abstract&4600&/&13 potent&andselective&c /cchemokine ... · vehicle flx:...

Potent and selective C-C chemokine receptor (CCR4) antagonists potentiate anti-tumor immune responses by inhibiting regulatory T cells (Treg)Oezcan Talay, Lisa Marshall, Cesar Meleza, Maureen K. Reilly, Omar Robles, Mikhail Zibinsky, Abood Okal, Lisa Seitz, Jenny McKinnell,

Scott Jacobson, Erin Riegler, Emily Karbaz, David Chian, Angela Wadsworth, Paul Kassner, David Wustrow, Jordan S. FridmanFLX Bio, Inc. South San Francisco, CA

Abstract FLX Antagonist can be Dosed to Cover the Mouse Treg Chemotaxis IC90

r

CCR4 Antagonism Blocks Treg Recruitment and Increase Teff in Tumors

NeNeed

C

Conclusions

Naturally suppressive CD4+ Foxp3+ Treg are essential for immune tolerance. Although Treg-mediated suppression of effector cells is important to control inflammation and prevent autoimmune diseases, 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. These chemokines are produced by tumor cells, tumor-associated macrophages and dendritic cells, as well as by effector T cells (Teff). 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. Further, recent longitudinal studies in patients receiving IO agents demonstrate an influx of Treg in responding patients which may dampen optimal anti-tumor responses. Therefore, CCR4 is an ideal target to selectively block Tregrecruitment into the TME.We have developed structurally unique series of small molecule antagonists of CCR4. These antagonists have cellular potencies in multiple assays (e.g. chemotaxis of primary human Treg in 100% serum) in the low double-digit nM range. Representative compounds are selective against other chemokine receptors, GPCRs and ion channels, including the hERG channel, and lack inhibition of common human CYP450 enzymes. Moreover, compounds have excellent in vitro and in vivo ADME properties, consistent with convenient oral dosing. In preclinical syngeneic tumor models, these CCR4 antagonists block Treg migration and support expansion of activated Teff. In contrast to the non-selective approach of depleting anti-CCR4 antibodies, our compounds reduce Treg in the tumor, but not in peripheral tissues such as blood, spleen or skin. In preclinical efficacy studies, CCR4 antagonists potentiate the anti-tumor effects of various checkpoint inhibitors and immune stimulators such as anti-PD-L1 and anti-CTLA-4 antibodies. We observe enhanced tumor growth inhibition and increased tumor regressions when these agents are combined with CCR4 antagonists, without any gross toxicity. Further characterization of these CCR4 antagonists and their anti-tumor effects will be described.

Abstract 4600 / 13

http

CCR4 is a Dominant Regulator of Treg in Human Tumors

FLX Antagonist are Highly Potent Against Mouse and Human Treg Chemotaxis

FLX CCR4 Antagonists Potentiate Various IO Agents

Effector Treg are the most suppressive subpopulation of Treg and they express the highest amount ofCCR4 (Tanaka and Sakaguchi, Cell Research, 2017). A) CCR4 is the most prevalent chemokinereceptor on human Treg. B) The expression of CCR4 ligands (CCL22 and CCL17) is elevated in aspectrum of human tumor tissues and ligand expression correlates with FOXP3 mRNA - a markerfor Treg. C) CCR4-ligand is upregulated in “ligand-low” syngeneic tumors (representative datashown in CT26) after treatment with checkpoint inhibitor.

CCR4 Antagonism is a Selective Approach to Blocking Treg Recruitment

TregTeff

CCR4CCL17CCL22

MΦDCs Treg

Blood

• Cytotoxic molecules• Granzyme B• Perforin

• Cytokines• IFN-y

• Chemokines

Tumor Microenvironment

Tumor

CCR4 Ligand Expression is Elevated In Multiple Human Cancer and Expression Correlates with FOXP3 mRNA Levels. Ligand Levels Increase with Clinically Relevant Rx

B)

A)

Serum Free Ca++Flux, IC50 (µM)

10% Serum Ca++Flux, IC50 (µM)

CTX 100% Serum, IC50 (µM)

Mouse IV t1/2 (hr)

Mouse Cl (L/hr/Kg)

Mouse Vss

(L/Kg)

MouseF(%)

FLX-‐A 0.055 0.103 0.164 2.1 12.6 21.1 7

FLX-‐B 0.021 0.093 0.065 7.6 0.72 5.7 26

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

Number of CD45.1+ T

reg cells

* * *


5 0 0 0

1 0 0 0 0

1 5 0 0 0

Number of CD45.1+ T

reg cells

* * * * *

SpleenTumor


2 0 0 0

4 0 0 0

6 0 0 0

Number of CD45.1+ T

reg cells

*

Blood

v e h F L X -B C C R 4 A b0

2 0 0

4 0 0

6 0 0

8 0 0

Number of GFP+ T

reg cells

*

Skin

* p<0.05 vs Veh or FLX-A vs CCR4-Ab** p<0.01 vs Veh*** p<0.005 vs Veh**** p<0.0001 vs Veh

0

2 0

4 0

6 0

8 0

% of PD1+ CD8+ T cells

V eh ic le FLX -A C C R 4 A b

* *

* *

*

0

1 0

2 0

3 0

4 0

5 0

% of CD69+ CD8+ T cells

C on tro l F LX -A C C R 4 A b

* * *

**

*

A) Ca2+ flux in CCR4-expressing Chem5-hCCR4 cells is induced by CCL22. B) Compound FLX-Ademonstrated dose-dependent inhibition of CCL22-induced Ca2+-flux. C, D) Chemotaxis of murine inducedTreg (miTreg) and human induced Treg (hiTreg) is stimulated by CCL22. E, F) FLX-A demonstrated dose-dependent inhibition of CCL22-induced chemotaxis of miTreg and hiTreg ,respectively. G,H) FLX-Bdemonstrated dose-dependent inhibition of CCL22-induced chemotaxis of miTreg and hiTreg ,respectively.

Exogenous GFP+ Treg were transferred into tumor-bearing mice and tumor-trafficking was analyzed.A) Pancreatic carcinoma Pan02 cells express high basal level of CCL22 and CCL17 (data not shown). B)Experimental outline Treg Tumor migration study 1. C) Significant and selective inhibition of Treg cellmigration into the tumor. D) Experimental outline Treg Tumor migration study 2. E) Decreased number ofTreg and increased number of activated CD8 T cells (shown as MFI for PD-1 and CD69) in the tumor ofCCR4-inhibitor treated animals.

A) Experimental outline. Animals were dosed with either FLX-A or depleting mouse anti-CCR4 Ab. B)CCR4 inhibitor selectively inhibits Treg migration into the tumor but not in the periphery. Anti-CCR4antibody systemically reduced Treg numbers. C) Both treatments show similar increase in activated CD8 Tcell numbers.

CCR4+ Treg cells treated with DMSO or CCR4 Inhibitor

Migration towards towards CCL22

Buffer with CCL22

Migratory cells are lysed and quantified using CellTiter-Glo® Assay

Tumor efficacy studies in Colon Carcinoma (CT26) tumor model using CCR4 inhibitor alone or incombinationA) Study outline. B) Mice treated with CCR4 inhibitor or anti-PD-L1 showed modest efficacywhile combination significantly reduced tumor growth. C) Mice treated with anti-CTLA-4 aloneshowed significant tumor growth inhibition (TGI) with one tumor-free animals. TGI wassignificantly improved when combined with CCR4 inhibitor with 6 tumor-free animals.

A)

C)

*

****

0 1 0 2 0 3 00

5 0 0

1 0 0 0

1 5 0 0

M e d ia n tum o r v o lum e

D a y s p o s t in n o c u la t io n

Tumor Volume (mm

3)

V e h ic le : 1 0% P E G 40 0

F LX -B

a -P D -L 1

a -P D -L 1 + F L X -B

* p > 0 .0 5 (a -P D -L 1 v s V e h )* * * * p > 0 .0 0 0 0 1 (a -P D -L 1 + F L X -B v s V e h )

B)

Rx

• CCR4 is the most prevalent chemokine receptor expressed on Treg and high CCR4 expressing Treg are the most potent suppressive sub-population

• CCL22 and CCL17, the cognate CCR4 ligands, are elevated in human tumors and their expression levels correlate with FOXP3, a Treg marker

• FLX CCR4 antagonists potently and selectively block agonist activation in both a calcium flux assay and chemotaxis assay run in 100% human serum

• FLX CCR4 antagonists selectively block Treg migration into PAN02 tumors

• Unlike depleting antibodies (e.g. aCCR4) Treg in skin, blood and spleen are not reduced with FLX CCR4 antagonists

• The addition of FLX CCR4 antagonists to clinically relevant antibodies, such as aPD-L1 or aCTLA4, results in dramatically improved tumor growth inhibition and regression rates

• These data demonstrate that a potent and selective CCR4 antagonist may be a tumor-selective and attractive approach to antagonize Treg function and support a productive anti-tumor immune response

• A FLX CCR4 antagonist is scheduled to begin clinical trials in 2017

0

5 0 0

1 0 0 0

1 5 0 0

CD69 (MFI)

* *

* * p < 0 .0 0 5 v s V eh

0

1 0 0 0

2 0 0 0

3 0 0 0

PD-1 (MFI)

* *

* * p < 0 .0 0 5 v s V ehVehicle FLX-A Vehicle FLX-A

CCR4 Is The Most Prevalent Chemokine Receptor On Human Effector Treg

R = 0.67CCL22

FOX

P3

R = 0.30

FOX

P3

TGFb1

Response(%Activity)

Response(%Activity)

ResponseFluorescence)

Response(%Activity)

Response(%Activity)

ResponseFluorescence)

Log [hCCL22] (M)Log [hCCL22] (M)

Log [FLX-A] (M) Log [FLX-A] (M)

Log [FLX-B] (M) Log [FLX-B] (M)

0 4 8 1 2 1 6 2 0 2 41

1 0

1 0 0

1 0 0 0

1 0 0 0 0

T im e (h rs )

Plasm

a Concentration

(nM

)Second dose

BID

QD

Mouse PK (FLX-A)

Chemotaxis (CTX) in 100% serum

A) B)

C)

Treg Tumor Migration Study 1

Treg Tumor Migration Study 2

IC90 mouse Tregchemotaxis

D)

A)

B)

CCL22 mRNA/Actin mRNA

CT26 B16 4T1 Pan02

C) D)

E) F)

G) H)TumorC)

CCL22Rx: Checkpoint inhibitor (CPI)

(i.e., a-PD_L1 or a-CTLA-4)

FLX-B (50 mg/kg, PO)

** No Matrigel used

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

Number of GFP+ Tregs

V eh ic le F LX -A(1 0 0 m g /k g B ID )

F L X -B(5 0 m g /k g Q D )

* * ** * * *

* * * p =0 .0 0 0 2 v s V eh* * * * p <0 .0 0 0 1 v s V eh

Tumor

Num

berofGFP

+T reg

0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

1 0 0 0 0 0

Number of GFP+ Tregs

V eh ic le F LX -A(1 0 0 m g /k g B ID )

F L X -B(5 0 m g /k g Q D )

*

* p < 0 .0 5 v s V eh

Spleen

Num

berofGFP

+T reg

0

2 0 0 0

4 0 0 0

6 0 0 0

Number of GFP+ T

reg

* * * *

* * p < 0 .0 0 5 v s V eh

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

Number of CD8+ T cells

*

* p < 0 .0 5 v s V eh

Pre-Dose Vehicle FLX-A Vehicle FLX-A

Num

berofGFP

+T reg

Num

berofCD8+Tcells

PD-1(MFI)

CD69(MFI)

TCGA Data for Ligand Expression in Human TumorsTCGA Data for FOXP3 correlation with CCL22 or TGF-b1

CCR4-Ligand Expression in CT26-model after I/O TreatmentC)

Calcium-Flux Assay

A) B)

Log [FLX-A] (M)Log [hCCL22] (M)

Chemotaxis (CTX) in 100% serum

CD8+ T cellsE)

Vehicle FLX-A(100mg/kg)

FLX-B(50mg/kg)

Vehicle FLX-A(100mg/kg)

FLX-B(50mg/kg)

Rx

0 5 1 0 1 5 2 0 2 50

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

M e d ia n tum o r v o lum e

D a y s p o s t in n o c u la t io n

Tumor Volume (mm

3)

V e h ic le : 1 0% P E G 40 0

a -C T LA -4

a -C T L A -4 + F L X -B

* * * *

* * * *

* * * * p < 0 .0 0 01 v s V e h ic le

0 5 1 0 1 5 2 0 2 5 3 0 3 50

5 0 0

1 0 0 0

1 5 0 0

D a y s p o s t in n o c u la tio n

Tumor Volume (mm

3)

0 5 1 0 1 5 2 0 2 5 3 0 3 50

5 0 0

1 0 0 0

1 5 0 0


Tumor Volume (mm

3)

0 5 1 0 1 5 2 0 2 5 3 0 3 50

5 0 0

1 0 0 0

1 5 0 0


Tumor Volume (mm

3)

Vehicle a-CTLA-4

a-CTLA-4 + FLX-B

1/10 TF

5/10 TF

Day post inoculation Day post inoculation

Day post inoculation

TumorVolume(mm3 )

TumorVolume(mm3 )

TumorVolume(mm3 )

0 5 1 0 1 5 2 0 2 5 3 00

5 0 0

1 0 0 0

1 5 0 0

V e h ic le


Tumor Volume (mm

3) g ro u p o u t

0 5 1 0 1 5 2 0 2 5 3 00

5 0 0

1 0 0 0

1 5 0 0

F 0 0 3 3 5 1


Tumor Volume (mm

3)

0 5 1 0 1 5 2 0 2 5 3 00

5 0 0

1 0 0 0

1 5 0 0

P D -L 1


Tumor Volume (mm

3)

0 5 1 0 1 5 2 0 2 5 3 00

5 0 0

1 0 0 0

1 5 0 0

P D -L 1 + F 0 0 3 3 5 1


Tumor Volume (mm

3)

Vehicle FLX-B

a-PD-L1 a-PD-L1 + FLX-B



TumorVolume(mm3 )

TumorVolume(mm3 )

TumorVolume(mm3 )

TumorVolume(mm3 )

Days 33 39

Days 33 39

abstract&4600&/&13 potent&andselective&c /cchemokine ... · vehicle flx:...

Documents