor cell
PS
PS
PS
M1 Mac
ADCC
• Effector cells engage bavi-coated PS
through Fcγ receptors
• Overrides upstream PS-mediated
immunosuppressive signal
• Inflammatory cytokines
• MDSC differentiation
• M2 to M1 macrophage polarization
• DC maturation
SOURCE: AACR 2013 Annual Meeting: Phosphatidylserine-targeting antibody reactivates tumor immunity and destroys tumor vasculature in miceYi Yin, Xianming Huang, Gustavo Barbero, Dan Ye, Philip E. Thorpe Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, TX.
Fcγ receptor
MDSCs, M2 macs,
Immature DCs
Y
PS receptor
Fcγ receptor
PS receptor
1. Bavituximab Treatment 2. Immune Signaling Changes 3. Immune Activation
• Tumor-specific cytotoxic
T-cells
• ADCC
Tumor blood vessel cell /Tumor cell
PS
PS
PS
PS
PS
Y Bavituximab Treatment
ImmunosuppressiveSignaling
Y = Bavituximab
Y
iNOS+
YY
Second-Line NSCLC Phase III Trial Initiated
N = ~ 600Stage IIIB/IVRandomized 1:1Placebo-Controlled
Bavituximab (3 mg/kg) Weekly
Docetaxel(75 mg/m2)
Day 1, of 21-day cycles up to 6
cycles
Primary Endpoint: OS
Secondary Endpoints:PFS, ORR, Safety
Docetaxel(75 mg/m2)
Day 1 of 21-day cycles for up to 6
cycles or until progression or
toxicities
PlaceboWeekly
• Initiated December 2013 • Single Phase III global registration trial• 582 target patient enrollment• >100 sites (U.S, E.U. and Asia Pacific)• Trial is adequately powered to support product
approval
• Patient Criteria:
• Non–squamous NSCLC• Only one prior systemic therapy for advanced
disease
• Unselected for genetic mutations
• Granted Fast Track Designation January 2014
23
MOA supports multiple opportunities with other immunotherapies
Upstream Immune
CheckpointInhibitor
Downstream Immune
Treatments
Bavituximab(PS-targeting
mAb)
Anti-PD-1
Anti-PD-L1
Anti-CTLA-4
IL-2
Vaccines
PAP-GMCSF
Key Messages - Summary
Phosphatidylserine (PS) is externalized in the tumor microenvironment and is a major immunosuppressive signal
PS is a global immune checkpoint
Antibody-mediated blockade of PS signaling breaks immune tolerance reactivating innate and adaptive immunity and results in durable anti-tumor responses in multiple pre-clinical models
Combination pre-clinical studies with other immune checkpoint inhibitors are underway and early phase clinical studies are planned
Acknowledgements and Collaborators
26
UT Southwestern Thorpe/Brekken Labs
http://www.utsouthwestern.edu/labs/brekken/
Philip E. Thorpe, Ph.D.
• Consistent, positive trend favoring 3mg/kg bavituximab arm:
• 60% improvement in median overall survival (11.7 months vs. 7.3 months)
• Positive safety database
Promising Phase II Second-Line NSCLC Data Supports Phase III Development
27
1
2
3
4
5
6
7
Release of cancer antigen
APC antigen presentation
Priming and activation
Effector phase Kill cancer cells
Recognition
Infiltrate into tumors
Traffic to tumors
Chen DS et al, Immunity 2013
Generation of antitumor immunity
Anti-CTLA-4
Anti-PD1/PD-L1
PS blockade repolarizes TAMs from M2 TO M1& induces nitric oxide production IN VITRO
Rela
tive
mRN
A
TNF-αIL-12
CD80CD86
MHC IIiN
OSYm
-1IL-
10Arg-
1
VEGF-A
CD206Fiz
z-1TG
F-β0.1
1
10
M1 markersM2 markers
%
2aG4C44
100
60
20
0
80
40
2aG4F(ab’)2
%
2aG4C44
100
60
20
0
80
40
2aG4F(ab’)2
NO
pro
ducti
on (µ
M)
2aG4C44
15
10
0
5
2aG4F(ab’)2
M2 M1 NO
Yi Y, et al CIR 2013
PS blockade (1N11) enhances the efficacy of anti-PD1 in K1735 melanoma model
Tu
mo
r V
olu
me
(mm
3)
PD-1
PD-1 +
1N11
0
200
400
600
800
P=0.0211
Days From Tumor Implant
Tu
mo
r V
olu
me
(m
m3)
10 20 30 40 50 60 700
500
1000
1500
PD-1
PD-1 + 1N11
PS blockade enhances the activity of anti-CTLA-4 in K1735 melanoma
ConfidentialDays Post Treatment Initiation
Pe
rce
nt
su
rviv
al
0 20 40 60 800
20
40
60
80
100
Control IgG
Ch1N11
Anti-CTLA4Ch1N11 + anti-CTLA-4
PPHM
AcknowledgmentsBrekken LabJason ToombsKatie LudwigRobin FrinkAshley BarrazaKristina AguileraMary TopalovskiNoah SorrelleTori BurtonMiao WangMoriah Hagopian
Sean DineenLaura SullivanAmanda Kirane
CollaboratorsJohn MinnaJoan SchillerDiego Castrilon Tom WilkieDavid GerberJoan Schiller
James Lorens – U BergenClinical collaborators
Peregrine PharmaceuticalsAffitechBerGenBio
Effie Marie Cain Scholarship in Angiogenesis Research, UTSW Department of Surgery, NCI/NIH, Simmons Cancer Center, NSF, Joan’s Legacy Foundation, Mary Kay Foundation, BerGenBio
Alan SchroitXianming HuangShuzhen LiLisa LiGustavo BarberoDan YeOlivier BelzileJanie IglehartKristi Lynn
Andrew (Tom) Ngo
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