Expanding the Therapeutic Potential of anti-PD-1 and anti-CTLA-4 Therapy with Innovative Fc Engineering and Rationale Combinations for the Treatment of Solid TumorsAntoine Tanne, Sylvia Vincent, Elena Paltrinieri, Sudesh Pawaria, Vidur Patel, Marques Marilyn, Margaret Wilkens, Shalu Kharkwal, Daniel Levey, David Savitsky, Jennifer Buell, Dhan Chand11Agenus Inc. or subsidiary thereof (current or former employee), Lexington, MA
AGEN1181ms has improved therapeutic potential against immunogenic tumors by enhancing Treg depletion and T cell priming
Hypothesis: Optimizing Fc - FcgR co-engagement enhances the activity of anti-CTLA-4antagonist antibodies1,2. CTLA-4 antibodies with increased binding affinities toactivating Fcg receptors FcgRIV (CD16-2, mouse) or FcgRIIIA (CD16a, human) augmentT cell priming by improving the quality of the immune synapse between a T cell and anantigen presenting cell (APC).
Table 1: anti-CTLA-4 Fc Isotype Fc mutations Blocking
Properties FcgR Binding
Characteristics
HumanParental IgG1 - + Low
Fc-Enhanced AGEN1181 IgG1.DLE S239D.A330L.I332E +
> FcgRIIIA binding(“Fc enhanced”)
MurineSurrogates
Parental 9D9 mIgG2b - + Low
AGEN1181ms
Fc-Enhanced 9D9 mIgG2b.DLE S241D.A332L.I334E + > FcgRIV binding
Fcүr binding characteristics of studied antibodies
1. Ligand blockade & CD28-dependent T cell activation
2. Depletion of intratumoralregulatory T cells (Tregs)
3. Effectory T cell priming and memory formation
FcγR-independent FcγR-dependent
Combination with anti-PD-1 and Focal Radiation promotes significant tumor control
Fc-enhanced CTLA-4 promotes tumor control in combination with Tumor Antigen Vaccine + QS-21 to enhance tumor control
Fc-enhanced CTLA-4 combines with Adoptive T Cell Therapy (ACT) to promote robust tumor control
AGEN1181 expands the therapeutic reach and activity of CTLA-4 therapy by improved binding to both low and high affinity FcγRIIIA allele
Combination with Alpha-GalCer as iNKT triggering therapy promotes robust tumor clearance in the lung
Fc-enhanced CTLA-4, AGEN1181, combines with anti-PD-1 or anti-TIGIT to further enhance T cell responsiveness
Concurrent treatment promotes curative responses in combination with HSC70 tumor antigen vaccine + QS-21
Agenus Inc. or subsidiary thereof (current orformer employee), Lexington, MA 02421, USA.
CorrespondenceAntoine Tanne [email protected] Chand [email protected] at The American Association for Cancer Research (AACR), June 22 - 24, 2020, Virtual Meeting
Figure 1: BALB/c and C57Bl6 mice with established CT26 and MC38 tumors received a single injection intraperitoneally (i.p.) of anti-CTLA4 clone 9D9 Parental or Fc-Enhanced or mIgG2a isotype control. A-B: Tumor growth are represented as average per group andsingle mice to emphasize the increased potency of the AGEN1181 “surrogate” C-D: CD8 Teff / CD4 Treg ratio changes over time isshown as average per group to emphasize the increased ability of the Fc-Enh. Antibody to deplete Treg and expend Teff. Briefly, tumorwere resected and were phenotyped by flow cytometry the effector CD8 T (CD3+ CD8+ CD44+) Vs CD4 regulatory T (CD3+ CD4+FoxP3+) ratio was calculated at different time points post treatment. E-F: The antitumoral memory response was assessed incomplete responder (CR) mice treated with the Fc Enh. Mice were bilaterally re-challenged with 100K primary tumor CT26 and MC38and non relevant tumors EMT 6 or TC1 .
Figure 5: C57Bl6 were challenged s.c. with B16F1::OVA cells. Mice were preconditioned with earlytreatment (D3) using AGEN1181 anti-CTLA-4 Fc-Enhanced “surrogate” and anti-PD-1 or thecorresponding isotype control. Eight days after tumor implantation, tumor-bearing mice were treatedintravenously with OT1-specific transgenic CD8+ T cells (ACT) or PBS. A: Tumor volumes weremeasured every 3-4 days and represented as average per group (N=10) B: Representation of theoverall survival per treatment group.
Sequential treatment with Fc-enhanced anti-CTLA-4 and PD1 promotes curative responses in combination with ACT
Figure 6: C57Bl6 were challenged s.c. with B16F1::OVA cells. Tumor-bearing mice weresubcutaneously treated with the Agenus preclinical heat shock protein vaccine targeting MHC-I/IIovalbumin, and MHC-I TRP2 epitopes (HSC70-OVA–TRP2) administered with Agenu’s QS21 Stimulonand CpG adjuvant. Mice were treated i.p. with anti-CTLA-4 and anti-PD-1 or corresponding isotypecontrol antibodies A: Average tumor volumes per group (n=10/group) B: survival curves and C:treatment response evaluation per treatment group (CR: Complete Responder; PR: partial responderdefined by relapsing pattern).
Conclusions AGEN1181 Fc-enhanced anti-CTLA-4 demonstrates superior single agent and broader IO & SOC combination activity than conventional CTLA-4 mAbs:
• Engages multiple mechanisms of action to promote optimal anti-tumor immunity• Promotes curative responses in checkpoint resistant preclinical cancer models in
combination with anti-PD-1 and Vaccine + QS-21, Adoptive T cell therapy, radiation therapy and iNKT activating Alpha-GalCer adjuvant therapy• Enhances T cell responsiveness in combination with anti-PD-1 or anti-TIGIT mAbs• Expands the therapeutic reach and activity of CTLA-4 therapy by improved binding to both
the low and high affinity FcyRIIIA and in turn expands the therapeutic reach of anti-CTLA-4 to an additional 40% of patients1,3,4 who express the low affinity FcyRIIIA allele and respond poorly to first generation anti-CTLA-4 mAbs.
AGEN1181ms Fc-Enhanced CTLA4 combines with adjuvant therapies
AGEN1181ms Fc-Enhanced CTLA4 & PD1 triple combination with ACT and Vaccines are curative in IO refractory models
Figure 2: C57BL/6 mice were injected i.p. with the staphylococcal enterotoxin B (SEB) superantigen together with anti-CTLA-4 parental, Fc-Enhanced AGEN1181 “surrogate” or isotype control antibodies. SEB-specific (TCR Vb8) CD8+ T cells (CD3+ CD8+ CD44+), theirreplicative status (Ki67) and their functional profile (GrZb) were evaluated by flow cytometry on day 6; demonstrating an increasepotency of the Fc-Enh. antibody to prime antigen specific effector CD8 T cells in a T reg depletion independent mechanism (notshown) . N=4 mice/group, and data are representative of at least three independent experiments.
Figure 3: C57Bl6 were challenged s.c. with B16F1::OVA cells. At day 10 tumor-bearing mice weretreated intravenously with OT1-specific transgenic CD8+ T cells (ACT) or PBS . CTLA4 treated groupsreceived three doses of i.p weekly injections of Fc-Enhanced AGEN1181 “surrogate” or the referencemolecule starting at D10 A: Tumor volumes were measured every 3-4 days and represented asaverage per group (N=10) B: Representation of the overall survival per treatment group. C: Tumorinfiltrating CD8 & CD4 Teff / CD4 Treg ratio evaluation D: Host and ACT Tumor infiltrating OT1 Tet +CD8 Teff / CD4 Treg ratio evaluation. Briefly, tumor were resected and phenotyped by flow cytometry.Effector CD8 T OT1 Tet +/- (CD3+ CD8+ CD44+ PD1+ GzB+) Vs CD4 regulatory T (CD3+ CD4+ FoxP3+)ratio was calculated for Host (CD45.1) and ACT (CD45.2).
Figure 4: C57Bl6 were injected s.c. with B16F1::OVA cells. Challenged mice were subcutaneouslytreated with Agenus preclinical heat shock protein vaccine targeting MHC-I/II ovalbumin epitopes,(HSC70-OVA OTI/II) administered with Agenu’s QS21 Stimulon and CpG adjuvant. Mice were treatedi.p. with anti-CTLA-4 and anti-PD-1 or corresponding isotype control antibodies A: Tumor volumeswere measured every 3-4 days and represented as average per group (N=10) B: survival curves pertreatment group are shown. C: In an independent study tumor were resected after euthanasia at day24 to prepare CD45+ enriched single cell solution and phenotyped by flow cytometry. Activated Eff.Cd8 T cells +/- Tetramer (OT1) (CD3+ CD8+ CD44+ PD1+ GzB+) and Eff. CD4 T cells (CD3+ CD4+CD44+) were characterized Vs host CD4 Tregs (CD3+ CD4+ FoxP3+)
Figure 7: C57BL6 mice, were challenged s.c. with B16F10 cells. Tumor bearing mice received noradiation or a single-dose 10 Gy radiation treatment administered using a Small Animal RadiationResearch Platform. Mice were subsequently treated i.p with anti-PD-1 and AGEN1181 anti-CTLA-4 FcEnhanced “surrogate” or the corresponding isotype control . A-B: Tumor growth are represented asaverage per group and single mice to emphasize the increased efficacy of the triple combo RT withFc-enhanced and PD1.
Figure 9: A: IL-2 production by human PBMCs stimulated with a suboptimal concentration of SEA peptide together with increasingconcentrations of Fc-enhanced anti-CTLA-4 AGEN1181, parental anti-CTLA-4 AGEN1884 (IgG1) or isotype control alone or incombination with 10 µg/ml anti-PD-1 (Pembrolizumab). B: IL-2 production (day 4) by human PBMCs stimulated with a suboptimalconcentration of SEA peptide together with 10 µg/ml of Fc-enhanced anti-CTLA-4, AGEN1181, alone or in combination with 5 µg/ml ofanti-TIGIT mAb.
AGEN1181, an Fc-Enhanced Anti-CTLA-4 Antibodies Engage Multiple Mechanisms of Action to Promote T Cell-Mediated Anti-Tumor Immunity
Background AGEN1181ms Fc Enhanced anti CTLA4 combines with ACT & Vaccines in CPM refractory tumor models AGEN1181 outperforms conventional IgG1 CTLA-4 mAbs
922Poster #
AGEN1181ms
demonstrates superior efficacy
10 15 20 250
500
1000
1500
Days post implantation
Tum
or v
olum
e (m
m3 )
Isotype
ParentalFc Enhanced
Anti-CTLA-4
0 10 20
CTLA4Parental
0 10 20
CTLA4Fc Enhanced
10 15 20 250
500
1000
1500
2000
Tum
or v
olum
e (m
m3 )
Isotype
Fc EnhancedParental
Anti-CTLA-4
0 10 20
CTLA4Parental
0 10 20
CTLA4Fc Enhanced
In vivo s.c. 100K CT26D8 50-80 mm3
Single dose:0.5mg/kg CTLA4
In vivo s.c. MC38
D8 50-80 mm3
Single dose:0.5mg/kg CTLA4
0 48 96 144 192 2400
20
40
60
80 CD8 Teff/Treg ratio
Hours post treatment
T eff/
T reg
ratio
0 50 100 150 200 2500
20
40
60
80CD8 Teff/Treg ratio
Hours post treatment
T eff/T
reg
ratio
0 20 400
500
1000
1500
2000
60 80 100
CR rechalenge
Days post primary implantation
Tum
or v
olum
e (m
m3 ) CT26 (left)
CR 7/8EMT6 (right)CR 0/8
0 10 20 30 400
500
1000
1500
50 60 70 80
CR rechalenge
Days post implantation
Tum
or v
olum
e (m
m3 ) MC38 (left )
CR=3/3TC1 (Right)CR=0/3
0
20
40
60
80
100TCR Vb8
CD8
T ce
lls (%
effe
ctor
) **
0
10
20
30
40 Ki-67
% o
f TCR
+ CD8
T ce
lls
****
0
20
40
60
80GrzB
GrzB
+ of
TCR+ C
D8 T
cells ****
In vivo Murine SEB Assay
SEB 150ug i.p.Single dose:
5 mg/kg CTLA4 0
20
40
60
80GrzB
ParentalFc Enhanced
ParentalFc Enhanced
Anti-CTLA-4
Isotypes
AGEN1181ms
demonstrates a superior CD8 effector T cell responses enhancing ability
In vivo s.c. 150K B16F1::Ova
D10: 120-150 mm3
OT1 ACT (1Dose i.v.1M)CTLA4 (3 Weekly Doses i.p. 2.5 mg/kg)
0 20 40 600
1000
2000
3000
Days post implantation
Tum
or V
olum
e (m
m3 )
NT + IsotypeACT + IsotypeACT + ParentalACT + Fc Enhanced
10 30 50 700
20
40
60
80
100
Study Days
Perc
ent s
urvi
val ACT + Fc Enhanced
ACT + ParentalACT + IsotypeNT + Isotype
*
Host T-CD8 Host T-CD4 0
2
4
6
8
10
25
50Activated Eff. T cells / T regs
Rat
io /
Treg
s
NT+IsotypeACT+IsotypeACT+ParentalACT+Fc Enhanced
ACT Host Host+ACT0.0
0.2
0.4
0.6Eff. CD8+ OT1 Tet + /Tregs
Rat
io /T
regs
NT+IsotypeACT+IsotypeACT+ParentalACT+Fc Enhanced
********
AGEN1181ms
unleashes the ACT antitumor effect
AGEN1181ms
significantly increases the effector T cells to T Reg ratio
Tet+ Act. Eff. CD8
Tet- Act. Eff. CD8
Act. Eff. CD4
0
2
4
6
8
10 CD8/Tregs
Rat
io /
Treg
s
NT+IsotypeVaccine+IsotypeVaccine + ParentalVaccine+ Fc Enhanced
In vivo s.c. 50K B16F1::Ova
D3 OVA Vax (3 Weekly Doses)+Qs21-CpGD3 CTLA4 (3 Weekly 2.5mg/kg Doses Doses)
D3 PD1 ( 6 Bi-Weekly 10mg/kg Doses)
AGEN1181ms
increases the frequency of tumor-specific effector T cell responses
In vivo s.c. 100K B16F1::Ova
D7: 60-80 mm3
D7 OVA Vax (3 Weekly Doses )+Qs21-CpG D3 CTLA4 (3 Weekly 2.5mg/kg Doses)
0 10 20 30 40 50 60 700
500
1000
1500
2000
2500
Days post tumor challenge
Tum
or V
olum
e (m
m3 )
NT + IsotypeVaccine + IsotypeVaccine + ParentalVaccine + Fc Enhanced
0 50 1000
50
100
Days post tumor challenge
Per
cent
sur
viva
l
NT + Isotype
Vaccine + Isotype
Vaccine + Parental
Vaccine + Fc Enhanced
1/9 CR
3/10 CR
AGEN1181ms
unleashes the antitumor vaccine effect
In vivo s.c. 150K B16F1::Ova
D8 OT1 ACT (1 Dose 1M )D3 CTLA4 (3 Weekly Doses i.p. 2.5 mg/kg) D3 PD1 (6 Bi-Weekly Doses i.p. 10 mg/kg )
0 20 40 600
1000
2000
3000
Days Post Tumor Challenge
Tum
or V
olum
e (m
m3 )
NT + IsotypesACT + Isotypes
ACT + Fc Enhanced & PD-1NT + Fc Enhanced & PD-1
0 20 40 600
50
100
Days Post Tumor Challenge
Perc
ent s
urvi
val
NT + Fc Enhanced & PD-1ACT + Fc Enhanced & PD-1
ACT + IsotypesNT + Isotypes
In vivo s.c. 50K B16F1::Ova
D3 OVA-TRP2 Vax (3 Weekly Doses )+Qs21-CpGD3 CTLA4 (3 Weekly Doses i.p. 2.5 mg/kg) D3 PD1 (6 Bi-Weekly Doses i.p. 10 mg/kg )
0 20 40 60 800
1000
2000
3000
4000
5000
Days post tumor challenge
Tum
or V
olum
e m
m3
NT + IsotypesVaccine + IsotypesNT + Fc Enhanced & PD-1Vaccine + Fc Enhanced & PD-1
0 20 40 60 800
50
100
Days post tumor challenge
Perc
ent s
urvi
val
NT+IsotypeIsotype+ TAA VaxNT+Fc Enhanced & PD1Vaccine+ Fc Enhanced & PD1
VaxIso.
VaxFc Enh. & PD-1
0
50
100
Per
cent
Ani
mal
s
CRPR
NTaGalCer NT
Fc Enh.& PD-1aGalCer
Fc Enh. & PD-1
0
10
20
30
40
% iN
KT o
f Live
CD4
5+ B22
0-
Liver
**** *****
NTaGalCer NT
Fc Enh.& PD-1aGalCer
Fc Enh. & PD-1
0
2
4
6
% iN
KT o
f Live
CD4
5+ B22
0-
Spleen
ns
NTaGalCer NT
Fc Enh.& PD-1aGalCer
Fc Enh. & PD-1
0
2
4
6
8
% iN
KT o
f Live
CD4
5+ B22
0-
Lungs***
*** ****
0
100
200
300
Nym
ber of
met
asta
ses
Control
Control + Fc enhanced CTLA-4 + PD-1
aGalCer
aGalCer + PD-1
aGalCer + Fc enhanced CTLA-4
aGalCer + Fc enhanced CTLA-4 + PD-1In vivo i.v. 500K B16F1::Ova
D2 aGAL-Cer ( 1 Dose i.p. 0.2 mg/kg)D3 CTLA4 (3 Weekly Doses i.p. 2.5 mg/kg) D3 PD1 (6 Bi-Weekly Doses i.p. 10 mg/kg)
Figure 8: C57BL6 mice, were injected i.v. with B16F1::OVA cells. Challenged mice were treated i.p. withthe iNKT activator lipid aGalCer or vehicle control. Mice were subsequently treated i.p with anti-PD-1and AGEN1181 anti-CTLA-4 Fc Enhanced “surrogate”. or the corresponding isotype control . A: Nodulecounting to evaluate Pulmonary Disease burden per group (N=8) B: Phenotyping of the lung, spleenand liver infiltrating leukocytes by flow cytometry focusing on the iNKT infiltration.
Isotyp
e
AGEN1181
anti-T
IGIT
AGEN1181
anti-T
IGIT
0
5
10
15
20
25
IL-2
Sec
retio
n(fo
ld c
hang
e)
In vitro PBMC SEA Assay
AGEN1181 and anti-PD-1 potentiate T cell signaling
-3 -2 -1 0 1 20
2000
4000
6000
8000
Antibody µg/ml [log]
IL-2
(pg/
ml)
Isotype controlAGEN1884 (parental)AGEN1181 (Fc enh.)
Isotype controlAGEN1884 (parental)AGEN1181 (Fc enh.)
+ Anti-PD-1
AGEN1181 and anti-TIGIT potentiate T cell signaling
In-vitro Cell Binding AssayAGEN1884 : IgG1
AGEN 1181: Fc enhanced IgG1
In-vitro SEA Assay
AGEN1884 : IgG1 AGEN 1181: Fc enhanced
IgG1
Figure 10: A: Binding profiles of the human Fc engineered antibody AGEN1181 Vs the parental AGEN1884 and Fc silentvariants of the anti-CTLA4 blocking antibody to cells stably expressing hFcγRIIIA V/V haplotype, hFcγRIIIA F/F haplotype.Binding was assessed by flow cytometry, mean fluorescence intensity normalized according to standard methods and theratio of the increase binding Vs parental was calculated. B: Evaluation of IL-2 production by human PBMCs donors thatare homozygous for the high affinity hFcγRIIIA V/V haplotype, or the low affinity hFcγRIIIA F/F haplotype andstimulated with staphylococcal enterotoxin A (SEA) peptide together with increasing concentrations of anti-CTLA-4hIgG1: AGEN1884, antiCTLA-4 Fc-enhanced hIgG1: AGEN1181 or an hIgG1 Fc-enhanced (Fc E) isotype controlantibody. Polymorphism in Fcg receptor was determined by PCR followed by Sanger sequencing.
AGEN1181ms
reshapes the tumor immune microenvironment and induces a durable memory response
On-Going TrialCombination of AGEN1181 with Agenus's balstilimab (anti-PD-1) is advancing in the clinic (NCT03860272).
A
B
C
D
C
A B
D C
A B
A B A B C
A B
E
F
In vivo s.c. 200K B16F10
D7 (100mm3) D7 10 Gy radiation (1 Dose)
D8 CTLA4 (5 Bi Weekly Doses i.p. 10 mg/kg)D8 PD1 (5 Bi Weekly Doses i.p. 10 mg/kg)
0 10 20 30 400
1000
2000
3000
4000
Days Post Tumor Challenge
Tum
or v
olum
e (m
m3 )
Isotype Controlanti-PD-1Fc-enhanced CTLA-4Fc-enhanced CTLA-4 + anti-PD-1
Isotype Controlanti-PD-1Fc-enhanced CTLA-4Fc-enhanced CTLA-4 + anti-PD-1
+ Focal Radiation (10Gy)
0 10 20 300
1000
2000
3000
4000
Focal Radiation+ Isotype Control
Tum
or v
olum
e (m
m3 )
0 10 20 30
Focal Radiation+ anti-PD1
0 10 20 30
Focal Radiation+ Fc-enhanced anti-CTLA-4
0 10 20 30 40
Triplecombination therapy
-3 -2 -1 0 1 20
10
20
30
40
50
Antibody [ug/mL]
Fold
cha
nge
(bin
ding
)
-3 -2 -1 0 1 20
50
100
150
Antibody [µg/mL]
Fold
cha
nge
(bin
ding
) IgG1 variantIsotype control
AGEN1181
FcγRIIIA V/V158 FcγRIIIA F/F158
-3 -2 -1 0 1 20
5000
10000
15000
Antibody (log µg/mL)
IL-2
pg/
mL
AGEN1181IgG1 variantIsotype control
-3 -2 -1 0 1 20
5000
10000
15000
Antibody (log µg/mL)
IL-2
pg/
mL
FcγRIIIA V/F158 FcγRIIIA F/F158
F - low affinity alleleV - high affinity allele
A
B
References:1Waight et al. Cancer Cell 20182Danbee et al. PNAS 20183Vargas et al. Cancer Cell 20184Romano et al. PNAS 2015
B
A A
B
For a more detailed presentation:
This version includes corrections to an error in the original presentation contained on the legend for Figure 10B.
