A Complex Pathway-A Feast of Possibilities New Immunology and New Immunotherapy of Type 1 Diabetes

A Complex Pathway-A Feast of Possibilities

New Immunology and New Immunotherapy of Type 1 Diabetes

Mark D. (The Other) Pescovitz, MD

Professor of Surgery and Microbiology/Immunology

Indiana University School of Medicine

2

POTENTIAL CONFLICT BASED ON FINANCIAL/CONSULTING/ RESEARCH

INTERACTIONS• ROCHE

• LILLY

• VICAL

• GENENTECH

• WYETH

• NOVARTIS

• ASTELLAS

• PFIZER

• US GOVERNMENT

Research SupportSpeaker’s Bureau Board Member/Advisory Panel Stock/Shareholder ConsultantTax Payer

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Clinical onset of Clinical onset of diseasedisease

100 %100 %

20%20%

PrimaryPrimary Prevention (genetically at risk) Prevention (genetically at risk)

STOP progression to autoimmunity/beta cell destruction

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Clinical onset of Clinical onset of diseasedisease

100 %100 %

20%20%

Secondary Prevention (antibody positive)Secondary Prevention (antibody positive)

STOP clinical disease

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TimeTime

100 %100 %

20%20%

Tertiary Prevention (early in clinical disease)Tertiary Prevention (early in clinical disease)

Clinical onset of disease

Preserve Beta cellsSTOP complications

6

The Immunobiology of Type 1 Diabetes

Activated Activated T cellsT cells

T-cell T-cell proliferationproliferation

Signal 2:Costimulation Signal 3:

IL-2R,IL-15R

RestingRestingDCDC

DC DC Maturation Maturation

Signal 1:MHC/peptides

Recognition by TCR

T-cellT-cellGrowthGrowthFactorsFactors

Normal blood sugar Diabetes

Immunosuppressive DrugsMechanisms of Action

T-Cell T-Cell ActivationActivation

T-Cell T-Cell ProliferationProliferation

Signal 2:Signal 2:CostimulationCostimulation Signal 3:Signal 3:

IL-2RIL-2RIL-15IL-15

RestingRestingDCDC

DCDCMaturationMaturation

DaclizumabDaclizumabBasiliximabBasiliximab

CCsAsATacrolimuTacrolimuss

Muromonab-CD3Muromonab-CD3

MMFMMF

Signal 1:Signal 1:MHC/peptMHC/peptidesides

Recognition by TCRRecognition by TCR

MHC TCR

MMFMMFSteroidsSteroids

MMFMMFSirolimusSirolimus

T-CellT-CellGrowthGrowthFactorsFactors

B7

CD40

CD28

CD40L

SirolimusSirolimus

Adapted with permission from Professor Dr. Walter Land and M. Schneeberger, University of Munich, Germany.

B

8

MONOCLONAL ANTIBODY STRUTURE

MouseMouse Human

HumanizedChimeric

9

MONOCLONAL ANTIBODY NOMENCLATURE

Rituximab

Daclizumab

MonoclonalAntibody

Chimeric

Humanized

Muromonab

Murine

10

Mechanisms of Action- Anti-CD3



Signal 2:costimulation Signal 3:

IL-2R,IL-15

RestingRestingDCDC


Anti-CD3Anti-CD3


Recognition by TCR

MHC TCRT-cellT-cell

GrowthGrowthFactorsFactors

B7

CD40

CD28

CD40L

11

HUMANIZED, MUTAGENIZEDANTI-CD3 MONOCLONAL ANTIBODY FOR

TREATMENT OF TYPE 1 DIABETES

12

Reduced insulin requirements in anti-CD3 treated subjects

Keymeulen, B. et al. N Engl J Med 2005;352:2598-2608

Example of Mixed Meal Tolerance TestExample of Mixed Meal Tolerance Test

Active Rx

Placebo

14

Better c-peptide response to MMTT in anti-CD3 treated subjects

Herold, K. et al. N Engl J Med 2002;346:1692-1698

Changes in C-Peptide Responses Changes in C-Peptide Responses During MMTT Over TimeDuring MMTT Over Time

0

20

40

60

80

100

120

140

Baseline 6 months 12 months

C-Peptide - Total AUC pmol/ml/240 min

Active RxComparison

Herold et al, NEJM 2002; 346:1692

16

Side effects of the anti-CD3 mAb

Symptom/sign Mild* Moderate* Severe*

Headache 33% 0 0

Fever 17% 58% 0

Nausea 8% 0 0

Vomiting 0% 8% 0

Diarrhea 0 0 0

Dyspnea 8% 0 0

Myalgias 17% 0 0

Arthralgias 8% 0 0

Rash 0% 83% 0

Hypotension 0 0 0

17

Status of anti-CD3 for Diabetes

Now in phase 2

and soon

phase 3 clinical trials

18

Anakinra

Phase II trial: Multiple doses Anti-CD3

Herold

Anti-CD3+ GLP1

19

Animal models: Animal models: GLP-1 blocks GLP-1 blocks Beta cell death Beta cell death and increases and increases growthgrowth

L. Baggio and D. DruckerAnnu. Rev. Med, 2006L. Baggio and D. DruckerAnnu. Rev. Med, 2006

Anti-CD3 and GLP-1 to increase Beta cell mass

20

EXENITIDE TO INCREASE ISLET MASS

21

Phase II trial: Thymoglobulin

Gitelman, UCSF

Randomized, placebo controlled trialRandomized, placebo controlled trial

Adults first; then ages 8-30Adults first; then ages 8-30

4 days of therapy in hospital/GCRC4 days of therapy in hospital/GCRC

22

Immunogen Immunogen ProductionProduction

Rabbit SeraRabbit SeraProductionProduction

PurificationPurification of IgGof IgG

Fill/FinishFill/Finish

Thymoglobulin: Anti-thymocyte Globulin (Rabbit) Production Process

23

CD1a CD3/TCR

CD4

CD6 CD7 CD8

CD16 CD19

CD20* CD25*

CD6 CD11a/CD18 (LFA-1)

CD44 CD49/CD29 (VLA-4)

CD50 (ICAM-3) CD51/61

CD54 (ICAM-1) CD56*

CD58 (LFA-3) LPAM-1(α4β7)

CD102 (ICAM-2) CD195 (CCR5) CD197 (CCR7)

CD184 (CXCR4)

CD2 CD5

CD11b CD29 CD38 CD40 CD45 CD52 CD95

CD126 CD138

Ankersmit HJ, et al. Am J Transplant. 2003;3:743. Bourdage JS, et al. Transplantation. 1995;59:1194. Michallet M-C, et al. Transplantation. 2003;75:657. Monti P, et al. Int Immunopharmacol. 2003;3:189. Pistillo MP, et al. Transplantation. 2002;73:1295. Préville X, et al. Transplantation. 2001;71:460. Rebellato LM, et al. Transplantation. 1994;57:685. Tsuge

I, et al. Curr Ther Res. 1995;56:671. Zand M, et al. Transplantation. 2005;79:1507. Zand MS, et al. Blood. 2006;107:2895.

CD28* CD30 CD32 CD40

CD80* CD86

CD152 (CTLA-4) HLA class I

HLA DR β2-M

Immune Response Antigens

Adhesion &Cell Trafficking

HeterogeneousPathways

* Results differ among laboratories due to inconsistencies in monoclonal competition assays.

Note: relative concentrations of antibodies targeting the listed antigens is not known.

Thymoglobulin:Anti-thymocyte Globulin (Rabbit)

Target Antigens

24

Mechanisms of Action- Anti-IL-2R




IL-2R,IL-15

RestingRestingDCDC




Recognition by TCR

MHC TCRT-cellT-cell


B7

CD40

CD28

CD40L

25

High Affinity IL-2 Receptor

SL-04

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PDPT: Study Design

Two year open label study

Randomized

Conventional therapy

Conventional therapy + DZB

• DZB infusions

– Q 2 wks X 5

– Q 3 wks X 4

– Q 1 mo X 19

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C-Peptide AUC

0

5

10

15

20

25

30

35

40

0 10 20 30 40 50 60 70 80 90 100Weeks

C-Peptide AUC

ControlDrug on-treatmentDrug off-treatment

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Insulin Requirement

0.0

0.5

1.0

1.5

2.0

0 10 20 30 40 50 60 70 80 90 100Weeks

Insulin Dose (u/kg/day)

ControlDrug on-treatmentDrug off-treatment

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Integrated

C-peptide

(ng/ml x min/wk)

Insulin

Requirement

(u/kg/day/wk)

Hgb A1C

(%/wk)

Control -0.0512 0.0051 -0.0087

Treatment 0.0248 -0.0012 -0.0331

Difference -0.0760 0.0063 0.0244

P-value0.0001 <.0001 0.006

Slope of Change over Time

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Mechanisms of Action-MMF/Anti-IL-2R




IL-2R,IL-15

RestingRestingDCDC



MMFMMF


Recognition by TCR

MHC TCRT-cellT-cell


B7

CD40

CD28

CD40L

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Phase II trial: MMF and DZB

P. Gottlieb, Denver

Ages 8-45 Diagnosed within past 3 monthsAges 8-45 Diagnosed within past 3 monthsRandomized trialRandomized trialN=126N=126Outcome: Insulin secretion at 2 yearsOutcome: Insulin secretion at 2 years

Oral MMF x 2 yearsOral MMF x 2 years

IV DZB x 2 dosesIV DZB x 2 doses

Oral MMF x 2 yearsOral MMF x 2 years

IV placeboIV placebo

Oral Placebo x 2 yearsOral Placebo x 2 years

IV placeboIV placebo

RECRUITMENT DONE- RESULTS HERE MONDAYRECRUITMENT DONE- RESULTS HERE MONDAY

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Mechanisms of Action- CD28 Blockade




IL-2R,IL-15

RestingRestingDCDC



Recognition by TCR

MHC TCR

BELATACEPTBELATACEPT

T-cellT-cellGrowthGrowthFactorsFactors

B7

CD40

CD28

CD40L

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Full T-cell activation requires 2 signals

APC

Signal 1

T Cell

Signal 2

34

CD28 is critical for T-cell activation

APC

T Cell

CD80 (B7-1) CD86 (B7-2)

CD28

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The absence of signal 2 results in T-cell anergy or apoptosis

APC

Signal 1

T Cell

Signal 2

Mechanisms of Action- B cells

T-Cell T-Cell ActivationActivation

T-Cell T-Cell ProliferationProliferation

Signal 2:Signal 2:CostimulationCostimulation Signal 3:Signal 3:

IL-2RIL-2RIL-15IL-15

RestingRestingDCDC


Signal 1:Signal 1:MHC/peptMHC/peptidesides

Recognition by TCRRecognition by TCR

MHC TCRT-CellT-Cell


B7

CD40

CD28

CD40L

Adapted with permission from Professor Dr. Walter Land and M. Schneeberger, University of Munich, Germany.

B

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B-CELLS IN DIABETES

• ANTIBODIES DETECTED IN TYPE 1 DIABETICS

• B-CELLS ARE PRESENT IN HISTOLOGIC SECTIONS (SIGNORE)

• B-CELL DEPLETION BY GENE KNOCKOUT OR ANTI-MU REDUCES DIABETES IN NOD (NOORDCHASM, YANG OTHERS)

• B-CELLS NEEDED FOR ANTIGEN PRESENTATION IN NOD MICE (FALCONE, SERREZE)

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Autoantibody Production by B Cells

• A variety of autoantibodies (antibodies directed against self antigens) are found in patients with diabetes

• Autoantibodies may act as self-perpetuating stimuli for B cells5,6

39

B-Cell Antigen Presentation

Step 1:

• High-affinity binding of antigen

– B cell binds antigen on B-cell receptor (BCR)1,2

References: 1. O’Neill SK et al. J Immunol. 2005;174:3781-3788. 2. Lund FE et al. Curr Dir Autoimmun. 2005;8:25-54.

40


Step 2:

• Internal processing of antigen

– Antigen processed by B cell1,2

– Antigen fragment presented on MHC-II molecule1,2

– Costimulatory molecule expressed on B cell1,2

Reference: 1. Dale DC et al. WebMD Scientific American Medicine. Chapter 6. WebMD ProfessionalPublishing; 2002. 2. Roitt I et al. Immunology. 6th ed. Chapter 8. Mosby; 2001.

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Step 3:

• Presentation of antigen to T cell1-4

– B cell presents antigen to T-cell receptor (TCR) and also provides costimulatory signal to T cell1-3

– Activated T cell produces proinflammatory cytokines that activate macrophages1-3

References: 1. Silverman GJ et al. Arthritis Res Ther. 2003;5(suppl 4):S1-S6. 2. Dale DC et al. WebMD Scientific American Medicine. Chapter 6. WebMD Professional Publishing; 2002. 3. Klippel JH et al. Primer on the Rheumatic Diseases. 12th ed. Chapter 9. Arthritis Foundation; 2001. 4. Roitt I et al. Immunology. 6th ed. Chapter 8. Mosby; 2001.

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Cytokine Production by B Cells May Be Stimulated by Multiple Pathways

• Antigen binding to the BCR stimulates cytokine production1,2

References: 1. Lund FE et al. Curr Dir Autoimmun. 2005;8:25-54. 2. Duddy ME et al. J Immunol. 2004;172:3422-3427.

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B Cells Express Specific Cell-Surface Molecules

References: 1. Roitt I et al. Immunology. 6th ed. Chapter 8. Mosby; 2001. 2. Sell S et al. Immunology, Immunopathology, and Immunity. 6th ed. Chapter 4. ASM Press; 2001. 3. Duddy ME et al. J Immunol. 2004;172:3422-3427.

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RITUXIMAB: AN ANTI-CD20 MONOCLONAL ANTIBODY

• Genetically engineered chimeric murine/human monoclonal antibody– Variable light- and heavy-

chain regions from murine anti-CD20 antibody IDEC-2B8

– Human IgG constant regions

• First monoclonal antibody to be approved by the FDA for treatment of cancer

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Rituximab: Mechanism of Action

Anderson et al. Biochem Soc Trans. 1997;25:705–708. Golay et al. Blood. 2000;95:3900–3908. Reff et al. Blood. 1994;83:435–445. Clynes et al. Nat Med. 2000;6:443–446. Shan et al. Cancer Immunol Immunother. 2000;48:673–683.

Rituximab selectively Rituximab selectively depletes B cells bearing depletes B cells bearing the CD20 surface the CD20 surface marker via:marker via:

• Antibody-dependent Antibody-dependent cellular cytotoxicity cellular cytotoxicity (ADCC)(ADCC)

• Complement-Complement-dependent cytotoxicity dependent cytotoxicity (CDC)(CDC)

• Induction of apotosisInduction of apotosis

46

Absolute CD19 (B cells) after Rituximab

0

50

100

150

200

250

300

350

400

0 7 21 42 73 181

Grp 1 (50mg)Grp 2 (150)Grp 3 (375)Control Avg.Con+SDCon -SD

Days after Rituximab Dose

Cel

ls/m

m3

Prevention/Treatment of Diabetes HuCD20-NOD

Hu et al. J Clin Invest 117:3857-67, 2007

Role of CD4+CD25+Foxp3+ Tregs in Immune Responses

Adapted from Najafian N, et al. Clin Dermatol. 2001;19:586.

CD4+

T cell

Apoptosis

Anergy

Memory

Effector

Termination

Regulation

Donor APC

Signal 1Signal 2

Activation

Indirect Pathway

Direct Pathway

B cell help

DTH

CTL help

CD4+CD25+Foxp3+

Self APC

49

Prevention of Diabetes in HuCD20-NOD

Xiu et al. The Journal of Immunology, 2008, 180: 2863–2875.

50

Treatment of Diabetes in HuCD20-NOD

Xiu et al. The Journal of Immunology, 2008, 180: 2863–2875

51

HYPOTHESIS FOR RITUXIMAB ACTION IN DIABETES

• B-CELLS ARE NECESSARY ANTIGEN PRESENTING CELL FOR MAINTENANCE OF ANTI-ISLET T-CELL MEDIATED DESTRUCTION

• RITUXIMAB DEPLETES ANTIGEN SPECIFIC CD27 MEMORY B-CELLS

• IMMUNE REACTION IS SUPPRESSED

52

RITUXIMAB IN TYPE 1 DIABETESSTUDY OUTLINE

• TYPE 1 DIABETICS AS PER TRIALNET DEFINTION

• AGE: 8 TO 40 YEARS

• N=87 2:1 RATIO BLINDED RITUXIMAB VS PLACEBO

• DOSE: 375mg/m2 Q WEEK x4

• ENDPOINT 2 HOUR MMTT C-PEPTIDE AUC AT 1 YEAR

• RITUXIMAB PK/PD

• IMMUNIZATION RESPONSE: phiX174, Hep A, TETANUS

• RECOVERY OF B-CELL SUBSETS

• MECHANISTIC STUDIES

53

RITUXIMAB DIABETES IMMUNIZATION SCHEDULE

0 6 12 52 583

WEEKS

DE NOVO DEPLETED TOLERANCE?

RECALL/PRESERVATION

DE NOVO POST RECOVERY

MMTTMMTT

54

NEXT GENERATION ANTI-B CELL AGENTS

BR3-FC

ANTI-BR3

55

TertiaryTertiary (then secondary?) Prevention (then secondary?) Prevention

Anti-CD3+GLP

Anti-CD3 multiple dose

MMF/DZB

CTLA4-Ig

Rituximab

Thymoglobulin

MetabolicIL-2+Rapamycin

GAD65alum

56

TrialNet Sites – North America

57

TrialNet International Sites

• AustraliaAustralia

• United KingdomUnited Kingdom

• FinlandFinland

• Italy & GermanyItaly & Germany

58

SUMMARY

• TYPE 1 DIABETES IS AN AUTOIMMUNE DISEASE

• MULITPLE CELL TYPES HAVE BEEN HYPOTHESIZED TO PLAY A ROLE IN THE PATHOPHYSIOLOGY

• IF THESE PILOT TRIALS SHOW A MODALITY IS SAFE AND EFFECTIVE, LARGER TRIALS INCLUDING PREDIABETICS, WOULD BE PLANNED

Documents

A Complex Pathway-A Feast of Possibilities New Immunology and New Immunotherapy of Type 1 Diabetes