Potent and selective C -Cchemokinereceptor 4(CCR4 ... · Younai , Hunter Shunatona, Abood Okal,...
1
Potent and selective C-C chemokine receptor 4 (CCR4) antagonists inhibit regulatory T cell recruitment, increase effector T cell numbers, and potentiate anti-tumor responses in mice Background Immunomodulatory agents induce CCR4 ligand upregulation C Results and Conclusions Regulatory T cells (Treg) are essential for immune tolerance and Treg-mediated suppression of effector T cells (T eff) is important to control inflammation and prevent autoimmune diseases. However, the presence of Treg in the tumor microenvironment (TME) has been shown to dampen anti-tumor immune responses. Human Treg express CCR4, the receptor for the chemokines CCL17 and CCL22. Preclinical and clinical data supports a role for CCR4-mediated recruitment and accumulation of Treg in the TME which can be associated with poor prognosis. Furthermore, patients receiving immunomodulatory agents demonstrate an influx of Treg in responding patients which may dampen optimal anti-tumor responses. CCR4 is an ideal target to selectively block Treg recruitment into the TME. We have developed a structurally unique series of small molecule antagonists of CCR4 with cellular potencies in multiple assays (e.g. chemotaxis of primary human Treg in 100% serum) in the low double-digit nM range. Moreover, these compounds have excellent in vitro and in vivo ADME properties, consistent with convenient oral dosing. These CCR4 antagonists were tested in murine syngeneic tumor models alone and in combination with immunomodulatory agents. During and following treatment, CCR4 ligand levels, tumor infiltrating lymphocytes, and tumor volumes were evaluated. C Potent CCR4 antagonists inhibit T reg recruitment to tumors C CCR4 antagonism increases T eff :T reg ratio in tumors Oezcan Talay, Lisa Marshall, Cesar Meleza, Maureen K. Reilly, Omar Robles, Mikhail Zibinsky, Minna Bui, John Ketcham, Dennis Hu, Ashkaan Younai, Hunter Shunatona, Abood Okal, Jenny McKinnell, Scott Jacobson, Erin Riegler, Sachie Marubayashi, Emily Karbarz, David Chian, Angela Wadsworth, David Wustrow, and Paul Kassner. FLX Bio, Inc., South San Francisco, CA A) Potency and PK parameters for select FLX Bio CCR4 antagonists. Three different FLX Bio CCR4 antagonists demonstrated dose-dependent inhibition of CCL22-induced chemotaxis of B) mouse iTreg and C) human iTreg ,respectively. Human Treg Chemotaxis (100% Serum), IC50(nM) Mouse Treg Chemotaxis (100% Serum), IC50(nM) Mouse IV t1/2 (hr) Mouse Cl (L/hr/Kg) Mouse Vss (L/Kg) Mouse F (%) FLX-A 154 192 2.1 12.6 21.1 7 FLX-B 33 42 7.6 0.72 5.7 26 FLX-C 31 63 19.6 0.23 6.2 22 A B C CCR4 antagonists potentiate anti-tumor effects of immune modulators CCR4 antagonism does not delete peripheral T reg cells A) Experimental outline. Animals were dosed with either FLX-A (100 mg/kg, PO/BID), FLX-B (50 mg/kg, PO/QD) or depleting mouse anti-CCR4 Ab (2G12, 300 µg). B) CCR4 inhibitor selectively inhibits Treg migration into the tumor but not in the periphery, spleen or skin. Anti-CCR4 antibody systemically reduced Treg numbers. CCR4 antagonism provides a potential safety advantage compared to a depleting antibody. Both treatments show similar increase in activated CD8 T cell numbers in the tumor (Data not shown). veh FLX-A CCR4 Ab 0 1000 2000 3000 4000 * Tumor Number of CD45.1 + T reg ** * veh FLX-A CCR4 Ab 0 5000 10000 15000 Spleen Number of CD45.1 + T reg * *** veh FLX-A CCR4 Ab 0 2000 4000 6000 Blood Number of CD45.1 + T reg veh FLX-B CCR4 Ab 0 200 400 600 800 Skin Number of CD45.1 + T reg * We have developed specific and potent CCR4 antagonists that block Treg migration and preclinically, these CCR4 antagonists block T reg migration and support expansion of activated T eff in the tumor. Our antagonists reduce T reg in the tumor, but not in peripheral tissues such as blood, spleen or skin; which presents a potential safety advantage to the non-selective approach of depleting anti-CCR4 antibodies. In preclinical efficacy studies, treatment with various checkpoint inhibitors and immune stimulators (e.g., anti-CTLA-4 or anti-CD137) induce the upregulation of CCR4 ligand expression. Combination therapy with CCR4 antagonist and immunomodulatory agents reduced intratumoral T reg number and increased number of activated and total T eff , resulting in an increase in the intratumoral ratios of both CD4 + and CD8 + T eff to T reg . The change in these T eff to T reg ratios is greater for our CCR4 antagonist in combination than with the immunomodulatory agent alone and correlates with enhanced tumor growth inhibition and increased tumor regression. Combination therapy with CCR4 antagonist and immunomodulatory agents overcome T reg -mediated suppression in tumors and tips the balance toward tumor rejection. PD-1+CD8+:Treg 0 1 2 3 4 Ratio Combo CTLA-4 CCR4 antagonist Vehicle CD4+:Treg 0 10 20 30 Ratio Combo CTLA-4 CCR4 antagonist Vehicle CD8+:Treg 0 10 20 30 Ratio Combo CTLA-4 CCR4 antagonist Vehicle CD44+CD8+:Treg 0 5 10 15 20 25 Ratio Combo CTLA-4 CCR4 antagonist Vehicle CD8 + :Treg CD44 + CD8 + :Treg PD-1 + CD8 + :Treg CD4 + :Treg Days 0 33 39 Pan02 tumor cell injection GFP+iTregtransfer Take down CD45.2+ mice FLX-C dosing PO QD Trough (24 hr) Concentrations on Day 6 of Study Vehicle 3mg/kg 10mg/kg 30mg/kg 0 20 40 60 80 100 Activated CD8 + Tells in Tumor *** **** iTreg = Regulatory T cells induced in cell culture B B) A C) A) 0 10 20 30 40 0 100 200 300 400 Days Post Inoculation Median Tumor Volume (mm 3 ) 10% PEG400 FLX-B αCD137 αCD137 + FLX-B Pan02-Ova CT26 Pan02 cells were engineered to express chicken ovalbumin (Pan02-Ova). C57BL/6 mice were immunized with ovalbumin (50 µg on day –14 and day -7) and then inoculated with Pan02-Ova (4x10 6 cells). On day 7 mice were randomized into groups and animals were dosed with either 10% PEG400, FLX-B (50 mg/kg PO/QD), αCD137 (LOB12.3, 50 µg per IP dose on days 0 and 7 post tumor inoculation) or combination of antibody and FLX-B. 0 10 20 30 40 0 100 200 300 400 Days Post Inoculation Tumor Volume (mm 3 ) FLX-B 0 10 20 30 40 0 100 200 300 400 Days Post Inoculation Tumor Volume (mm 3 ) 10% PEG400 0 10 20 30 40 0 100 200 300 400 Days Post Inoculation Tumor Volume (mm 3 ) αCD137 0 10 20 30 40 0 100 200 300 400 Days Post Inoculation Tumor Volume (mm 3 ) αCD137 + FLX-B -9 -5 0 25 50 75 125 FLX-C FLX-B FLX-A -9 -5 0 25 50 75 100 125 FLX-A FLX-B FLX-C Vehicle 3mg/kg 10mg/kg 30mg/kg 0.0 0.5 1.0 1.5 In vivo Treg trafficking into tumor. D) Study design: Murine pancreatic tumor cells (Pan02) were subcutaneously inoculated into C57BL/6 mice. Tumor-bearing mice were dosed with FLX-C or vehicle prior to transfer of in vitro-induced Treg (iTreg) (I.V.). E) Analysis of TILs: Dose-dependent inhibition of Treg trafficking into tumor but not in periphery (data not shown). Activated CD8 + T cell numbers (measured by PD-1 + staining) increased with higher dose of FLX-C. D E Both CCL17 and CCL22 ligand levels are increased after dosing with IO agents in colon tumor (CT26)-bearing mice. Anti-PD-1(J43; 200 µg/mouse first dose, then 100ug), anti-CD137 (LOB12.3; 150 µg/mouse first dose, then 100ug) or anti-CTLA-4 (9H10; 150 µg/mouse/first dose, then 100ug) were given on days 4, 7 and 11 following tumor inoculation. Tumors were harvested at day 20 and ligands measured by ELISA. CCR4 antagonism augments the immune cell ratio toward an anti-tumor activity. CT26-tumor bearing mice treated with anti-CTLA-4 Ab (9H10, 50 µg day 7, 10, 14) in combination with FLX-B (50 mg/kg PO/QD starting day 7) have highest change in T eff:Treg ratio in tumors. Data from 21 day time point. % Activity % Activity [FLX Cmpd], Log M [FLX Cmpd], Log M Unless otherwise indicated, significance tests and p-values refer to test compared to vehicle control group. P values represented as follows: 0.05 > * > 0.01 > ** > 0.001 > *** > 0.0001 > **** *** *** * * * * ** *** *** CT26 cells inoculated into BALB/c mice and grown for 7 days and randomized into groups (40-70 mm 3 ). Antibody treatment on days 7, 10, 14, FLX-B treatment (50 mg/kg QD/PO) starting at day 7. (TF=Tumor free). B) PD-L1 antibody treatment (10F.9G2), 100 µg each dose) C) CTLA-4 antibody treatment (9H10, 50 µg each dose) D) Animals that recovered from antibody/FLX-B combination treatment were re-challenged with CT26 tumor inoculation and tumors were measured at 20 days post inoculation. 8/8 TF Naïve mice Previously treated and tumor free D) ™ Tumor infiltrating Treg [Plasma FLX-A], nM PD-1 + (% of CD8 + ) T reg (% of live lymphocytes) Chemotaxis IC90
Transcript of Potent and selective C -Cchemokinereceptor 4(CCR4 ... · Younai , Hunter Shunatona, Abood Okal,...
Potent and selective C-C chemokine receptor 4 (CCR4) antagonists inhibit regulatory T cell recruitment, increase effector T cell numbers, and potentiate anti-tumor responses in mice
Background
Immunomodulatory agents induce CCR4 ligand upregulation
C
Results and Conclusions
Regulatory T cells (Treg) are essential for immune tolerance and Treg-mediated suppression of effector T cells (Teff) is important to control inflammation and prevent autoimmune diseases. However, the presence of Treg in the tumor microenvironment (TME) has been shown to dampen anti-tumor immune responses. Human Treg express CCR4, the receptor for the chemokines CCL17 and CCL22. Preclinical and clinical data supports a role for CCR4-mediated recruitment and accumulation of Treg in the TME which can be associated with poor prognosis. Furthermore, patients receiving immunomodulatory agents demonstrate an influx of Treg in responding patients which may dampen optimal anti-tumor responses. CCR4 is an ideal target to selectively block Treg recruitment into the TME.
We have developed a structurally unique series of small molecule antagonists of CCR4 with cellular potencies in multiple assays (e.g. chemotaxis of primary human Treg in 100% serum) in the low double-digit nM range. Moreover, these compounds have excellent in vitro and in vivo ADME properties, consistent with convenient oral dosing. These CCR4 antagonists were tested in murine syngeneic tumor models alone and in combination with immunomodulatory agents. During and following treatment, CCR4 ligand levels, tumor infiltrating lymphocytes, and tumor volumes were evaluated.
C
Potent CCR4 antagonists inhibit Tregrecruitment to tumors
C
CCR4 antagonism increases Teff:Treg ratio in tumors
Oezcan Talay, Lisa Marshall, Cesar Meleza, Maureen K. Reilly, Omar Robles, Mikhail Zibinsky, Minna Bui, John Ketcham, Dennis Hu, AshkaanYounai, Hunter Shunatona, Abood Okal, Jenny McKinnell, Scott Jacobson, Erin Riegler, Sachie Marubayashi, Emily Karbarz, David Chian, Angela Wadsworth, David Wustrow, and Paul Kassner. FLX Bio, Inc., South San Francisco, CA
A) Potency and PK parameters for select FLX Bio CCR4 antagonists. Three differentFLX Bio CCR4 antagonists demonstrated dose-dependent inhibition of CCL22-inducedchemotaxis of B) mouse iTreg and C) human iTreg ,respectively.
HumanTregChemotaxis
(100%Serum),IC50(nM)
MouseTregChemotaxis
(100%Serum),IC50(nM)
MouseIVt1/2(hr)
MouseCl(L/hr/Kg)
MouseVss(L/Kg)
MouseF(%)
FLX-A 154 192 2.1 12.6 21.1 7
FLX-B 33 42 7.6 0.72 5.7 26
FLX-C 31 63 19.6 0.23 6.2 22
A
B C
CCR4 antagonists potentiate anti-tumor effects of immune modulators
CCR4 antagonism does not delete peripheral Treg cells
A) Experimental outline. Animals were dosed with either FLX-A (100 mg/kg, PO/BID),FLX-B (50 mg/kg, PO/QD) or depleting mouse anti-CCR4 Ab (2G12, 300 µg). B) CCR4inhibitor selectively inhibits Treg migration into the tumor but not in the periphery,spleen or skin. Anti-CCR4 antibody systemically reduced Treg numbers. CCR4antagonism provides a potential safety advantage compared to a depleting antibody.Both treatments show similar increase in activated CD8 T cell numbers in the tumor(Data not shown).
v e h F L X -A C C R 4 A b0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
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mb
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45
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ells
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v e h F L X -A C C R 4 A b0
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2 0 0
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f G
FP
+ T
reg
cel
ls
*
SkinN
umbe
r of
CD
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+ T r
eg
*
We have developed specific and potent CCR4 antagonists that block Treg migration and preclinically, these CCR4 antagonists block Treg migration and support expansion of activated Teff in the tumor. Our antagonists reduce Treg in the tumor, but not in peripheral tissues such as blood, spleen or skin; which presents a potential safety advantage to the non-selective approach of depleting anti-CCR4 antibodies. In preclinical efficacy studies, treatment with various checkpoint inhibitors and immune stimulators (e.g., anti-CTLA-4 or anti-CD137) induce the upregulation of CCR4 ligand expression. Combination therapy with CCR4 antagonist and immunomodulatory agents reduced intratumoral Treg number and increased number of activated and total Teff, resulting in an increase in the intratumoral ratios of both CD4+ and CD8+ Teff to Treg. The change in these Teff to Treg ratios is greater for our CCR4 antagonist in combination than with the immunomodulatory agent alone and correlates with enhanced tumor growth inhibition and increased tumor regression.
Combination therapy with CCR4 antagonist and immunomodulatory agents overcome Treg-mediated suppression in tumors and tips the balance toward tumor rejection.
P D -1 + C D 8 + :T re g0
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tio C o m b oC T L A -4C C R 4 a n ta g o n is tV e h ic le
C D 4 + :T re g0
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2 0
3 0
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C D 8 + :T re g0
1 0
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C D 4 4 + C D 8 + :T re g0
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C o m b oC T L A -4
C C R 4 a n ta g o n is tV e h ic le
CD8+:Treg CD44+CD8+:Treg PD-1+CD8+:Treg
CD4+:Treg
Days 0 33 39
Pan02 tumor cell injection
GFP+ iTreg transfer
Take down
CD45.2+ mice
FLX-C dosing PO QD
Trough (24 hr) Concentrations on Day 6 of Study
V e h ic le 3 m g /k g 1 0 m g /k g 3 0 m g /k g 0
2 0
4 0
6 0
8 0
1 0 0
% P
D-1
+ C
D8
T c
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Activated CD8+ Tells in Tumor
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iTreg = Regulatory T cells induced in cell culture
B
B)
A
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A)
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100
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400
Days Post Inoculation
Med
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Tum
or V
olum
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m3 )
10% PEG400
FLX-B
αCD137
αCD137 + FLX-B
Pan02-Ova
CT26
Pan02 cells were engineered to express chicken ovalbumin (Pan02-Ova). C57BL/6 mice were immunized with ovalbumin (50 µg on day –14 and day -7) and then inoculated with Pan02-Ova (4x106 cells). On day 7 mice were randomized into groups and animals were dosed with either 10% PEG400, FLX-B (50 mg/kg PO/QD), αCD137 (LOB12.3, 50 µg per IP dose on days 0 and 7 post tumor inoculation) or combination of antibody and FLX-B.
0 10 20 30 400
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Days Post Inoculation
Tum
or V
olum
e (m
m3 )
FLX-B
0 10 20 30 400
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Days Post Inoculation
Tum
or V
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m3 )
10% PEG400
0 10 20 30 400
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m3 )
αCD137
0 10 20 30 400
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Days Post Inoculation
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or V
olum
e (m
m3 )
αCD137 + FLX-B
-9 -8 -7 -6 -50
2 5
5 0
7 5
1 0 0
1 2 5
L o g [F L X C m p d ] (M )
% A
cti
vit
y
F L X -C
F LX -B
F LX -A
-9 -8 -7 -6 -50
2 5
5 0
7 5
1 0 0
1 2 5
L o g [F 0 0 3 4 7 5 ] (M )
% A
cti
vit
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F LX -B
F L X -C
V e h ic le 3 m g /k g 1 0 m g /k g 3 0 m g /k g 0 .0
0 .5
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% T
reg
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liv
e l
ym
ph
oc
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s
In vivo Treg trafficking into tumor. D) Study design: Murine pancreatic tumor cells(Pan02) were subcutaneously inoculated into C57BL/6 mice. Tumor-bearing mice weredosed with FLX-C or vehicle prior to transfer of in vitro-induced Treg (iTreg) (I.V.). E)Analysis of TILs: Dose-dependent inhibition of Treg trafficking into tumor but not inperiphery (data not shown). Activated CD8+ T cell numbers (measured by PD-1+
staining) increased with higher dose of FLX-C.
D
E
Both CCL17 and CCL22 ligand levels are increased after dosing with IO agents in colon tumor (CT26)-bearing mice. Anti-PD-1(J43; 200 µg/mouse first dose, then 100ug), anti-CD137 (LOB12.3; 150 µg/mouse first dose, then 100ug) or anti-CTLA-4 (9H10; 150 µg/mouse/first dose, then 100ug) were given on days 4, 7 and 11 following tumor inoculation. Tumors were harvested at day 20 and ligands measured by ELISA.
CCR4 antagonism augments the immune cell ratiotoward an anti-tumor activity. CT26-tumor bearingmice treated with anti-CTLA-4 Ab (9H10, 50 µg day7, 10, 14) in combination with FLX-B (50 mg/kgPO/QD starting day 7) have highest change in Teff:Treg
ratio in tumors. Data from 21 day time point.
% A
ctiv
ity
% A
ctiv
ity
[FLX Cmpd], Log M [FLX Cmpd], Log M
Unless otherwise indicated, significance tests and p-values refer to test compared to vehicle control group. P values represented as follows: 0.05 > * > 0.01 > ** > 0.001 > *** > 0.0001 > ****
*** ****
* * *
**
***
***
CT26 cells inoculated into BALB/c mice and grown for 7 days and randomized into groups (40-70 mm3). Antibody treatment on days 7, 10, 14, FLX-B treatment (50 mg/kg QD/PO) starting at day 7. (TF=Tumor free).
B) PD-L1 antibody treatment (10F.9G2), 100 µg each dose)C) CTLA-4 antibody treatment (9H10, 50 µg each dose)D) Animals that recovered from antibody/FLX-B combination treatment were re-challenged with CT26 tumor inoculation and tumors were measured at 20 days post inoculation.
8/8 TF
Naïve mice
Previously treated and tumor free
D)
™
Tumor infiltrating Treg
[Pla
sma
FLX-
A],
nM
PD-1
+(%
of
CD
8+ )
T reg
(% o
f li
ve l
ymph
ocyt
es)
Chemotaxis IC90
