- - 0.1 1 3 - - 0.1 1 3 - - 0.1 1 3 - - 0.1 1 3 T CM /T EM ratio in OT-II Isotype Ab Anti-PD-1 Ab Anti-PD-L1 Ab Anti-CTLA4 Ab eFT508, µM OT-II peptide stim - + + + + - + + + + - + + + + - + + + + 0 1 2 3 T CM bias T EM bias - - 0.1 1 3 - - 0.1 1 3 - - 0.1 1 3 - - 0.1 1 3 T CM /T EM ratio in OT-I Isotype Ab Anti-4-1BB Ab Anti-OX40 Ab Anti-GITR Ab eFT508, µM OT-I peptide stim - ++++ - ++++ - ++++ - ++++ 1 0 2 3 T CM bias T EM bias Vehicle eFT508 0 2 4 6 8 10 CD8 + /FOXP3 + T cells Vehicle eFT508 0.0 0.5 1.0 1.5 % M2 macrophages in tumor 0 50 100 150 200 250 Tumor Volume (mm 3 ) anti-PD-1 Naïve eFT508 anti-PD-1 eFT508 8 12 15 19 22 26 29 0 500 1000 1500 2000 2500 3000 3500 Time, days Tumor Volume (mm 3 ) Vehicle eFT508 1 mg/kg QD anti-PD-1 0.5 mg Q4D eFT508 1mg/kg QD + anti-PD-1 0.5 mg Q4D Dosing 0.0 0.5 1.0 1.5 Day 24 post-transfer % CD8 + Memory T cells p < 0.0001 eFT508 (d1-7): - + - + Immunization (d1): - - + + Boost (d21): - - + + 0 20 40 60 Day 3 post-transfer % CM CD8 + T cells eFT508 (d1-3): - + - + Immunization (d1): - - + + p = 0.016 ns - - 0.01 0.1 1 3 - - 0.01 0.1 1 3 0 20 40 60 80 % Divided Cells eFT508, µM: T-cells T-cells + MΦ T-cells + MΦ T-cells CD4 + CD8 + Blood Spleen Lymph Node 0 10 20 30 % CD11c + MHC I-A/I-E + cells eFT508 1 mg/kg vehicle ** ns ns 0 0.01 0.1 1 3 10 0 2 4 6 CCR7 Fold change % CCR7 + cells imMo-DC Mo-DCs (µM eFT508) HLA-DR (MHC II) 0 0.01 0.1 1 10 0 100 200 300 400 500 HLA-DR expression, MFI Mo-DCs (µM eFT508) CD 14 imMo-DC - 0.1 1 3 10 0.0 0.5 1.0 1.5 MARCH1 MARCH1 expression fold change from vehicle eFT508, µM: -9 -8 -7 -6 -5 -4 0 20 40 60 80 100 Log [eFT508], M % of Activated Control PD-1 LAG3 4-1BB TIM3 PD-L1 IL-10 Viability eFT508, A Potent and Highly Selective Inhibitor of MNK 1/2, Regulates T Cell Differentiation Promoting an Anti-tumor Immune Response Rajesh 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 Abstract An effective and durable T cell response is a cornerstone of current immunotherapies. We show that eFT508, a potent, selective inhibitor of MNK1 and MNK2, establishes a regulatory program that promotes multiple steps in the cancer immunity cycle including expansion of memory T cells and prevention of T cell exhaustion. Using OT-I and OT-II transgenic systems, we show that eFT508 shifts the distribution of T cells towards a CD62L high CD44 high central memory (CM) phenotype in both CD4 and CD8 T cells upon activation with SIINFEKL peptide in vitro without adverse effects on T cell proliferation, interferon-g production or cytotoxic function. Similar effects are seen in vivo, where eFT508 treatment also enriches the CM T cell pool in a SIINFEKL vaccine-induced OT-I adoptive T cell transfer model, which results in increased persistence as demonstrated by a higher memory-recall T cell response upon re-challenge. In addition, the CM bias elicited by 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, LAG3 and TIM3 leading to increased cytotoxic T cell function. eFT508 is currently under evaluation as a single agent in two phase 1/2 clinical trials for patients with advanced solid tumors and patients with advanced lymphoma. In addition, a phase 2 study evaluating eFT508, alone or in combination with avelumab, a PD-L1 immune checkpoint inhibitor, in microsatellite stable relapsed or refractory CRC patients is ongoing. The pre-clinical studies presented here provide further evidence that eFT508 may combine well with additional immunotherapies beyond checkpoint blockade. Introduction Results Conclusions • eFT508 promotes antigen presentation and formation of the T CM 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 in MSS 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 immune response 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 is plotted. D) CT-26 allografts were treated as in (A) for seven days. Tumors were 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 scored as CD206 + /MHC class II low and plotted as a percentage of the total cell count. Figure 8. eFT508 triggers anti-tumor immunity and enhances the efficacy of PD-1 immune checkpoint blockade. A) CT-26 allografts were treated with eFT508, anti-PD-1 antibody, or the combination of eFT508 and anti-PD-1 at day 7 post-implant for the indicated time. Tumor volumes were measured and plotted as a function of time. B) Naïve animals or animals from (A) which exhibited regression of tumors at d29 were re-challenged with CT-26 allografts in the absence of any further treatment. Tumors were measured at d10 post-implant. C) CT-26 allografts were treated as in (A) for four days. Tumors were harvested, dissociated into single cell suspensions and stained for immune cell surface markers Figure 7. eFT508 increases cytotoxic T cell function. Splenocytes from OT-I mice were stimulated with SIINFEKL peptide in the presence of the indicated concentrations of eFT508 for 3 d. OT-I splenocytes were washed and mixed at a 10:1 ratio with B6.SJL splenocytes (1:1 mix of SIINFEKL-pulsed CellTrace high and unpulsed CellTrace low populations) for 16 h in the absence of eFT508. B6.SJL cells were gated by CD45.1 expression and analyzed for CellTrace Violet levels by flow cytometry. The % cell killing relative to target 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 with co-stimulatory agonists or checkpoint antagonists. CellTrace Violet-labeled OT-I or OT-II splenocytes were incubated 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) CD44 and CD62L expression in OT-I CD8 + cells. CD44 high CD62L low define T effector memory cells (T EM ) and CD44 high CD62L high define central T memory cells (T CM ). C) CM/EM ratio of CD8 + T cell populations in OT-I cells. 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/kg eFT508 and/or immunized/boosted with 50 µg SIINFEKL peptide (days post-transfer). A) Spleens were harvested at day 3 and CD45.2 + CD8 + T cells were scored for CD44 high CD62L high (CM) expression by flow cytometry. Bars, average from animals in group (n=3); Error bars, SEM. B) Spleens were harvested on day 24 and processed for flow cytometry analysis. CD45.2 + CD8 + CD44 + Memory T cells are plotted as a percentage of total lymphocytes from two independent experiments. Bars, average from animals in group (n=12 over two experiments); Error bars, SEM. A. B. + Anti-4-1BB Ab 6 60 66 61 OT-I Cells Alone SIINFEKL + 0.1 µM eFT508 + 1 µM eFT508 + 3 µM eFT508 58 59 57 61 63 B. Figure 1. eFT508 selectively downregulates expression of key immunosuppressive factors in activated T cells. Primary human T cells were stimulated with α-CD3/CD28 antibodies in the presence of the indicated concentrations of compound. A) Whole cell lysates from T cells incubated for 24 h with eFT508 were immunoblotted with the indicated antibodies. B) Activated T cells were treated for 24 h with eFT508 and analyzed for 4-1BB, PD-1, PD-L1, TIM3, LAG3, and cell viability by flow cytometry (% positive cells) or IL-10 secretion (pg/ml) by ELISA. Values plotted are % inhibition of each marker relative to the activated vehicle (DMSO) control cells. E. Figure 3. eFT508 drives T cell activation and proliferation in the MLR setting. Peritoneal macrophages isolated from BALB/c mice were incubated with the indicated concentrations of eFT508 for 24 h, washed and then mixed with panned CellTrace Violet-labeled splenocytes isolated from C57BL/6 mice in an MLR reaction for an additional 4 days. Cells were analyzed for CD4, CD8 and CellTrace Violet by flow cytometry. Figure 4. eFT508 regulates T cell differentiation and enhances the formation of the T cell central memory pool. A) Peritoneal macrophages isolated from BALB/c mice were incubated with the indicated concentrations of eFT508 for 24 h and then mixed with panned splenocytes isolated from C57BL/6 mice in 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 SIINFEKL peptide in the presence of the indicated concentrations of eFT508 for 4 days. In both experiments, cells were analyzed for CD8, CD44 and CD62L expression by flow cytometry. Representative scatter plots for CD44 and CD62L expression in CD8 + cells are shown. CD44 high CD62 low define T effector memory cells (T EM ) and CD44 high CD62L high define T central memory cells (T CM ). A. B. CD44 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/kg eFT508 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 of MHC class II. F) mRNA from imMo-DCs differentiated into Mo-DCs in the presence of the indicated concentrations of eFT508 for 24 h was isolated and MARCH1 transcript levels were analyzed by qRT- PCR. Figure 2. eFT508 increases key dendritic cell activation markers and trafficking in vivo.A) CD14 + cells isolated from human PBMCs were differentiated into mature monocyte-derived dendritic cells (Mo-DCs) using the schema shown. Immature monocyte- derived-DCs (imMo-DCs) were differentiated in the presence of the indicated concentrations of