Antitumor efficacy of anti-TIGIT antagonist antibody EOS884448 is mediated by a dual mechanism ofLGO action involving restoration of T cell effector functions and preferential depletion of Tregs. Catherine Hoofd, Julia Cuende, Virginie Rabolli, Julie Preillon, Noémie Wald, Lucile Garnero, Florence Lambolez, Shruthi Prassad, Marjorie Mercier, Florence Nyawame, Margreet Brouwer, Erica Houthuys, Véronique Bodo, Xavier Leroy, Michel Detheux and Gregory Driessens. HIGH TIGIT EXPRESSION ON IMMUNE CELLS, FURTHER SUMMARY TIGIT-DRIVEN IMMUNOSUPPRESSION INCREASED IN CANCER PATIENTS • T cell Immunoreceptor with Ig and ITIM domains (TIGIT) is a negative 1 A. Healthy donors B. Cancer patients costimulatory receptor that inhibits Teff and NK cell function and marks a highly 100 suppressive Treg subset. 2 80 CD4+ T cells CD8+ T cells 60 • TIGIT ligands belong to the PVR/nectin family, among which PVR (CD155) shows Naïve Tr e g 40 the highest affinity and is commonly expressed on antigen presenting cells (APC) Memory % TIGIT+ cells%TIGIT+ % of TIGIT expression Effector and tumor cells. 20 0 • CD226, a co-stimulatory receptor also expressed on NK and T cells, competes γδ TIGIT TCR with TIGIT for PVR binding but with a lower affinity. CD4 Treg CD4 naive T cells CD8 naive T cells CD4 effectorCD4 T cells memory T cells CD8 effectorCD8 T cells memory T cells 100 • TIGIT expression is increased on T and NK(T) cells from cancer patients and is Iguchi-Manaka et al. PLoS One 2016 Johnston RJ et al. Cancer Cell, 2014 80 Killing + correlated to poor outcome and response to aPD1 therapy in some indications. CD4+ T cells CD8 T cells 60 TIGIT phenotyping TIGIT Naïve Tr e g • iTeos developed an anti-human/cyno TIGIT blocking mAb (EOS884448) and a 40 1 drug = multiple anti-tumor mechanisms of action Memory % TIGIT+ cellsTIGIT+ % surrogate anti-mouse TIGIT mAb with comparable properties. % of TIGIT expression Effector 20 1. Reactivation of immune response by • EOS884448 properties and functionality make it an attractive Immuno-Oncology 0 • Suppressing TIGIT-mediated inhibitory signaling γδ TIGIT therapy candidate: TCR • Increasing ligand availibility for CD226 co-stimulatory receptor CD4 Treg ü Strong binding to primary human and cyno T cells (sub-nM Kd) CD4 naive T cells CD8 naive T cells CD4 effectorCD4 T cells memory T cells CD8 effectorCD8 T cells memory T cells ü Competition with natural ligands with IC50 in sub-nM range 2. Depletion of TIGIT+ highly suppressive Treg subpopulation and TIGIT+ tumor ü Increase of primary T cell functions in healthy donors and cancer patients Fig. 1. Flow cytometry analysis of TIGIT expression, gating on different lymphocyte subsets in healthy ü Strong antitumor efficacy in monotherapy and combination with aPD(L)-1 cells with ADCC-triggering isotype (FcyR dependent) donor (A) PBMCs (n=7). Frequency of TIGIT expressing cells is highest on CD4+ Tregs and effector and (with surrogate mAb) in mouse CT26 and Hepa 1-6 models. memory T cells and (B) further increases in cancer patient PBMCs or tumor infiltrated lymphocytes (TILs) (n=12). Infiltrated Tregs display both the highest frequency and density of TIGIT receptor. EOS884448 BINDS WITH HIGH AFFINITY TO HUMAN AND EOS884448 INCREASES IFNγ SECRETION IN T CELLS FROM EOS884448 PREFERENTIALLY DEPLETES TREG IN HUMAN CYNO TIGIT AND PREVENTS LIGAND BINDING TO TIGIT HEALTHY AND CANCER PATIENTS PBMCs FROM HEALTHY DONORS AND CANCER PATIENTS A. Primary CD8+ T cells A. Healthy donor CD8+ T cells Cancer patient CD3+ T cells A. Healthy donor PBMC Fortessa HD263 2.0 Ab at 66.6nM 150 EOS884448 affinity summary L 1600 a-TIGIT IgG2 µ 80 + 1200 400 a-TIGIT IgG1 Ab at 0.66 nM 800 Ab at 0.0066 nM 1.5 a-TIGIT IgG4 60 350 400 T cells 100 Iso at 66.6nM + 300 Isotype IgG2 200 300 Isotype IgG1 40 Iso at 0.666nM 1.0 100 CD8 100 Iso at 0.00666nM + 250 Isotype IgG4 Kd (nM) IFNg (pg/ml) 75 20 50 50 No Ab % of specific lysis T cells T 25 0.5 200 EC50 = 0.11nM Absolute number cells/ 0 Kd=0.08nM Total Memory Tregs Total Memory 0 of TIGIT Normalized frequency (on gated immune populations) Total Memory Tregs Total Memory -3 -2 -1 0 1 2 3 assay BindingCD8to 0 0.0 Log Ab concentration (nM) CD4+ CD8+ CD4+ CD8+ 0 2 4 6 8 10 HD CD8+ CP CD8+ Cyno CD8+ Antibody concentration (nM) invitro B. Cancer patient PBMC B. wuxi uprot 313M32 4.1.D3 B. Cancer patient PBMC or Dissociated tumor cells (DTC) Cancer type 1 Cancer type 3 Bmax 109.6 Bmax 113.1 Bmax 139.5 Bmax5000 146.1 100 Cancer type 2 Cancer type 4 Kd 0.08425 Kd 0.09253 n.s. Kd 1.267 Kd4000 0.8441 Functionalassays IFNγ TNFα IL-2 80 80 80 50 ADCC * 22G2 31C6 Arcus hIgG1_Isotype 60 * 3000 40 60 60 + EOS884448 + Bmax 121.9 Bmax 114.5 Bmax 104.1 + α γ 40 2000 30 Kd 0.4507 Kd 0.09426 Kd 0.1378 40 40 20 20 % IL-2 % of specific lysis 1000 % IFN 20 % TNF 20 MFI (CD155 signal) IC50 = 0.04nM 10 0 0 hCD155 0 0 0 (on gated immune populations) PBMC (CD3) DTC (CD4) DTC (CD8) PBMC (CD3) DTC (CD4) DTC (CD8) PBMC (CD3) DTC (CD4) DTC (CD8) Total Tregs Total B cells -6 -4 -2 0 2 4 memory memory CD19+ Competition with Competition Log Ab concentration (nM) (Rituximab CD4+ CD8+ activity) Fig. 2. EOS884448 displays a subnM affinity to primary T cells from healthy donors and cancer + + patients and potently prevents binding of TIGIT ligands. (A) Human PBMCs from healthy donors and Fig. 3. EOS884448 potently restores pro-inflammatory cytokines in presence of CD155 ligand. (A) Human Fig. 4. EOS884448, as an hIgG1, shows preferential depletion of Tregs over memory CD4 and CD8 T primary CD3+ or CD8+ T cells were stimulated with APC-like cells (CHO-TCR-CD155). Addition of aTIGIT during cells. (A). Absolute cell counts and depletion of different T cell populations expressing TIGIT is shown when cancer patients, as well as cynomolgous PBMCs were incubated with dose escalating concentrations of EOS884448. Kd of binding was calculated based on normalized frequency of TIGIT+CD8+ at the different stimulation prevents CD155-induced inhibition and increases IFNγ production in T cells from healthy donors and PBMCs are incubated in vitro in presence of EOS884448 for 20h and quantified by FACS. EOS884448 cancer patients. (B) EOS884448 restores intracellular cytokine release from frshly isolated cancer patient PBMC shows a preferential depletion of Treg cells at 66.6 nM or lower concentrations in healthy donors. Similar concentrations tested. (B) EOS884448 prevents CD155 ligand binding to TIGIT overexpressing Jurkat cells, or infiltrated lymphocytes after an overnight stimulation with aCD3/CD28. preferential depletion is observed in PBMC from cancer patients suffering from different solid tumor types (B). showing IC50 values at subnM range. iTEOS SURROGATE Ab SHOWS STRONG ANTITUMOR ITEOS SURROGATE Ab INCREASES CD8+ T CELLS AND ADENOSINE-DRIVENCONCLUSIONS IMMUNOSUPPRESSION EFFICACY IN MONOTHERAPY OR COMBO WITH a-PD-1 CYTOTOXICITY SIGNATURES IN VIVO A. CT26 syngeneic tumor model CT26 syngeneic tumor model EOS884448 is a highly potent antagonist aTIGIT mAb that: 30 treatment IFNg+CD8+ T cells 2000 **p=0.005 ** 3 100 mIgG2a iso ctrl 200 ug mIgG2a isotype 200 ug * + rando : TV=45 mm TV=45 : rando T cellsT v Binds with high affinity to human and cynomolgus TIGIT T cells. ) aTIGIT mIgG2a 200 ug + 3 1500 aTIGIT mIgG2a 200 ug 20 aPD-1 + mIgG2a iso ctrl 200 ug aPD-1 + mIgG2a isotype 200 ug aPD-1 + aTIGIT mIgG2a 200 ug aPD-1 + aTIGIT mIgG2a 200 ug v Competes for binding with TIGIT ligands. 1000 CD8 total in 50 + 10 CD8/Treg ratio CD8/Treg 3 IFNg P<0.0001 (vs isotype) hepa 1-6 median TV + 500 analysis v Increases IFNγ secretion on human primary T cells from healthy donors and Tumor volume (mm Survival t r e a t m e n t 1000 % CD8 rando : TV=60 mm TV=60 : rando % mice with tumor <1000mm 0 0 ) Flow cytometry a-TIGIT a-TIGIT 3 Ctrl P<0.0001 (vs PD-1) 20 25 30 35 40 0 mIgG1 mIgG2a cancer patients in vitro Days post-tumor cell implant Vehicle aPD-1 aPD-1+aTIGIT aPD-1+aTIGIT 10 15 20 25 30 35 mIgG1 mIgG2a Days post-tumor cell implant 500 ⇒ All at sub-nM concentrations B. Hepa1-6 syngeneic tumor3 model treatment hepa 1-6 median TV v Preferentially depletes human Tregs in vitro. 2000 PBS Tumor volume (mm aTIGIT mIgG1 200 ug t r e a t m e n t 1000 0 ) ) rando : TV=97 mm TV=97 : rando 3 aPD-1 3 rando : TV=60 mm TV=60 : rando 0 5 10 15 20 25 1500 aPD-1 + aTIGIT mIgG1 200 ug Days post-tumor cell implant Mediangrowthtumor 1000 PBS 500 p=0,0009 aTIGIT mIgG2a When replaced by a mouse surrogate with identical properties: 3 500 signatures Nanostring Tumor volume (mm 3 + + Tumor volume (mm v Inverts the immunosuppressive Treg/CD8 ratio and restores CD8 T cell 0 0 10 15 20 25 30 35 0 5 10 15 20 25 IFNg secretion in vivo. Days post-tumor cell implant Days post-tumor cell implant p=0,0009 PBS Fig. 6. Antitumor efficacy is associated with increased effector and cytotoxic functions. (A) Flow v Promotes antitumor immunity as monotherapy or in combination with Fig. 5. Surrogate mouse anti-TIGIT demonstrates potent isotype-dependentaTIGIT mIgG2aantitumor efficacy. The cytometry analysis of dissociated CT26 tumors, done 1 day after the last antibody treatment, demonstrated efficacy of mouse surrogate aTIGIT mAb antagonist was evaluated in established CT26 (A) and Hepa 1-6 strong increase of IFNγ producing CD8+ T cells and of the CD8+/ Tregs ratio within tumors when treated with the aPD-1 in CT26 colon carcinoma and Hepa 1-6 liver hepatoma mouse models.