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 antoine.tanne@agenusbio.comDhan Chand dhan.chand@agenusbio.comPresented 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

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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

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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.