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
POTENTIAL CONFLICT BASED ON
FINANCIAL/CONSULTING/ RESEARCH
INTERACTIONS
• ROCHE
• LILLY
• VICAL
• GENENTECH
• WYETH
• NOVARTIS
• ASTELLAS
Research Support
Speaker’s Bureau
Board Member/Advisory
Panel
Stock/Shareholder
Consultant
Tax Payer
• PFIZER
• US GOVERNMENT
2
Primary Prevention (genetically at risk)
Beta cell function
100 %
STOP progression to autoimmunity/beta cell destruction
Clinical onset of
disease
20%
Time
Secondary Prevention (antibody positive)
Beta cell function
100 %
STOP clinical disease
Clinical onset of
disease
20%
Time
Tertiary Prevention (early in clinical disease)
Beta cell function
100 %
Preserve Beta cells
STOP complications
Clinical onset of disease
20%
Time
The Immunobiology of Type 1 Diabetes
Normal blood sugar
Diabetes
Resting
DC
T-cell
proliferation
DC
Maturation
Activated
T cells
Signal 2:
Costimulation
Signal 3:
IL-2R,
IL-15R
T-cell
Growth
Factors
Signal 1:
MHC/peptides
Recognition by TCR
6
Immunosuppressive Drugs
Mechanisms of Action
Resting
DC
MMF
Steroids
B
T-Cell
Proliferation
DC
Maturation
T-Cell
Activation
MMF
MMF
Sirolimus
Signal 2:
Costimulation
B7
CD28
CD40
CD40L
MHC
TCR
Signal 3:
IL-2R
IL-15
T-Cell
Growth
Factors
Sirolimus
Signal 1:
MHC/peptides
Recognition by TCR
CsA
Tacrolimus
Muromonab-CD3
Daclizumab
Basiliximab
Adapted with permission from Professor Dr. Walter Land and M. Schneeberger, University of Munich, Germany.
MONOCLONAL ANTIBODY STRUTURE
Mouse
Chimeric
Human
Humanized
8
MONOCLONAL ANTIBODY
NOMENCLATURE
Murine
Chimeric
Rituximab
Muromonab
Monoclonal
Antibody
Daclizumab
Humanized
9
Mechanisms of Action- Anti-CD3
Resting
DC
T-cell
proliferation
DC
Maturation
Activated
T cells
Signal 2:
costimulation
B7
CD40
MHC
Signal 3:
IL-2R,
IL-15
CD28
CD40L
TCR
Signal 1:
MHC/peptides
Recognition by TCR
T-cell
Growth
Factors
Anti-CD3
10
HUMANIZED, MUTAGENIZED
ANTI-CD3 MONOCLONAL ANTIBODY FOR
TREATMENT OF TYPE 1 DIABETES
11
Reduced insulin requirements in anti-CD3 treated
subjects
Keymeulen, B. et al. N Engl J Med 2005;352:2598-2608
12
Example of Mixed Meal Tolerance Test
Active Rx
Placebo
Better c-peptide response to MMTT in anti-CD3
treated subjects
Herold, K. et al. N Engl J Med 2002;346:1692-1698
14
Changes in C-Peptide Responses
During MMTT Over Time
140
C-Peptide - Total AUC
pmol/ml/240 min
120
100
80
Active Rx
Comparison
60
40
20
0
Baseline
6 months
12 months
Herold et al, NEJM 2002; 346:1692
Side effects of the anti-CD3 mAb
Symptom/sign
Headache
Fever
Nausea
Vomiting
Diarrhea
Dyspnea
Myalgias
Arthralgias
Rash
Hypotension
Mild*
33%
17%
8%
0%
0
8%
17%
8%
0%
0
Moderate*
0
58%
0
8%
0
0
0
0
83%
0
Severe*
0
0
0
0
0
0
0
0
0
0
16
Status of anti-CD3 for Diabetes
Now in phase 2
and soon
phase 3 clinical trials
17
Anakinra
Phase II trial:
Multiple doses Anti-CD3
Herold
Anti-CD3+ GLP1
18
Anti-CD3 and GLP-1 to increase Beta cell mass
Animal models:
GLP-1 blocks
Beta cell death
and increases
growth
L. Baggio and D. DruckerAnnu. Rev. Med, 2006
19
EXENITIDE TO INCREASE ISLET MASS
20
Phase II trial: Thymoglobulin
Gitelman, UCSF
Randomized, placebo controlled trial
Adults first; then ages 8-30
4 days of therapy in hospital/GCRC
21
Thymoglobulin:
Anti-thymocyte Globulin (Rabbit)
Immunogen
Production
Rabbit Sera
Production
Production Process
Purification
of IgG
Fill/Finish
22
Thymoglobulin:
Anti-thymocyte Globulin (Rabbit)
Immune Response
Antigens
CD1a
CD3/TCR
CD4
CD6
CD7
CD8
CD16
CD19
CD20*
CD25*
CD28*
CD30
CD32
CD40
CD80*
CD86
CD152 (CTLA-4)
HLA class I
HLA DR
β2-M
Target Antigens
Adhesion &
Cell Trafficking
Heterogeneous
Pathways
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
* Results differ among laboratories due to inconsistencies in monoclonal competition assays.
Note: relative concentrations of antibodies targeting the listed antigens is not known.
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.
23
Mechanisms of Action- Anti-IL-2R
Resting
DC
T-cell
proliferation
DC
Maturation
Activated
T cells
Signal 2:
costimulation
B7
CD40
MHC
CD28
Signal 3:
IL-2R,
IL-15
CD40L
TCR
Signal 1:
MHC/peptides
Recognition by TCR
T-cell
Growth
Factors
Daclizumab
Basiliximab
24
High Affinity IL-2 Receptor
b
g
a
SL-04
25
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
26
C-Peptide AUC
40
Control
Drug on-treatment
Drug off-treatment
35
C-Peptide AUC
30
25
20
15
10
5
0
0
10
20
30
40
50 60
Weeks
70
80
90 100
27
Insulin Requirement
Insulin Dose (u/kg/day)
2.0
Control
Drug on-treatment
Drug off-treatment
1.5
1.0
0.5
0.0
0
10
20
30
40
50 60
Weeks
70
80
90 100
28
Slope of Change over Time
Integrated
Insulin
C-peptide
Requirement
Hgb A1C
(ng/ml x min/wk)
(u/kg/day/wk)
(%/wk)
Control
-0.0512
0.0051
-0.0087
Treatment
0.0248
-0.0012
-0.0331
Difference
-0.0760
0.0063
0.0244
0.0001
<.0001
0.006
P-value
29
Mechanisms of Action-MMF/Anti-IL-2R
Resting
DC
T-cell
proliferation
DC
Maturation
MMF
Activated
T cells
Signal 2:
costimulation
B7
CD40
MHC
CD28
Signal 3:
IL-2R,
IL-15
CD40L
TCR
Signal 1:
MHC/peptides
Recognition by TCR
T-cell
Growth
Factors
Daclizumab
Basiliximab
30
Phase II trial: MMF and DZB
P. Gottlieb, Denver
Ages 8-45 Diagnosed within past 3 months
Randomized trial
N=126
Outcome: Insulin secretion at 2 years
Oral MMF x 2 years Oral MMF x 2 years Oral Placebo x 2 years
IV DZB x 2 doses
IV placebo
IV placebo
RECRUITMENT DONE- RESULTS HERE MONDAY
31
Mechanisms of Action- CD28 Blockade
Resting
DC
T-cell
proliferation
DC
Maturation
Activated
T cells
Signal 2:
costimulation
B7
CD40
MHC
CD28
Signal 3:
IL-2R,
IL-15
CD40L
TCR
T-cell
Growth
Factors
Signal 1:
MHC/peptides
Recognition by TCR
BELATACEPT
32
Full T-cell activation requires 2 signals
APC
Signal 1
T Cell
Signal 2
33
CD28 is critical for T-cell activation
APC
CD80 (B7-1)
CD86 (B7-2)
CD28
T Cell
34
The absence of signal 2 results in
T-cell anergy or apoptosis
APC
Signal 2
Signal 1
T Cell
35
Mechanisms of Action- B cells
Resting
DC
B
T-Cell
Proliferation
DC
Maturation
T-Cell
Activation
Signal 2:
Costimulation
B7
CD28
CD40
CD40L
MHC
TCR
Signal 3:
IL-2R
IL-15
T-Cell
Growth
Factors
Signal 1:
MHC/peptides
Recognition by TCR
Adapted with permission from Professor Dr. Walter Land and M. Schneeberger, University of Munich, Germany.
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 ANTIMU REDUCES DIABETES IN NOD (NOORDCHASM,
YANG OTHERS)
• B-CELLS NEEDED FOR ANTIGEN PRESENTATION IN
NOD MICE
(FALCONE, SERREZE)
37
Autoantibody Production by B Cells
• A variety of autoantibodies
(antibodies directed against self
antigens) are found in patients
with diabetes
• Autoantibodies may act as selfperpetuating stimuli for B cells5,6
38
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.
39
B-Cell Antigen Presentation
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.
40
B-Cell Antigen Presentation
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.
41
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.
42
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.
43
RITUXIMAB: AN ANTI-CD20
MONOCLONAL ANTIBODY
• Genetically engineered
chimeric murine/human
monoclonal antibody
– Variable light- and heavychain regions from murine
anti-CD20 antibody IDEC2B8
– Human IgGk constant
regions
• First monoclonal antibody to
be approved by the FDA for
treatment of cancer
44
Rituximab: Mechanism of Action
 Rituximab selectively
depletes B cells bearing
the CD20 surface
marker via:
• Antibody-dependent
cellular cytotoxicity
(ADCC)
• Complementdependent cytotoxicity
(CDC)
• Induction of apotosis
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.
45
Absolute CD19 (B cells) after Rituximab
400
Cells/mm3
350
300
Grp 1 (50mg)
Grp 2 (150)
Grp 3 (375)
Control Avg.
Con+SD
Con -SD
250
200
150
100
50
0
0
7
21
42
73
181
Days after Rituximab Dose
46
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
Indirect
Pathway
Self APC
Activation
Memory
Direct
Pathway
Donor
APC
Signal 1
Signal 2
CD4+
T cell
B cell help
DTH
Effector
CTL help
Apoptosis
Termination
Anergy
CD4+CD25+Foxp3+
Regulation
Adapted from Najafian N, et al. Clin Dermatol. 2001;19:586.
Prevention of Diabetes in HuCD20-NOD
Xiu et al. The Journal of Immunology, 2008, 180: 2863–2875.
49
Treatment of Diabetes in HuCD20-NOD
Xiu et al. The Journal of Immunology, 2008, 180: 2863–2875
50
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
51
RITUXIMAB IN TYPE 1 DIABETES
STUDY 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
52
RITUXIMAB DIABETES
IMMUNIZATION SCHEDULE
0
3
6
WEEKS
12
52
58
MMTT
DE NOVO DEPLETED
TOLERANCE?
RECALL/PRESERVATION
DE NOVO POST RECOVERY
53
NEXT GENERATION ANTI-B CELL
AGENTS
BR3-FC
ANTI-BR3
54
Tertiary (then secondary?) Prevention
GAD65alum
Thymoglobulin
MMF/DZB
Rituximab
Anti-CD3+GLP
Anti-CD3 multiple dose
CTLA4-Ig
IL-2+Rapamycin
Metabolic
55
TrialNet Sites – North America
56
TrialNet International Sites
• Australia
• United Kingdom
• Finland
• Italy & Germany
57
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
58