A Fully Human anti-VISTA Antibody as a Promising Therapy Against Poorly Immunogenic Tumors Thierry Guillaudeux, Eric Tarcha, Robert Bader, Nathan Eyde, Emily Frazier, David Jurchen, Remington Lance, Cristina Loomis, Kurt Lustig, Yulia Ovechkina, David Peckham, Jeff Posakony, Shaarwari Sridhar, Mei Xu and Shawn Iadonato. Kineta Inc. 219 Terry Ave. North, Seattle, WA 98109. Background Kineta’s Anti-VISTA Antibodies Induce Monocyte Activation Kineta’s Anti-VISTA Antibodies Reverse MDSC Suppression of T Cells • VISTA (V-domain Ig Suppressor of T cell Activation) is a unique B7 family member, mainly expressed on myeloid cells, neutrophils, NK cells and Treg. Abs Enhance Activation of Monocytes & DCs • VISTA is highly expressed on MDSC and Treg in the TME and play an important role in immune modulation. PBMCs • VISTA is a negative regulator that directly suppresses T-cell activation and proliferation. KVA Ab IFNγ CFSE Dilution High VISTA expression correlates with poor survival in cancer patients. GM-CSF • 50 2000 • VISTA is a unique Immune checkpoint inhibitor for tumor immunotherapy. IL-6 /mL) 40 /mL) 1500 pg pg MDSCs Increasing Patient Survival Remains a Significant Unmet Need for Cancer Patients + CD14+ 30 1000 (pg/ml) CXCL10 ( CXCL10 ( Hot Tumors Cold Tumors PBMC Enriched CD14+ + 20 γ • Current Checkpoint therapies are effective and improve patient survival: Only for 20% • Not responsive to current and T cell centered checkpoint therapies IFN KVA Ab 500 of the Patient population. • Poor patient survival: 80% of the Patient population 10 % Proliferating Cells % Proliferating ↑ Anti-cancer T cells Solution = T cell targeted therapies ↓ Anti-cancer T cells : Anergy, MHCneg, No T cells Solution = TME targeted therapies 0 0 5.1 5.1 PD1 PD1 Monocyte activation 12.1 17.1 23.1 27.1 12.1 17.1 23.1 27.1 ↑ Immunosuppressive TME No Tx No Tx CEACAM 6000 500 Measure T Cell Pos Ctrl Pos Ctrl PD-L1 ↑ VISTA expression high and driver of Immunosuppression IgG1 Ctrl IgG1 Ctrl ILT4 27.1 KVA 27.1 LAG-3 5.1 KVA 5.1 Suppression TIM-3 TIGIT 5000 12.1 HVEM 400 KVA 12.1 ICOS BTLA IDO1 4000 PD-1 4-1BB GITR VISTA CTLA-4 300 Figure 7. 6E7 PBMCs were cultures for 7 days with GM-CSF and IL6 (10 ng/mL ea.) after which cells were collected and CD11b+ or CD33+ CD27 β 3000 OX40 CD47 TGF cells were isolated and evaluated for VISTA expression by flow cytometry. 1E5 MDSCs were added to 1E5 PBMCs labeled with Cell Trace in (CD80) MFI (HLA-DR) MFI IgG1 200 the presence or absence of 4 µg/mL anti-CD3 antibody and 10 µg/mL anti-VISTA antibody and incubated for 4 days. Cell proliferation of 2000 100ug/ml IgG1 100ug/ml CD3+ T cells was measured by flow cytometry and IFNγ secretion was measured using ProQuantum. 1000 100 Objectives 0 0 0 0 HLA-DR • Screen for specific fully human anti-VISTA antibodies. Figure 4. 0.5E5 CD14+ enriched human PBMCs were co-cultured with 2E5 human PBMCs in the presence of KVA Abs or KVA12 Antibody Induces Strong Anti-tumor Response as a • Evaluate and select an antagonist anti-VISTA antibody (KVA) as clinical candidate from in vitro and in vivo studies. control Abs tested at 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001 µg/ml for 24 hrs. Upregulation of activation markers on CD14+ • Characterize KVA12.1, our lead candidate for IND submission. cells at 24 hrs was quantified by Flow Cytometry on gated CD14+ live cells (MFI). CXCL10 production was measured by Single Agent or in Combo-therapies ELISA. Data are mean ± SD of n=3. Mean Tumor Volume Mean Tumor Volume IgG Control Mean Tumor Volume Results Anti-mPD1 2000 Anti-hVISTA (KVA12.2a) ) 3 2000 Control MsIgG2a ) KVA Mediated Monocyte Activation is Dependent on NK Cells 3000 Anti-mPD-1 + Anti-hVISTA (KVA12.2a) 3 Control MsIgG2a ) 3 KVA-12.2a WT KVA-12.2a WT 1500 1500 Kineta’s Anti-VISTA Antibodies (KVA) Bind to Cell-Surface Human and Monkey VISTA KVA-12.2a LALA KVA-12.2a LALA 2000 150 nM 1 nM 0.05 nM 1000 1000 1000000 150 nM 1 nM 0.05 nM 1000000 1000 100000 500 500 100000 Avg.tumor volume(mm Avg.tumor volume (mm Tumor Volume (mm 10000 MFI 0 0 10000 0 MFI 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 0 10 20 30 40 0 2 4 6 8 10 12 14 16 18 20 22 24 26 1000 Days Post Implantation 1000 Study Day Days Post Implantation 100 VISTA h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m 100 orthologue VISTA h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m h c m 3500 KVA-12.2a WT orthologue KVA-12.2a WT Control MsIgG2a ) Vehicle Anti-PD1 Control Ms IgG2a 3500 3 3000 3000 1.1 27.1 Pos Ctrl 1.1 27.1 Pos Ctrl ) 3 huIgG1 5000 5000 3000 ) 3 3000 Pos Ctrl KVA 1.1 KVA 4.1 KVA 5.1 KVA 6.1 KVA 7.1 KVA 8.1 KVA 9.1 ) ) ) 3 KVA 11.1 KVA 12.1 KVA 13.1 KVA 14.1 KVA 16.1 KVA 17.1 KVA 18.1 3 2500 2500 3 Neg Ctrl Neg Ctrl 2500 huIgG4 4000 4000 2500 KVA 19.1 KVA 20.1 KVA 21.1 KVA 22.1 KVA 23.1 KVA 25.1 KVA 26.1 KVA 27.1KVA 29.1 KVA 30.1 KVA 12.4 KVA 17.4 KVA 19.4 KVA 25.4 KVA 27.4 2000 2000 2000 2000 Antibody 3000 3000 1500 Antibody 1500 1500 1500 2000 2000 “h c m” indicates CHO cells expressing human, cyno and mouse VISTA, respectively. 1000 1000 1000 1000 1000 1000 500 Tumor Volume (mm Tumor Volume (mm 500 500 Tumor Volume (mm 500 Tumor Volume (mm Tumor Volume (mm Tumor Volume (mm 0 0 0 0 0 0 2 4 6 8 101214161820222426283032 0 0 10 20 30 40 0 2 4 6 8 101214161820222426283032 Figure 1. Anti-human VISTA antibodies titrate against CHO-K1 cell lines stably expressing human and cynomolgus, but not mouse VISTA. 1.5e5 VISTA CHO-K1 cells 0 10 20 30 40 Days 0 2 4 6 8 101214161820222426283032 0 2 4 6 8 101214161820222426283032 Study Day Study Day Days Days Days CHO were indirectly labeled with titrating amounts of human IgG1 or human IgG4 KVA series anti-VISTA antibodies and Gt anti-Human IgG (H+L-specific)-PE. PE Mean VISTA VISTA Fluorescence Intensity was determined on viable cells by flow cytometry. h=human, c=cynomolgus, m=mouse. Anti-PD-1 + Anti-VISTA KVA-12.2a LALA Anti-VISTA KVA-12.2a LALA 3500 5000 3000 ) 5000 ) 3 ) 3 3 ) 3000 3 4000 2500 4000 2500 2000 3000 KVA Antibodies Bind Only To VISTA and Not Other B7 Family Proteins 3000 2000 1500 2000 2000 1500 1000 1000 B7-1 (CD80) B7-2 (CD86) B7-H1 (PD-L1, CD274) B7-H4 (B7S1) B7-H3 (CD276) VISTA 1000 Tumor Volume (mm 1000 B7-H2 (ICOS Ligand) Tumor Volume (mm 500 B7-DC (PD-L2, CD273) 0 Tumor Volume (mm 2.5 2.5 2.5 2.5 2.5 3.0 0 Tumor Volume (mm 500 2.5 2.5 0 10 20 30 40 0 1.1 27.1 Pos Ctrl 1.1 27.1 Pos Ctrl 0 10 20 30 40 2.5 Study Day 0 2 4 6 8 101214161820222426283032 0 2.0 2.0 2.0 2.0 Study Day 2.0 2.0 2.0 Days 0 2 4 6 8 101214161820222426283032 Neg Ctrl Neg Ctrl 2.0 Days 1.5 1.5 1.5 1.5 1.5 1.5 1.5 450 450 450 450 450 450 450 450 1.5 OD OD OD OD OD OD OD 1.0 1.0 1.0 1.0 OD 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Figure 5. CD14+ enriched human PBMCs were co-cultured with either human PBMCs, or NK-depleted PBMCs from the 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 µ MC38 Tumor Model: 1E6 MC38 tumor cells were MB49 Tumor Model: 5E5 MB49 tumor cells were EG-7 Tumor Model: 1E6 EG-7 tumor cells were 1.1 5.1 8.1 1.1 5.1 8.1 1.1 5.1 8.1 1.1 5.1 8.1 1.1 5.1 8.1 1.1 5.1 8.1 1.1 5.1 8.1 1.1 5.1 8.1 same donor with KVA Abs or control Abs tested at 3, 0.3, 0.03 g/ml for 24 hrs. Upregulation of activation markers on 12.1 17.1 23.1 27.1 65.1 18.1 25.1 IgG1 12.1 17.1 23.1 27.1 65.1 18.1 25.1 IgG1 12.1 17.1 23.1 27.1 65.1 18.1 25.1 IgG1 12.1 17.1 23.1 27.1 65.1 18.1 25.1 IgG1 12.1 17.1 23.1 27.1 65.1 18.1 25.1 IgG1 12.1 17.1 23.1 27.1 65.1 18.1 25.1 IgG1 12.1 17.1 23.1 27.1 65.1 18.1 25.1 IgG1 12.1 17.1 23.1 27.1 65.1 18.1 25.1 IgG1 inoculated subcutaneously in the right rear flank region inoculated subcutaneously to female hVISTA inoculated subcutaneously to female hVISTA VSTB174 VSTB174 VSTB174 VSTB174 VSTB174 VSTB174 VSTB174 VSTB174 + + BackgroundPos.