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April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
1
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
I have the following financial relationships to disclose:
Full-time employee of Apogenix
Disclosures Meinolf Thiemann
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
2AACR Annual Meeting, Washington DC
HERA-Technology Platform:Targeting major pathways in Immuno-Oncology
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
AACR Annual Meeting, Washington DC 3
• HERA = Hexavalent TNF SF Receptor Agonist• Proprietary technology platform• Agonists targeting co-stimulatory receptors
HERA-Technology Platform:Presentations at AACR
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
AACR Annual Meeting, Washington DC 4
Poster 1688 / 7HERA-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-LappeApril 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
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
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
5AACR Annual Meeting, Washington DC
CD40
TNF SF receptors
TNF SF ligands
Stimulation
In vivo
ADCCCDC
Backsignaling
Stimulation
Next generation -HERA-ligands
ADCC: antibody dependent cellular cytotoxicity; CDC: complement dependent cytotoxicity
Immunecells
Molecular design of HERA-GITRL
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
6AACR Annual Meeting, Washington DC
• Receptor agonist with defined trimerizationcapabilities 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 toFc-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
12
3 12
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-Anon-
reduced3 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
100857060
50
40
30
2520
15
10
1 2 3 4 5 6 7
SDS-PAGE
Human and murine HERA-GITRL constructs: Functional binding to GITR from different species
7
0
1
2
3
0,1 1 10 100
ELIS
A s
ign
al
[OD
45
0 n
m]
GITRL [ng/ml]
Binding to human GITR-Fc
0
1
0,1 1 10 100
ELIS
A s
ign
al
[OD
45
0 n
m]
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
ELIS
A s
ign
al[O
D 4
50
nm
]
GITRL [ng/ml]
Binding to monkey GITR-Fc
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
AACR Annual Meeting, Washington DC
BindingHERA-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 ofbinding constants
QCM measurement (Attana)
QCM: Quartz Crystal Microbalance
Human HERA-GITRL: Cellular in vitro activity assay
8April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
AACR Annual Meeting, Washington DC
• 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
Luci
fera
sesi
gnal
[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
Binding of HERA-GITRL to PBMCs
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
9AACR Annual Meeting, Washington DC
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)
Ce
ll co
un
t
GITR expression
Day 0 stimulation, day 2 FCM (ligand binding; detection via StrepTag)
No ligand HERA-GITRL-A HERA-GITRL-B HERA-GITRL-C
Cel
l co
un
t
HERA-GITRL binding
Unstimulated (Medium)
Stimulated with anti-CD3 (HIT3a)
HERA-GITRL treatment enhances proliferation and differentiation in stimulated human T cell cultures
T cells alone anti-CD3anti-CD3
+ HERA-GITRL-C 10 ng/ml
anti-CD3+ HERA-GITRL-C
100 ng/ml
CFSE (proliferation) (log scale) (gated on T cells)
FSC
(si
ze)
(lin
ear)
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)
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
AACR Annual Meeting, Washington DC 10
HERA-GITRL enhances proliferationin 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
CD
45
RO
CD45RA
Medium anti-CD3 anti-CD3+ HERA-GITRL-A
13.9% 49.0% 61.2%
73.3% 31.0% 14.1%
CD
45
RO
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
HERA-GITRL treatment enhances activation marker expression and production of pro-inflammatory cytokines in stimulated human T cell cultures
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
AACR Annual Meeting, Washington DC 11
Medium anti-CD3/anti-CD28 anti-CD3/anti-CD28+ HERA-GITRL-C
45.9% 47.5% 60.2%
Ce
ll co
un
t
TNFα (intracellular) (log scale)
14.3% 33.8% 40.9%
Ce
ll co
un
t
IFNγ (intracellular) (log scale)
HERA-GITRL increases production ofpro-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
Ce
ll co
un
t
2.6% 43.5% 50.6%
Total T lymphocytes
CD25
Medium anti-CD3 anti-CD3 + HERA-GITRL-A
Ce
ll co
un
t
9.4% 41.2% 46.5%
Day 0 stimulation with anti-CD3 (OKT3) + HERA-GITRL-A, day 6 FCM
AACR Annual Meeting, Washington DC 12
Anti-tumor activity of PBMCs by combinatorial treatmentwith HERA-ligands in vitro (RTCA)
• HERA-CD40L treated PBMCs induce killing activity against tumor cells in co-cultures
• HERA-GITRL in combination withHERA-CD40L increases in vitro killing
-0,001
0,001
0,003
0,005
0,007
MDA-MB231human breast adenocarcinoma cell line
Tum
or
cell
gro
wth
[slo
pe
1/h
r]
0.0
PBMC - + + + +
HERA-CD40L - - + - +
HERA-GITRL-C - - - + +
-0,0025
0,0025
0,0075
0,0125
0,0175
HCT 116human colorectal carcinama cell line
Tum
or
cell
gro
wth
[slo
pe
1/h
r]
PBMC - + + + +
HERA-CD40L - - + - +
HERA-GITRL-C - - - + +
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
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 assayco-culture of
tumor cells andPBMC
RTCA: Real time cell analyzer
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
AACR Annual Meeting, Washington DC 13
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
Seru
m c
on
cen
trat
ion
[µg/
ml]
Time after injection [h]
Single i.v. dose of 10 mg/kg bw HERA-GITRL-A
HERA-GITRL-B
HERA-GITRL-C
CompoundDosing
[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
Seru
m c
on
cen
trat
ion
[µ
g/m
l]
Time after injection [h]
Single i.v. dose of 1 mg/kg bwHERA-GITRL-A
HERA-GITRL-B
HERA-GITRL-C
CompoundDosing
[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)
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
14
mmHERA-GITRL enhances antigen-specific clonal expansionin both CD4+ and CD8+ T cell populations in vivo
OVA peptide-specificCD8+ “OT-1” + CD4+ “OT-2” CD45.2+
i.v. 2 x 106 (each) day -1
OVA proteini.p. 5 mg day 0
mmHERA-GITRLi.v. day 0
Serial blood samplesDays 6, 10, 13
CD4+CD45.2+ & CD8+CD45.2+
T cells enumerated
B6/Ly5.1CD45.1+
CD45.2+ (congenic marker)
2%
1.3% 11.3%
4.2%
3.5% 5.1%
7.7%10.7%
CD
8+
CD
4+
OT-1
OT-2
PBSmmHERA-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 10 150
5
10
15APG1585 (1 mg/kg)
APG1585 (5 mg/kg)
DTA-1 (1 mg/kg)
PBS
No OVA
OT-2
0 5 10 150
5
10
15APG1585 (1 mg/kg)
APG1585 (5 mg/kg)
DTA-1 (1 mg/kg)
PBS
No OVA
CD
45
.2+
% o
f C
D8
+C
D4
5.2
+%
of
CD
4+
OT-1
0 5 10 150
5
10
15mmHERA-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 cellsin a dose dependent manner
OT-1/OT-2 mouse model
mmHERA-GITRL shows anti-tumor efficacy in syngeneic mouse models
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
15AACR Annual Meeting, Washington DC
• 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-CEAmmHERA-GITRL (1mg/kg)
days (d)
tum
or
vo
lum
e [
mm
³] +
/-S
EM
0 5 10 15 20 250
200
400
600
800
1) PBS
2) mm HERA-GITRL
TGI: 42.2%
MC38-CEAControl (PBS)
day [d]
tum
or
vo
lum
e [
mm
³] +
/-S
EM
0 5 10 15 20 250
200
400
600
800A1
A2
A3
A4
A5
A6
MC38-CEAmmHERA-GITRL (1mg/kg)
days (d)
tum
or
vo
lum
e [
mm
³] +
/-S
EM
0 5 10 15 20 250
200
400
600
800A1
A2
A3
A4
A5
A6
MC38-CEA syngeneic mouse model
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
AACR Annual Meeting, Washington DC 16
Human HERA-GITRL shows anti-tumor activity in xenograft mouse modelsinjected 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)
tum
or
vo
lum
e [
mm
³] +
/-S
EM
0 5 10 150
50
100
150
200Control
1mg/kg
TGI: 35.8%
Kyse270 (H&N)HERA-GITRL-C
days (d)
tum
or
vo
lum
e [
mm
³] +
/-S
EM
0 5 10 150
100
200
300
400Control
1mg/kg
TGI: 31.9%
TGI d2: 43.1%
HCC827 (NSLC)HERA-GITRL-C
days (d)
tum
or
vo
lum
e [
mm
³] +
/-S
EM
0 5 10 150
50
100
150
200Donor 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)
tum
or
vo
lum
e [
mm
³] +
/-S
EM
0 5 10 150
100
200
300
400Donor 1 (Control)
Donor 1 (1mg/kg)
Donor 2 (Control)
Donor 2 (1mg/kg)
TGI d2: 37.1%
HERA-GITRL are superior over agonistic GITR antibodies
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
17AACR Annual Meeting, Washington DC
• 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 activityYes
Defined mechanism of actionNo
Unclear mechanism of action
Treg depletion No Yes
Other activities No
YesDepletion of GITR expressing cells
(ADCC, CDC)Fc-receptor mediated back-signaling
Toxicity /Immunogenicity
Potentially lowLow immunogenic potential
Autoimmune reaction (Treg depletion)Potential ADA response to CDRs
Pharmacokinetics Adjustable half-life (days) Long half-life (weeks)
DynamicsFast-In / fast-Out
-> No exhaustion of T cellsLong-term activation
-> Exhaustion of T cells; effect on Tregs
Combination withother immune-oncology (I-O) compounds
Sequential combination with otherI-O compounds is possible and
addresses the dynamic nature of anti-tumor response
Sequential combination with otherI-O compounds is presumably difficultdue to long half-life and toxicity issues
Thanks for the commitment of all Apogenix colleagues involved in the HERA projects!
Acknowledgements
April 2017 - © Copyright 2017 Apogenix AG. All rights reserved
18AACR Annual Meeting, Washington DC
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www.apogenix.com
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