• 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

TRA-8/TRAIL

TRAIL - R1 R2 R3 R4 Osteoprotegerin

Decoy

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

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?

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

Strategies Used to Determine the Arthritis

Therapeutic Effects of TRA-8 in Mice

3

both general (Ubiquitous C) and macrophage specific (Lysozyme M) expression Cre mice.

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

Human DR5 Mouse DR5 Chimeric DR5

Human, mouse and chimeric DR5

Generation of human/mouse Chimeric

DR5 Transgenic Mice

Not recognized

by anti-hu DR5Recognized by

anti-hu DR5

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

Trigger apoptosis

in mouse cells

Mo 3kb promoter

Generation of human/mouse Chimeric

DR5 Transgenic Mice

5

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

CD8+CD4+ CD19+

CD11b+

Gr1+

CD11b+

Ly6C+

CD11b-

Ly6C+

Co

un

tB6

Ubc.Cre

DR5 Ubc.Cre

Ly6C+CD11b+

CD11cGr1

CD19

CD8

CD4

Ly6C+CD11b+

CD4+

CD8+

CD19+

CD11b+

Gr1+

B6

Ubc.Cre

DR5 Tg+ Ubc.Cre

CD11b+

Ly6C+

Tg DR5 Expression in Draining LN of

Ubc.Cre DR5 Mice (2-mo post CII)

6

Hu/mo DR5

Co

un

t

0 20 40 60 80 100

Ly6C-CD11b+

CD11b+

hu/mo DR5+ (%)

0 20 40 60 80 100

CD11b-

Ly6C+

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.

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

hritis S

co

res

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-8

Late TRA-8

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

7

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

Day 60 post 1°°°° cCII

DR5Tg- DR5Tg+

Late TRA-8

TRA-8 Treatment Depletes Macrophages

8

Question: What is the TRA-8 effect if TgDR5

expression is restricted in macrophages?

Inflammatory Anti-Inflammatory

GM-CSF

LPS

IFN-γ

TNF-α

M-CSF

IL-4

IL-13

IL-10

TNF-α IL-4,10,IL-1Ra,Tyrosine kinase

Tyrosine phosphatase

Anti-TNF-α

Anti-GM-CSF Anti-M-CSF

Clodoronate Liposomes

Imatinib

mesylate

M1/M2 Imbalance in Rheumatoid Arthritis -

Can It Be Corrected by TRA-8?

Current therapeutic targets related to macrophages

JAK

Tofacitinib

Osteoclast

TNF-αCD80,86

IL-1, 6, 12, 23

IFN-I

RNI

ROI

CXCL9,10,11

CCL2,3,4,5,15,20

iNOS

IRF5 IRF4

DR5

IL-4,10,IL-1Ra,

CD163, MR, GR

CD200R

TGF-β

Ym1/2

Fizz1

CCL1,16,17,18,22

Arg1

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

Tyrosine phosphatase

TRA-8

CD200-Fc

RANK

RANKL

Denosumab

Blocker

Ligand or

agonistic antibody

Adapted from J. Li, et al 2012

Mo 3kb promoter

Generation of Chimeric DR5 Transgenic LysM.Cre Mice

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

TRA-8 Treatment Eliminates Inflammatory

Macrophages in DR5 Transgenic LysM.Cre CIA Mice

Before TRA-8 treatment

DR5 Tg- DR5 Tg+

After TRA-8 treatment

DR5 Tg- DR5 Tg+

Caspase Activity

Apoptosis Induced by TRA-8 in Joints of DR5 Transgenic

LysM.Cre Mice with CIA

12

0

5

1 0

1 5

2 0

2 5

T g

h u D R 5 -

T g

h u D R 5 +

T g

h u D R 5 -

T g

h u D R 5 +

B e f o re T R A -8 A f te r T R A -8

**

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

Before TRA-8 After TRA-8

AB

50-C

y5

Inte

nsity/J

oin

t 2520

15

10

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.

DR5 Tg-

DR5 Tg+

H&E TUNEL Mac-3

HE

E

H

H

E H

H

E H

E H

H

EH

TU M

M

Apoptosis Induced by TRA-8 in Joints of DR5 Transgenic

LysM.Cre Mice with CIA

13

TU

EN

L+

cells

in the s

ub

synovia

l lin

ing layer(

%)

0

10

20

30

40

50

Total Macrophages Fibroblasts

Tg huDR5-Tg huDR5+

**

**

**

Total Mφ Fibroblasts

DR5 Tg-

DR5 Tg+50

40

30

20

10

0

H H H

TU

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.

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

hritis s

co

re

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

hritis s

co

re

cCII cCIIcCII cCII

TRA-8

TRA-8 Treatment Ameliorates the Severity of CIA in DR5

Transgenic LysM.Cre Mice

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

TRA-8

Immuno-modulatory Effects of TRA-8 in DR5 Transgenic

LysM.Cre Mice with CIA

15

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

Summary I

16

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.

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

Joint

Joint

Mac-3H.E.

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

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.

Lung

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.autoimmune diseases.

Naïve CD4

M2 Mφφφφ

Treg

Monocytes

TRA-8

Th1/17

IL-6, IL-23

M1 Mφφφφ

GM-CSF

TRA-8RA inflammation

positive feedback loop

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

DR5+GM-CSF+

IL-17+ [IFNγ+]IL-23R+

RORγt+

IL-10+

Foxp3+

IL-10+

TGF+

M-CSFR+

IRF-4+

Naïve CD4

M2 Mφφφφ

Treg

Monocytes

TRA-8

Th1/17

IL-6, IL-23

M1 Mφφφφ

GM-CSF

TRA-8RA inflammation

positive feedback loop

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

DR5+GM-CSF+

IL-17+ [IFNγ+]IL-23R+

RORγt+

IL-10+

Foxp3+

IL-10+

TGF+

M-CSFR+

IRF-4+

β

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

Acknowledgement

Thank you for your supportThank you for your support

Dr. David M. Spalding

Dr. W. Winn Chatham

Dr. Laura Hughes