d i b potent tumor-directed t-cell activation and tumor
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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
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Tc
ell
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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
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(m
m3
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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).
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