novel hexavalent gitr agonists stimulate t cells and enhance memory formation

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


2AACR Annual Meeting, Washington DC

HERA-Technology Platform:Targeting major pathways in Immuno-Oncology


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



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



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



• 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



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


• 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



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)



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



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


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 - - - + +


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



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)


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


• 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



• 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



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



• 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



Apogenix AGIm Neuenheimer Feld 584D-69120 HeidelbergGermany

Telephone +49 (0)6221 586080Fax +49 (0)6221 5860810

www.apogenix.com

novel hexavalent gitr agonists stimulate t cells and enhance memory formation

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