Bispecific Antibody Constructs Mediate Immunotherapeutic Retargeting of
Effector Cells Towards HBV Infected Target Cells
Bohne F.1, Hasreiter J.1, Quitt O.1, Meyer C.1, Dhamodaran A 2, Alvarez L.2, Bruss V.3, Moldenhauer G.2, Momburg F.2, Protzer U.1
(1) Institute of Virology, Helmholtz Zentrum Muenchen, Trogerstr. 30, 81675 München, Germany
(2) German Cancer Research Center (DKFZ), National Center for Tumor Diseases, Im Neuenheimer Feld 460, 69120 Heidelberg, Germany
(3) Institute of Virology, Helmholtz Zentrum Muenchen, Ingolstaedter Landstr. 1, 85764 Neuherberg, Germany
1
infectious
Introduction
HBV-infected hepatocytes carrying a cccDNA display viral
surface proteins on their extracellular membrane. This
occurs because the mature capsid buds into intracellular
membranes of multivesicular bodies with inserted small,
middle and large surface proteins for envelopment. Since
this process does not consume all surface proteins, excess
proteins reside in the MVB-membrane. In the course of
vesicle transport processes necessary for export of viral
particles these membranous HBV surface proteins reach the
extracellular membrane and are present on the
surface of infected hepatocytes.
The immunotherapeutic retargeting of effector cells is a promising approach to circumvent
the immunotolerant state found in malignancies and chronic viral infections. Effector cells
are supplied with designed specificities and retargeted towards antigens reverting the
immunocompromised situation.
To elaborate existing retargeting strategies, we constructed tetravalent bispecific antibody
constructs harboring two different binding moieties of immunoglobulins. The first binding
site is designed to target HBV-infected cells by binding the small HBV surface proteins on
the plasma membrane of the infected cells. The second binding motif engages immune
effector cells with specificities for T cells (CD3-T cell receptor and CD28 for
costimulatory signals).
3
2
subviral
particle
attachment
particles
export
ER/MVB
envelopment
re-import
cccDNA
reverse transcription
RNA
pregenomic RNA
HBV-infected
hepatocyte
subgenomic RNAs
Experimental Background
4
Properties of bispecific antibody constructs
Binding of bispecific antibody constructs to effector and target
cells mediates T-cell activation and secretion of cytokines
12000
CD3/CD28
IFNγ
10000
IFNγ [pg/ml]
anti-huCD3/CD28 scFv
bispecific tetravalent
antibody (BTA)
8000
6000
4000
2000
0
/CD28
4
8
12
24
48
ctr
dimerization
4
8
TNFa+ IL-2+
IFNg+ TNFa+ IL-2+
10
ctr
12
24
48
ctr
TNFα [pg/ml]
1200
800
0
4
20
48
400
40
30
24
TNFα
1600
HuH7
IFNg+
IL-2+
TNFa+
IFNg+IL-2+
IFNg+TNFa+
12
2000
PB
24
M
h
C
s
on
ly
12
h
8h
PB
24
M
h
Cs
on
ly
12
h
s
8
For a first binding control, bispecific antibody constructs were used for immune effector cell stimulation in
co-cultures of HBV-transfected Huh7-S hepatoma cells and human primary PBMCs. The cross-linking of T
cells and target cells led to marked secretion of proinflammatory cytokines over time. All three measured
cytokines, IFNg, IL-2 and TNFa showed a time dependent induction with maximum production after 1224h of co-culture. While IFNg and IL-2 reached a plateau at 48h, TNFa production started declining after
24h.
HuH7-S
HuH7
PB
24
M
h
C
s
on
ly
12
h
8h
on
ly
M
C
s
24
h
12
h
0
PB
8h
ly
6
Bispecific
antibody
constructs
mediate
cytotoxic
degranulation as assessed by the translocation of Lamp-1.
HuH7
F
HuH7
CD8+
negative control
anti-huCD3/CD28
Place a graph here:
 Double-click, or
 Drag a graph from
the navigator
CD8+
CD4+
12
h
8h
PB
24
M
h
C
s
on
ly
HuH7-S
Place a graph here:
 Double-click, or
 Drag a graph from
Conclusion
the navigator
12
12
h
h
PB
24
24PB
M
M
h C h
C
s
s
on
on
ly
ly
8h
on
ly
C
s
24
h
PB
M
Cs
PB
HuH7
In co-cultures of HBV-transfected HuH7-S target cells, expressing
HBV surface protein, the co-administration of PBMC and bispecific
antibody constructs resulted in target cell killing of up to 90% (as
analysed by xCELLigence viability assay). The target cell lysis was
dose dependent in cocultures of PBMCs and Huh7-S target cells
upon administration of both, the anti-huCD3/CD28 (A) and the antihuCD3DADCC/CD28DADCC constructs (B).
800
0
on
ly
on
ly
C
s
24
h
12
h
8h
HBV+
HuH7-S
+
IL-2+
 Drag a graphTNFa
from
IFNg+ TNFa+ IL-2+
the navigator
10
PB
M
PB
M
C
s
24
h
on
ly
0
20
s
5
30
M
C
10
D
IFNg+
IL-2+
TNFa+
Place a graphIFNg+IL-2+
here:
 Double-click,IFNg+TNFa+
or
24
h
IFNg+ IL-2+
IFNg+ TNFa +
TNFa + IL-2+
IFNg+ TNFa+ IL-2+
40
12
h
HBV-
HuH7-S
8h
15
% cytokine+ CD8+ T cells
IL-2
TNFa+
on
ly
s
D
+
20
12
h
0
M
C
HuH7
IFNg+
7 Summary &
4
0
2
D
25
HuH7-S
5
6
HuH7-S
PB
M
C
PB
HuH7-S
8h
% cytokine+ CD4+ T cells
on
24
h
12
h
8h
ly
HBV+
s
on
24
h
12
h
0
TNF-a+
IFNg+ TNFa
TNFa +CD154
IL-2+
IFNg+ TNFa+ IL-2+
12
h
1
IL-2
TNFa+IFN-g
IL-2+
IFNg+ IL-2
12
h
24PB
hM
2
+
20
20
15
15
10
10
8h
3
aCD3/aCD28IFNg+
8h
TNF-a
CD154
+ +
+ +
% cytokine/CD154
CD8
T cells
% cytokine
CD4
T cells
IFN-g
IL-2HBV-
8h
% cytokine/CD154+ CD4+ T cells
30
25
20
15
10
C
CD4+
B 25
aCD3/aCD28
HuH7
1200
0
0
E
IL-2
400
2
M
C
HuH7-S
C
A
PB
C
A
on
24
h
8h
ly
PB
M
C
s
on
24
h
12
h
8h
Bispecific antibody constructs mediate cytotoxic
elimination
0
of HBV-positive and HBV-infected target cells with a
polyfunctional T-cell activation.
ly
1
8h
2
8h
3
% cytokine/CD154+ CD8+ T cells
B
% cytokine+ CD8+ T cells
A
The binding moieties of bispecific antibody constructs consist of scFv which are
30
generated by fusing the variable domains of IgG heavy and light chains (VH and V25
L)
using a flexible linker like (Gly4Ser)3 or YOL. These scFvs can be further integrated
20
in a modular polypeptide to generate a bispecific antibody construct harboring 15
to
10
distinct and designed specificities for target-antigens.
5
IL-2 [pg/ml]
Bispecific tetravalent antibody constructs are generated fusing two distinct single chain variable-fragments
(scFv) using a IgG1 Fc-domain (CH3, CH2, Hinge). C-terminally, the constructs bear a first scFv designed
aCD3/aCD28
to bind to a cognate antigen of the designated effector T cells.aCD3/aCD28
This first scFv is connected to a human
Fc-domain with mutated residues to either decrease or abrogate (DADCC) binding through
IFN-g ADCC. Further,
20
IFN-g providing
CD16 (FcgIIR) and minimize
the Fc-domain contains the CH2-hinge,
IL-2
IL-2
for the two cysteine residues,
mediating Bisulfide bridging of IgG-heavy chains.
This results
15
in a dimerizationTNF-a
of the bispecific antibody, rendering it tetravalent. N-terminally,
TNF-a
the constructs CD154
possess a scFv specific for HBV surface proteins as target cell
CD154
10
antigens. Binding of both antigens produces a spatial proximity between
6
effector- and target cell sufficient
to initiate a cytotoxic response,
eliminating 4the HBV-infected cell.
12
h
% cytokine/CD154+ CD4+ T cells
1600
HuH7
In co-cultures of HBV-producing HuH7 target cells and
PBMCs treated with bispecififc constructs we performed
intracellular cytokine staining for IFNg, IL-2 and TNFa to
determine the functional properties of (C) CD4+ T cells
or (D) CD8+ T cells over time. This showed a
polyfunctional activation of effector cells retargeted by
bispecific antibodies directed against HBV surface
proteins.
Place a graph here:
In co-cultures of Huh7-S and PBMCs, also the single
administration of one bispecific antibody lead to target cell
killing (E). Additionally, co-administration at the same
concentration resulted in synergistic enhancement. In cocultures of HBV-infected HepaRG cells with PBMCs and
the respective bispecific antibody constructs directed
against T-cell antigens, activation of effector cells resulted
in the specific cytotoxic elimination of HBV-infected
HepaRG cells (F).
LAMP-1-positive CD8
LAMP-1-positive CD4
Upon stimulation with coated HBsAg and bispecific antibody constructs, T cells translocate Lamp-1
(CD107a), a marker of cytotoxic degranulation to the cellular surface. This was detected for CD8+ T
cells, but also to a lesser extend for CD4+ Th cells. Stimulation of PBMC with NK cell directed bispecific
antibody constructs did not result in translocation of Lamp-1, indicating a possible different mechanism
of cytotoxicity for NK cell mediated target cell lysis.
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The newly designed bispecific tetravalent antibody constructs are capable of binding to HBV
surface proteins on the membrane of HBV-producing hepatocytes. The retargeting of T cells
employing bispecific tetravalent antibody constructs resulted in synergistic polyfunctional
activation and cytotoxicity upon co-culture with HBV-transfected HuH7 target cells. Furthermore,
bispecific antibody constructs mediated specific elimination of HBV-infected HepaRG cells. The
activation upon administration of CD3 and CD28 specific constructs was accompanied by
cytotoxic degranulation and translocation of Lamp-1 to the cellular membrane of effector cells.
In the future these new therapeutic tools have to be further evaluated, both in vitro and in
vivo. For this purpose, the bispecific antibodies will be produced in high yields and purified
and further tested for bioavailabilty, safety and efficacy.
Taken together, retargeting of immune effector cells towards HBV-infected cells using
bispecific antibody constructs is a promising new therapeutic approach for the elimination of
HBV infected hepatocytes in chronic hepatitis B.