Claudin 18.2 - 4-1BB bispecific antibody induced potent tumor inhibition through tumor-specific 4-1BB activation.

Wenqing Jiang1, Lei Fang1, Zhengyi Wang1, Taylor B Guo1. Eunyoung Park2, Eunsil Sung2, Jaeho Jung2.

1. I-MAB BIOPHARMA, Shanghai, China 2. ABL Bio, INC., Gyeonggi-do, Republic of Korea.

Introduction

Claudin 18.2 (CLDN18.2) is a gastric-specific membrane protein. In the healthy

tissue, CLDN18.2 expression is restricted to the gastric mucosa as a component of

tight junction. However, it is ectopically expressed at significant levels in a variety of

primary lesion and metastases of epithelial tumor entities, including gastric,

pancreatic, esophageal, and lung adenocarcinoma cells. Taken together, CLDN18.2

is believed to be an ideal tumor antigen for immunotherapy. Here, we reported the

development of a bispecific antibody CLDN18.2-4-1BB (TJ-CD4B). TJ-CD4B showed

stronger binding capability to CLDN18.2 when compared with benchmark CLDN18.2

monoclonal antibody (IMAB362). Functional evaluation of TJ-CD4B indicated the

activation of 4-1BB signaling was solely dependent on CLDN18.2 expression on the

cell. Furthermore, TJ-CD4B showed superior 4-1BB activity than benchmark 4-1BB

monoclonal antibody (Urelumab) in the presence of cells expressing a wide range of

CLDN18.2 level including CLDN18.2-low cells. Using a humanized 4-1BB mouse

model, TJ-CD4B showed strong tumor growth inhibition (TGI) of CLDN18.2

expressing tumor cells, with an increase of tumor infiltrating lymphocytes. Our data

suggested TJ-CD4B activated 4-1BB in a CLDN18.2-dependent manner, thus

addressing the safety concern associated with 4-1BB-based therapies. TJ-CD4B is a

promising IO therapeutic option for gastric and other CLDN18.2-positive tumors.

RESULTS

Design of anti-CLDN18.2-4-1BB BsAb

CONCLUSIONS

▪ TJ-CD4B is a bispecific antibody that can recognize cells expressing wide range of CLDN18.2.

▪ TJ-CD4B activated 4-1BB signaling to enhance T cell activation in a CLDN18.2-dependent manner.

▪ TJ-CD4B showed significant inhibition of tumor growth (7/7 tumor free) in a syngeneic mouse model. Furthermore, TJ-CD4B can induce long-term protective immunological

memory.

▪ Ex vivo analysis suggested TJ-CD4B increased CD8+ T cell number in tumor microenvironment with no impact on peripheral lymphocytes.

Anti-CLDN18.2 mAbAnti-CLDN18.2 mAb

Anti-4-1BB scFvAnti-4-1BB scFv

▪ Highly potent anti-CLDN18.2 mAb

▪ Benefit patients with wide range of CLDN18.2

expression.

▪ IgG1(N297A)

▪ No ADCC or CDC

▪ Minimize FcgR mediated 4-1BB clustering

▪ Unique 4-1BB binding epitope

▪ CLDN18.2 dependent 4-1BB activation

▪ Potent 4-1BB agonism

Superior CLDN18.2 binding potency than benchmark antibody

0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

C H O -K 1 C 1 8 .2 H ig h *

C o n c e n tra t io n (n M )

MF

I

T J -C D 4 B

IM A B 3 6 2

0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

C H O -K 1 C 1 8 .2 L o w **

C o n c e n tra t io n (n M )

MF

I

T J -C D 4 B

IM A B 3 6 2

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

S N U 6 2 0

(E n d o g e n o u s C 1 8 .2 )

C o n c e n tra t io n (n M )

MF

I

T J -C D 4 B

IM A B 3 6 2

CLDN18.2-dependent 4-1BB agonism

CLDN18.2- cells

-6 -4 -2 0 2 4

0

5 0 0 0 0 0

1 0 0 0 0 0 0

1 5 0 0 0 0 0

2 0 0 0 0 0 0

2 5 0 0 0 0 0

C o n c e n tra t io n [n M ]

RL

U

CLDN18.2+ cells

-8 -6 -4 -2 0 2 4

0

5 0 0 0 0 0

1 0 0 0 0 0 0

1 5 0 0 0 0 0

2 0 0 0 0 0 0

2 5 0 0 0 0 0

C o n c e n tra t io n [n M ]

RL

U

T J -C D 4 B

U re lu m a b

1 A 1 0

4-1BB reporter assay

Ig G U re lu m a b T J -C D 4 B

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

IL2

(n

g/m

l)

CLDN18.2- cells

Ig G U re lu m a b T J -C D 4 B

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

IL2

(n

g/m

l)

2 0 n M

4

0 .8

0 .1 6

0 .0 3 2

0 .0 0 6 4

CLDN18.2+ cells

Primary CD8+ T / CHO-K1 coculture assay

CLDN18.2– cells

CD8+ T cell

CLDN18.2+ cells/Tumor

CD8+ T cell

Significant tumor growth inhibition and increased TIL

0 3 6 9 1 2 1 5 1 8 2 1 2 4 2 7 3 0 3 3 3 6 3 9 4 2 4 5 4 8 5 1 5 4 5 7 6 0

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

Tu

mo

r V

olu

me

mm

3

D a ys a fte r 1 s t c h a lle n g e

h Ig G (1 0 m p k )

T J -C D 4 B (1 3 .3 m p k )

C L D N 1 8 .2 m A b (1 0 m p k )

4 -1 B B m A b (1 0 m p k )

C o m b o (1 0 + 1 0 m p k )

IM A B 3 6 2 (1 0 m p k )

N a ivet u m o r r e c h a l le n g etu m o r c h a l le n g e

B IW * 6 d o s e s

Hu4-1BB mice

MC38-

huCLDN18.2 TV=102mm3

BIW × 6 doses

BIW × 2 doses TIL analysis (n=3)

TGI, Tumor Re-challenge （n=7）

MC38-

huCLDN18.2

hIg

G

TJ-C

D4B

CL

DN

18.2

mA

b

4-1

BB

mA

b

Co

mb

o

IMA

B362

0

2 0

4 0

6 0

% C

D8

+ i

n C

D3

+ T

**

C D 8

hIg

G

TJ-C

D4B

CL

DN

18.2

mA

b

4-1

BB

mA

b

Co

mb

o

IMA

B362

3 0

3 5

4 0

4 5

5 0

% C

D8

in

CD

3+

T

C D 8

hIg

G

TJ-C

D4B

CL

DN

18.2

mA

b

4-1

BB

mA

b

Co

mb

o

IMA

B362

0

1 0

2 0

3 0

4 0

% C

D4

5 i

n l

ive

ce

lls

*C D 4 5

hIg

G

TJ-C

D4B

CL

DN

18.2

mA

b

4-1

BB

mA

b

Co

mb

o

IMA

B362

8 5

9 0

9 5

1 0 0

% C

D4

5 i

n L

ive

ce

lls

C D 4 5

Tumor infiltrating lymphocytes

Peripheral blood

Cell-based binding of CLDN18.2 by CLDN18.2-4-1BB bispecific antibodies. Antibodies were 3-fold serially diluted and incubated

with 1X105 CHO-K1-C18.2 and SNU620 for 30 minutes at 4 °C in FACS buffer. Then, APC conjugated-anti-human IgG antibody was

added at 4°C for another 30 minutes. After washing, MFI of APC was evaluated by FACS. * FACS-sorted CLDN18.2 higher expression

clone；** FACS-sorted CLDN18.2 lower expression clone； ¶ SNU620 is a gastric carcinoma cell line with very low CLDN18.2

expression on the cell surface. IMAB362 is the benchmark CLDN18.2 monospecific antibody.

Generation of anti-CLDN18.2-4-1BB bispecific antibody. Lead CLDN18.2 mAbs were identified for binding to human CLDN18.2.

Binders with high affinity were fused with anti-4-1BB antibody to generate different formats of anti-CLDN18.2-4-1BB bispecific

antibodies. Final candidates were selected based on in vitro and in vivo functional evaluation and drug developability.

4-1BB reporter assay. GloResponseTM NFkB-luc2/4-1BB Jurkat cell at a density of 2.5X104 cells per well were mixed with 2.5X104

target cells in a white 96-well plate. Antibodies were serially diluted and added to a white 96-well assay plate. After 6 hours’ incubation

at 37 C, luminescence was obtained by adding the substrate of luciferase and measured by a microplate reader. Urelumab is the

benchmark 4-1BB mono-specific antibody. 1A10 is a 4-1BB human IgG, the sequence of which was used to generate TJ-CD4B.

Primary CD8+T assay. Human CD8+ T cells (1×105) stimulated with 0.5 mg/ml human anti-CD3 antibody were co-cultured with CHO-

K1-CLDN18.2 or CHO-K1. Serially diluted antibodies were added and incubated with co-culture. After 48 hours, levels of IL2 in the

culture medium were measured using IL-2 LANCE Ultra TR-FRET Detection Kit (PerkinElmer).

Primary PBMC assay. Gastric adenocarcinoma(GA) PDX-derived cells were coculture with human PBMC (1×105) from the same

donor. Coculture was stimulated with 0.5 mg/ml human anti-CD3 antibody and incubated with serially diluted antibodies. After 48 hours,

levels of IL2 in the culture medium were measured using IL-2 LANCE Ultra TR-FRET Detection Kit (PerkinElmer).

0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0

0

5

1 0

1 5

3 0

4 5

C o n c e n tra t io n (n M )

IL2

(n

g/m

l)

G A 0 0 0 6

G A 0 0 4 4

G A 0 0 6 0

G A 3 0 5 5

P rim a ry P B M C + P D X C o c u ltu reGA0006 GA0044 GA0060

C1

8.2

IHC

IHC H score

223 156 74

In vivo efficacy study. MC38-hCLDN18.2 cells were subcutaneously implanted into hu4-1BB mice. When the tumor grew to an

average of 102 mm3, mice (n=7/group) were intraperitoneally treated with human IgG, CLDN18.2 mAb and 4-1BB mAb alone or in

combination, IMAB362 and BsAb at equal molar amount twice weekly for 6 doses. For the re-challenge study, a second dose of

MC38-hCLDN18.2 cells were injected in the contralateral flank after the initial tumor challenge. Tumor growth was monitored by

volumetric measurement. Ex vivo analysis of FACS analysis of intratumoral and peripheral CD45+ cells and CD8+ T cells were

performed in a subgroup (n=3/group). * n<0.05; **n <0.01

Primary PBMC / PDX coculture assay

4-1BB

4-1BB

CLDN18.2

¶