- - 0.1 1 3 - - 0.1 1 3 - - 0.1 1 3 - - 0.1 1 3

T CM

/TEM

ratio

in O

T-II

Isotype AbAnti-PD-1 Ab

Anti-PD-L1 AbAnti-CTLA4 Ab

eFT508, µM OT-II peptide stim - ++ ++ - ++ ++ - ++ ++ - ++ ++

0

1

2

3

TCM bias

TEM bias

- - 0.1 1 3 - - 0.1 1 3 - - 0.1 1 3 - - 0.1 1 3

T CM

/TEM

ratio

in O

T-I

Isotype AbAnti-4-1BB Ab

Anti-OX40 AbAnti-GITR Ab

eFT508, µMOT-I peptide stim - + + + + - + + + + - + + + + - + + + +

1

0

2

3

TCM bias

TEM bias

Vehicle eFT5080

2

4

6

8

10

CD

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Vehicle eFT5080.0

0.5

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Tu

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a n t i-P D -1N a ïv e e F T 5 0 8a n ti-P D -1

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500

1000

1500

2000

2500

3000

3500

Time, days

Tum

or V

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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

0.0

0.5

1.0

1.5

Day 24 post-transfer

% C

D8+

Mem

ory

T ce

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p < 0.0001

eFT508 (d1-7): - + - +Immunization (d1): - - + +

Boost (d21): - - + +

0

20

40

60

Day 3 post-transfer

% C

M C

D8+ T

cells

eFT508 (d1-3): - + - +Immunization (d1): - - + +

p = 0.016

ns

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

20

40

60

80

% D

ivid

ed C

ells

eFT508, µM:

T-ce

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T-ce

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CD4+ CD8+

Blood Spleen Lymph Node0

10

20

30

% C

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cells

eFT508 1 mg/kgvehicle

**

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Mo-DCs (µM eFT508)

HLA-DR (MHC II)

0 0.01 0.1 1 100

100

200

300

400

500

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exp

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ion,

MFI

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40

60

80

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Log [eFT508], M

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PD-1

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4-1BB

TIM3PD-L1

IL-10

Viability

eFT508, A Potent and Highly Selective Inhibitor of MNK 1/2, Regulates T Cell Differentiation Promoting an Anti-tumor Immune ResponseRajesh K Sharma, Vikas K Goel, Jocelyn Staunton, Maria Barrera, Ana Parra, Eric Sung, Gary G Chiang and Kevin R Webster eFFECTOR Therapeutics, San Diego, CA

AbstractAn effective and durable T cell response is a cornerstone of current immunotherapies. Weshow that eFT508, a potent, selective inhibitor of MNK1 and MNK2, establishes aregulatory program that promotes multiple steps in the cancer immunity cycle includingexpansion of memory T cells and prevention of T cell exhaustion. Using OT-I and OT-IItransgenic systems, we show that eFT508 shifts the distribution of T cells towards aCD62LhighCD44high central memory (CM) phenotype in both CD4 and CD8 T cells uponactivation with SIINFEKL peptide in vitro without adverse effects on T cell proliferation,interferon-g production or cytotoxic function. Similar effects are seen in vivo, whereeFT508 treatment also enriches the CM T cell pool in a SIINFEKL vaccine-induced OT-Iadoptive T cell transfer model, which results in increased persistence as demonstrated by ahigher memory-recall T cell response upon re-challenge. In addition, the CM bias elicitedby eFT508 remains dominant when combined with agonists of co-stimulatory molecules,such as 4-1BB, OX-40 and GITR, or checkpoint inhibitors, such as PD-1, PD-L1 and CTLA-4,suggesting that eFT508 can affect the rate of T cell differentiation in these combinations.eFT508 treatment also reduces the expression of exhaustion markers such as PD-1, LAG3and TIM3 leading to increased cytotoxic T cell function. eFT508 is currently underevaluation as a single agent in two phase 1/2 clinical trials for patients with advanced solidtumors and patients with advanced lymphoma. In addition, a phase 2 study evaluatingeFT508, alone or in combination with avelumab, a PD-L1 immune checkpoint inhibitor, inmicrosatellite stable relapsed or refractory CRC patients is ongoing. The pre-clinical studiespresented here provide further evidence that eFT508 may combine well with additionalimmunotherapies beyond checkpoint blockade.

Introduction

Results

Conclusions• eFT508 promotes antigen presentation and formation of the TCM pool while

enhancing cytotoxic T cell function• eFT508 promotes central memory bias in combination with co-stimulatory

agonists or checkpoint antagonists• eFT508 modulates anti-tumor immunity and effectively synergizes with

immune checkpoint blockade in vivo• eFT508 is currently being evaluated in phase 1/2 clinical trials as a single

agent in patients with solid tumors (NCT02605083) and lymphoma(NCT02937675), and as a single agent and in combination with avelumab inMSS colorectal cancer (NCT03258398).

• eFFECTOR Therapeutics has designed eFT508, a potent, highly selective, small molecule inhibitor of MNK1 and MNK2 activity

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

• MNK selectively controls the translation of key regulators of the anti-tumor immuneresponse

5546

DO NOT POST

A. B.

A. B.

C. D.

(CD45, CD3, CD8, CD4, FOXP3) followed by flow cytometry analysis. The ratio of CD8+ to FOXP3+ cells isplotted. D) CT-26 allografts were treated as in (A) for seven days. Tumors were harvested, dissociated intosingle-cell suspensions and stained for immune cell surface markers (CD45, F4/80, CD206, MHC class II)followed by flow cytometry analysis. M2 macrophages were scored as CD206+/MHC class IIlow and plottedas a percentage of the total cell count.

Figure 8. eFT508 triggers anti-tumor immunityand enhances the efficacy of PD-1 immunecheckpoint blockade. A) CT-26 allografts weretreated with eFT508, anti-PD-1 antibody, or thecombination of eFT508 and anti-PD-1 at day 7post-implant for the indicated time. Tumorvolumes were measured and plotted as a functionof time. B) Naïve animals or animals from (A)which exhibited regression of tumors at d29 werere-challenged with CT-26 allografts in the absenceof any further treatment. Tumors were measuredat d10 post-implant. C) CT-26 allografts weretreated as in (A) for four days. Tumors wereharvested, dissociated into single cell suspensionsand stained for immune cell surface markers

Figure 7. eFT508 increases cytotoxic T cell function. Splenocytes from OT-I mice were stimulated withSIINFEKL peptide in the presence of the indicated concentrations of eFT508 for 3 d. OT-I splenocyteswere washed and mixed at a 10:1 ratio with B6.SJL splenocytes (1:1 mix of SIINFEKL-pulsed CellTracehigh

and unpulsed CellTracelow populations) for 16 h in the absence of eFT508. B6.SJL cells were gated byCD45.1 expression and analyzed for CellTrace Violet levels by flow cytometry. The % cell killing relative totarget cells alone is listed in red.

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

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

11% 49% 52%

53% 64% 73% 81%

A.

B. C. D.

Results

Figure 6. eFT508 induced central memory bias is retained in T cell stimulation assays when combined withco-stimulatory agonists or checkpoint antagonists. CellTrace Violet-labeled OT-I or OT-II splenocytes wereincubated with the indicated concentrations of eFT508 and corresponding peptides (5 µg/ml) and co-stimulatory or checkpoint or isotype antibodies (5 µg/ml) for 4 days. Cells were analyzed for CD4, CD8,CD44 and CD62L expression by flow cytometry. A) Representative scatter plots for proliferation. B) CD44and CD62L expression in OT-I CD8+ cells. CD44highCD62Llow define T effector memory cells (TEM) andCD44highCD62Lhigh define central T memory cells (TCM). C) CM/EM ratio of CD8+ T cell populations in OT-Icells. D) CM/EM ratio of CD4+ T cell populations in OT-II cells

OT-I Cells Alone SIINFEKL + 0.1 µM eFT508 + 1 µM eFT508 + 3 µM eFT508

+ Anti-4-1BB Ab

55 36 43 45 35 55 29 57

28 67 28 58 37 41 55

50 35 40 44 34 46 23 63+ Isotype Ab

A.

C. D.

Figure 5. eFT508 enhances T cell central memory pool in vivo and leads to higher memory-recall responses.OT-I T cells were adoptively transferred into B6.SJL mice (day 0). Mice were treated as indicated with 1 mg/kgeFT508 and/or immunized/boosted with 50 µg SIINFEKL peptide (days post-transfer). A) Spleens wereharvested at day 3 and CD45.2+CD8+ T cells were scored for CD44highCD62Lhigh (CM) expression by flowcytometry. Bars, average from animals in group (n=3); Error bars, SEM. B) Spleens were harvested on day 24and processed for flow cytometry analysis. CD45.2+CD8+CD44+ Memory T cells are plotted as a percentage oftotal lymphocytes from two independent experiments. Bars, average from animals in group (n=12 over twoexperiments); Error bars, SEM.

A. B.

+ Anti-4-1BB Ab

6 606661

OT-I Cells Alone SIINFEKL + 0.1 µM eFT508 + 1 µM eFT508 + 3 µM eFT508

58

5957 61 63

B.

Figure 1. eFT508 selectively downregulates expression of key immunosuppressive factors in activated Tcells. Primary human T cells were stimulated with α-CD3/CD28 antibodies in the presence of theindicated concentrations of compound. A) Whole cell lysates from T cells incubated for 24 h with eFT508were immunoblotted with the indicated antibodies. B) Activated T cells were treated for 24 h witheFT508 and analyzed for 4-1BB, PD-1, PD-L1, TIM3, LAG3, and cell viability by flow cytometry (% positivecells) or IL-10 secretion (pg/ml) by ELISA. Values plotted are % inhibition of each marker relative to theactivated vehicle (DMSO) control cells.

E.

Figure 3. eFT508 drives T cell activation andproliferation in the MLR setting. Peritonealmacrophages isolated from BALB/c mice wereincubated with the indicated concentrations ofeFT508 for 24 h, washed and then mixed withpanned CellTrace Violet-labeled splenocytesisolated from C57BL/6 mice in an MLR reactionfor an additional 4 days. Cells were analyzedfor CD4, CD8 and CellTrace Violet by flowcytometry.

Figure 4. eFT508 regulates T cell differentiation and enhances the formation of the T cell centralmemory pool. A) Peritoneal macrophages isolated from BALB/c mice were incubated with the indicatedconcentrations of eFT508 for 24 h and then mixed with panned splenocytes isolated from C57BL/6 micein an MLR reaction for an additional 4 days in the presence of the indicated concentrations of eFT508. B)Splenocytes from OT-I mice (C57BL/6-Tg(TcraTcrb)1100Mjb) were stimulated with 5 µg/ml SIINFEKLpeptide in the presence of the indicated concentrations of eFT508 for 4 days. In both experiments, cellswere analyzed for CD8, CD44 and CD62L expression by flow cytometry. Representative scatter plots forCD44 and CD62L expression in CD8+ cells are shown. CD44highCD62low define T effector memory cells(TEM) and CD44highCD62Lhigh define T central memory cells (TCM).

A.

B.

CD

44

CD62L

68

F.

eFT508 for 24 h. B-C) Cells were harvested and the CD14+ cells were analyzed for cell surface markers(HLA-DR, and CCR7) by flow cytometry analysis. D) BALB/c mice were dosed with vehicle or 1 mg/kgeFT508 daily for 2 days. Blood, spleen, and lymph node were harvested on day 3 and CD11c+ MHC-II (I-A/I-E)+ cells were analyzed by flow cytometry. E) Schematic for the MARCH1-dependent regulation ofMHC class II. F) mRNA from imMo-DCs differentiated into Mo-DCs in the presence of the indicatedconcentrations of eFT508 for 24 h was isolated and MARCH1 transcript levels were analyzed by qRT-PCR.

Figure 2. eFT508 increases keydendritic cell activation markersand trafficking in vivo. A) CD14+

cells isolated from human PBMCswere differentiated into maturemonocyte-derived dendritic cells(Mo-DCs) using the schemashown. Immature monocyte-derived-DCs (imMo-DCs) weredifferentiated in the presence ofthe indicated concentrations of