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%CD4+
ofCD3+
0
20
40
60
Vehicle IPI-549
%CD8+
ofCD3+
0
20
40
60
80
100
Vehicle IPI-549
The PI3 kinases belong to a family of signal transducing enzymes that mediate
key cellular functions in cancer and immunity. The PI3K-g isoform plays an
important role in immune cell function and migration and a role for PI3K-g in
tumor growth and immune tolerance has been established in studies utilizing
PI3K-g-/-
KO mice1-4
. Syngeneic tumors implanted into PI3K-g-/-
mice showed
decreased growth rates and reduced tumor vascularization.1
Tumor
associated macrophages (TAMS) isolated from PI3K-g-/-
mice show decreased
expression of genes which promote tumor growth, and importantly, immune
suppression.2,3,4
Therefore, targeting PI3K-g in immune cells could alter the
balance from an immune suppressive to an immune competent environment
leading to tumor growth inhibition.
The Potent and Selective Phosphoinositide-3-Kinase (PI3K)-γ Inhibitor, IPI-549, Inhibits Tumor Growth in Murine Syngeneic Solid Tumor Models
through Alterations in the Immune Suppressive Microenvironment
Jeffery Kutok, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Karen McGovern, Somarajan Nair, Melissa Pink,
Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Katherine Walsh, Kerry White, David Winkler, Vito Palombella
Infinity Pharmaceuticals, Inc., Cambridge, MA
Background
Assay PI3K-a PI3K-b PI3K-g PI3K-d
Binding Affinity Kd = koff
/kon
(nM) [n] 17 [2] 82 [2] 0.29 [3] 23 [3]
Biochemical IC50
(nM) ± SDM [n] 3241 ± 1206 [4] 3468 ± 1282 [4] 15.7 ± 6.6 [4] >8421 [4]
Cellular IC50
(nM) [n] 247.7 [4] 241.0 [2] 1.2 [2] 175.8 [2]
Bone marrow derived macrophages (BMDM) were differentiated
with MCSF1, then polarized towards an M2 phenotype with IL-4
and MCSF1 while simultaneously treating with varying concen-
trations of IPI-549. M2 macrophage polarization, as measured
by Arg1 gene expression, was inhibited in a dose-responsive
manner compared to untreated control.
REFERENCES 1. Schmid et al. 2011, Cancer Cell 19:715; 2. Joshi et al. 2014, Mol Canc Res12:1520;
3. Kaneda et al., 2014 AACR meeting; 4. Varner J. 2015 Keystone PI3K; 5. Winkler et al. 2013,
Chemistry & Biology 20:1364.
ACKNOWLEDGEMENTS: Gabriel Birrane, Beth Israel Deaconess Medical Center, Boston, MA and
Thomas Tibbits, Infinity Pharmaceuticals, for co-crystal structure
IPI-549 Inhibits Macrophage Function in vitro
IPI-549 inhibits murine macrophage
polarization to an M2 phenotype
IPI-549 inhibits migration of murine
bone marrow derived macrophages
Bone marrow derived macrophages were stimulated to
migrate towards 100 μg/ml CXCL12 for 3 hours through
a 5 micron Boyden Chamber with or without DMSO or
increasing concentrations of IPI-549. Migrated BMDMs
were counted and compared to control.
IPI-549 is Active in Syngeneic Solid Tumor Models
Cancer Type Cell Line % tumor growth inhibition [n experiments]
Lung LLC-luc brei 28-52% [8]
Colon MC38 49% [1]
Breast 4T1-luc 23-38% [5]
Colon CT26 31-45% [8]
IPI-549 Increases Tumor Associated T Cells
IPI-549 increases intratumoral T-cells
The growth inhibitory effect of IPI-549 is
lost in the absence of mature T cells
CD8+
T cells are required for
IPI-549 activity in mice
The administration of IPI-549 dosed orally to BALB/c nu/nu
mice that lack a thymus and mature T cells had no impact on
the growth of CT26 colon carcinoma tumors (n=15/group) as
compared to the vehicle control group (p = 0.47)
IPI-549 administered at 15 mg/kg/day to immunocom-
petent BALB/c mice for 13 days resulted in a significant
inhibition of CT26 colon carcinoma tumor growth com-
pared to vehicle treated mice (p=0.0022). In contrast, oral
administration of IPI-549 at 15 mg/kg/day for 13 days to
CT26 tumor bearing CD8+
T cell depleted BALB/c mice
had no impact on tumor growth (p = 0.20)
IPI-549 Combination Activity with Checkpoint Inhibitors
Conclusions
• 	 PI3K-g plays a key role in supporting the immune suppressive tumor
microenvironment
• 	 IPI-549 is a potent, selective inhibitor of PI3K-g
• 	 IPI-549 blocks murine macrophage M2 polarization and migration in vitro
• 	 IPI-549 has favorable pharmacokinetic properties to allow potent and
selective inhibition of PI3K-g in vivo
• 	 IPI-549 inhibits tumor growth in a dose responsive manner as a single agent
in multiple syngeneic murine tumor models
• 	 IPI-549 treatment reduces tumor associated M2 myeloid cells and increases
tumor associated CD8+ T-cells in murine models
• 	 IPI-549 mediated tumor growth inhibition is T-cell dependent
• 	 IPI-549 in combination with checkpoint inhibitors demonstrates enhanced
anti-tumor effects compared to monotherapies in murine syngeneic models
• 	 IPI-549 is being developed as an orally administered therapeutic in multiple
cancer indications
IPI-549 is a Potent, Selective Inhibitor of PI3K-g
T Cells are Required for IPI-549 Tumor Growth Inhibition
Binding Affinity (Kd) of IPI-549 for Class I PI3K isoforms were determined as previously reported5
. Biochemical IC50s were
determined (Promega ADP-Glo assay) at physiological ATP concentrations (3 mM). Activity against all Class II PI3K isoforms
was >2000nM. Cellular IC50s of IPI-549 for Class I PI3K-α, PI3K-β, PI3K-γ, and PI3K-δ were determined in SKOV-3, 786-O,
RAW 264.7, and RAJI cells, respectively, by monitoring inhibition of pAKT S473 by ELISA.5
IPI-549Vehicle
Intratumoral T-cell number correlates with final tumor volume after
IPI-549 treatment
IPI-549 treated MC38 colon cancer tumors show a correlation between decreasing tumor volume and increasing CD3+
T-cell
infiltrates. This correlation was not observed in the vehicle treated group.
Greater tumor growth inhibition with IPI-549 treatment in combination
with anti-PD-L1 antibody than either treatment alone
CT26 colon carcinoma cells were implanted in BALB/c female mice and treatment was initiated 3 days post-implant. Groups of
mice (n=13 for vehicle; n=15 for all other groups) were treated with 15 mg/kg IPI-549 or vehicle PO daily and either anti-PD-L1
antibody (200 mg) or isotype control antibody IP every third day for a total of 5 doses. Growth inhibition was not significant
between anti-PD-L1 vs. vehicle (p=0.057) and between anti-PD-L1 vs. combination (p=0.070). Growth inhibition was
significant between IPI-549 vs. vehicle (p=0.009) and between the IPI-549 vs. combination (p=0.002).
IPI-549 has favorable pharmacokinetic (PK) properties to allow potent
and selective inhibition of PI3K-g in vivo in the mouse
Murine Pharmacokinetics of IPI-549
PK plasma time profile of a 3 mg/kg, 7.5 mg/kg and 15 mg/kg IPI-549 single PO dose in Lewis Lung Carcinoma
(LLC) tumor bearing C57/B6 Albino mice.
To date, potent and selective PI3K-g
inhibitors with drug-like properties
have not been available to test this
hypothesis. We now report the
properties of a potent and selective,
small molecule inhibitor of PI3K-g,
IPI-549, and provide data to support
the therapeutic potential of breaking
tumor immune tolerance through
PI3K-g inhibition.
MC38 tumors stained for CD3 protein by immuno-
histochemistry (brown). IPI-549 treated tumors had
significantly increased CD3+
T cells as compared to
vehicle treated tumors (p value = 0.01)
IPI-549 Decreases Tumor Associated Myeloid Cells
IPI-549 leads to decreased myeloid cells measured by both
CD11b and CD68 immunohistochemistry
IPI-549 treatment decreases TAMS
(CD11b+
Ly6C–
Ly6G–
)
LLC tumors from vehicle or IPI-549 treated mice were dissociated into single cell suspension at the end of an efficacy study.
Tumor associated cell populations were measured by flow cytometry for both TAMS and CD206+
M2 cells.
IPI-549 treatment decreases CD206+
M2
myeloid cells
Quantitation of IHC staining for CD11b or CD68 in 4T1
breast cancer tumors from mice treated with IPI-549
compared to vehicle (day 13 of study)
Representative images showing decreased CD11b+
myeloid cell staining (brown) in viable regions of 4T1
breast cancer tumors from mice treated with IPI-549
compared to vehicle (day 13 of study).
LLC tumors implanted in C57/B6 Albino male mice. Daily, oral
treatment with varying doses of IPI-549 was initiated at average
tumor volume of 100 mm3
. All groups showed significant growth
inhibition compared to vehicle (p-value < 0.05).
MC38 was implanted into C57Bl/6 mice. Treatment of
mice with IPI-549 at 15 mg/kg PO started when tumors
reached 80-100 mm3
and led to significant growth
inhibition (p=0.017).
Lewis lung carcinoma model MC38 colon carcinoma model
Greater survival with IPI-549 treatment in combination with
anti-CTLA4 antibody than either treatment alone
LLC brei tumors were implanted in C57/B6 Albino male mice and treatment was initiated at an average size of 50-100 mm3
.
Groups of mice were treated with 15 mg/kg IPI-549 or vehicle PO daily and either anti-CTLA4 antibody (100 mg) or isotype
control antibody IP every third day for a total of 5 doses. By log-rank test, a difference in survival was statistically significant
between IPI-549 and vehicle (p=0.0086), combination vs. vehicle (p=0.0002), combination vs. CTLA4 (p=0.0078) and
combination vs. IPI-549 (p=0.0106).
IPI-549 treated murine tumors have decreased CD4+
T cells and
increased CD8+
T cells
CT26 colon cancer tumors from mice treated with 15 mg/kg daily of IPI-549 for 9 days demonstrated significantly decreased
CD4+
T-cells and increased CD8+
cytotoxic T cells compared to vehicle treated mice. In addition, the overall CD3+
levels
increased significantly (p = 0.0121) in IPI-549 treated CT26 tumors (data not shown).
Summary of IPI-549 activity in syngeneic solid tumor models
Log IPI-549 [M]
Arg1mRNAexpression
Percentofcontrol
-10 -9 -8 -7 -6 -5
0
20
40
60
80
100
120
140
-11
Log IPI-549 [M]
BMDMmigration
Percentofcontrol
0
20
40
60
80
100
120
140
-10 -9 -8 -7 -6 -5 -4
IC50
= 88 nM IC50
= 85 nM
Vehicle + Isotype Control
Tumorvolume(mm3
)
3500
0
Days after Implant
2800
2100
1400
700
0
5 10 15 20 25
IPI-549 + Isotype Control
Tumorvolume(mm3
)
3500
0
Days after Implant
2800
2100
1400
700
0
5 10 15 20 25
anti-PD-L1 + Vehicle
Tumorvolume(mm3
)
Days after Implant
3500
0
2800
2100
1400
700
0
5 10 15 20 25
anti-PD-L1 + IPI-549
Tumorvolume(mm3
)
Days after Implant
3500
0
2800
2100
1400
700
0
5 10 15 20 25
Survival to 2000 mm3
Percentsurvival
100
50
0
0 10 20 30
Treatment day
Vehicle + Isotype (n=14)
IPI-549 + Isotype (n=14)
Vehicle + Anti-CTLA4 (n=13)
IPI-549 + Anti-CTLA4 (n=10)
Time (h)
UnboundConcentrationofIPI-549(ng/mL)
0 4 8 12 16 20 24
1
0
10
100
1000
IPI-549 15 mg/kg
IPI-549 7.5 mg/kg
IPI-549 3 mg/kg
IPI-549 PI3K-α
Cellular IC50 IPI-549 PI3K-β and δ
Cellular IC50
IPI-549 PI3K-γ
Cellular IC50
*
* Student t-Test
p-value < 0.05 compared
to Vehicle + Isotype
*
CD11b CD68
0
10
20
30
40 Vehicle + Isotype
IPI-549 + Isotype
n=10
n=6-9
%Marker+Area/TotalViableArea
IPI-549
Vehicle
IPI-549
Vehicle
*p value <0.05 compared to vehicle
Tumorvolume(mm3
)
0
500
1000
1500
2000
2500
3000
0 3 6 9 12
Days on Study
*
*
*
Vehicle PO n=14
IPI-549 3 mg/kg PO n=14
IPI-549 7.5 mg/kg PO n=11
IPI-549 15 mg/kg PO n=13
0
500
1000
1500
2000
Tumorvolume(mm3
)
Vehicle
IPI-549 (15 mg/kg)
*p = 0.017
0 10
Days on Study
20 30
*
* p value <0.05 compared to vehicle
%CD11b+
Ly6C–
Ly6G–
oftotalcells
%CD206+
ofCD11b+
0
5
10
15
0
10
20
30
40
50
* p value <0.05 compared to vehicle
Vehicle IPI-549 Vehicle IPI-549
Vehicle IPI-549
0
5
10
15
PercentofCD3positivecellspertotal
nucleiwithinviableareasoftumor
* p = 0.01
TumorVolume(mm3
)
Percent of CD3+
cells per total
nuclei within viable areas of tumor
0
500
1000
1500
2000
2500
3000
3500
4000
0 1 2 3 4 5 6 7
-500
0
500
1000
1500
2000
2500
0 5 10 15 20
Percent of CD3+
cells per total
nuclei within viable areas of tumor
TumorVolume(mm3
)
R2
= 0.02
R2
= 0.79
* p = 0.03 * p = 0.02
p = 0.0022 compared to vehicle control
Tumorvolume(mm3
)
1000
2000
3000
0
Vehicle n=15
IPI-549 n=15
Vehicle + anti-CD8 n=15
IPI-549 + anti-CD8 n=13
0 5
Days on Study
10 15
Vehicle
IPI-549 (15 mg/kg)
1000
2000
3000
0
Tumorvolume(mm3
)
Days on Study
0 3 9 126
IPI-549 = 15 mg/kg PO, QD
M1
Treg
Treg
M1
M1
Treg
DC
CTL
CTL
CTL
CTL
NK
Cell
CTL
CD4+
T-cell
B Cell
CTL
CD4+
T-cell
B Cell
CTL
CTL
Treg
DC
Treg
M2/TAM/
MDSC
Immune Activation-
Tumor EliminationPro-Tumor immune
suppressive cells
Activated Anti-tumor
immune cells
Inhibited anti-tumor
immune cells
M2/TAM/
MDSC
M2/TAM/
MDSC
Treg
DC
NK
Cell
Immune Suppression-
Tumor Growth
IPI-549 mediated inhibition of PI3K-γ
Working model for IPI-549 in anti-tumor immunity
*
*
*
*
CRI-CIMT-EATI-AACR - The Inaugural International Cancer Immunotherapy Conference • Friday September 18, 2015 • New York, NY
X-ray crystal structure of IPI-549
bound to PI3K-g

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AACR_ImmunotherapyNYCmeeting_549unveiling_POSTER

  • 1. %CD4+ ofCD3+ 0 20 40 60 Vehicle IPI-549 %CD8+ ofCD3+ 0 20 40 60 80 100 Vehicle IPI-549 The PI3 kinases belong to a family of signal transducing enzymes that mediate key cellular functions in cancer and immunity. The PI3K-g isoform plays an important role in immune cell function and migration and a role for PI3K-g in tumor growth and immune tolerance has been established in studies utilizing PI3K-g-/- KO mice1-4 . Syngeneic tumors implanted into PI3K-g-/- mice showed decreased growth rates and reduced tumor vascularization.1 Tumor associated macrophages (TAMS) isolated from PI3K-g-/- mice show decreased expression of genes which promote tumor growth, and importantly, immune suppression.2,3,4 Therefore, targeting PI3K-g in immune cells could alter the balance from an immune suppressive to an immune competent environment leading to tumor growth inhibition. The Potent and Selective Phosphoinositide-3-Kinase (PI3K)-γ Inhibitor, IPI-549, Inhibits Tumor Growth in Murine Syngeneic Solid Tumor Models through Alterations in the Immune Suppressive Microenvironment Jeffery Kutok, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Karen McGovern, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Katherine Walsh, Kerry White, David Winkler, Vito Palombella Infinity Pharmaceuticals, Inc., Cambridge, MA Background Assay PI3K-a PI3K-b PI3K-g PI3K-d Binding Affinity Kd = koff /kon (nM) [n] 17 [2] 82 [2] 0.29 [3] 23 [3] Biochemical IC50 (nM) ± SDM [n] 3241 ± 1206 [4] 3468 ± 1282 [4] 15.7 ± 6.6 [4] >8421 [4] Cellular IC50 (nM) [n] 247.7 [4] 241.0 [2] 1.2 [2] 175.8 [2] Bone marrow derived macrophages (BMDM) were differentiated with MCSF1, then polarized towards an M2 phenotype with IL-4 and MCSF1 while simultaneously treating with varying concen- trations of IPI-549. M2 macrophage polarization, as measured by Arg1 gene expression, was inhibited in a dose-responsive manner compared to untreated control. REFERENCES 1. Schmid et al. 2011, Cancer Cell 19:715; 2. Joshi et al. 2014, Mol Canc Res12:1520; 3. Kaneda et al., 2014 AACR meeting; 4. Varner J. 2015 Keystone PI3K; 5. Winkler et al. 2013, Chemistry & Biology 20:1364. ACKNOWLEDGEMENTS: Gabriel Birrane, Beth Israel Deaconess Medical Center, Boston, MA and Thomas Tibbits, Infinity Pharmaceuticals, for co-crystal structure IPI-549 Inhibits Macrophage Function in vitro IPI-549 inhibits murine macrophage polarization to an M2 phenotype IPI-549 inhibits migration of murine bone marrow derived macrophages Bone marrow derived macrophages were stimulated to migrate towards 100 μg/ml CXCL12 for 3 hours through a 5 micron Boyden Chamber with or without DMSO or increasing concentrations of IPI-549. Migrated BMDMs were counted and compared to control. IPI-549 is Active in Syngeneic Solid Tumor Models Cancer Type Cell Line % tumor growth inhibition [n experiments] Lung LLC-luc brei 28-52% [8] Colon MC38 49% [1] Breast 4T1-luc 23-38% [5] Colon CT26 31-45% [8] IPI-549 Increases Tumor Associated T Cells IPI-549 increases intratumoral T-cells The growth inhibitory effect of IPI-549 is lost in the absence of mature T cells CD8+ T cells are required for IPI-549 activity in mice The administration of IPI-549 dosed orally to BALB/c nu/nu mice that lack a thymus and mature T cells had no impact on the growth of CT26 colon carcinoma tumors (n=15/group) as compared to the vehicle control group (p = 0.47) IPI-549 administered at 15 mg/kg/day to immunocom- petent BALB/c mice for 13 days resulted in a significant inhibition of CT26 colon carcinoma tumor growth com- pared to vehicle treated mice (p=0.0022). In contrast, oral administration of IPI-549 at 15 mg/kg/day for 13 days to CT26 tumor bearing CD8+ T cell depleted BALB/c mice had no impact on tumor growth (p = 0.20) IPI-549 Combination Activity with Checkpoint Inhibitors Conclusions • PI3K-g plays a key role in supporting the immune suppressive tumor microenvironment • IPI-549 is a potent, selective inhibitor of PI3K-g • IPI-549 blocks murine macrophage M2 polarization and migration in vitro • IPI-549 has favorable pharmacokinetic properties to allow potent and selective inhibition of PI3K-g in vivo • IPI-549 inhibits tumor growth in a dose responsive manner as a single agent in multiple syngeneic murine tumor models • IPI-549 treatment reduces tumor associated M2 myeloid cells and increases tumor associated CD8+ T-cells in murine models • IPI-549 mediated tumor growth inhibition is T-cell dependent • IPI-549 in combination with checkpoint inhibitors demonstrates enhanced anti-tumor effects compared to monotherapies in murine syngeneic models • IPI-549 is being developed as an orally administered therapeutic in multiple cancer indications IPI-549 is a Potent, Selective Inhibitor of PI3K-g T Cells are Required for IPI-549 Tumor Growth Inhibition Binding Affinity (Kd) of IPI-549 for Class I PI3K isoforms were determined as previously reported5 . Biochemical IC50s were determined (Promega ADP-Glo assay) at physiological ATP concentrations (3 mM). Activity against all Class II PI3K isoforms was >2000nM. Cellular IC50s of IPI-549 for Class I PI3K-α, PI3K-β, PI3K-γ, and PI3K-δ were determined in SKOV-3, 786-O, RAW 264.7, and RAJI cells, respectively, by monitoring inhibition of pAKT S473 by ELISA.5 IPI-549Vehicle Intratumoral T-cell number correlates with final tumor volume after IPI-549 treatment IPI-549 treated MC38 colon cancer tumors show a correlation between decreasing tumor volume and increasing CD3+ T-cell infiltrates. This correlation was not observed in the vehicle treated group. Greater tumor growth inhibition with IPI-549 treatment in combination with anti-PD-L1 antibody than either treatment alone CT26 colon carcinoma cells were implanted in BALB/c female mice and treatment was initiated 3 days post-implant. Groups of mice (n=13 for vehicle; n=15 for all other groups) were treated with 15 mg/kg IPI-549 or vehicle PO daily and either anti-PD-L1 antibody (200 mg) or isotype control antibody IP every third day for a total of 5 doses. Growth inhibition was not significant between anti-PD-L1 vs. vehicle (p=0.057) and between anti-PD-L1 vs. combination (p=0.070). Growth inhibition was significant between IPI-549 vs. vehicle (p=0.009) and between the IPI-549 vs. combination (p=0.002). IPI-549 has favorable pharmacokinetic (PK) properties to allow potent and selective inhibition of PI3K-g in vivo in the mouse Murine Pharmacokinetics of IPI-549 PK plasma time profile of a 3 mg/kg, 7.5 mg/kg and 15 mg/kg IPI-549 single PO dose in Lewis Lung Carcinoma (LLC) tumor bearing C57/B6 Albino mice. To date, potent and selective PI3K-g inhibitors with drug-like properties have not been available to test this hypothesis. We now report the properties of a potent and selective, small molecule inhibitor of PI3K-g, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-g inhibition. MC38 tumors stained for CD3 protein by immuno- histochemistry (brown). IPI-549 treated tumors had significantly increased CD3+ T cells as compared to vehicle treated tumors (p value = 0.01) IPI-549 Decreases Tumor Associated Myeloid Cells IPI-549 leads to decreased myeloid cells measured by both CD11b and CD68 immunohistochemistry IPI-549 treatment decreases TAMS (CD11b+ Ly6C– Ly6G– ) LLC tumors from vehicle or IPI-549 treated mice were dissociated into single cell suspension at the end of an efficacy study. Tumor associated cell populations were measured by flow cytometry for both TAMS and CD206+ M2 cells. IPI-549 treatment decreases CD206+ M2 myeloid cells Quantitation of IHC staining for CD11b or CD68 in 4T1 breast cancer tumors from mice treated with IPI-549 compared to vehicle (day 13 of study) Representative images showing decreased CD11b+ myeloid cell staining (brown) in viable regions of 4T1 breast cancer tumors from mice treated with IPI-549 compared to vehicle (day 13 of study). LLC tumors implanted in C57/B6 Albino male mice. Daily, oral treatment with varying doses of IPI-549 was initiated at average tumor volume of 100 mm3 . All groups showed significant growth inhibition compared to vehicle (p-value < 0.05). MC38 was implanted into C57Bl/6 mice. Treatment of mice with IPI-549 at 15 mg/kg PO started when tumors reached 80-100 mm3 and led to significant growth inhibition (p=0.017). Lewis lung carcinoma model MC38 colon carcinoma model Greater survival with IPI-549 treatment in combination with anti-CTLA4 antibody than either treatment alone LLC brei tumors were implanted in C57/B6 Albino male mice and treatment was initiated at an average size of 50-100 mm3 . Groups of mice were treated with 15 mg/kg IPI-549 or vehicle PO daily and either anti-CTLA4 antibody (100 mg) or isotype control antibody IP every third day for a total of 5 doses. By log-rank test, a difference in survival was statistically significant between IPI-549 and vehicle (p=0.0086), combination vs. vehicle (p=0.0002), combination vs. CTLA4 (p=0.0078) and combination vs. IPI-549 (p=0.0106). IPI-549 treated murine tumors have decreased CD4+ T cells and increased CD8+ T cells CT26 colon cancer tumors from mice treated with 15 mg/kg daily of IPI-549 for 9 days demonstrated significantly decreased CD4+ T-cells and increased CD8+ cytotoxic T cells compared to vehicle treated mice. In addition, the overall CD3+ levels increased significantly (p = 0.0121) in IPI-549 treated CT26 tumors (data not shown). Summary of IPI-549 activity in syngeneic solid tumor models Log IPI-549 [M] Arg1mRNAexpression Percentofcontrol -10 -9 -8 -7 -6 -5 0 20 40 60 80 100 120 140 -11 Log IPI-549 [M] BMDMmigration Percentofcontrol 0 20 40 60 80 100 120 140 -10 -9 -8 -7 -6 -5 -4 IC50 = 88 nM IC50 = 85 nM Vehicle + Isotype Control Tumorvolume(mm3 ) 3500 0 Days after Implant 2800 2100 1400 700 0 5 10 15 20 25 IPI-549 + Isotype Control Tumorvolume(mm3 ) 3500 0 Days after Implant 2800 2100 1400 700 0 5 10 15 20 25 anti-PD-L1 + Vehicle Tumorvolume(mm3 ) Days after Implant 3500 0 2800 2100 1400 700 0 5 10 15 20 25 anti-PD-L1 + IPI-549 Tumorvolume(mm3 ) Days after Implant 3500 0 2800 2100 1400 700 0 5 10 15 20 25 Survival to 2000 mm3 Percentsurvival 100 50 0 0 10 20 30 Treatment day Vehicle + Isotype (n=14) IPI-549 + Isotype (n=14) Vehicle + Anti-CTLA4 (n=13) IPI-549 + Anti-CTLA4 (n=10) Time (h) UnboundConcentrationofIPI-549(ng/mL) 0 4 8 12 16 20 24 1 0 10 100 1000 IPI-549 15 mg/kg IPI-549 7.5 mg/kg IPI-549 3 mg/kg IPI-549 PI3K-α Cellular IC50 IPI-549 PI3K-β and δ Cellular IC50 IPI-549 PI3K-γ Cellular IC50 * * Student t-Test p-value < 0.05 compared to Vehicle + Isotype * CD11b CD68 0 10 20 30 40 Vehicle + Isotype IPI-549 + Isotype n=10 n=6-9 %Marker+Area/TotalViableArea IPI-549 Vehicle IPI-549 Vehicle *p value <0.05 compared to vehicle Tumorvolume(mm3 ) 0 500 1000 1500 2000 2500 3000 0 3 6 9 12 Days on Study * * * Vehicle PO n=14 IPI-549 3 mg/kg PO n=14 IPI-549 7.5 mg/kg PO n=11 IPI-549 15 mg/kg PO n=13 0 500 1000 1500 2000 Tumorvolume(mm3 ) Vehicle IPI-549 (15 mg/kg) *p = 0.017 0 10 Days on Study 20 30 * * p value <0.05 compared to vehicle %CD11b+ Ly6C– Ly6G– oftotalcells %CD206+ ofCD11b+ 0 5 10 15 0 10 20 30 40 50 * p value <0.05 compared to vehicle Vehicle IPI-549 Vehicle IPI-549 Vehicle IPI-549 0 5 10 15 PercentofCD3positivecellspertotal nucleiwithinviableareasoftumor * p = 0.01 TumorVolume(mm3 ) Percent of CD3+ cells per total nuclei within viable areas of tumor 0 500 1000 1500 2000 2500 3000 3500 4000 0 1 2 3 4 5 6 7 -500 0 500 1000 1500 2000 2500 0 5 10 15 20 Percent of CD3+ cells per total nuclei within viable areas of tumor TumorVolume(mm3 ) R2 = 0.02 R2 = 0.79 * p = 0.03 * p = 0.02 p = 0.0022 compared to vehicle control Tumorvolume(mm3 ) 1000 2000 3000 0 Vehicle n=15 IPI-549 n=15 Vehicle + anti-CD8 n=15 IPI-549 + anti-CD8 n=13 0 5 Days on Study 10 15 Vehicle IPI-549 (15 mg/kg) 1000 2000 3000 0 Tumorvolume(mm3 ) Days on Study 0 3 9 126 IPI-549 = 15 mg/kg PO, QD M1 Treg Treg M1 M1 Treg DC CTL CTL CTL CTL NK Cell CTL CD4+ T-cell B Cell CTL CD4+ T-cell B Cell CTL CTL Treg DC Treg M2/TAM/ MDSC Immune Activation- Tumor EliminationPro-Tumor immune suppressive cells Activated Anti-tumor immune cells Inhibited anti-tumor immune cells M2/TAM/ MDSC M2/TAM/ MDSC Treg DC NK Cell Immune Suppression- Tumor Growth IPI-549 mediated inhibition of PI3K-γ Working model for IPI-549 in anti-tumor immunity * * * * CRI-CIMT-EATI-AACR - The Inaugural International Cancer Immunotherapy Conference • Friday September 18, 2015 • New York, NY X-ray crystal structure of IPI-549 bound to PI3K-g