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Decoy. TRAIL - R1 R2 R3 R4 Osteoprotegerin. Cell Res. 2004 Oct;14(5):359-72. TRAIL, Death Receptors, TRA-8, and Apoptosis. 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.

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slide1

Decoy

TRAIL - R1 R2 R3 R4 Osteoprotegerin

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

TRAIL, Death Receptors, TRA-8, and Apoptosis

  • DR5is 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-8is a monoclonal anti-human DR5 antibody, which induces apoptosis upon binding to its receptor, DR5.

TRA-8/TRAIL

DR5

Apoptosis

slide2

Anti-human DR5 Antibody Triggers Apoptosis in RA Synovial Fibroblasts

  • TRA-8 can induce apoptosis in human 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?
slide3

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

slide4

Generation of human/mouse Chimeric DR5 Transgenic Mice

Human DR5 Mouse DR5 Chimeric DR5

Not recognized by anti-hu DR5

Recognized by anti-hu DR5

Doesn’t trigger apoptosis in mouse cells

Trigger apoptosis in mouse cells

Human, mouse and chimeric DR5

Generation of hu/mo chimeric DR5 Tg mice

slide6

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

B6

Ubc.Cre

DR5 Ubc.Cre

CD19+

CD4+

CD8+

CD4+

B6

Ubc.Cre

DR5 Tg+ Ubc.Cre

CD8+

CD19+

CD11b+ Gr1+

CD11b+ Ly6C+

CD11b-Ly6C+

CD11b+Gr1+

CD11b+ Ly6C+

Ly6C+

CD11b+

CD11b-

Ly6C+

Count

0 20 40 60 80 100

Hu/mo DR5

hu/mo DR5+ (%)

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.

slide7

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

DR5 Tg+ UbcCre Isotype

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

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

**

Arthritis Scores

Early TRA-8

cCII

cCII

Late TRA-8

DR5Tg- DR5Tg+

H&E Mac-3

H&E Mac-3

Day 60 post 1 cCII

slide8

TRA-8 Treatment Depletes Macrophages

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

slide9

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

Current therapeutic targets related to macrophages

Anti-Inflammatory

Inflammatory

Anti-GM-CSF

Anti-M-CSF

M-CSF

IL-4

IL-13

IL-10

GM-CSF

LPS

IFN-γ

TNF-α

Imatinib mesylate

Anti-TNF-α

Clodoronate Liposomes

Tofacitinib

TNF-α

CD80,86

IL-1, 6, 12, 23

IFN-I

RNI

ROI

CXCL9,10,11

CCL2,3,4,5,15,20

iNOS

Tyrosine kinase

Tyrosine phosphatase

IL-4,10,IL-1Ra,

CD163, MR, GR

CD200R

TGF-β

Ym1/2

Fizz1

CCL1,16,17,18,22

Arg1

CD200-Fc

JAK

IRF4

IRF5

RANK

DR5

TRA-8

Fas

IL-4R IL-10R

RANKL

TLR2,4

Denosumab

Blocker

Osteoclast

Ligand or agonistic antibody

Adapted from J. Li, et al 2012

slide12

25

**

20

15

AB50-Cy5 Intensity/Joint

10

5

0

DR5 Tg-DR5 Tg+

DR5 Tg-DR5 Tg+

Before TRA-8

After TRA-8

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

Caspase Activity

Before TRA-8 treatment

DR5 Tg- DR5 Tg+

After TRA-8 treatment

DR5 Tg- DR5 Tg+

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.

slide13

H&E TUNEL Mac-3

H

H

E

H

E

E

H

M

H

H

DR5

Tg-

DR5

Tg+

E

H

H

H

E

E

M

TU

H

H

H

DR5 Tg-

DR5 Tg+

50

**

40

30

**

TUENL+ cells in the sub synovial lining layer(%)

20

**

10

0

Total Mf Fibroblasts

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

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.

slide14

DR5 Tg- Isotype

DR5 Tg+ Isotype

12

DR5 Tg- TRA-8

DR5 Tg+ TRA-8

8

4

0

12

**

8

Arthritis score

Arthritis score

4

0

10 20 30 40 50 60 70

10 20 30 40 50 60 70

cCII

cCII

cCII

cCII

DR5 Tg TRA-8 Treatment

DR5 Tg+ TRA-8 Treatment

H&E

Mac-3

H&E

Mac-3

TRAP

TRAP

20 X 10 X

E

TR

H

M

H

H

E

E

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

TRA-8

slide16

Summary I

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

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

slide17

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

H.E.

Mac-3

Joint

Joint

Lung

  • Prominent disorders of SHP-1 (PTPN6) deficient mice:
  • Myeloid hyper-proliferation and inappropriate activation.
  • Rapidly progressive patchy dermatitis, limb necrosis, recurrent infections, and premature death consequent to hemorrhagic pneumonitis.
slide21

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

M1 signature genes

Th17 signature genes

slide22

Summary II

Summary

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

β

slide23

Acknowledgement

Division of clinical rheumatology, UAB

Dr. John D. Mountz and lab members

Dr. Hui-Chen Hsu and lab members

Dr. Robert P. Kimberly Dr. S. Louis Bridges Dr. David M. Spalding Dr. W. Winn Chatham Dr. Laura Hughes

Thank you for your support

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