Highly Potent Fully Human anti-VISTA Antibodies

A New Target Checkpoint Inhibitor against Immunosuppressive Myeloid Cells

Thierry Guillaudeux, PhD, Vice-President Immuno-Oncology

Kineta Inc., 219 Terry Av. North, Seattle, WA 98109. Seattle, USA

Thierry Guillaudeux, Eric Tarcha, Tina Albershardt, Robert Bader, Benjamin Dutzar, Nathan Eyde, Emily Frazier, David Jurchen, Remington Lance, Cristina Loomis, Kurt Lustig,

Yulia Ovechkina, David Peckham, Jeff Posakony, Rebecca Reeves, Shaarwari Sridhar, Mei Xu and Shawn Iadonato.

Kineta Inc. focuses on targets that integrate innate and adaptive immune response

VISTA a new Target Checkpoint Inhibitor

in Immunotherapy

VISTA a B7 family ligand

VISTA :

- Membrane protein of the Ig SF member of the B7 family

- Strongest similarity with PD1-L1

- Express on Myeloid cells, Neutrophils, NK cells and Treg

- Highly express on MDSC and Treg in the TME

Function

VISTA :

- Inhibitory effect on T cell activation

- APC-expressed VISTA interacts as a ligand with an unknown

receptor on activated T cells that results in co-inhibition

- Function as a co-inhibitory receptor expressed on T Cells

Ling Ni & Chen Dong Immunol. Reviews 2020

VISTA is predominantly expressed in the myeloid lineage within peripheral blood

M1 M2

PBMC + GMCSF + IL6 (7days)

CD33+, CD14+, HLA-DRdim

MDSC

PBMC + GMCSF + IFNg/LPS (5days) PBMC + MCSF + IL4/IL10 (5days)

Neutrophils

CD56lo NK

Classical Monocytes Intermediate Monocytes

B Cells CD4 CD8 Treg

Eosinophils BasophilsCD14+/CD16- CD14+/CD16+

Human Blood

VISTA is Highly expressed on Immune Cells

VISTA Has a Profound Negative Regulatory Effect on

T Cell Activation and Proliferation

0.1 1 10 100 1000

0

2000

4000

6000

mcg/ml Fc

IFN (

pg

/ml)

IgG1-Fc

VISTA ECD-Fc

0.1 1 10 100 1000

0

20

40

60

80

mcg/ml Fc

% P

rolife

rati

ng

Cells IgG1-Fc

VISTA ECD-Fc

Effect of VISTA-Fc on anti-CD3-induced T cell proliferation. Immobilized VISTA-Fc or IgG1-Fc was incubated on plates with

immobilized anti-CD3 (OKT3), and the ability of VISTA to suppress anti-CD3-induced T cell proliferation and cytokine production

was assessed. 96-well flat-bottomed plates were coated with 2 mcg/mL anti-CD3 +/- 0, 2, 6, 20 or 60 mcg/ml VISTA-Fc or IgG1-Fc.

Human pan-T cells from a healthy donor were labeled with CellTrace Violet and plated at 2E5 cells/well. After 4 days of incubation,

T cell proliferation was analyzed via flow cytometry; IFNγ concentrations were measured by ELISA. Representative graphs are

shown. Means of % proliferating cells or IFNγ production ± SEM are graphed.

T cell Proliferation (Flow Cytometry)T cell Proliferation (Flow Cytometry)

Cytokine Production (ELISA)

Kineta Is Developing Human Anti-VISTA Antibodies

▪ We have screened 107 fully human ScFv antibodies directed against VISTA

▪ We have evaluated and selected highly potent VISTA antagonist antibodies in vitro and in vivo

▪ Immunization of humanized Trianni® mice

⁻ In vivo affinity maturation in mouse leads to high affinity antibodies⁻ VDJ recombination allows unique fully human variable domains

▪ Microfluidics-based sequencing of single B cell clones

▪ Native pairing of heavy and light chains

▪ Flow-based enrichment using yeast ScFv display libraries

CD

R3H

Sim

ila

rity

Gro

up

sC

DR

3L

Gro

up

s

▪ 15 VH diversity groups

▪ 15 VL diversity groups

▪ Highest diversity in CDR3H

Kineta’s anti-VISTA Antibodies are Highly Potent

Concentration (ug/ml)

OD

450

0.00

10.

010.111010

0

0

1

2

317.1

26.1

1.1

4.1

6.1

7.1

9.1

11.1

14.1

16.1

22.1

27.1

29.1

Pos Ctrl

Concentration (ug/ml)

OD

450

0.00

10.

010.111010

0

0

1

2

3

435.1

43.1

63.1

65.1

74.1

77.1

96.1

36.1

38.1

39.1

98.1

34.1

91.1

102.1

Pos Ctrl

33.1

Concentration (ug/ml)

OD

450

0.00

10.

010.111010

0

0

1

2

3

437.1

48.1

95.1

106.1

66.1

81.1

82.1

Pos Ctrl

ELISA Binding

250125

62.53115

Association Dissociation500

27.1

Octet Binding

Log nM Ab

Lo

g M

FI

0.0001 0.001 0.01 0.1 1 10 100

2

3

4

5

KVA 1.1

KVA 4.1

KVA 5.1

KVA 6.1

KVA 7.1

KVA 8.1

KVA 9.1

KVA 11.1

KVA 12.1

KVA 13.1

KVA 14.1

KVA 16.1

KVA 17.1

KVA 18.1

KVA 19.1

KVA 20.1

KVA 21.1

KVA 22.1

KVA 23.1

KVA 25.1

KVA 26.1

KVA 27.1

KVA 29.1

KVA 30.1

KVA 12.4

KVA 17.4

KVA 27.4

KVA 25.4

KVA 19.4

huIgG1

huIgG4

Classical Monocytes

CD14+/CD16-Kasumi-3 (AML)

Flow Cytometry Binding

Log nM Ab

Lo

g M

FI

0.0001 0.001 0.01 0.1 1 10 100

2

3

4

5

KVA 1.1

KVA 4.1

KVA 5.1

KVA 6.1

KVA 7.1

KVA 8.1

KVA 9.1

KVA 11.1

KVA 12.1

KVA 13.1

KVA 14.1

KVA 16.1

KVA 17.1

KVA 18.1

KVA 19.1

KVA 20.1

KVA 21.1

KVA 22.1

KVA 23.1

KVA 25.1

KVA 26.1

KVA 27.1

KVA 29.1

KVA 30.1

KVA 12.4

KVA 17.4

KVA 27.4

KVA 25.4

KVA 19.4

huIgG1

huIgG4

Kineta’s anti-VISTA Antibodies Bind to

Cynomolgus Monkey VISTA-ECD

Forty-two (42) fully human monoclonal antibodies were

tested for cross-species binding to human and cynomolgus

monkey VISTA. Scatter plot of log mean OD450 values of

anti-VISTA antibodies (2 mcg/mL) with 1 mcg/mL plate

bound human or cyno VISTA ECD.

0.0 0.5 1.0 1.5 2.0 2.5

0.0

0.5

1.0

1.5

2.0

2.5

Cyno VISTA (OD450)

Hu

ma

n V

IST

A (

OD

45

0)

R2=0.9892Slope=1.042R2=0.99

Slope=1.0

ELISA Binding

▪ Kineta’s Anti-VISTA Antibodies are Highly Specific to Human and Cynomolgus Monkey VISTA but donot recognize Mouse VISTA

0.0 0.5 1.0 1.5 2.0

0.0

0.5

1.0

1.5

2.0

Cyno KD (nM)

Hu

ma

n K

D (

nM

)

R2=0.87Slope=1.4

Octet Binding

Lead monoclonal antibodies were tested for

cross-species binding to human, mouse and

cynomolgus monkey VISTA-ECD using Octet.

Scatter plot of mean KD values of anti-VISTA

antibodies immobilized on AHC biosensors and

interrogated with human or cyno VISTA ECD

(50nM).

HsV

IST

A

HsV

IST

A

KVA Ab

MfV

IST

A

MfV

IST

A

KVA Ab

KVA Ab VISTAAHC Sensor

VISTA Antagonist Antibody Induces Strong Anti-Tumor

Response as a Single Agent or in Combo-Therapies

Mean Tumor Volume(Error Bars represent SEM)

Days Post Implantation

Avg

. tu

mo

r vo

lum

e (

mm

3)

0 2 4 6 8 10 12 14 16 18 20 220

200

400

600

800

1000 Non-Treated

Ham-IgG+Rat-IgG

Anti-mPD-L1

Anti-mVISTA

Combo (aVISTA+aPD-L1)

Mean Tumor Volume(Error Bars represent SEM)

Days Post Implantation

Avg

. tu

mo

r vo

lum

e (

mm

3)

0 2 4 6 8 10 12 14 16 18 20 220

200

400

600

800

1000 Non-Treated

Anti-mVISTA

Ham-IgG+Ms-IgG

Anti-CTLA-4

Combo (aVISTA+aCTLA-4)

Flow Analysis of Whole Blood

Increases in Dendritic Cells Decreases in Myeloid Cells (MDSC)

Ctrl Anti-mVISTA

0.0

0.5

1.0

1.5

Blood - CD11c+ DCs

% L

ive

C

ells

**

Ctrl Anti-mVISTA

0

1

2

3

4

Blood - CD11b+ DCs

% L

ive

C

ells

**

Ctrl Anti-mVISTA

0

10

20

30

40

50

Blood - CD11b+

Myeloid Cells

% L

ive

C

ells

CT26 mouse model

VISTA and/or PDL-1

VISTA and/or CTLA-4

▪ 107 fully human ScFv anti-VISTA antibodies were generated and analyzed

▪ Kineta’s anti-VISTA antibodies carry unique sequences and the selected lead candidatesexhibit potencies in the subnanomolar range

▪ Kineta’s human anti-VISTA antibodies are highly specific and cross-react with Cyno-VISTAbut not Mouse-VISTA

▪ VISTA antagonist antibody induces strong anti-tumor response as a single agent or incombo-therapies with anti-PDL1 or anti-CTLA-4 in CT26 tumor models

▪ VISTA antagonist antibody activates Dendritic Cells in the blood and reduces MDSCs

Conclusions