1. No category

Expansion and Activation of T-cells via the
Targeting of the Immunosuppressive Ligand
Phosphatidylserine: Combination Strategy
with Other Checkpoint Inhibitors
Jeff T. Hutchins, PhD
Vice President, Pre-Clinical Research
Peregrine Pharmaceuticals, Inc.
March 25, 2015
Immune Checkpoint Inhibitors:
Validate Novel Pathways, Discover Predictive Biomarkers, Optimize Clinical Strategy
Combination Therapy is the Future
More Patients Responding to Therapy
Combination Treatment *
* Hypothetical ± for illustrative purposes only
Checkpoint Blockade
Yervoy® and Opdivo® are registered trademarks of Bristol Meyers Squibb.
Keytruda® is a registered trademark of Merck.
Targeted Therapy
Conventional Therapy
1.
Why do only a sub-set of patients respond to IO Therapy?
ƒ
Role of the immunosuppressive tumor microenvironment
ƒ
Lack of T-cell activation and myeloid cell differentiation
2. How can this patient pool be expanded?
ƒ
Ways a PS blockade changes this environment
ƒ
Consequences of PS targeting -- activation and expansion of T-cells
Immune Activity in the Tumor Environment
Tumor-Promoting Phenotype:
Tumor-Rejecting Phenotype:
Patients unable to benefit from checkpoint inhibitors
Patients more likely to respond to checkpoint inhibitors
MDSC
Myeloid-Derived Suppressor Cell
‡ Highly immunosuppressive
‡ Secrete anti-inflammatory cytokines
M2
Mac
M2 Macrophage
‡ Secrete anti-inflammatory cytokines
and growth factors
DC
Immature Dendritic Cell
‡ Incapable of antigen presentation
to T-cells
T-reg
TGF-ǃ
IL-10
T-Regulatory Cell
‡ Inhibits cytotoxic T-cells
Cytokines
‡ Inhibit anti-tumor immune responses
‡ Keep Immune cells in quiet state
MDSC
M1
Mac
Myeloid-Derived Suppressor Cell
‡ Reduction is current goal of
immunotherapy
M1 Macrophage
‡ Secrete inflammatory cytokines
‡ Kill tumor cells through ADCC
DC
Mature Dendritic Cell
‡ Present tumor antigens to T-cells
‡ Co-stimulatory signaling for T-cells
CD8
T-cell
Cytotoxic (CD-8+) T-cell
‡ Receptor specific tumor cell killing
‡ Increased PD-1 expression
Cytokines
‡ Stimulate anti-tumor immunity
TNF-Į
IL-12
Immune Suppression in the Tumor Environment
Creates a High Hurdle for Immunotherapies
MDSCs
M2 Macrophages
‡ Maintain an immunosuppressive tumor environment
TGF-ȕ
IL-10
‡ Lack of antigen presentation
‡ T-cells do not activate
‡ Low levels of PD-1 expression
Immature DC
Naïve T-cells
‡ Ineffective response to tumors
M1
Macrophage
MDSC
TGF-ȕ
!"
M2
Immature
DC
IL-10
Naïve
T cell
Poor
Response
to Tumor
Bavituximab:
Immune System Activation with Broad Applicability
Immune Signaling
Target
ƒ Monoclonal antibody targeting PS
Broad
Applicability
ƒ Activity in multiple cancer types and
infectious disease
Significant
Clinical
Experience
Immune
Activation MOA
ƒ 19 studies, 500+ patients treated with
bavituximab, currently in Phase III
ƒ Good safety profile alone and in combination
with other therapies (no IR safety events)
ƒ 5HPRYHVWKH³%UDNHV´
and
ƒ +LWVWKH³$FFHOHUDWRU´
PS Ligand Engages Multiple Receptors
BAI1
PS
PS
PS Expressing
Cells
Stabilin-2
TIM-1
PS
Tumor cell
Tumor microvesicle
Tumor blood vessel
PS
TIM-3
PS
TIM-4
Immune Cells
MDSC
M2 Macrophage
Immature DC
T-Cells
‡ Wu, Y., Tibrewal, N., and Birge, R.B. (2006).
Phosphatidylserine recognition by phagocytes: a view to
a kill. Trends Cell Biol. 16, 189±197Park et al, (2007).
‡ BAI1 is an engulfment receptor for apoptotic cells
upstream of the ELMO/Dock180/Rac module. Nature
450, 430±434.
‡ Hanayama, R., Tanaka, M., Miwa, K., and Nagata, S.
(2004). Expression of developmental endothelial locus-1
in a subset of macrophages for engulfment of apoptotic
cells. J. Immunol. 172, 3876±3882
‡ Park et al, (2007). Rapid cell corpse clearance by
stabilin-2, a membrane phosphatidylserine receptor. Cell
Death Differ 15, 192±201.
ĮȞȕ3 RUĮȞȕ5
PS
MFG-E8
PS
Tyro-3, Axl, Mer (TAM)
Gas6, Protein S
‡ Ishimoto Y, Ohashi K, Mizuno K, Nakano T. Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific
gene, gas6. J Biochem (Tokyo) 2000;127:411±7.
‡ Scott RS, McMahon EJ, Pop SM, et al. Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 2001;411:207-11.
‡ Miyanishi M, Tada K, Koike M, Uchiyama Y, Kitamura T, Nagata S. Identification of Tim4 as a phosphatidylserine receptor. Nature 2007;450:
435-9.
‡ Hanayama R, Miyasaka K, Nakaya M, Nagata S. MFG-E8-dependent clearance of apoptotic cells, and autoimmunity caused by its failure. Curr
Dir Autoim- mun 2006;9:162-72.
‡ Jinushi M, Nakazaki Y, Dougan M, Carrasco DR, Mihm M, Dranoff G. MFG-E8 mediated uptake of ap- optotic cells by APCs links the pro- and
antiinflammatory activities of GM-CSF. J Clin Invest 2007;117: 1902-13
‡ Donna L. Bratton and Peter M. Henson, Apoptotic Cell Recognition: Will the Real Phosphatidylserine Receptor(s) Please Stand up? Current
Biology Vol 18 No 2
Mechanisms of PS Immunosuppression Mediated
by TAM and TIMs Receptor Signaling
TIM Receptors
Apoptotic cells and
debris with exposed PS
TAM Receptors
Apoptotic cells polarize to M2 macrophages
CD4
Tim-1
Th2 bias
CD8
Tim-3
pTh1 response
T cell exhaustion
Innate
immunosuppressive
actions stimulated
by ligand PS
complexes
CD8
!I"
Tim-4
Tolerance
Freeman et al, Immunol Rev. 235: 172±189 (2010)
Graham et. al. Nature Reviews 14: 769-785 (2014)
TAM
signaling
inhibits NK
antimetastatic
effects
Activated T
cell
feedback
inhibits
innate
immunity
Strong TAM Kinase Domain Activation Requires PS
Courtesy of Lew et. al., and Lemke; eLife 2014;3:e03385
PS Signaling Pathway
Placing the ³%UDNHV´ on the Immune System
‡ Exposed PS is a checkpoint that places brakes on
the immune system
PS Signaling
Immune System
Signaling
‡ PS receptor signaling causes increased anti-inflammatory
cytokine expression
‡
‡
‡
‡
Immune
Suppression
MDSCs accumulate
DCs fail to mature
T-cells remain naïve
M1 Macrophages fail to develop
PS is engaged
MDSC
By PS receptors
PSR
Tumor
Cell
M1
MDSC
PS
PS
PSR
= Exposed PS
M2
TGF-ȕ
2. Immune System Signaling
IL-10
MDSC
Naive
3. ImmuneT-cell
Suppression
Immature
DC
MDSC Naive
T-cell
Multiple Pre-&OLQLFDO6WXGLHV6XSSRUW%DYLWX[LPDE¶V02$
2aG4 treatment of PC3 tumor-bearing mice increases production of immunostimulatory IL-12 and TNF-ĮLQWKHWXPRU
microenvironment and decreases the production of immunosuppressive TGF-ȕDQG,/-10. 1
#$%&'(%)'"*+"',(*$-.%&$/)0"(/1%"
/)1$%&2%2"(&',$%"342 5"
2aG4 treatment induces expression of T-cell co-stimulatory molecules on CD11b+ cells in PC3 tumors1
Prolonged survival time and cures in rats bearing orthotopic F98 tumors treated with
irradiation and 2aG4. Rats bearing orthotopic F98 gliomas were treated with 10 Gy
radiation therapy (RT), 2aG4, RT + 2aG4, or RT + C44 5 d after injection of tumor
cells as in Fig. 1. The survival in the RT + 2aG4 group was significantly longer than in
all other groups (P < 0.001). Thirteen percent of rats treated with RT + 2aG4 were
rendered disease free. After 230 d, the disease-free rats were rechallenged by
intracerebral injection of 5 î 104 into the contralateral hemisphere. None of the rats
developed disease, indicating that their prior treatment had caused them to develop
immunity.3
Tumor-specific cytotoxic T-cells
3
Cure of castration resistant prostate cancer in TRAMP mice treated with mchN11 and androgen
deprivation therapy. Survival time (until sacrifice) was analyzed by the Log-rank method. Mice treated
with Cx+ mch1N11 had longer survival times than mice treated with Sx+ mch1N11, Cx+C44, or
Sx+C44. * Significant for mch1N11 or castration alone versus C44 control; ** Significant for Cx+
mch1N11 versus mch1N11 or Cx or alone. Cx, castration. Sx, sham-operated. N = 20-33. 2
()*+,-,+"./0,0""
1"
#$%&""""""""""""
'&&"
2aG4 directly induces repolarization of TAMs by binding to PS on their cell surface and activating them in an Fc -dependent manner. (a) FACS analyses showing that F4/80+
TAMs from disaggregated PC3 tumors acquire an M1-like phenotype when cultured for 4 days with 2aG4. The M2-like (F4/80+, Arg-1+) population and the M1-like (F4/80+,
iNOS+) were quantified. (b) Treatment of F4/80+ TAMs with 2aG4 induces NO production in an Fc-dependent manner. (c) RT-PCR showing that F4/80+ TAMs lose M2 markers
and acquire M1 markers when cultured with 2aG4. 1
2aG4 directly induces repolarization of TAMs by binding to PS on their cell
surface and activating them in an Fc-dependent manner. (a) FACS analyses
showing that F4/80+ TAMs from disaggregated PC3 tumors acquire an M1-like
phenotype when cultured for 4 days with 2aG4. The M2-like (F4/80+, Arg-1+)
population and the M1-like (F4/80+, iNOS+) were quantified. (b) Treatment of
F4/80+ TAMs with 2aG4 induces NO production in an Fc-dependent manner. (c)
RT-PCR showing that F4/80+ TAMs lose M2 markers and acquire M1 markers
when cultured with 2aG4. 1
Vascular damage is caused by M1-like TAMs generated by 2aG4 treatment. a) Representative
frozen sections showing TAM-mediated disintegration of vascular endothelium in PC3 tumors from
mice treated for 2 weeks with 2aG4, alone or in combination with docetaxel. b) TAMs congregating
around damaged vessels in 2aG4-treated mice costained for iNOS (blue) and F4/80 (green),
indicating they were of M1-like phenotype. c) Representative sections showing that 2aG4 treatment
causes a shift in the predominant polarity of TAMs from M2-like (F4/80+, green; Arg-1+, red) in C44
mice (upper) to M1-like (F4/80+, green; iNOS+, blue) in 2aG4-treated mice (lower). d) Histograms
show the area of PC3 tumor sections occupied by M1 (F4/80+, iNOS+) or M2 (F4/80+, Arg-1+)
TAMs. (e) RT-PCR of RNA from PC3 tumor homogenates reveals a general increase in M1 markers
and a decrease in M2 markers in tumors from 2aG4-treated mice relative to C44-treated mice. (f)
TAMs from 2aG4-treated mice manufacture NO . (g). TAMs from 2aG4-treated mice are tumoricidal. 1
Damage to tumor vasculature induced by treatment with 2aG4. a)Denudation of vascular endothelium in PC3 tumors in mice treated
with 2aG4. b) Reduction of tumor vascularity and perfusion in PC3 tumor-bearing mice treated with 2aG4 alone or in combination
with docetaxel. c) 3-D power Doppler ultrasound showing a reduction in blood flow in PC3 tumors in mice treated with 2aG4 alone or
in combination with docetaxel. 1
1. AACR 2013 Annual Meeting: Phosphatidylserine-Targeting Antibody Induces Differentiation of Myeloid-Derived Suppressor Cells into M1-like Macrophages Yi Yin, Xianming Huang, Dan Ye, Yi Yin, Philip E. Thorpe Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, TX.
2. AACR 2012 Annual Meeting: Yin et al, Cure of castration-resistant prostate cancer in TRAMP mice by reactivating tumor immunity with a phosphatidylserine-targeting antibody. Department of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX
3. 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
Bavituximab Facilitates Induction of Tumor-Specific
Cytotoxic T-Cells
MHCII
CD40
Antigen
presentation
Mature DC
CD80
CD86
T
T
Cytotoxic T-cells
Tumor blood
vessel cell /
Tumor cell /
Microvesicle
Generation of Antitumor T-cell Immunity in
Syngeneic F98 Glioma in Rat2
T
Long-term
Immunity
Tumor-specific
cytotoxicicity
Cure of TRAMP Mice by Anti-PS Combination1
Cytotoxic T-cell Generation2
1.
AACR 2012 Annual Meeting: Yin et al, Cure of castration-resistant prostate cancer in TRAMP mice by reactivating tumor immunity with a phosphatidylserine-targeting
antibody. Department of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX
2.
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
Tumor Response to Bavi Equivalent Involves
Increased CD8 T cells and Decreased MDSCs
(PS Targeting)
EMT-6 breast tumor model in Balb/c mice
(N=10/group; mean “ sem)
200
10
G ro u p
M ean
C 44
1 .4 7
ch1N 11
4 .1 9
p v a lu e = 0 .0 1 8
5
% M D S C /C D 8
% C D 8 T IL s
15
150
100
G ro u p
M ean
C 44
7 6 .6 0
ch1N 11
3 0 .8 9
p v a lu e = 0 .0 1 4
50
0
0
0
1000
2000
3000
3
T u m o r V o lu m e ( m m )
4000
0
1000
2000
3000
3
T u m o r V o lu m e (m m )
4000
Bavituximab Blocks Immunosuppression (Removes Brakes)
Activates Immune Response (Hits Accelerator)
Blocks PS
Signaling
‡ Blocks PS receptor immunosuppressive signal
‡ IL-10 and TGF-ȕ levels decrease
Activates Immune
Response
‡
‡
‡
‡
Sustained
Immune
Response
‡ Increased Inflammatory cytokines
‡ M1 macrophages kill tumor cells via ADCC
‡ Activated T-cells kill tumor cells
Fc / )FȖ receptor signaling
MDSC differentiation
M2 to M1 macrophage polarization
DCs mature, present tumor antigens to T-cells
Yi Yin, Xianming Huang, Kristi D. Lynn, and Philip E. Thorpe, Phosphatidylserine-Targeting Antibody Induces M1
Macrophage Polarization and Promotes Myeloid-Derived Suppressor Cell Differentiation. Cancer Immunology Research
October 2013 1:256-268
Promising and Consistent Pre-Clinical Data Support
Phase III Development in Combination with Docetaxel
Rationalebased
Combination
ƒ Docetaxel has been shown
to increase PS exposure
(AACR 2013 Poster #2850)
ƒ Docetaxel has
immunostimulatory effects
(Kodumudi et al, Clin Cancer Res
2010;16:4583-4594)
Compelling
Pre-clinical
Data
ƒ Tumor models show greater
than 90% reduction in tumor
growth with bavi equivalent
plus docetaxel
(Cancer Research 2005; 65: (10) 4408-4416)
Final Phase II 2nd Line NSCLC
Overall Survival Data with Docetaxel
4*(./)%6"4*)'$*7"
4*(./)%6"4*)'$*7"
Global Phase III
Registration Trial
N = 582
Randomized 1:1
Placebo-Controlled
Bavituximab
(3 mg/kg)
Weekly
Placebo
Weekly
Docetaxel
Docetaxel
Day 1, of 21-day
cycles up to 6
cycles
Day 1 of 21-day
cycles for up to 6
cycles
ƒ Positive EOP2 discussion with FDA and Ex-US
Regulatory Agencies
ƒ Single Phase III global registration trial
ƒ > 150 sites (U.S., Europe and Asia Pacific)
ƒ Est. 24 month enrollment and 12 month followup
ƒ Powered to show approx. 2 month
improvement in median overall survival
ƒ Enrollment initiated January 2014
ƒ Patient Criteria:
Primary Endpoint:
OS
Secondary Endpoints:
PFS, ORR, Safety
ƒ Stage IIIb/IV Non±squamous NSCLC
ƒ Only one prior systemic therapy for advanced
disease
ƒ Unselected for genetic mutations
ƒ Granted Fast Track Designation January 2014
Promising ORR, PFS and Survival Data in Breast
Cancer
Single-Arm Trials
n=
ORR
PFS
OS
46
61%
7.4
20.7
29
41%
4.5
11.4
Bavituximab + Carbo/Paclitaxel
46
74%
6.9
23.2
Carboplatin/Paclitaxel
100
62%
4.8
16.0
14
85%
7.3
--
Advanced/MBC ± All Types (1 Prior Chemo)
Bavituximab + Docetaxel
Docetaxel
Advanced/MBC ± All Types (Front-Line)
MBC (Her2 neg)
Bavituximab + Paclitaxel
Breast Docetaxel: ORR: Burstein, H., et al (2000). JCO. PFS/OS: Nabholtz, J., et al (1999). JCO. Peregrine ASCO (2010), 8/11.
Breast Paclitaxel/Carboplatin: Loesch D., et al (2002). JCO. Peregrine ASCO (2010).
Bavi IST: Alison Stopeck
A HCC Responsive Patient Treated with
Bavituximab in Combination with Sorafenib
Pre
Post
Pre
Post
CD68
Pt ID #P2-23
(7.98)
IHC score: 3
IHC score: 3
IHC score: 1
IHC score: 0
IHC score: 1
IHC score: 2
FoXP3
H&E
CD4
IHC score: 3
CD8
IHC score: 2
IHC score: 3
GranzymeB
IHC score: 2
Increase in tumor immune activity: increase CD8+ & CD4+ T cells
Hypothesis: Bavituximab Can Potentiate Additional
anti-PD-1 Responders
Phase II HCC Data: Increased T-cell tumor infiltration
‡ Best immune responses observed in patients with greatest % increase
in PD-1 and CD8+ cells
= poor immune responders
PD-L1/PD-1 Signaling = Suppression of T-Cell Function
IL-10
APC
MDSC
PD-L1
+
PD-L1
=
PD-1
Exhausted
or
Regulatory
Cell
T-reg
PD-L1
PD-1
PD-1
Endothelial
Cell PD-L1
Conversion
into T-reg cell
PD-1
CD8+
T-cell
PD-1
PD-L1
Tumor Cell
Adapted from Nguyen et al, Nature Reviews: Immunology 15, 45±56 (2015)
and Lee et al, Advances in Bioscience and Biotechnology; 2013, 4, 19-29
PD-L1
PD-1
T-reg
PD-1
PD-L1
M1
Mac
Conversion
into Reg. Mac
M2
Mac
Anti-inflammatory
cytokine production
Relationship of PD-L1 Expression and Activity in Tumor
Biopsies Evaluable by IHC
NSCLC
PD-L1 Tumor
Expression
N=
Responses
PFS at 6 mo.
OS at 6 mo.
High
7
4 (57%)
67%
89%
Low
21
1 (5%)
11%
33%
AACR 2014 Annual Meeting, abstract CT105
MK-3475 (anti-PD-1 monoclonal antibody) for non-small cell lung cancer (NSCLC): Antitumor activity and association with tumor PD-L1 expression
Melanoma
PD-L1 Tumor
Expression
N=
Responses
median PFS
OS at 6 mo.
Positive
60
29 (48%)
10.6
93%
Negative
22
1 (5%)
2.9
75%
AACR 2014 Annual Meeting, abstract CT104
Antitumor activity of the anti-PD-1 monoclonal antibody MK-3475 in melanoma (MEL): Correlation of tumor PD-L1 expression with outcome
Upstream & Downstream Checkpoint Combinations
CTLA-4
= Exposed
PS
Tumor
Cell
MDSC
TGF-ȕ
PSR
PS
B7-H3L
T
PD-1
PD-L1
PS
B7-H3L
PSR
IL-10
M2
Macrophage
PS / PSR: Upstream
³,PPXQH&KHFNSRLQW´
‡ PS
PS Receptors:
‡ Axl
‡ TIM-1
"
"
Immunosuppressive
Cytokines/ Enzymes
‡ TGF-ȕ
‡ IL-10
‡ IL-21
‡ TIM-4 Hokkaido University, Japan
‡ TIM-3
Immature
Dendritic
Cell
‡ IDO
TIM-3
PD-L1
KIR
NK
B7-H3
Downstream
³,PPXQH&KHFNSRLQWV´
‡ PD-1
‡ PD-L1
‡ CTLA-4
‡ TIM-3
PS Blockade Synergizes with Anti-PD-1
Day 42
Response in Individual Mice
89:;:<55"
3
T u m o r V o lu m e ( m m )
600
400
200
a n t i- P D - 1
1500
1000
500
0
0
0
10
20
30
40
50
60
70
60
70
PS
E x p e r im e n t a l D a y s
Mice bearing K1735 melanoma tumors were treated
with anti-PD-1 or mch1N11 + anti-PD-1,
2.5 mg/kg 2x per week. "
3
T u m o r V o lu m e ( m m )
an
tiP
D
-1
+
an
ti-
D
-1
an
tiP
Tumor Volume (mm3)
800
1500
a n t i- P D - 1 + a n t i- P S
1000
500
0
0
10
20
30
40
50
E x p e r im e n t a l D a y s
Combination Treatment Repolarizes Immune Profile
in Spleen
Results of PS Blockade + anti-PD-1 Versus anti-PD-1 Alone (K1735 melanoma model)
8"9
CD8+ Cells
CD8+
T-cell
MDSC
CD8 cells
40%
Mac
DC
0.056
PD-1+
No Change
IFN-Ȗ
40%
0.065
IL-2+
80%
0.034
CD4+
T-cell
CD4 cells
No Change
T-reg
No Change
PD-1+
No Change
IL-2+
150%
0.018
8"9
MDSC / Macs
CD11b+
8"9
CD4+ Cells
No Change
MDSC ( via CD11b)
45%
PD-L1+ CD11b cells
No Change
PD-L1+ CD11b/Ly6G-
No Change
PD-L1+ CD11b/Ly6G-
No Change
DC (CD11chi/F480lo)
30%
Mac (CD11b/F480hi)
No Change
0.001
0.020
ƒ PS Blockade + Anti-PD-1shifts
spleenocytes to immune active
phenotype
‡ Data support potentiating
additional PD-1 responders via
PS blockade.
PS Targeting Treatment Dramatically Reduces Expression
of PDL-1 on Tumor Isolated Leukocytes
4*)'$*7"
CD45
30.5%
30.5%
&)'/-8="
7.9%
7.94%
DE;<B"*+"83-C5">"4%772"
?@;AB"*+"83-C5">"4%772"
&)'/-835"
&)'/-835>&)'/-8="
5.0%
4.97%
31.4%
31.4%
?5;?B"*+"83-C5">"4%772"
?5;FB"*+"83-C5">"4%772"
PD-L1
85 to 90% of PD-L1+
cells are CD45+
leukocytes, less than
10-20% PDL-1+ cells
are tumor cells in this
analysis
Combination Treatment Repolarizes Immune Profile
in Tumor
Results of PS Blockade + Anti-PD-1 Versus anti-PD-1 Alone (K1735 melanoma model)
8"9
CD8+ Cells
CD8 cells
< 0.01
PD-1+
Lag-3+
90%
< 0.01
IFN-Ȗ
70%
0.017
TNF-Į
70%
0.007
Granzyme-B+
70%
0.017
8"9
MDSC / Macs
Mac
MDSC ( via CD11b)
40%
0.02
iNOS+ CD11b cells
90%
0.001
80%
<0.01
PD-L1+ TIL
CD4 /T-reg Ratio
CD4+
T-cell
0.001
MDSC
8"9
CD4+ Cells
0.001
" 100%
" 50%
CD8 /T-reg Ratio
CD8+
T-cell
30%
PD-1+
‡
< 0.01
No change
CD137+ (41BB)
60%
0.03
IFN-Ȗ
30%
< 0.05
TNF-Į
100%
< 0.01
60%
0.02
IL-2+
‡
100%
PS Blockade + Anti-PD-1 increases TIL ;
shifting TIL and cytokines towards tumorrejecting phenotype.
Data support potentiating additional PD-1
responders via PS blockade.
Key Takeaway: PS Blockade Optimizes Tumor
Environment for Additional Anti-PD-1 Responders
Results of PS Blockade + anti-PD-1 versus anti-PD-1 alone (K1735 melanoma model)
MDSC
CD8+
T-cell
PD-1
PD-L1
on TILs
ƒ
40% reduction* of myeloid-derived
suppressor cells
ƒ
ƒ
30% increase* in CD8+ T-cells
50% increase* in PD-1 expression
ƒ
80% decrease* PD-L1 expression
on TIL (CD45+)
* Statistically significant (p = < 0.02)
Positive
environment
for increasing
anti-PD-1
Responders
PS Targeted Combination Therapy in PD-1 Resistant
Melanoma Increases the Anti-tumor Response Rate
C o n tr o l Ig G (C 4 4 )
T u m o r V o lu m e (m m
3500
C o n tro l Ig G
3
3500
3000
2500
2000
1500
1000
500
0
C h1N 11
3000
(Anti-PS)
0
A n t i- P D -1
2500
5
10
15
20
E x p e r im e n t a l D a y s
p = 0 .0 0 7
C h 1N 11
4000
T u m o r V o lu m e (m m
3
)
C h 1 N 1 1 + A n ti-P D -1
2000
1500
3500
3000
2500
2000
1500
1000
500
0
0
p = 0 .0 1 8
5
10
15
20
E x p e r im e n t a l D a y s
1000
A n ti-P D -1
T u m o r V o lu m e (m m
3
)
4000
500
0
0
5
10
15
3500
3000
2500
2000
1500
1000
500
0
20
0
5
10
15
20
E x p e r im e n t a l D a y s
E x p e r im e n ta l D a y s
A n ti-P D -1 + 1 N 1 1
3
)
4000
B16F10 Melanoma Tumor Model in C57BL Mice (10/group)
T u m o r V o lu m e (m m
M e d ia n T u m o r V o lu m e (m m )
3
)
4000
3500
3000
2500
2000
1500
1000
500
0
0
5
10
15
20
E x p e r im e n t a l D a y s
28
Immune Cell Changes in the Tumor
Microenvironment in the Mouse B-16F10 Model
Immune Cell Type (%)
Treatment Type
(Day 23, after 4
treatments)
CD45
CD3*
CD8**
(Cyto T-
CD4**
T reg
cells)
(helper Tcells)
Tumor
Size (mm3)
(WBC)
(mature
T-cells)
Control mAb (2)
<:;5!
FG;D!
AA;E!
<:;D!
5G;E!
FF<A!
ch1N11 (anti- PS) (7)
<F;G!
EF;G!
AF;D!
FF;G!
5:;G!
<5@A!
Anti-CTLA-4 (5)
5@;?!
F?;D!
EA;5!
E:;E!
?;F!
<<:E!
Anti-PD-1 (6)
<G;F!
E<;<!
E?;E!
FF;?!
5?;<!
5G?G!
ch1N11 + Anti-PD-1 (5)
2&3&!
4535!
6738!
<:;A!
5G;F!
967!
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HH"8%$1%)'&0%"*+"43F"I(&',$%"#-1%77K"L*2/'/M%"1%772"
(:$:,0:,+$;;/"0,<=,-,+$=:"+>$=<*"
Key Observations - Summary
ƒ Phosphatidylserine (PS) is externalized in the tumor
microenvironment and is a major immunosuppressive signal
ƒ PS is a global immune checkpoint
ƒ 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
ƒ Combination pre-clinical studies in immuno-competent models
with PD-1 and CTLA-4:
ƒ increase CD8 T cells and M1 macrophages
ƒ decrease MDSCs and M2 macrophages
ƒ demonstrate statistically significant anti-tumor responses
Powerful science. Novel therapies. Dedicated to
=L%1/&7"#N&)O2"'*P"
patients.
Xianming Huang, Ph.D.
Rolf Brekken, Ph.D.
Dmitry Gabrilovich, M.D., Ph.D.
Bruce Freimark, Ph.D.
Nikoletta Kallinteris