Highly Potent Fully Human anti-VISTA Antibodies A New Target Checkpoint Inhibitor against Immunosuppressive Myeloid Cells. Thierry Guillaudeux1, Eric Tarcha1, Tina Albershardt1, Robert Bader1, Benjamin Dutzar1, Nathan Eyde1, Emily Frazier1, David Jurchen1, Remington Lance1, Cristina Loomis1, Kurt Lustig1, Yulia Ovechkina1, David Peckham1, Jeff Posakony1, Rebecca Reeves1, Shaarwari Sridhar1, Mei Xu1 and Shawn Iadonato1. 1 Kineta Inc. 219 Terry Av. North, Seattle, WA 98109.

Background Results . VISTA (V-domain Ig Suppressor of Activation) is a unique family member that functions as both and Exceptional Antibody Diversity in Both Heavy and Light Chain Kineta’s anti-VISTA Antibodies Bind Cyno VISTA-ECD but not Mouse VISTA-ECD . VISTA is mainly expressed in Immune cells and mostly on Myeloid cells, Neutrophils, NK cells and Treg 107 fully human ScFv antibodies directed . VISTA is highly expressed on MDSC and Treg in the TME 2.5 RR22=0.9892=0.99 ELISA Binding Figure 5 : Forty-two (42) fully . VISTA is a negative regulator that directly suppresses T cell activation and proliferation against Human VISTA were generated Slope=1.042Slope=1.0 2.0 human monoclonal antibodies were . High VISTA expression correlates with poor survival in patients tested for cross-species binding to 1.5 . VISTA is a unique inhibitor for tumor immunotherapy . 15 VH diversity groups human and cynomolgus monkey VISTA. Scatter plot of log mean CDR3H Similarity Groups Similarity CDR3H . 15 VL diversity groups 1.0 OD450 values of plate-bound human VISTA is predominantly expressed in the myeloid lineage within peripheral blood . Highest diversity in CDR3H 0.5

Human VISTA (OD450) Human VISTA or cyno VISTA ECD (1mcg/mL) with

0.0 anti-VISTA antibodies (2mcg/mL) Human PBMCs Human Blood 0.0 0.5 1.0 1.5 2.0 2.5 are represented. Cyno VISTA (OD450) VISTA+

Classical MonocytesIntermediate CDR3L Groups (29.3%) -

- Neutrophils Eosinophils Basophils VISTA+ CD14+/CD16- CD14+/CD16+ A A CD3+ T SSC SSC cells (5.2%) VISTA CD3 Kineta’s anti-VISTA Antibodies are Highly Potent VISTA Antagonist Antibody Induces Strong Anti-tumor ELISA Binding Response as a Single Agent or in Combo-therapies CD3- Myeloid cells 3 3 90% 1.1 17.1 5.1 25.1 CT26 mouse model 4.1 22.1 8.1 30.1 6.1 26.1 12.1 12.4 CD4 CD8 2 2 B Cells Treg CD56lo NK 7.1 27.1 13.1 17.4 450 450 CD14 CD11c CD14 Mean Tumor Volume Mean Tumor Volume - Others + 9.1 29.1 18.1 19.4 OD CD14 CD14 OD (Error Bars represent SEM) (Error Bars represent SEM) Myeloid DCs 6.3% Monocytes 1 11.1 Pos Ctrl 1 19.1 25.4 20.1 27.4 (6.5%) CD11b (82.0%) 14.1 ) 16.1 21.1 Pos Ctrl ) 1000 Non-Treated 1000 Non-Treated 3 3 0 0 23.1 Ham-IgG+Rat-IgG Anti-mVISTA 1 1 MDSC 10 0.1 10 0.1 M1 M2 100 0.01 100 0.01 0.001 0.001 800 Anti-mVISTA 800 Ham-IgG+Ms-IgG Concentration (ug/ml) Concentration (ug/ml) Anti-mPD-L1 Anti-CTLA-4 4 4 37.1 . 63.1 35.1 600 Combo (aVISTA+aPD-L1) 600 Combo (aVISTA+aCTLA-4) Granulocytes, Monocytes, Dendritic cells, Treg and NK cells highly 65.1 98.1 48.1 3 3 74.1 43.1 66.1 PBMC + GMCSF + IFNg/LPS (5days) PBMC + MCSF + IL4/IL10 (5days) PBMC + GMCSF + IL6 (7days) express VISTA on their surface 77.1 34.1 81.1 450 CD33+, CD14+, HLA-DRdim 450 400 2 96.1 91.1 2 82.1 400 OD . M2 and MDSC differentiated cells in vitro highly express VISTA OD 36.1 102.1 95.1 1 38.1 33.1 1 106.1 200 39.1 Pos Ctrl Pos Ctrl 200

0 0 Avg. tumorvolume (mm Avg. tumorvolume (mm 1 1 10 10 0.1 VISTA Has Profound Negative Regulatory Effect on T Cell Activation and Proliferation 0.1 100 0.01 100 0.01 0 0.001 0.001 0 Concentration (ug/ml) Concentration (ug/ml) 0 2 4 6 8 10 12 14 16 18 20 22 0 2 4 6 8 10 12 14 16 18 20 22 T cell Proliferation (Flow Cytometry) T cell Proliferation (Flow Cytometry) Figure 2 : Anti-VISTA antibodies were evaluated for binding to human VISTA-ECD. Antibodies were Days Post Implantation Days Post Implantation

80 tittered (10 mcg/ml – 0.003 mcg/ml, 67 – 0.020 nM) on a nickel ELISA plate coated with 50 ng/well IgG1-Fc human VISTA-ECD (his-tag). Biotinylated anti-human IgG(H+L) followed by HRP-streptavidin were 60 VISTA ECD-Fc Flow Analysis of Whole Blood used to detect the anti-VISTA antibodies. Plot of OD450 values of anti-VISTA antibodies. Increases in Dendritic Cells Decreases in Myeloid Cells (MDSC) 40

20 Octet Kinetic Blood - CD11b+ DCs Blood - CD11c+ DCs Blood - CD11b+

% Proliferating Cells 4 Myeloid Cells 27.1 Association Dissociation ** 1.5 ** 0 500 50 0.1 1 10 100 1000 250 3 125 mcg/ml Fc 40 62.5 1.0 31 15 2 30 Cytokine Production (ELISA) 0.5 20 % Live Cells

1 % Live Cells % Live Cells 10 6000 IgG1-Fc 0.0 VISTA ECD-Fc Figure 3 : Kinetic binding studies were performed as 0 0 follows: the purified hu.Abs were captured at 25 mcg/ml Ctrl Anti-mVISTA Ctrl Anti-mVISTA Ctrl Anti-mVISTA Figure 1: Effect of VISTA-Fc on anti-CD3-induced T cell proliferation. Immobilized VISTA-Fc or 4000 using Anti-human IgG Fc Capture (AHC) dip and read

IgG1-Fc was incubated on plates with immobilized anti-CD3 (OKT3), and the ability of VISTA to (pg/ml) γ biosensors. Sensors were dipped human or cyno VISTA- 2000 suppress anti-CD3-induced T cell proliferation and cytokine production was assessed. 96-well IFN Figure 6 : CT26 Tumor Model. 5.E5 CT26 cells were inoculated in PBS subcutaneously to female BALB/c mice. flat-bottomed plates were coated with 2 mcg/mL anti-CD3 +/- 0, 2, 6, 20 or 60 mcg/ml VISTA-Fc ECD in assay buffer (PBS, pH7.4) at 500, 250, 125, 62.5 Tumor volume (L x W2 x 0.5) and bodyweight were measured 3X/wk. Beginning on Day 3, animals were 31.3, 15.6 and 0 nM. Probes were dipped into assay or IgG1-Fc. Human pan-T cells from healthy donor were labeled with CellTrace Violet and plated 0 treated by IP injection of 200 mcg control antibodies (hamster + rat or mouse isotype ctrl), anti-mVISTA 0.1 1 10 100 1000 buffer and the dissociation rate was measured. 1:1 curve at 2E5 cells/well. After 4 days of incubation, T cell proliferation was analyzed via flow cytometry; mcg/ml Fc (13F3, hamster) and/or anti-mPD-L1 or anti-CTLA-4 3X/wk for 3 weeks. Mean tumor volume is plotted. IFNγ concentrations were measured by ELISA. Representative graphs are shown. Means of % fitting analysis was performed to determine kon, koff, KD. Whole blood was collected for immunophenotyping on Day 14 by flow cytometry for the following immune proliferating cells or IFNγ production ± SEM are graphed. N=1 each. Scouting data at 50 nM are included. populations: CD45, CD3, CD4, CD8, CD11b, CD11c, and NKp46.

5 Flow Cytometry Binding Objectives 27.1-AF647 Conclusions 4 Log nM Ab

. Screen for specific fully human anti-VISTA antibodies KVA 1.1 KVA 12.1 KVA 21.1 KVA 12.4 KVA 4.1 KVA 13.1 KVA 22.1 KVA 17.4 . 107 fully human ScFv anti-VISTA antibodies were generated and analyzed KVA 5.1 KVA 14.1 KVA 27.4 3 KVA 23.1 . Evaluate and select highly potent VISTA antagonist antibodies in vitro and in vivo MFILog KVA 6.1 KVA 16.1 KVA 25.1 KVA 25.4 KVA 7.1 KVA 17.1 KVA 26.1 KVA 19.4 KVA 8.1 KVA 18.1 KVA 27.1 huIgG4 . Kineta’s anti-VISTA antibodies carry unique sequences and the selected lead candidates huIgG1 KVA 9.1 KVA 19.1 KVA 29.1 KVA 11.1 KVA 20.1 KVA 30.1 . Immunization of humanized Trianni® mice 2 exhibit potencies in the subnanomolar range

⁻ In vivo affinity maturation in mouse leads to high affinity antibodies 0.0001 0.001 0.01 0.1 1 10 100 . Classical Monocytes Kasumi-3 (AML) Kineta’s human anti-VISTA antibodies are highly specific and cross-react with Cyno-VISTA ⁻ VDJ recombination allows unique fully human variable domains Log nM Ab CD14+/CD16- but not Mouse-VISTA Figure 4: Dose dependent binding of Kineta’s mAbs to CHO cell surface expressed human and cyno

VISTA by flow cytometry (Left) . Prototypic Kineta’s mAb binding on Human Classical Monocytes as . . Microfluidics-based sequencing of single B cell clones VISTA antagonist antibody induces strong anti-tumor response as a single agent or in

well as Kasumi -3 cell line (Right). . Native pairing of heavy and light chains combo-therapies with anti-PDL1 or anti-CTLA4 in CT26 tumor models

. Flow-based enrichment using yeast ScFv display libraries . VISTA antagonist antibody activates Dendritic Cells in the blood and reduces MDSCs