10-10 10-9 10-8

0

20

40

60

80

concentration [M]

cyto

toxi

city

[%]

136.2

EC50: 620 pM

10-10 10-9 10-8

0

20

40

60

80

concentration [M]

cyto

toxi

city

[%]

A375

EC50: 550 pM

Isotype control bTCE

AT1413 bTCE

AT1002 bTCEAT1413 bTCEAT1002 KiHAT1413 KiH

10-11 10-10 10-9 10-8 10-7-20

0

20

40

60

80

concentration [M]

cyto

toxi

city

[%]

EC50: 27 nM

EC50: 500 pM

Isotype control bTCE

AT1413 bTCE

Isotype control KiH

AT1413 KiH

AT1002 bTCEAT1413 bTCEAT1002 KiHAT1413 KiH

AT1002 bTCEAT1413 bTCEAT1002 KiHAT1413 KiH

0 7 14 21 28 350

50

100

150

200

250

Days since tumor engraftment

Nor

mal

ized

tum

or g

row

th

0

50

100

150

200

250

Norm

aliz

ed tu

mor

gro

wth Isotype control

bTCE2.5 mg/kg

AT1413 bTCE2.5 mg/kg

Isotype controlKiH1.5 mg/kg

0 7 14 21 28 35Days since tumor engraftment

AT1413KiH1.5 mg/kg

0 357 14 21 28

biweekly I.V. antibody injection

sacrifice

A375, S.C.2x106 cells

caliper measurement of tumor size

Nor

mal

ized

tum

or g

row

thDays since tumor engraftment

anti-humanIgG-AF647

A375melanoma

Isotype controlAT1413

Untreated

Neuraminidase

136.2melanoma

THP-1AML

Isotyp

e ctr

AT14

13

MXinput

A37

5

IP

150kDa

100kDa75kDa

I

III

II

IV

50μm

50μm

50μm

50μm

50μm

50μm

100μm

100μm

Isotype control

AT1413 AT1413

Isotype control

AT1413

Isotype control

AT1413

Isotype control

BM PB SPL TUM0

20

40

60

80

100

% o

f liv

e ce

lls

FACS - human CD38+CD69+ T cells (by SSC - CD45 bright)BM PB SPL TUM

0

20

40

60

T ce

lls/ l

ive

cells

FACS - human T cells (negative selection) % live cells

AT1002 bTCEAT1413 bTCE

AT1413 KiHAT1002 KiH

** *

BLM

MEL06.33

MEL12.07

MEL04.01

MEL06.07

MEL07.16

MEL00.08A

MEL93.15.2

MEL518A2

MEL93.03

MEL03.10

MEL06.04

MEL93.04C

MEL96.11

MEL90.07

MEL94.03

MEL99.08

MEL05.18

MEL05.06

MEL93.08

MEL04.07

MEL93.050

50

100

150

patient-derived melanoma

delta

MFI

positive

MEL12.07

MEL06.07

MEL99.08

Isotype controlAT1413

Melanoma

Leukemia

Coloncarcinoma

Pancreascarcinoma

anti-human IgG-AF647

Mammacarcinoma

136.2 A375 BLM WBO

SH-2 THP-1 K-562 Jurkat

Caco-2 COLO 205 HCT 116 SW480

Capan-2 PANC-1 PANC 08.13 PDX derived

BT-20BT-549 T-47DZR-75-1

1 AIMM Therapeutics, Amsterdam, The Netherlands, 2 Department of Hematology, Academic University Medical Centers, location AMC, Amsterdam, The Netherlands3 Cancer Center Amsterdam (CCA), Amsterdam, The Netherlands, 4 Amsterdam Infection and Immunity Institute (AI&II), Amsterdam, The Netherlands5 Leiden University Medical Center, Leiden, The Netherlands*These authors contributed equally to this work.

G. de Jong*1,2,3,4, L. Bartels*1,4, M. Kedde1, E. Verdegaal5, M.A. Gillissen1,2,3,4, S.E. Levie1, M.G. Cercel1, S.E. van Hal-van Veen1, C. Fatmawati1, D. van de Berg1, E. Yasuda1, Y. Claassen1, A.Q. Bakker1, S.H. van der Burg5, R. Schotte, J. Villaudy1, K. Wagner1, H. Spits1,4, M.D. Hazenberg2,3,4, P.M. van Helden1.

Background

‣ Antibody AT1413 was isolated from a high-risk AML patient who successfully cleared the leukemia after allogeneic stem cell transplantation ‣ AT1413 targets CD43s, a unique sialylated form of CD43 expressed by all of >70 primary AML and MDS isolates tested‣ AT1413 constructed into a bispecific T-cell engaging format (AT1413 bTCE) was effective against primary AML in vitro and in vivo‣ Here we show that CD43s is also present and can be targeted on non-hematopoietic tumors

1. CD43s expression is not restricted to hematologic malignancies

(A) AT1413 binds to melanoma cell lines and a subset of mama carcinoma cell lines as determined by flow cytometry

(B) AT1413 binds 18/21 short-term cultured patient-derived melanoma samples as determined by flow cytometry

(C) Immunohistochemistry staining by AT1413 on a tissue micro array containing 39 patient melanoma samples was positive in 72% of cases.

2. Confirmation of sialylated CD43 (CD43s) as the target of AT1413 on melanoma cells

3. AT1413 T-cell engagers induce T cell-mediated lysis of melanoma cells in vitro

(A) Schematic view of T-cell engager (TCE) formats of AT1413(B) The AT1413-bTCE directed T-cell mediated lysis against two

melanoma cell lines, 136.2 and A375(C) AT1413-KiH induced lysis with lower potency (EC50: 27 nM)

compared to AT1413-bTCE (EC50: 500 pM) on A375

Conclusion‣ AT1413 recognizes a sialylated epitope on CD43 (CD43s) shared by melanoma, AML and MDS‣ Two bispecific AT1413 T-cell engagers targeting CD43s x CD3 redirected T-cell cytotoxicity against melanoma

cells with different potencies in vitro‣ AT1413 TCEs induced strong anti-tumor cytotoxic activities in vivo and T-cell infiltration into the tumor‣ These data indicate a broad therapeutic potential of AT1413.

4. AT1413 T-cell engagers inhibit melanoma tumor growth and increase tumor infiltration in vivo

T-cell engager bispecific formats of an AML patient-derived antibody targeting a unique sialylated CD43 epitope induce kill of melanoma cells in vitro and in vivo

A B

A B

Conflict of interest disclosure:

MK, SL, MC, SvH, CF, DvB, EY, YC, AB, RS, JV and PvH are employees of AIMM Therapeutics.

MK, SL, MC, SvH, CF, DvB, EY, YC, AB, RS, JV and PvH have equity ownership in AIMM Therapeutics.

MK, MG, AB, HS, MH and PvH are inventors on a patent relevant to this subject.

pmvanhelden@aimmtherapeutic.com

Abstract #4745 Poster #542

A B

(A) Detection of CD43 on Western blot afterimmunoprecipitation of lysed A375melanoma cells using AT1413. M = marker, X= empty lane

(B) Neuraminidase treatment of melanoma andAML cells, which removes sialyl groups fromglycosylated proteins, resulted in loss ofAT1413 binding as detected by flowcytometry. This confirms the AT1413 epitopeis sialylated.

C

(A) Human Immune System mice were engrafted subcutaneously with luciferase-expressing A375 melanoma cells andwere treated as indicated. Normalized tumor growth is depicted over time; AT1413 TCEs inhibited tumor growth by73-77% compared to negative control TCEs.

(B) AT1413-bTCE treatment induced T-cell infiltration into the tumor (C) and a larger lymphocyte infiltrate with anactivated phenotype (CD38+CD69+) compared to mice treated with bTCE control.

C

A B

C Evaluated by two-way ANOVA/mixed-effect analysis, corrected for multiple testing. Error bars represent mean with standard deviation of the mice per treatment group. *: P < 0.05, **: P < 0.01

I. stage III tumor on the upper arm II. stage II tumor on the sole of the footIII. stage III tumor of the scalpIV. Adjacent healthy skin.

Detection by mouse anti-CD43 (clone MEM59)

bTCE (2+2)Bispecific T-cell

engaging antibody

KiH (1+1)Knob-in-hole monovalent T-cell engaging antibody

No FcgR interaction

Knob-into-hole mutations to favor HC heterodimerization

anti-CD3 scFv (UCHT1) to circumvent LC pairing problem

anti-CD3 scFv (UCHT1)

Isotype control bTCE

AT1413 bTCEIsotype control KiH

AT1413 KiH

LegendLegendLegendLegend