More Related Content Similar to Novel hexavalent GITR agonists stimulate T cells and enhance memory formation (20) Novel hexavalent GITR agonists stimulate T cells and enhance memory formation1. April 2017 - ©
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TNF Superfamily Modulators –
Next Generation Immunotherapy
Novel hexavalent GITR agonists stimulate T cells and
enhance memory formation
AACR Annual Meeting, Washington DC
Meinolf Thiemann, PhD
Director Assay Development
April 4, 2017
2. I have the following financial relationships to disclose:
Full-time employee of Apogenix
Disclosures Meinolf Thiemann
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2AACR Annual Meeting, Washington DC
3. HERA-Technology Platform:
Targeting major pathways in Immuno-Oncology
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AACR Annual Meeting, Washington DC 3
• HERA = Hexavalent TNF SF Receptor Agonist
• Proprietary technology platform
• Agonists targeting co-stimulatory receptors
4. HERA-Technology Platform:
Presentations at AACR
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AACR Annual Meeting, Washington DC 4
Poster 1688 / 7
HERA-CD40L: A novel hexavalent CD40
agonist with superior biological activity.
C. Merz et al.
April 3, 2017, 8:00 - 12:00 PM
Talk DDT01-03
ABBV-621: A best-in-class TRAIL-receptor agonist
fusion protein that enhances optimal clustering
for the treatment of solid and hematologic
tumors. S. Morgan-Lappe
April 2, 2017, 1:48 - 2:12 PM
Poster 4690 / 6
Hexavalent CD27 agonists show single
agent anti-tumor activity and enhanced
memory formation in mouse syngeneic
tumor models. C. Gieffers et al.
April 4, 2017, 1:00 - 5:00 PM
Licensed to
GITR agonists
• Activation of effector T cells
• Suppression of tumor-associated Tregs
5. 1st generation - antibodies
Fc-receptor
Stimulation
Immune cells
• Apogenix´ HERA-ligands efficiently induce receptor multimerization
• Agonistic antibodies require Fc clustering associated with cell depletion via ADCC and/or CDC
HERA-ligands: best-in-class TNF receptor SF agonists
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5AACR Annual Meeting, Washington DC
CD40
TNF SF
receptors
TNF SF ligands
Stimulation
In vivo
ADCC
CDC
Backsignaling
Stimulation
Next generation -
HERA-ligands
ADCC: antibody dependent cellular cytotoxicity; CDC: complement dependent cytotoxicity
Immune
cells
6. Molecular design of HERA-GITRL
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6AACR Annual Meeting, Washington DC
• Receptor agonist with defined trimerization
capabilities and adjustable PK
• Dimer of a single chain polypeptide comprising
three receptor binding domains (RBD) fused to
human IgG1-Fc
silenced Fc:
no ADCC
no binding to
Fc-receptors
Hexavalent RBD:
High clustering
capacity (6 receptors)
Molecular design: HERA-GITRL
RBD 1 RBD 2 RBD 3 IgG1 (Fc)
Linker Linker Hinge + Linker
Human
GITR-Ligand-
RBD Monomers
Hinge+Linker
Human IgG1-Fc
1
2
3 1
2
3
Hexavalent TNF SF Receptor Agonist = HERA
RBD: receptor binding domain
• Different molecular design
• Antibody-like molecular weight: 140-170 kDa
• Lab scale production and purification results in
protein batches with excellent stability and
purity (> 99% monomer content)
HPLC-SEC
Lane Sample
1 Marker
2 HERA-GITRL-A
non-
reduced
3 HERA-GITRL-B
4 HERA-GITRL-C
5 HERA-GITRL-A
reduced6 HERA-GITRL-B
7 HERA-GITRL-C
Biochemical features: 3 different HERA-GITRL molecules
HERA-GITRL-A
HERA-GITRL-B
HERA-GITRL-C
A214nm
A214nm
A214nm
time [min]
kDa
100
85
70
60
50
40
30
25
20
15
10
1 2 3 4 5 6 7
SDS-PAGE
7. Human and murine HERA-GITRL constructs:
Functional binding to GITR from different species
7
0
1
2
3
0,1 1 10 100
ELISAsignal
[OD450nm]
GITRL [ng/ml]
Binding to human GITR-Fc
0
1
0,1 1 10 100
ELISAsignal
[OD450nm]
GITRL [ng/ml]
Binding to mouse GITR-Fc
HERA-GITRL-A
HERA-GITRL-B
HERA-GITRL-C
mmHERA-GITRL-A
mmHERA-GITRL-B
0
1
2
0,1 1 10 100
ELISAsignal
[OD450nm]
GITRL [ng/ml]
Binding to monkey GITR-Fc
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AACR Annual Meeting, Washington DC
Binding
HERA-GITRL
mouse human
human GITR-Fc - +
mouse GITR-Fc + -
monkey GITR-Fc - +
• Since human HERA-GITRL does not bind to mouse GITR, a murine surrogate
(mmHERA-GITRL) is needed for functional mouse studies
• Cynomolgus monkey is a relevant species
kD
GITR-receptor
human monkey
HERA-GITRL-A 0.2 nM 0.2 nM
HERA-GITRL-B 0.4 nM 0.6 nM
HERA-GITRL-C 0.9 nM 0.4 nM
Determination of
binding constants
QCM measurement (Attana)
QCM: Quartz Crystal Microbalance
8. Human HERA-GITRL:
Cellular in vitro activity assay
8
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• HERA-GITRL shows full activity without crosslinking
• Activity of trimeric GITRL is clearly enhanced by crosslinking
NFkB-luc2/GITR
transfected Jurkat cells
Assay kit from Promega
0
100000
200000
300000
400000
500000
600000
0,1 1 10 100 1000
Luciferasesignal[RLU]
GITRL [ng/ml]
GITR luciferase assay
HERA-GITRL
trimeric GITRL
HERA-GITRL + X-link
trimeric GITRL + X-link
Assay design
Effector cell
GITR receptor
GITR ligand
RE luciferase
9. Binding of HERA-GITRL to PBMCs
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HERA-GITRL binds to PBMCs previously activated with anti-CD3GITR is upregulated on
activated CD4+ cells
Day 0 stimulation, day 6 FCM
Unstimulated (Medium)
Stimulated with anti-CD3 (HIT3a)
Cellcount
GITR expression
Day 0 stimulation, day 2 FCM (ligand binding; detection via StrepTag)
No ligand HERA-GITRL-A HERA-GITRL-B HERA-GITRL-C
Cellcount
HERA-GITRL binding
Unstimulated (Medium)
Stimulated with anti-CD3 (HIT3a)
10. HERA-GITRL treatment enhances proliferation and differentiation in
stimulated human T cell cultures
T cells alone anti-CD3
anti-CD3
+ HERA-GITRL-C
10 ng/ml
anti-CD3
+ HERA-GITRL-C
100 ng/ml
CFSE (proliferation) (log scale) (gated on T cells)
FSC(size)(linear)
2.9% 55.6% 65.1% 65.5%
staining intensity decreases with every cell division,
i.e., undivided cells are most positive for CFSE (carboxyfluorescein succinimidyl ester)
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HERA-GITRL enhances proliferation
in stimulated human T cells
Day 0 stimulation with anti-CD3 (OKT3) + HERA-GITRL-C, day 5 FCM
Naïve CD4+ T lymphocytes
Day 0 stimulation with anti-CD3 (OKT3) + HERA-GITRL-A, day 6 FCM
Total T lymphocytes
CD45RO
CD45RA
Medium anti-CD3 anti-CD3
+ HERA-GITRL-A
13.9% 49.0% 61.2%
73.3% 31.0% 14.1%
CD45RO
CD45RA
Medium anti-CD3 anti-CD3
+ HERA-GITRL-A
3.9% 26.5% 32.5%
80.8% 36.2% 23.2%
• HERA-GITRL induces memory formation (CD45RO and CD45RA)
in total T lymphocytes and CD4+ T lymphocytes
11. HERA-GITRL treatment enhances activation marker expression and production
of pro-inflammatory cytokines in stimulated human T cell cultures
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Medium anti-CD3/anti-CD28 anti-CD3/anti-CD28
+ HERA-GITRL-C
45.9% 47.5% 60.2%
Cellcount
TNFα (intracellular) (log scale)
14.3% 33.8% 40.9%
Cellcount
IFNγ (intracellular) (log scale)
HERA-GITRL increases production of
pro-inflammatory cytokines
in stimulated naïve human CD4+ T cells
Day 0 stimulation with anti-CD3 (OKT3)/anti-CD28 (CD28.2) + HERA-GITRL-C,
day 3 fresh medium, day 6 re-stimulation with PMA/Ionomycin/Brefeldin A
for 5 hours, then FCM
Naïve CD4+ T lymphocytes
CD25
Medium anti-CD3 anti-CD3
+ HERA-GITRL-A
Cellcount
2.6% 43.5% 50.6%
Total T lymphocytes
CD25
Medium anti-CD3 anti-CD3
+ HERA-GITRL-A
Cellcount
9.4% 41.2% 46.5%
Day 0 stimulation with anti-CD3 (OKT3) + HERA-GITRL-A, day 6 FCM
12. AACR Annual Meeting, Washington DC 12
Anti-tumor activity of PBMCs by combinatorial treatment
with HERA-ligands in vitro (RTCA)
• HERA-CD40L treated PBMCs induce
killing activity against tumor cells in
co-cultures
• HERA-GITRL in combination with
HERA-CD40L increases in vitro killing
-0,001
0,001
0,003
0,005
0,007
MDA-MB231
human breast adenocarcinoma cell line
Tumorcellgrowth
[slope1/hr]
0.0
PBMC - + + + +
HERA-CD40L - - + - +
HERA-GITRL-C - - - + +
-0,0025
0,0025
0,0075
0,0125
0,0175
HCT 116
human colorectal carcinama cell line
Tumorcellgrowth
[slope1/hr]
PBMC - + + + +
HERA-CD40L - - + - +
HERA-GITRL-C - - - + +
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Isolate immune cells
(PBMC) PBMC pretreatment
(CD40L, GITRL)
Seed tumor cells
Tumor cell recovery
(re-adherence/growth)
add PBMC
Observation period
(tumor cell death/detachment, re-growth)
20 – 24 h
72 – 168 h
> 48 h
Assay design:
RTCA assay
co-culture of
tumor cells and
PBMC
RTCA: Real time cell analyzer
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PK studies of human HERA-GITRL variants in mice and monkeys
0,01
0,1
1
10
100
0 50 100 150 200 250 300 350
Serumconcentration
[µg/ml]
Time after injection [h]
Single i.v. dose of 10 mg/kg bw HERA-GITRL-A
HERA-GITRL-B
HERA-GITRL-C
Compound
Dosing
[mg/kg]
Cmax
[µg/ml]
Cl
[ml/h]
Vd
[ml/kg]
AUC0-inf
[µg · h/ml]
t1/2
[h]
HERA-GITRL-A 10 171 3.35 968 2990 200.6
HERA-GITRL-B 10 123 15.1 2119 663 97.3
HERA-GITRL-C 10 173 16.8 1493 597 61.7
PK study in CD-1 mice PK study in Cynomolgus monkeys
0,01
0,1
1
10
100
0 50 100 150 200
Serumconcentration
[µg/ml]
Time after injection [h]
Single i.v. dose of 1 mg/kg bw
HERA-GITRL-A
HERA-GITRL-B
HERA-GITRL-C
Compound
Dosing
[mg/kg]
Cmax
[µg/ml]
Cl
[ml/h]
Vd
[ml/kg]
AUC0-inf
[µg · h/ml]
t1/2
[h]
HERA-GITRL-A 1 20.6 2.87 157 348 38.0
HERA-GITRL-B 1 29.4 8.12 428 123 36.5
HERA-GITRL-C 1 22.5 23.6 813 42.3 23.8
• Modular PK – terminal half-life: between 2.6 and 8.5 days in mice and between 1.0 and 1.6 days in monkeys
• Exposure in monkeys: exposure of HERA-GITRL-A is 2.8 times higher than HERA-GITRL-B and 8.2 times higher than HERA-GITRL-C
• Pilot tolerability study in monkeys with 1 and 3 mg/kg bw HERA-GITRL-C: in-life phase completed (well tolerated; no side effects)
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mmHERA-GITRL enhances antigen-specific clonal expansion
in both CD4+ and CD8+ T cell populations in vivo
OVA peptide-specific
CD8+ “OT-1” + CD4+ “OT-2” CD45.2+
i.v. 2 x 106 (each) day -1
OVA protein
i.p. 5 mg day 0
mmHERA-GITRL
i.v. day 0
Serial blood samples
Days 6, 10, 13
CD4+CD45.2+ & CD8+CD45.2+
T cells enumerated
B6/Ly5.1
CD45.1+
CD45.2+ (congenic marker)
2%
1.3% 11.3%
4.2%
3.5% 5.1%
7.7%10.7%
CD8+CD4+
OT-1
OT-2
PBS
mmHERA-GITRL
(1 mg/kg)
mmHERA-GITRL
(5 mg/kg)
αGITR (clone DTA-1)
(1 mg/kg)
Day 6
Time [d]
Time [d]
OT-1
0 5 10 15
0
5
10
15
APG1
APG1
DTA-1
PBS
No OV
OT-2
0 5 10 15
0
5
10
15
A
A
D
P
N
CD45.2+%ofCD8+CD45.2+%ofCD4+
OT-1
0 5 10 15
0
5
10
15
mmHERA-GITRL (1 mg/kg)
mmHERA-GITRL (5 mg/kg)
GITR (1 mg/kg)
PBS
No OVA
OT-2
OT-1
AACR Annual Meeting, Washington DC
• A single dose of mmHERA-GITRL enhances clonal expansion of CD4+ and CD8+ T cells
in a dose dependent manner
OT-1/OT-2 mouse model
15. mmHERA-GITRL shows anti-tumor efficacy in syngeneic mouse models
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• Pilot efficacy in the syngeneic mouse tumor model MC38-CEA with 2 groups (n=6 each):
control (PBS) and mmHERA-GITRL (1mg/kg)
• Dosing: twice weekly (6 dosings in total)
• mmHERA-GITRL is well tolerated
• mmHERA-GITRL shows anti-tumor activity: tumor growth inhibition of 42.2 %
• A further pilot efficacy in the syngeneic mouse tumor model CT26 also shows anti-
tumor activity for mmHERA-GITRL
MC38-CEA
mmHERA-GITRL (1mg/kg)
days (d)
tumorvolume[mm³]+/-SEM
0 5 10 15 20 25
0
200
400
600
800
1) PBS
2) mm HERA-GITRL
TGI: 42.2%
MC38-CEA
Control (PBS)
day [d]
tumorvolume[mm³]+/-SEM
0 5 10 15 20 25
0
200
400
600
800
A1
A2
A3
A4
A5
A6
MC38-CEA
mmHERA-GITRL (1mg/kg)
days (d)
tumorvolume[mm³]+/-SEM
0 5 10 15 20 25
0
200
400
600
800
A1
A2
A3
A4
A5
A6
MC38-CEA syngeneic mouse model
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Human HERA-GITRL shows anti-tumor activity in xenograft mouse models
injected with human PBMCs
• Study is ongoing; further tumor entities are included
Anti-tumor activity of HERA-GITRL-C in MiXeno models MiXeno tumor model
• Efficacy screening of MiXeno tumor models
• Implantation of different human xenograft
tumors to NOG or NOD/SCID mice
• Injection of human PBMCs from 2 different
donors
• Treatment with PBS (control) or 1 mg/kg
HERA-GITRL-C twice weekly (6 dosings in
total)
• 8 mice per model (for both groups: 2 mice
each with PBMCs from donor 1 and donor 2)
• Tumor growth inhibition (TGI) is monitored
• Results:
• HERA-GITRL-C shows anti-tumor activity in
HCC827 tumors (NSCLC) and Kyse270 tumors
(head & neck)
HCC827 (NSLC)
HERA-GITRL-C
days (d)
tumorvolume[mm³]+/-SEM
0 5 10 15
0
50
100
150
200
Control
1mg/kg
TGI: 35.8%
Kyse270 (H&N)
HERA-GITRL-C
days (d)
tumorvolume[mm³]+/-SEM
0 5 10 15
0
100
200
300
400
Control
1mg/kg
TGI: 31.9%
TGI d2: 43.1%
HCC827 (NSLC)
HERA-GITRL-C
days (d)
tumorvolume[mm³]+/-SEM
0 5 10 15
0
50
100
150
200
Donor 1 (Control)
Donor 1 (1mg/kg)
Donor 2 (Control)
Donor 2 (1mg/kg)TGI d1: 29.1%
Kyse270 (H&N)
HERA-GITRL-C
days (d)
tumorvolume[mm³]+/-SEM
0 5 10 15
0
100
200
300
400
Donor 1 (Control)
Donor 1 (1mg/kg)
Donor 2 (Control)
Donor 2 (1mg/kg)
TGI d2: 37.1%
17. HERA-GITRL are superior over agonistic GITR antibodies
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• HERA-GITRL activates T cells in vitro
and in vivo and induces memory
formation
• HERA-GITRL activity is independent of
Fc-receptor crosslinking
• Adjustable short PK
• HERA-GITRL has demonstrated anti-
tumor activity in several mouse tumor
models (xenograft and syngeneic)
• Outlook: GMP-cell line development
will be started soon
Summary: HERA-GITRL Advantages of HERA-GITRL over agonistic GITR antibodies
Stimulation
HERA-GITRL GITR antibodies
Agonistic activity
Yes
Defined mechanism of action
No
Unclear mechanism of action
Treg depletion No Yes
Other activities No
Yes
Depletion of GITR expressing cells
(ADCC, CDC)
Fc-receptor mediated back-signaling
Toxicity /
Immunogenicity
Potentially low
Low immunogenic potential
Autoimmune reaction (Treg depletion)
Potential ADA response to CDRs
Pharmacokinetics Adjustable half-life (days) Long half-life (weeks)
Dynamics
Fast-In / fast-Out
-> No exhaustion of T cells
Long-term activation
-> Exhaustion of T cells; effect on Tregs
Combination with
other immune-
oncology (I-O)
compounds
Sequential combination with other
I-O compounds is possible and
addresses the dynamic nature of
anti-tumor response
Sequential combination with other
I-O compounds is presumably difficult
due to long half-life and toxicity issues
18. Thanks for the commitment of all Apogenix colleagues involved in the HERA projects!
Acknowledgements
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