Antibodies

• Anti-human KIT mAb

(KTN0158, Kolltan Pharmaceuticals)

• Anti-mouse KIT mAb (ACK2, Biolegend)

• Anti-CTLA-4 (UC10-4F10-11, BioXCell)

• Anti-PD-1 (RMP1-14, BioXCell)

• Anti-mast cell tryptase (AA1, Dako)

Immune Cell Flow Cytometry

T-Cell Panel: CD45, CD3, CD4, CD8, CD25, FoxP3.

Myeloid Cell Panel: CD45, CD3, CD11b,

Ly6G (g-MDSC), Ly6C (m-MDSC).

Dosing and Sampling Schedules (Flow

Cytometry Pharmacodynamic Studies)

Tumors were staged to 150-250 mm3, dosed q3d x2,

then sampled for flow cytometry at day 7.

Dosing Schedules (Efficacy Studies)

ACK2: 15 or 3 mg/kg, biweekly x2 (from day 3).

Anti-CTLA-4: 5 mg/kg x1 (day 8), 2.5 mg/kg (days

11, 14).

Anti-PD-1: 5 mg/kg biweekly x2 (from day 3).

Colon26 (cell line) and Pan02 (tumor fragments)

dosed from day 3 of implant.

CloudmanS91 (cell line) dosed from day 3 after

growth to 140-160 mm3.

CR indicates number of animals

exhibiting complete responses (no

measurable tumor mass) within

a treatment group.

• KTN0158 is a potent humanized anti-KIT mAb in clinical

development. Tumor-infiltrating mast cells represent a potential

target for anti-KIT antibodies within the tumor microenvironment.

• The combination of an anti-KIT antibody with immune checkpoint

inhibitors showed enhanced anti-tumor activity in the Colon26,

Pan02 and CloudmanS91 models.

• High pre-treatment m-MDSC counts are associated with reduced

survival in melanoma patients treated with checkpoint inhibitors.

• Anti-KIT treatment in vivo reduced m-MDSC numbers, which may

result in reduced suppression of anti-tumor immunity.

• The data support clinical evaluation of KTN0158 in combination

with anti-PD-(L)1 and/or anti-CTLA-4 for the treatment of cancer.

• KTN0158 is a humanized anti-KIT IgG1 monoclonal antibody that

binds to the extracellular domain of KIT, and is being developed as

a potential therapy for cancer (Phase 1 study: NCT02642016) and

other mast cell-related diseases such as neurofibromatosis type 1.

• Expression of KIT in immune cell types, including mast cells,

suggests the potential for additional roles of KIT in indirect

modulation of tumor progression.

• KIT may be involved in modulating the activity of mast cells and

MDSC’s in tumors (Danelli et al, Cancer Immunol Res. 2015;

Saleem et al, J Immunol. 2012; Pan et al, Blood 2008).

• In melanoma patients, prolonged overall survival is associated with

lower numbers of monocytic myeloid-derived supressor cells in

peripheral blood prior to treatment with ipilimumab or nivolumab

(Kitano et al, Cancer Immunol Res. 2014; Weber et al, Cancer

Immunol Res. 2016).

• The ability of anti-KIT mAb treatment to relieve immune

suppression and enhance anti-tumor activity of immune checkpoint

inhibitors was evaluated in a panel of preclinical tumor models.

Inhibition of KIT In Vivo Modifies Immune Cell Populations to Improve

The Efficacy of Checkpoint Inhibitors in Syngeneic Mouse Tumor Models

Anti-KIT Treatment Enhances the Anti-Tumor Activity of Anti-PD-1

in the CloudmanS91 Mouse Melanoma Tumor Model

Immune Cell Profiling of Colon26 Tumors Following Dosing

with Antibodies Targeting KIT, PD-1 and CTLA-4

Anti-KIT Treatment Enhances the Anti-Tumor Activity of Immune

Checkpoint Inhibitors in the Colon26 Tumor Model

Anti-KIT Treatment Reduces Monocytic Myeloid-Derived

Suppressor Cell Counts In Multiple Mouse Tumor Models

Andrew J. Garton, Lori Lopresti-Morrow, Scott Seibel, Theresa LaVallee, Richard Gedrich

Kolltan Pharmaceuticals, Inc., New Haven, CT

Introduction Results

Conclusions

4020

0 2 0 4 0 6 0

0

8 0 0

1 6 0 0

2 4 0 0

S tu d y D a y

Tu

mo

r V

olu

me

(m

m3

)

CTLA-4

CTLA-4 + KIT

PD-1

PD-1 + KIT

0 CR

2 CR

Control IgG

KIT

Control IgG

CTLA-4 + PD-1

Control IgG

CTLA-4

Control IgG

PD-1

0 2 0 4 0 6 0

0

8 0 0

1 6 0 0

2 4 0 0

S tu d y D a y

Tu

mo

r V

olu

me

(m

m3

)

0 2 0 4 0 6 0

0

8 0 0

1 6 0 0

2 4 0 0

S tu d y D a y

Tu

mo

r V

olu

me

(m

m3

)

0 2 0 4 0 6 0

0

8 0 0

1 6 0 0

2 4 0 0

S tu d y D a y

Tu

mo

r V

olu

me

(m

m3

)

0 2 0 4 0 6 0

0

8 0 0

1 6 0 0

2 4 0 0

S tu d y D a y

Tu

mo

r V

olu

me

(m

m3

)

0 2 0 4 0 6 0

0

8 0 0

1 6 0 0

2 4 0 0

S tu d y D a y

Tu

mo

r V

olu

me

(m

m3

)

Dosing Dosing Dosing

Dosing Dosing Dosing

CD4

T-Cells

CD8

T-Cells Treg g-MDSC m-MDSC

Control IgG

CTLA-4

PD-1

CTLA-4 + PD-1

KIT

CTLA-4 + KIT

PD-1 + KIT

Antibody Treatments

Treatment Group

Treatment Group

0

1 0

2 0

3 0

4 0

5 0

Tu

mo

r C

ell

Co

un

ts

(% o

f L

ive

Ce

lls

)

0 1 0 2 0 3 00

6 0 0

1 2 0 0

1 8 0 0

S tu d y D a y

Tu

mo

r V

olu

me

(m

m3

)

0 1 0 2 0 3 00

6 0 0

1 2 0 0

1 8 0 0

S tu d y D a y

Tu

mo

r V

olu

me

(m

m3

)

0 1 0 2 0 3 00

6 0 0

1 2 0 0

1 8 0 0

S tu d y D a y

Tu

mo

r V

olu

me

(m

m3

)

Dosing Dosing Dosing

CTLA-4

CTLA-4 + KIT (3 mg/kg)

Control IgG

CTLA-4

CTLA-4

CTLA-4 + KIT (15 mg/kg)

-1 4 -1 2 -1 0 -8 -6

0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

IC50

KTN0158

2.851e-010

ACK2

4.180e-010

lo g [A b ] (M )

Mo

us

e m

as

t c

ell

p-K

IT (

RL

U)

Hu

ma

n m

as

t ce

ll p-K

IT (R

LU

)

KTN0158 (human KIT)

ACK2 (mouse KIT)

IC50 285 pM

IC50 418 pM

Mast Cell Staining

of Colon26 Tumors

Toluidine Blue

Mast Cell Tryptase

Inhibition of Human and Mouse KIT in

SCF-Treated Mast Cells

• The potency of KIT inhibition in mouse mast cells by ACK2 was

comparable to KTN0158 inhibition of KIT in human mast cells.

• The immune cell content of Colon26 tumors included mast cells

identified by ex vivo staining of tumor tissue with toluidine blue and an

anti-mast cell tryptase antibody.

• Anti-CTLA-4 and anti-PD-1 were both efficacious as single agents in

the Colon26 model.

• Anti-KIT enhanced activity of both anti-CTLA-4 and anti-PD-1, but had

no activity as a single agent in the Colon26 model.

• Combination of anti-KIT and anti-PD-1, or anti-CTLA-4 and anti-PD-1,

yielded additional anti-tumor activity in the CloudmanS91 melanoma

model compared to single agent treatments.

The Combination of Anti-KIT and Anti-CTLA-4 Exhibits Anti-Tumor

Activity in the Pan02 Mouse Pancreatic Tumor Model

• The combination of anti-KIT and anti-CTLA-4 treatment yielded anti-

tumor activity in the Pan02 pancreatic tumor model.

• Anti-CTLA-4 and anti-PD-1 did not exhibit strong anti-tumor activity in

the Pan02 model when dosed as single agents or in combination.

• Anti-KIT treatment reduced monocytic myeloid-derived suppressor

cell (m-MDSC) counts in Colon26 tumors.

• Anti-KIT treatment did not further enhance CD8+ T-cell infiltrates

induced by anti-CTLA-4 or anti-PD-1 treatments.

• m-MDSC numbers were reduced in the spleens of tumor-bearing

mice following dosing with an anti-KIT mAb, both in the absence

and presence of anti-CTLA-4 or anti-PD-1.

Potential Mechanism for Enhanced Efficacy of Immune

Checkpoint Inhibitors in Combination with a KIT mAb

KIT mAb reduced m-MDSC

KIT and SCF Expression in Mouse Tumor Cell Lines

Methods

Co

lon

26

4T

1

Clo

ud

ma

n

S91

MC

38

mK

IT-C

HO

KIT Protein Expression

in Mouse Cell Lines Secretion of SCF by Mouse Cell Lines

Colon26 4T1 Cloudman

S91

MC38 0

2 5 0

5 0 0

7 5 0

1 0 0 0

[SC

F](

pg

/ml)

Colon26 4T1 CloudmanS91 MC38

Lack of Effect of SCF and KIT Inhibition on Cell Growth In Vitro

0 .0 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

Re

lati

ve

Ce

ll G

ro

wth

(vs

. M

ed

ium

Co

ntr

ol)

L o w S e ru m - S C F

L o w S e ru m + S C F

L o w S e ru m + S C F + K IT m A b

G ro w th M e d iu m

G ro w th M e d iu m + K IT m A b

• No evidence for KIT-dependent proliferation in mouse tumor cell

lines in vitro, regardless of KIT or SCF expression levels

Growth Medium

Conditioned

Medium

Anti-KIT Treatment Inhibits KIT Phosphorylation in Mast Cells and

Mast Cells are Present in the Tumor Microenvironment

Colon26 (Tumor Fragments)

Pan02 (Tumor Fragments)

Colon26 (Cell line)

Antibody Treatments

Control IgG

CTLA-4 + PD-1

PD-1

CTLA-4 ▲

Control mice

(No-tumors)

KIT ▼

PD-1 + KIT

CTLA-4 + KIT

0

5

1 0

1 5

T re a tm e n t G ro u p s

M-M

DS

C's

(%

of

Liv

e C

ell

s)

Sp

leen

m-M

DS

C C

ou

nts

(% o

f L

ive C

ells)

0 CR

2 CR 1 CR

2 CR

0 CR

0 CR

0 CR

0 CR

0 CR

1 CR

0 1 0 2 0 3 0

0

2 5 0

5 0 0

7 5 0

1 0 0 0

S tu d y D a y

Me

dia

n T

um

or V

olu

me

(m

m3)

C o n tro l Ig G

A n ti-P D -1

A n ti-C T L A -4

A n ti-C T L A -4 + A n ti-P D -1

A n t i-K IT 3 m g /k g

A n ti-K IT 1 5 m g /k g

A n ti-C T L A -4 + A n ti-K IT 3 m g /k g

A n ti-C T L A -4 + A n t i-K IT 1 5 m g /k g

A n ti-P D -1 + A n ti-K IT 1 5 m g /k g

Study Day

Dosing