12 24 36 48 60 72 84 96

0

50

100

150

Hours

Cyto

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ity

Ob

ject

Are

a u

m2

red

co

lor

(m

ed

ium

on

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ALG.APV-527 + CD3-EpCAM

CD3-EpCAM

ALG.APV-527

0 .1 1 1 0 1 0 0

0

2 0

4 0

6 0

A L G .A P V -5 2 7 c o n c (n M )

CD

8+

%IF

N-g

0

C T 2 6 -5 T 4

w t C T 2 6

A

0 7 1 4 2 1 2 8

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

D a y P o s t T u m o r C h a lle n g eMe

an

Tu

mo

r V

olu

me

(m

m3)

M B 4 9 -5 T 4

M B 4 9

M B 4 9 -5 T 4

M B 4 9

N a iv e

R e -ch a lle n ge

0 .1 1 1 0 1 0 0

0

2 0

4 0

6 0

8 0

1 0 0

A L G .A P V -5 2 7 C o n c (n M )

CD

8+

% D

ivid

ing

Tc

ell

s

Is o ty p e C o n tro l

A L G .A P V .5 2 7

0

About ALG.APV-527

ModifiedFc

Anti-4-1BB scFv

Anti-5T4 scFv

Summary and Conclusions

Michelle Nelson1, Robert Miller1, Anneli Nilsson2, Lill Ljung2, Allison Chunyk1, Catherine McMahan1, David Bienvenue1, Maria Askmyr2, Gabriela Hernandez-Hoyos1, Sara Fritzell2

Potent Tumor-Directed T-cell Activation and Tumor Inhibition Induced by a 4-1BB x 5T4 ADAPTIR™ Bispecific Antibody

1Aptevo Therapeutics Inc., Seattle, WA, USA 2Alligator Bioscience AB, Medicon Village, 223 81 Lund, Sweden Presenting author

• ALG.APV-527 is an ADAPTIR™ bispecific therapeutic containing two

sets of scFv binding domains targeting 5T4 and 4-1BB, linked to an

effector-null Ig Fc domain

• The scFvs originate from the Alligator Gold® human scFv library

(Alligator Bioscience) and have been optimized for use in the

bispecific ADAPTIR™ format (Aptevo Therapeutics)

• ALG.APV-527 features target-driven T cell stimulation, optimized

stability, good manufacturing properties with potential for better risk-

benefit in humans than other monospecific 4-1BB antibodies

• ALG.APV-527 is cross-reactive to 4-1BB and 5T4 from cynomolgus

monkey. It enhances stimulation of CD3-activated human and

cynomolgus T cells in vitro

• ALG.APV-527 has an antibody-like in vivo half-life

Figure 1. ALG.APV-527 augments CD8+ T cells and NK cells

ALG.APV-527 augments T cell proliferation.

PBMC were stimulated with anti-CD3 Ab in solution and

serial dilutions of ALG.APV-527 in the presence of

human 5T4-expressing CT26 cells. Representation of

the percentage of proliferating.

(A) The percentage of proliferating CD8+ T cells were

calculated on day 5 via flow cytometry. One

representative donor from 4 donors.

(B) CD8+ T cells producing IFN-g were analyzed at 48

hours via flow cytometry following treatment with

Brefeldin A. Two representatives from 4 donors shown.

• 4-1BB (CD137) is an activation-induced costimulatory immune receptor expressed on

tumor-infiltrating T cells and NK cells

• Stimulation of 4-1BB leads to enhanced proliferation, increased survival, intensified

cytolytic activity, and induced IFN-g production of T and NK cells

• 4-1BB-targeting immunotherapies have shown promising anti-tumor effects clinically

however, a monospecific 4-1BB agonist induced dose-limiting hepatic toxicities

• 5T4 is a tumor-associated antigen expressed in a variety of malignancies, including

NSCLC, head and neck, mesothelioma, renal, pancreas, bladder, breast, colorectal,

gastric, ovarian and cervical cancers

ALG.APV-527 Mode of Action

Introduction

• Augments CD8+ T cell proliferation & IFN-g production & the cytotoxic profile of NK cells in the presence of 5T4+ tumor cells

• Inhibits growth of 5T4+ tumor cells in a human 4-1BB KI murine model and induce tumor-specific memory cells

• Induced cytotoxic killing of 5T4-expressing tumor cells when CD8+ T cells were stimulated with a sub-optimal concentration

of CD3-EpCAM showing that the ALG.APV-527 induced tumor cell killing is dependent on CD3/TCR activation of T cells.

ALG.APV-527 induces the generation of memory cells. (A) Day 0, MB49 cells expressing human 5T4 were injected SQ into 4-

1BB knock-in mice. Starting on day 7, treatments of ALG.APV-527 were administered IP twice weekly until day 24, (8 mice/

treatment). (B) Surviving mice that had cleared their primary tumor were re-challenged with MB49 tumor cells on day 80. Naïve mice

were used as controls. No further therapy was given.

NK cell

IL-2

CD8 + T cell

Anti-CD3 (soluble

or bead-bound)

B C

Figure 3. ALG.APV-527 induces rejection of established

tumors and promotes anti-tumor memory response

• ALG.APV-527 has a favorable non-clinical safety profile with no indications of systemic activation or liver toxicity in NHP or murine models

• The anti-4-1BB x anti-5T4 targeting ADAPTIR molecule, ALG.APV-527, has the potential to be a unique anti-cancer therapeutic agent with

an improved safety profile for the treatment of numerous 5T4-expressing solid tumors with unmet medical need

• CTA documents are prepared for filing of a phase 1 clinical trial

ALG.APV-527 directs

the stimulation of CD8+

T and NK cells by 5T4+

tumors and is designed

to minimize the toxicity

observed with other 4-

1BB therapeutics

CD8+ T cellsALG.APV-527 enhances NK

cells’ effector functions.

(C) IL-2 pre-stimulated primary NK

cells co-cultured with 5T4-

expressing HCT116 cells and

ALG.APV-527. Granzyme B were

measured in the supernatant at

72h using ELISA. 4

representatives from 12 donors

shown.

IFN-g

GrzB

IFN-γ

TCR/CD3

5T4 antigen

Upregulation

4-1BB

Increased number

and potency of

tumor-specific

cytotoxic T cellsCD8+ T cell

IL-2 upregulates 4-1BB expression on NK cells.

Titration of ALG.APV-527 in the presence of 5T4-

expressing tumor cells enhances secretion of

cytolytic molecules such as IFN-g and Granzyme B

(GrzB) and promotes proliferation.

Stimulation of T cells with anti-CD3 induces the

upregulation of 4-1BB. Addition of ALG.APV-527

and 5T4+ tumors augments primarily CD8+ T cells’

proliferation and secretion of IFN-g.

CD8+ T cells

Figure 4. ALG.APV-527 has a favorable

safety profile in a murine study

0 .0 1 0 .1 1

0

2

4

6

A L G .A P V -5 2 7 c o n c (n M )

Gra

nz

ym

e B

(n

g/m

l)

A L G .A P V -5 2 7

w / IL -2

A L G .A P V -5 2 7

w /o IL -2

Is o ty p e c o n tro l

ALG.APV-527:

ALG.APV-527

Recall response

B

NK cells

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

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 6 0 0

D a y P o s t T u m o r C h a lle n g e

Me

an

Tu

mo

r V

olu

me

(m

m3

)

Evaluate

Systemic

T-cell

stimulation

ALG.APV-527

Urelumab Analogue

Hu IgG

200mg (IV)

7 14 21D0

hu4-1BB

KI mice

(9-10 mice/gr)

0 7 1 4 2 1

8 5

9 0

9 5

1 0 0

1 0 5

1 1 0

D a y

% o

f In

itia

l B

od

y W

eig

ht

Body Weight

ALG.APV-527Urelumab

Analogue

Ulcerative

Dermatitis

0/10 7/9

Hu IgG

0/10

0

1 0

2 0

3 0

4 0

5 0

%K

i67

+ c

ell

s

****

**** P=<0.0001

ALG.APV-527 has a favorable safety profile. Surviving human 4-1BB KI mice previously treated with therapy were later treated with 200 μg (IV).

Mice were sacrificed on D21. (A) Ulcerative dermatitis severity was scored

by a blinded observer at D20 using the published scoring system (Hampton J Am

Assoc Lab Anim Sci 2012). (B) Body weight was monitored. (C) Spleens were stained

for Ki67+ CD8+ T cells. (D) Serum cytokines were collected on D21. (E-G)

Livers were processed for H&E and IHC for CD8+ infiltrate expression. IHC

sections was quantified using Visiopharm software: results are represented

as the Ratio of CD8 (µm2)/Total area scanned (µm2).

Liver- Centrilobular

InflammationLiver Histopathology

CD8+ cells

B

A

E

20 μg

PBS

6.6 μg2.0 μg

0.66 μg

Dose-Dependent Tumor Clearance

MB49 /

hu5T4 hu4-1BB

ALG-APV-527 dosing

7 10 13 17 20 24Day 0

MB49 cell

re-challenge

80

A

Hu IgG

ALG.APV-527

Urelumab Analogue

H&E

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

Se

ru

m I

L-6

(pg

/mL

)

0 .0 0 0

0 .0 0 4

0 .0 0 8

0 .0 1 2

0 .0 1 6

0 .0 2 0

CD

8 I

HC

are

a r

ati

o

0

1

2

3

4

His

tolo

gy

Sc

ore

CD8 area/Total area

C

F G

ALG.APV-527 Urelumab

AnalogueHu IgG ALG.APV-527 Urelumab

AnalogueHu IgG

ALG.APV-527 Urelumab

AnalogueHu IgG

ALG.APV-527 Urelumab

AnalogueHu IgG

IL-6 serum cytokine Proliferating CD8+

T cells

D

A B

Figure 2. ALG.APV-527 promotes increased in vitro tumor lysis

****

****

****

****

ALG.APV-527 promoted

increased tumor lysis in a

CD8+ T cell cytotoxic

killing assay.

(A) Representative live cell

images of cytotoxic primary

CD8+ T cells co-cultured with

HT29 human tumor cells

expressing endogenous

levels of 5T4 in combination

with an antibody targeting

CDCD3 x EpCAM. Cytotox Red was measured at 0 and 96 hours (Cytation 5).

Sub-optimal concentrations of CD3-EpCAM were used to mimic TCR/MHC:

peptide signaling, essential for CD8+ T cell activation and 4-1BB upregulation.

Green viability stain faded as cells divided. (B) Cytotoxicity was quantified

over time by measuring the total red color object area (background with

medium only was subtracted).