• DR5 is a pro-apoptotic molecule, which can induce cell death in a variety of cells by engaging the it ligand, TRAIL or anti-DR5 antibody.

• TRA-8 is a monoclonal anti-human DR5 antibody, which induces apoptosis upon binding to its receptor, DR5.

TRAIL, Death Receptors, TRA-8, and Apoptosis

TRAIL - R1 R2 R3 R4 Osteoprotegerin

Decoy

Cell Res. 2004 Oct;14(5):359-72.

TRA-8/TRAIL

DR5

Apoptosis

• TRA-8 can induce apoptosis in human RA synovial fibroblasts.

Anti-human DR5 Antibody Triggers Apoptosis in RA Synovial Fibroblasts

Rheumatoid Arthritis Synovial Fibroblasts

Isotype control TRA-8

• Advantages of TRA-8 compared to TRAIL: Specificity to DR5; Long half-life; No hepatocellular toxicity.

• Can TRA-8 induce apoptosis of pathogenic macrophages and T cells in RA?

3

1. Creation of a mouse model with a humanized DR5 reactive with TRA-8.

2. Control of expression through a Cre/loxp system, using both general (Ubiquitous C) and macrophage specific (Lysozyme M) expression Cre mice.

3. Murine models of inflammatory arthritis (Collagen II-induced arthritis).

Strategies Used to Determine the Arthritis Therapeutic Effects of TRA-8 in Mice

Human DR5 Mouse DR5 Chimeric DR5

Human, mouse and chimeric DR5

Generation of human/mouse Chimeric DR5 Transgenic Mice

cIAP2

c-FLIPL

cIAP2

c-FLIPL

Mouse DR5 Promoter Floxed STOP

cFLIP or cIAP2 Chimeric DR5

Mouse DR5 Promoter Floxed STOPFloxed STOP

cFLIP or cIAP2 Chimeric DR5cIAP2

c-FLIPL

cIAP2

c-FLIPL

Mouse DR5 Promoter Floxed STOP

cFLIP or cIAP2 Chimeric DR5

Mouse DR5 Promoter Floxed STOPFloxed STOP

cFLIP or cIAP2 Chimeric DR5cIAP2

c-FLIPL

cIAP2

c-FLIPL

Mouse DR5 Promoter Floxed STOP

cFLIP or cIAP2 Chimeric DR5

Mouse DR5 Promoter Floxed STOPFloxed STOP

cFLIP or cIAP2 Chimeric DR5cIAP2

c-FLIPL

cIAP2

c-FLIPL

Mouse DR5 Promoter Floxed STOP

cFLIP or cIAP2 Chimeric DR5

Mouse DR5 Promoter Floxed STOPFloxed STOP

cFLIP or cIAP2 Chimeric DR5

Generation of hu/mo chimeric DR5 Tg mice

Doesn’t trigger apoptosis in mouse cells

Not recognized by anti-hu DR5

Recognized by anti-hu DR5

Trigger apoptosis in mouse cells

5

Mo 3kb promoter

x

x

Floxed STOP hu/mo DR5 Ubc/Cre

Floxed STOP hu/mo DR5 LysM/Cre

xx

xx

Floxed STOP hu/mo DR5 Ubc/Cre

Floxed STOP hu/mo DR5 LysM/CreLysM.Cre

Ubc.Cre

Generation of human/mouse Chimeric DR5 Transgenic Mice

6

CD8+CD4+ CD19+

CD11b+

Gr1+

CD11b+ Ly6C+

CD11b-

Ly6C+

Hu/mo DR5

Co

un

tB6

Ubc.Cre

DR5 Ubc.Cre

0 20 40 60 80 100

Ly6C-CD11b+

Ly6C+CD11b+

CD11cGr1

CD19

CD8

CD4

Ly6C+CD11b+

CD4+

CD8+

CD19+

CD11b+

Gr1+

hu/mo DR5+ (%)

B6

Ubc.Cre

DR5 Tg+ Ubc.Cre

0 20 40 60 80 100

CD11b-

Ly6C+

CD11b+

Ly6C+

Induction of CIA. CIA was induced using DR5 Tg mice of C57BL/6 background that were 8- to 16-weeks old. Mice were immunized by intradermal administration of chicken Type II collagen in complete CFA, followed by injection of chicken CII in IFA on day 30 after the primary injection. To ensure a higher incidence of CII arthritis, an adenovirus expressing mouse IL-17 (2x109 pfu/mouse) was administered IV to all mice 2 days prior to the primary immunization with CII.

TRA-8 treatment of CIA mice. TRA-8 (dissolved in PBS, 0.2 mg per mouse) or IgG1 isotype control was administered i.v. or i.p. twice/week starting on day 0 (early treatment) or on day 30 (late treatment) until mice were sacrificed.

Tg DR5 Expression in Draining LN of Ubc.Cre DR5 Mice (2-mo post CII)

7

H&E Mac-3 H&E Mac-3

Day 60 post 1 cCII

DR5Tg- DR5Tg+

0

1

2

3

4

5

6

7

8

9

0 20 23 25 27 29 31 33 35 37 39 41 43 45

Art

hrit

is S

core

s

DR5 Tg+ UbcCre Isotype

DR5 Tg+ UbcCre Early TRA-8 (from day 0)

DR5 Tg+ UbcCre Late TRA-8 (from day 30)**

cCII cCII

Early TRA-8Late TRA-8

TRA-8 Suppresses the Development of CIA in Ubc.Cre DR5 Mice

8

TRA-8 Treatment Depletes Macrophages

Question: What is the TRA-8 effect if TgDR5 expression is restricted in macrophages?

Inflammatory Anti-Inflammatory

Osteoclast

GM-CSFLPSIFN-γTNF-α

M-CSFIL-4

IL-13IL-10

TNF-αCD80,86IL-1, 6, 12, 23IFN-IRNIROICXCL9,10,11CCL2,3,4,5,15,20iNOS

IRF5 IRF4

DR5

IL-4,10,IL-1Ra,CD163, MR, GRCD200RTGF-βYm1/2Fizz1CCL1,16,17,18,22Arg1

Fas TLR2,4 IL-4R IL-10R

Tyrosine kinaseTyrosine phosphatase

TRA-8

Anti-TNF-α

Anti-GM-CSF Anti-M-CSF

Clodoronate Liposomes

CD200-Fc

RANK

RANKL

Denosumab

Imatinib mesylate

Blocker

Ligand or agonistic antibody

Adapted from J. Li, et al 2012

M1/M2 Imbalance in Rheumatoid Arthritis - Can It Be Corrected by TRA-8?

Current therapeutic targets related to macrophages

JAK

Tofacitinib

Mo 3kb promoter

x

x

Floxed STOP hu/mo DR5 Ubc/Cre

Floxed STOP hu/mo DR5 LysM/Cre

xx

xx

Floxed STOP hu/mo DR5 Ubc/Cre

Floxed STOP hu/mo DR5 LysM/CreLysM.Cre

Ubc.Cre

Generation of Chimeric DR5 Transgenic LysM.Cre Mice

TRA-8 Treatment Eliminates Inflammatory Macrophages in DR5 Transgenic LysM.Cre CIA Mice

12

Before TRA-8 treatment

DR5 Tg- DR5 Tg+

After TRA-8 treatment

DR5 Tg- DR5 Tg+

Caspase Activity

0

5

10

15

20

25

TghuDR5-

TghuDR5+

TghuDR5-

TghuDR5+

Before TRA-8 After TRA-8

**

DR5 Tg- DR5 Tg+ DR5 Tg- DR5 Tg+

Before TRA-8 After TRA-8

AB

50-

Cy5

In

tens

ity/J

oint

25201510

50

Measurement method: Mice were induced to develop CIA. At 8 week after the primary cCII injection, Mice were then treated with TRA-8 (0.2 mg on day 0 and day 3) and apoptosis imaging using AB50-Cy5 was performed on the same mice on day 6 after initiating TRA-8 treatment.

Apoptosis Induced by TRA-8 in Joints of DR5 Transgenic LysM.Cre Mice with CIA

13

TU

EN

L+ c

ells

in t

he s

ub

syno

vial

lini

ng la

yer(

%)

0

10

20

30

40

50

Total Macrophages Fibroblasts

Tg huDR5-Tg huDR5+

**

**

**

Total M Fibroblasts

DR5 Tg-

DR5 Tg+50

40

30

20

10

0

DR5 Tg-

DR5 Tg+

H&E TUNEL Mac-3

H

H

E

E

H

H

E H

H

E H

H

E H

H

EH

H

TU M

M

Measurement method: Mice were induced to develop CIA. At 8 week after the primary cCII injection, Mice were then treated with TRA-8 (0.2 mg on day 0 and day 3) and were sacrificed for in situ staining of joint apoptosis (TUNEL) and joint macrophage (Mac-3) infiltration.

Apoptosis Induced by TRA-8 in Joints of DR5 Transgenic LysM.Cre Mice with CIA

14

DR5 Tg+ TRA-8 Treatment

H&E Mac-3 TRAP

DR5 Tg TRA-8 Treatment

H&E Mac-3 TRAP

H

E

E

HM

H

E

TR

20

X

1

0 X

0123456789

101112

0 10 19 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68

DR5 Tg- Isotype

DR5 Tg- TRA-8

Art

hrit

is s

core

12

8

4

0

10 20 30 40 50 60 70

DR5 Tg- Isotype

DR5 Tg- TRA-8

0123456789

101112

0 10 19 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68

DR5 Tg+ Isotype

DR5 Tg+ TRA-8

**

10 20 30 40 50 60 70

12

8

4

0

DR5 Tg+ Isotype

DR5 Tg+ TRA-8

Art

hrit

is s

core

cCII cCIIcCII cCII

TRA-8

TRA-8 Treatment Ameliorates the Severity of CIA in DR5 Transgenic LysM.Cre Mice

15

Immuno-modulatory Effects of TRA-8 in DR5 Transgenic LysM.Cre Mice with CIA

16

1. In human/mouse DR5 Tg Ubc.Cre mice with CIA, Tg DR5 is expressed on CD11b+ macrophages and CD4+ T cells; In DR5 Tg LysM.Cre mice, Tg DR5 is restrictively expressed on macrophages;

2. TRA-8 (anti-human DR5) treatment results in depletion of pathogenic macrophages and Th17 cells, increased Treg cells in draining LNs of DR5 Tg CIA mice;

3. TRA-8 is potential novel biologic agent for rheumatoid arthritis.

Summary I

QUESTION: What is the TRA-8 effect in a systemic autoimmune disease model?

Prominent disorders of SHP-1 (PTPN6) deficient mice:1. Myeloid hyper-proliferation and inappropriate activation.2. Rapidly progressive patchy dermatitis, limb necrosis, recurrent infections,

and premature death consequent to hemorrhagic pneumonitis.

Joint

Joint

Lung

Mac-3H.E.

Chimeric DR5 Transgenic x Viable Motheaten Mice (mev/mev)

DR5 expression and TRA-8 Induced Depletion of Inflammatory Macrophages in DR5 Tg mev/mev Mice

DR5 expression and TRA-8 Induced Depletion of Pathogenic CD4 T Cells in DR5 Tg mev/mev Mice

TRA-8 Treatment Reduces Tissue Inflammation and Increases Lifespan of DR5 Tg mev/mev Mice

TRA-8 Eliminates Inflammatory M1 Macrophages and CD4 T Cells in Human RA Patients with Low PTPN6

M1 signature genes Th17 signature genes

SummarySummary II

1. DR5 expression is upregulated in inflammatory macrophages and pathogenic CD4 T cells in DR5 Tg mev/mev Mice;

2. TRA-8 selectively eliminates IRF5+ IL-23+ pathogenic macrophages and IL-17+ GM-CSF+ CD4 T cells in both human and mouse autoimmune conditions, resulting in immune homeostasis and resolution of inflammation.

3. Anti-human DR5 (TRA-8) can deplete inflammatory cell populations that result from a hyperactive GM-CSF/IRF5 AXIS, and it is a novel biologic agent for RA and other autoimmune diseases.

Naïve CD4

M2 M

Treg

Monocytes

TRA-8

Th1/17

IL-6, IL-23

M1 M

GM-CSF

TRA-8RA inflammation

positive feedback loop

DR5+IRF-5+IL-23+IL-23R+GM-CSFR+

DR5+GM-CSF+IL-17+ [IFN+]IL-23R+RORt+

IL-10+Foxp3+

IL-10+TGF+M-CSFR+IRF-4+

Naïve CD4

M2 M

Treg

Monocytes

TRA-8

Th1/17

IL-6, IL-23

M1 M

GM-CSF

TRA-8RA inflammation

positive feedback loop

DR5+IRF-5+IL-23+IL-23R+GM-CSFR+

DR5+GM-CSF+IL-17+ [IFN+]IL-23R+RORt+

IL-10+Foxp3+

IL-10+TGF+M-CSFR+IRF-4+

β

Thank you for your supportThank you for your support

Dr. John D. Mountz and lab members Dr. Hui-Chen Hsu and lab members

Division of clinical rheumatology, UAB

Dr. Robert P. Kimberly Dr. S. Louis Bridges

Dr. David M. Spalding Dr. W. Winn Chatham Dr. Laura

Hughes

Acknowledgement