eFT508, A Potent and Highly Selective Inhibitor of MNK1 and MNK2, is an Activator of Anti-Tumor Immune ResponseKevin R Webster1, Rajesh Sharma1 , Vikas K Goel1, Craig R. Stumpf1, Jocelyn Staunton1, Peggy A Thompson1, Gary G Chiang1, Yichen Xu2, Hyun Yong Jin2, and Davide Ruggero2

1eFFECTOR Therapeutics, San Diego, CA; 2Departments of Urology, Cellular and Molecular Pharmacology, and the Hellen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA

Abstract Results

Introduction

• eFFECTOR Therapeutics has discovered eFT508, a potent, highlyselective, small molecule inhibitor of MNK1 and MNK2 activity

• MNK1 and MNK2 are S/T protein kinases that integrate signals fromseveral oncogenic and immune signaling pathways, such as RAS andT-cell receptor (TCR), at the level of translational control

• MNK selectively controls the translation of key regulators of theanti-tumor immune response

AKT

RTK

PI3K

PTEN

AKT

CD4 TCR/CD3

PI3K

MEK1/2

Lck

CD28

RAS

ZAP-70

IRAK1/4

MyD88

IL1/TLR

RAS

RAF

mTOR

eIF4E

eIF4G

PABP

eIF4AP

AAAAAUAUUUAUUUAAAAAAAAAAAAAA…

RNABPsP

MNK

eIF3

P4EBP1

m7G

eFT508

MAPKs MAPKs

P

mTOR

4EBP1

Selective regulation of • Checkpoint receptors• Cytokines

Conclusions• eFT508 selectively inhibits immune checkpoint receptors (PD-1,

LAG3) and cytokine (IL-10) expression in T-cells • MNK1/2 control mRNA fate through specific 5’ and 3’ UTR

elements• eFT508 increases MHC class II expression on the surface of antigen-

presenting cells and affects DC trafficking in vivo• eFT508 can block induced T regulatory cell differentiation and

enhance central memory pool formation and cytotoxic function inT effector cells

• eFT508 modulates anti-tumor immunity and effectively synergizeswith immune checkpoint blockade in vivo

• eFT508 is currently being evaluated in phase 1/2 clinical trials as asingle agent in patients with solid tumors (NCT02605083) andlymphoma (NCT02937675), and as a single agent and incombination with avelumab in MSS colorectal cancer(NCT03258398).

Background: eFT508 is a potent and highly selective inhibitor of MNK1and MNK2, kinases that function to mediate tumor immune evasiondownstream of MEK and MAPK signaling. eFT508 treatment establishes aregulatory program that promotes multiple steps in the cancer immunitycycle including antigen presentation and T cell priming, expansion ofmemory T cells, and prevention of T cell exhaustion. Methods: Theimmunological effects of eFT508 have been evaluated in the context ofnormal human immune cells in vitro and in immunocompetent syngeneicand genetically engineered mouse models in vivo. Results: eFT508treatment of normal donor T cells has no deleterious effect onαCD3/αCD28 stimulated T cell proliferation or T cell viability in contrastto inhibitors acting upstream of MAPK signaling. eFT508 selectively downregulates key immune checkpoint proteins and the production of a subsetof pro-inflammatory and immunosuppressive cytokines. In vitromechanism of action studies have demonstrated that MNK selectivelyregulates gene expression at the level of mRNA translation via specificsequence elements in the 5’- and 3’-untranslated regions. In addition,eFT508 activates antigen presenting cells leading to more effective T cellpriming. eFT508 also affects T cell memory formation, both in the contextof specific peptide antigen stimulation and in a mixed lymphocytereaction, shifting the distribution of T cells towards a CD62L+CD44+ centralmemory T cell population. eFT508 also enhances the cytotoxic functionof T cells from OT-I mice stimulated with SIINFEKL peptide demonstratinga dose-dependent increase of cell killing. Consistent with themechanisms elaborated upon in vitro, eFT508 shows significant anti-tumor activity mediated through tumor infiltrating lymphocytes in theCT26 syngeneic tumor model as well as genetically engineered mousemodels of NSCLC and HCC. Conclusions: eFT508 treatment establishes aregulatory program that promotes anti-tumor immunity. eFT508 iscurrently under evaluation as a single agent in two phase 1/2 clinical trialsfor patients with advanced solid tumors and patients with advancedlymphoma. A biomarker driven proof of concept study, includingmandatory pre- and on-treatment biopsies, to evaluate theimmunological mechanism of action of the drug is planned to be initiatedlater this year. In addition, a phase 2 study evaluating eFT508, alone orin combination with avelumab, a PD-L1 immune checkpoint inhibitor, inmicrosatellite stable relapsed or refractory CRC patients is planned.

Figure 2. eFT508 selectively downregulates expressionof key immunosuppressive factors in activated T cells.Primary human T cells were stimulated with α-CD3/CD28antibodies in the presence of the indicated concentrationsof compound. A) Whole cell lysates from T cellsincubated for 48 h with eFT508 were immunoblotted withthe indicated antibodies. B) Activated T cells were treatedfor 24 h with eFT508 and analyzed for PD-1, TIM3, LAG3,4-1BB, and cell viability by flow cytometry (% positivecells) or IL-10 secretion (pg/ml) by ELISA. Values plottedare % inhibition of each marker relative to the activatedvehicle (DMSO) control cells. C) Activated T cells wereincubated with the indicated concentrations of eFT508 orthe MEK inhibitor cobimetinib for 5 days and proliferationwas assessed by CellTrace Violet.

A. B.

Results

C.

Figure 3. MNK1/2 controls mRNA fate through specificsequences in the 5’ and 3’ UTRs. A) Schematicrepresentation of LAG3 5’ UTR region. Jurkat T cells weretransfected with the indicated reporter RNAs and treatedwith eFT508 for 24 h. Luciferase activity was assessed and% expression was calculated relative to vehicle control.eFT508 IC50 values for Jurkat T cells transfected with RNAfrom the indicated LAG3 UTR reporter constructs arelisted in the table. B) Schematic representation of the IL-10 3’ UTR region. MDA-MB-231 cells were transfectedwith the indicated reporter RNAs and treated witheFT508 for 24 h. Luciferase activity was assessed and %expression was calculated relative to vehicle control.

A. B.

LAG3 UTR construct

eFT508IC50, µM

5’ and 3’ UTR 1Full 5’ UTR 2.95’ UTR A 7.45’ UTR B > 305’ UTR C > 30

Tubulin 5’ UTR > 30

P368

A.

Figure 4. eFT508 increases key dendritic cell markers and trafficking in vivo. A) CD14+ cells isolatedfrom human PBMCs were differentiated into mature monocyte-derived dendritic cells (Mo-DCs) usingthe schema shown. B-D) Immature monocyte-derived-DCs (im-MoDCs) were differentiated in thepresence of the indicated concentrations of eFT508 for 24 h. Cells were harvested and the CD14+ cellswere analyzed for cell surface markers (HLA-DR, CD83, and CCR7) by flow cytometry analysis. E) BALB/cmice were dosed with vehicle or 1 mg/kg eFT508 daily for 2 days. Blood, spleen, and lymph node wereharvested on day 3 and CD11c+ MHC-II (I-A/I-E)+ cells were analyzed by flow cytometry.

Figure 7. eFT508 enhances the formation of the T cell central memory pool. A) Peritonealmacrophages isolated from BALB/c mice were incubated with the indicated concentrations of eFT508for 24 h and then mixed with panned splenocytes isolated from C57BL/6 mice in an MLR reaction foran additional 4 days in the presence of the indicated concentrations of eFT508. B) Splenocytes fromOT-I mice (C57BL/6-Tg(TcraTcrb)1100Mjb) were stimulated with 5 µg/ml SIINFEKL peptide in thepresence of the indicated concentrations of eFT508 for 4 days. In both experiments, cells wereanalyzed forCD8, CD44 and CD62L expression by flow cytometry. Representative scatter plots forCD44 and CD62L expression in CD8+ cells are shown. CD44highCD62low define effector memory cells(EM) and CD44highCD62Lhigh define central memory cells (CM).

Figure 8. eFT508 increases cytotoxic T cell function. Splenocytes from OT-I mice (C57BL/6-Tg(TcraTcrb)1100Mjb) were stimulated with SIINFEKL peptide in the presence of the indicatedconcentrations of eFT508 for 3 days. OT-I splenocytes were washed and mixed at a 10:1 ratio withB6.SJL splenocytes (1:1 mix of SIINFEKL-pulsed CellTracehigh and unpulsed CellTracelow populations) for16 h in the absence of eFT508. B6.SJL cells were gated by CD45.1 expression and analyzed forCellTrace Violet levels by flow cytometry. The % cell killing relative to target cells alone is listed in red.

Figure 6. eFT508 blocks induced Treg (iTreg) differentiation and IL-10 production A) Human PBMCswere stimulated with α-CD3/CD28 antibodies and 5 ng/ml TGF-β for 7 days in the presence of theindicated concentrations of eFT508. Cells were analyzed for the expression of CD3, CD4 and FOXP3 byflow cytometry. The CD4+FOXP3+ cells were scored as the iTreg population. Data are from 3 separatedonors. B) Human PBMCs were treated as in (A) and the supernatants were analyzed for IL-10 byELISA. Data are from 2 separate donors.

iTreg IL-10

A. B.

Figure 5. eFT508 drives T cell activation and proliferation in the MLR setting. Peritonealmacrophages isolated from BALB/c mice were incubated with the indicated concentrations of eFT508for 24 h and then mixed with panned CellTrace Violet-labeled splenocytes isolated from C57BL/6 micein an MLR reaction for an additional 4 days in the presence of the indicated concentrations of eFT508.Cells were analyzed for CD4, CD8 and CellTrace Violet by flow cytometry.

B. C.

D. E.

A.

B.

A. B.

C. D.

Response rate

50 %

100 %

in (A) for 4 days. Tumors were harvested, dissociated into single cell suspensions and stained forimmune cell surface markers (CD45, CD3, CD8, CD4, FOXP3) followed by flow cytometry analysis. Theratio of CD8+ to FOXP3+ cells is plotted. D) CT-26 allografts were treated as in (A) for 7 days. Tumorswere harvested, dissociated into single-cell suspensions and stained for immune cell surface markers(CD45, F4/80, CD206, MHC class II) followed by flow cytometry analysis. M2 macrophages were scoredas CD206+/MHC class IIlow and plotted as a percentage of the total cell count.

Figure 9. eFT508 triggers anti-tumorimmunity and enhances the efficacy of PD-1 immune checkpoint blockade. A) CT-26allografts were treated with eFT508, anti-PD-1 antibody, or the combination ofeFT508 and anti-PD-1 at day 7 post-implantfor the indicated time. Tumor volumeswere measured and plotted as a function oftime. B) Naïve animals or animals from (A)which exhibited regression of tumors at d29were re-challenged with CT-26 allografts inthe absence of any further treatment.Tumors were measured at day 10 post-implant. C) CT-26 allografts were treated as

Figure 1. eFT508 is a potent and highly selective inhibitor of MNK1 and MNK2 kinases. A) Chemicalstructure of eFT508. B) The IC50 of eFT508 was determined against MNK1 and MNK2 by in vitro kinaseassays. eFT508 was also profiled at 1 µM against 414 kinases using the Invitrogen SelectScreen kinaseprofiling service. The IC50 of eFT508 was determined against the hits with >50% inhibition from thisscreen, DRAK1 and CLK4, by LanthaScreen kinase binding assay. n.d., not determined.

Kinase eFT508IC50, nM

% inhibition @1 µM eFT508

MNK1 2.4 100%MNK2 1 100%

STK17A/DRAK1 131 82%CLK4 787 60%

Remaining 410protein kinases n.d. < 40%

NNH

NH

NN

H2NO

O

A.

eFT508

B.

WTLung

KRASG12D

Adenoma

P-eIF4E

eIF4E

A. B.

C. D.

Figure 10. eFT508 prolongs survival and increases anti-tumor immune response in a KRASG12D;p53-/-

(KP) NSCLC model A) WT or KP mice were intra-nasally infected with adenoviral-Cre. 16 weeks post-infection, normal and tumor lesions were micro-dissected and immunoblotted for expression of P-eIF4E and eIF4E. B) Animals treated as in (A) were dosed with 10 mg/kg eFT508 at 13 weeks post-infection for an additional 3 weeks. Animals were sacrificed at 16 weeks and lung sections werestained with hematoxylin and eosin (H&E). C) Kaplan-Meier survival plot for animals treated as in (A).D & E) KP mice infected with adenoviral-Cre were treated with vehicle or 10 mg/kg eFT508 at 10 weekspost-infection for an additional 8 weeks. Lungs were harvested and tumor infiltrating lymphocyteswere analyzed by flow cytometry for CD8 and PD-1 expression.

H&ELung

Vehicle 10 mg/kg eFT508

E.

A.

C.

B.

D.

triangle). A) Kaplan-Meier survival curves. Animal numbers are denoted in parentheses. B)Representative H&E staining of lungs from animals after 7 days of treatment. Lung metastases aredenoted by red arrows. C) Mouse livers from vehicle or eFT508-treated animals (n=3/group) wereanalyzed for infiltrating CD4+ and CD8+ T cells by flow cytometry after 7 days of treatment. D) Liversections from vehicle or eFT508-treated animals (n=3/group) were analyzed for CD107a expression byimmunofluorescence after 7 days of treatment.

Figure 11. eFT508 prolongssurvival and increases anti-tumorimmune response in aMYCTg;KRASG12D orthotopic HCCmodel. MYCTg;KRASG12D HCC cellswere injected orthotopically intothe liver. Animals were treated with10 mg/kg eFT508 starting at 7 dayspost-injection (denoted by green

H&ELung

Vehicle 10 mg/kg eFT508

Tumor cell T cell

-9 -8 -7 -6 -5 -40

20

40

60

80

100

Log [eFT508], M

% o

f Act

ivat

ed C

ontr

ol

PD-1

LAG3

4-1BB

TIM3

IL-10

Viability

-10 -9 -8 -7 -6 -5 -40

20

40

60

80

100

Log [Compound], M

% C

ell P

rolif

erat

ion

CobimetnibeFT508

-10 -9 -8 -7 -6 -5 -40

25

50

75

100

125

Log [eFT508], M

Rep

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ress

ion,

%

Lag3 5'+3' UTR Tubulin 5' UTR

-10 -9 -8 -7 -6 -5 -40

25

50

75

100

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Log [eFT508], M

Rep

orte

r Exp

ress

ion,

%

IL-10 3' UTR Tubulin 5' UTR

0 0.01 0.1 1 100

100

200

300

400

500

HLA

-DR

exp

ress

ion,

MFI

Mo-DCs (µM eFT508)CD14im

-MoD

C 0 0.01 0.1 1 100

100200300400500600700800900

CD

83 e

xpre

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n, M

FI

Mo-DCs (µM eFT508)CD14im

-MoD

C

0 0.01 0.1 1 3 100

2

4

6

Fold

cha

nge

% C

CR

7+ cel

lsim

-moD

C

moDC eFT508, µM

Blood Spleen Lymph Node0

10

20

30

% D

Cs

in ti

ssue

eFT508 1 mg/kgvehicle

**

ns ns

- - 0.01 0.1 1 3 - - 0.01 0.1 1 30

20

40

60

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% D

ivid

ed C

ells

eFT508, µM:

T-ce

lls T-cells + MΦ T-cells + MΦ

T-ce

lls

CD4+ CD8+

- - 0.01 0.1 1 3 100

2

4

6

8

10

% C

D4+ FO

XP3+

Treg

s

α-CD3/CD28 + TGFβ

eFT508, µM: - 0.01 0.1 1 3 100

20

40

60

80

100

120

IL-1

0, %

of s

timul

ated

con

trol

α-CD3/CD28 + TGFβ

eFT508, µM:

8 12 15 19 22 26 290

500

1000

1500

2000

2500

3000

3500

Time, days

Tum

or V

olum

e (m

m3 )

VehicleeFT508 1 mg/kg QDanti-PD-1 0.5 mg Q4DeFT508 1mg/kg QD+ anti-PD-1 0.5 mg Q4D

Dosing

0

5 0

1 0 0

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2 0 0

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Tu

mo

r V

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(m

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Vehicle eFT5080.0

0.5

1.0

1.5

% M

2 m

acro

phag

es in

tum

or

0 20 40 60 80 1000

25

50

75

100

Days post treatment

Perc

ent s

urvi

val

Vehicle (9)eFT508 (11)

Vehicle eFT5080.00

0.05

0.10

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CD8+ cells

Frac

tion

of v

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n-tu

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0 10 20 300

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Days

Perc

ent s

urvi

val

Vehicle (10)eFT508 (6)

***

Day 0 Vehicle eFT5080

1

2

3

4

5

CD

4/C

D8

ratio

of C

D3+ T

ILs

*

Vehicle eFT5080

2

4

6

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10

CD

8+/ F

OXP

3+ T c

ells

Vehicle eFT5080

10

20

30

CD8+PD-1+ T cells

% p

ositi

ve C

D8+ P D

-1+ T

cel

ls

Vehicle eFT5080

200

400

600

800

CD

107a

+ cel

ls p

er H

PF

**

55 36 43 45CM

30 27EM

CD

44

CD62L

17 6329 5735 55

OT-I T cells alone SIINFEKL 0.1 µM eFT508

3 µM eFT508 10 µM eFT5081 µM eFT508

8 2116 15CM

7 8EM

CD

44

CD62L

17 2311 2610 29

MLRCD8 T cells alone 0.1 µM eFT508

10 µM eFT5083 µM eFT5081 µM eFT508

Target alone Unstim. OT-I SIINFEKL OT-I 0.01 µM eFT508

0.1 µM eFT508 1 µM eFT508 3 µM eFT508 10 µM eFT508

10.6% 49.1% 52.2%

52.6% 63.8% 72.9% 81.2%