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

0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

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

NK

(C

D3

35

+)

ce

ll c

ou

nts

0

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

stability, good manufacturing properties with potential for improved

risk-benefit in humans than other monospecific 4-1BB antibodies

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

monkey. It binds to human and cynomolgus 5T4 and 4-1BB

expressing cells and enhances activation of CD3-stimulated human

and cynomolgus T cells

• Demonstrated an extended antibody-like serum half-life of 9 days

About ALG.APV-527

ModifiedFc

Anti-4-1BB scFv

Anti-5T4 scFv

ALG.APV-527 enhances NK cell effector

function in presence of 5T4+ cells

Summary and Conclusions

Michelle Nelson1,*, Gabriele Blahnik-Fagan1, Robert Bader1, Doreen Werchau2, Anneli Nilsson2, Lill Ljung2, Jeannette Bannink1, Danielle Mitchell1, Lynda Misher1,

Catherine McMahan1, David Bienvenue1, Maria Askmyr2, Anna Dahlman2, Peter Ellmark2, Gabriela Hernandez-Hoyos1, Sara Fritzell2,*

Potent Tumor-Directed T cell Activation and Tumor Inhibition Induced by a

4-1BB x 5T4 ADAPTIR™ Bispecific Antibody

ALG.APV-527 inhibits tumor growth

of a human HCT116 colon carcinoma

Day 0, HCT116 colon carcinoma

cells injected SQ into the flank of

SCID-beige mice. Day 1, Human

PBMC’s from 4 donors injected IP.

5 mice/ group/ donor total 20 mice/

treatment. Treatments of ALG.APV-

527 at 10ug were administered IP

twice weekly starting on day 6.

Significant decreases in tumor size

observed from day 13 vs. vehicle,

Mann-Whitney, non-parametric 2-

tailed t test.

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

• ALG.APV-527 is a bispecific therapeutic in the

ADAPTIR™ format containing two sets of

binding domains, scFv, targeting 5T4 and 4-

1BB which are linked to a silent Ig Fc domain,

providing an antibody-like in vivo half-life

• The scFv originate from the Alligator Gold®

human scFv library (Alligator Bioscience)

• Each scFv has then been optimized and

developed for use in the bispecific

ADAPTIR™ format (Aptevo Therapeutics)

ALG.APV-527 augments CD8+ T cell proliferation

and IFN-g production in the presence of 5T4+ cells

ALG.APV-527 augments CD8+T cell proliferation. Primary PBMC were stimulated with α-CD3 Ab in

solution and serial dilutions of ALG.APV-527 in the presence of 5T4-expressing CHO-K1 cells. (A)

Representation of the proliferation of CD8+ T cells. The number of CD8+ T cells (B) and CD4+ T cells (C)

were calculated at 96 hours via flow cytometry. Three healthy donors are represented here.

• 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 but one monospecific 4-1BB agonist has induced

dose-limiting hepatic toxicities

• 5T4 is a tumor-associated antigen expressed in patients 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

0 .0 1 0 .1 1

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 )

No

rm

ali

ze

d I

FN

-g

re

lea

se

(%

)E C 5 0 = 0 .1 (0 .1 4 -0 .2 1 ) n M

Introduction

> ALG.APV-527:

• Augments CD8+ T cell proliferation and IFN- g production but only in the presence of 5T4+ expressing cells

• Enhances the cytotoxic profile of NK cells via an increase in CD25high & NKD2G expression and the

production of IFN-g and Granzyme B

• Effectively localizes to 5T4+ tumor in vivo

• Inhibits colon carcinoma HCT116 tumor growth in a xenograft murine model

A

M e d iu m

Is o ty p e c o n trol

0 .0 0 40 .0 1 6

0 .0 6 40 .2 5

0

1 0

2 0

3 0

A L G .A P V -5 2 7 (g /m l)

CD

25

ex

pre

ss

ion

% o

ut

of

CD

56

+C

D3

-c

ells

Heart (negative)Liver (negative)

Malignant epithelial mesothelioma

(pleura): 1-3+, occas to freq

Squamous cell lung cancer

1-3+ frequent

Squamous cell carcinoma (larynx)

1-2+ rare to occas

5T4 expression in human normal and tumor tissue. Formalin-fixed paraffin-embedded tissue micro-

arrays (TMAs) were acquired from US Biomax Inc, and stained for 5T4 detection (clone MAB4975, R&D

Systems). 5T4 expression was detected in tumors from NSCLC, Head and Neck, mesothelioma, pancreatic,

bladder, renal and ovarian cancer with variable frequency and intensity, as exemplified above. Occasional

membranous staining of 5T4 was detected in specific cell populations in normal tissue, specifically on the

smooth myocytes of the esophagus, osteocytes in bone marrow and epithelial cells in the adenohypophysis.

There was no 5T4 staining on cells from any major organ system such as cardiovascular, respiratory or

hepatic systems.

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

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

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

CD

8+

T c

ell

co

un

ts

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

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

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

CD

4+

T c

ell

co

un

ts

0

NK cell

IL-2

CD8 + T cell

αCD3 (soluble or bead-bound)

0 .0 1 0 .1 1

0 .0 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

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

IFN

g (

ng

/ml)

0 .0 1 0 .1 1

0

1

2

3

4

5

6

7

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 (+ IL -2 p re -s tim )

A L G .A P V -5 2 7 (n o IL -2 p re -s tim )

Iso typ e co n tro l

ALG.APV-527 enhances CD8+ T cells’ ability

to secrete IFN-g. (D) Primary CD8+ T cells and

anti-CD3 abs coated on beads at a 1:1 T cell/

beads ratio were incubated with ALG.APV-527 in

the presence of Mitomycin C treated HCT116

tumor cells expressing endogenous levels of 5T4

(6.2 x 104/ 5T4 receptors/ cell). IFN-g production

was measured in the supernatant after 72h

using ELISA. Normalized IFN-g levels (mean of

12 donors) and nonlinear curve fit log (agonist)

vs. normalized response variable slope) is

plotted here using GraphPad Prism.

A L G .A P V -5 2 7 V e hic le

0

1

2

3

4

5

% h

IgG

+ b

ind

ing

to

via

ble

CD

45

- tu

mo

r c

ell

s

B 1 6 -5 T 4

B 1 6 -w t

ALG.APV-527 localizes to 5T4+ tumors

5T4-dependent localization of ALG.APV-527 in a B16 twin tumor model

Day 0: each mouse received one 5T4 negative and one 5T4 positive B16 tumor injected subcutaneously

(SQ, 1x105 cells in 100 µL) at each side of the hind flank/back. Intraperitoneal (IP) treatment of ALG.APV-

527 (100 µg) was given on days 6 and 13 and mice were sacrificed on day 14 (24 h after the final

treatment). Tumors were collected and the levels of hIgG positive cells were assessed either by IHC or

flow cytometry using an antibody detecting human IgG. IHC of ALG.APV-527 binding to either

A 5T4-negative tumors or B 5T4-positive tumors. C Flow cytometry of ALG.APV-527 binding to

dissociated 5T4-positive and -negative tumor cells (percentage of hIgG+ cells out of live CD45- cells)

ALG.APV-527 enhances the expression of cytolytic

markers CD25, IFN-g and granzyme B. (D-F) Primary NK

cell assays and Mitomycin C-treated HCT116 tumor cells

expressing endogenous levels of 5T4 (6.2 x 104/ 5T4

receptors/ cell). IL-2 pre-stimulated NK cells were

incubated with a dilution of ALG.APV-527 and surface

expression of (D) CD25 was measured via flow cytometry.

The secretion of (E) IFN-g and (F) Granzyme B was

measured in the supernatant after 72h of culture using

ELISA.

Cell Trace

NK

G2D

0nM

10nM

0nM

0.05

50

Cell Trace

B C

E

B

D

F

D

High 5T4 expression in TMA of different tumor

indications but low expression in normal tissue

> 5T4 is expressed in a wide range of tumor indications, but not in any vital organs such as

the heart or the liver

>The α-4-1BB x α-5T4 ADAPTIR molecule, ALG.APV-527, has the potential to be a unique α-

cancer therapeutic agent with an improved safety profile for the treatment of numerous

5T4-expressing solid tumors with high unmet medical need

ALG.APV-527 directs the activation of CD8+ T and NK cells to 5T4+ tumors and is

designed to minimize or eliminate the toxicity observed with other 4-1BB therapeutics

C CD4+ T cellsCD8+ T cells

0 5 1 0 1 5 2 0 2 5

0

2 5 0

5 0 0

7 5 0

1 0 0 0

T im e a fte r tu m o r in o c u la tio n (d a y s )

Tu

mo

r v

olu

me

(m

m3

)

A L G .A P V -5 2 7

V e h ic le

*

***

***

**

ALG.APV-527 promotes the proliferation and upregulation of NKD2G on NK cells.

(A-C) Primary NK cells were stimulated with IL-2 and serial dilutions of ALG.APV-527 in

the presence of 5T4-expressing CHO-K1 cells. (A) Representation of the proliferation of

NK cells. (B) The number of CD335+ NK cells were assessed in 2 healthy samples on

day 6 by flow cytometry. (C) Representative expression of NKG2D on treated NK cells.

IFN-γGrzB

IFN-γ

TCR/CD3

5T4 antigen

Upregulation

4-1BB

Increased

number and

potency of

tumor-specific

cytotoxic

T cellsCD8+ T cell

The addition of 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-γ and granzyme B and promotes

proliferation.

α-CD3 added to PBMC cultures or α-CD3-coated beads

cultured with enriched CD8+ T cells stimulate through the

TCR to induce the upregulation of 4-1BB. The addition of

ALG.APV-527 in culture with 5T4+ tumors augment the

cells’ proliferation and secretion of IFN-γ.

CA B

Cell Trace

A

0nM

0.005

5

Pancreatic duct adenocarcinoma

1-2+ rare to occas