Biomedical Research Models, Inc
Contract Discovery Research
Presentation Overview • BRM Experience and Collaborations
• Background on Systemic Lupus Erythematosus (SLE)
• BRM’s Models for SLE
• In Vivo and In Vitro Endpoints
BRM Experience and Collaborations
• BRM initiated the NZB/W F1 mouse model in 2011 with the award of a $540K, 2-year NIAMS STTR grant to develop small molecule therapeutics for lupus.
• Scientists and technical staff have received training from our collaborators (Drs. Betty Diamond and Thomas Coleman) at the Feinstein Institute for Medical Research.
• NZB/W F1 mouse model for prevention and remission was validated in 2011 - 2012 at BRM, including in vitro assays.
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Nat Rev Rheumatol 6:13-20, 2010
Pathogenesis of SLE
NZB/W F1 Mouse• Originally developed by
Helyer at Howie at in 1963 and subsequently transferred to JAX.
• Develops anti-dsDNA (auto)antibodies at ≥ 16 weeks of age.
• Develops 3+ proteinuria after 20 weeks of age.
• The most commonly used preclinical model for SLE.
• NZM lines: genetic susceptibility loci (sle1,2,3)
Clinical Measures
• Proteinuria (semi-quantitative measurement with clinistix)◦ Scores based on a 0 - 4+ scale
• Body Weight
• Glomerular Filtration Rate (GFR, inulin clearance method)
• IDEXX clinical analyzer (e.g., BUN/creatinine, protein/creatinine [urine], ALT/AST)◦ Metabolic caging or pan-catch urine collection available
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Anti-dsDNA Ab Levels Increase at 16 Weeks of Age
Anti-dsDNA Ab ELISA( SEM)
M26
A (12
wks 0
+)
M26
B (12
wks 0
+)
M26
C (12
wks 0
+)
M26
D (16
wks 0
+)
M26
E (16
wks 0
+)
M26
F (16
wks 0
+)
M26
I (20
wks
0+)
M26
S (28
wks 1
+)
M26
R (40
wks 4
+)0.0
0.5
1.0
1.5
2.0
1:500
1:1000
OD
40
5 n
m
Prevention vs. remission:Prevention = proteinuria 0+ with elevated anti-dsDNA Abs (start at 20 wks of age)
Remission = proteinuria 1 - 2+ with elevated auto anti-dsDNA Abs (start at 28 wks of age)
Progressive Immune Complex Deposition in Glomeruli
Proteinuria = 0+at 12 weeks of age
Proteinuria = 3+at 28 weeks of age
Proteinuria = 0+at 16 weeks of age
100x
Treatment of NZB/W F1 Mice to Prevent Onset of Lupus
PREVENTION STUDY DESIGN• N=10/group with proteinuria = 0+ at 20 weeks of age• Dose once every two weeks from 20 - 46 weeks of age• Measure body weights once weekly and proteinuria once every two weeks.• Remove animals from study at onset (defined as ≥ 3+ proteinuria, ≥ 20%
body weight loss, or prostration).
Results similar to those reportedBy Wang et al., Arthritis and Rheumatism 48:495-506, 2003 andEarly et al., JI 157:3159-3164, 1996
0 5 10 15 20 25 30 35 40 45 50 550
20
40
60
80
100
No RxControl IgGanti-CD40L
P = 0.0265
Weeks of Age
% <
300
mg
/dL
Pro
tein
uri
a
Decreased Anti-dsDNA Ab Levels in MiceTreated with Anti-CD40L mAb
Results similar to those reportedBy Wang et al., Arthritis and
Rheumatism 48:495-506, 2003
Anti-dsDNA Ab ELISA(Sera from 28 weeks of age SEM)
1:50
0
1:10
000.0
0.5
1.0
1.5
No Rx)Control IgGanti-CD40L
Sera Dilution
OD
405
nm
Anti-dsDNA Ab ELISA(Sera from Nx SEM)
1:50
0
1:10
000.0
0.5
1.0
1.5No RxControl IgGanti-CD40L
Sera DilutionO
D 4
05 n
m
Reduced Immune Complex Deposition with anti-CD40L
Treatment: anti-CD40LProteinuria = 2+
at 52 weeks of age
Treatment: control IgGProteinuria = 3+
at 28 weeks of age
Results similar to those reportedBy Wang et al., Arthritis and
Rheumatism 48:495-506, 2003
100x
Treatment of NZB/W F1 Mice with Moderate Proteinuria (Remission Model)
NZB/W F1 female mice withmoderate proteinuria (1 - 2+)were entered into groups at 28 weeks of age (n = 10/group)and initiated treatment (bar).
Body weight was measuredonce weekly and proteinuria(Uristix) was measured onceevery two weeks (the followingweek to confirm a ≥ 3+ reading).
Humane survival end points:≥ 3+ proteinuria on twoconsecutive weeks; ≥ 20% bodyweight loss; or prostration.
00
20
40
60
80
100
28 30 32 34 36 38 40 42 44 46
ControlPrednisolone
Weeks of Age
Per
cen
t D
isea
se-f
ree
Su
rviv
al
Flow cytometric analysis for:
B cells (follicular, marginal zone,
T1, T2, switched, plasma,
activation, MHCII, CD80, CD86,
germinal center), T cells (CD4,
CD8, activation, CD25, Foxp3,
IFN-g, IL-4, IL-17), Monocytes
(CD11b, Ly-6c, MHCII),
dendritic cells (CD11c),
NK cells, NKT cells; apoptosis
(annexin V/PI, live-dead violet).
Experienced with multicolor (up to 4-5 color) panel design and validation with
human (PMBCs), and rat and mouse (whole blood, splenocytes, lymph node cells) cells.
Control Prednisolone
CD
21
CD23Gated on CD19+ splenocytes
Prednisolone Effects on Splenic B-cell Localization
Con
trol
Pre
dnis
olon
e
CD
4
CD69Gated on CD3+ splenocytes
Prednisolone Effects on Splenic T-cell Activation
CD
8CD4
CD
8
Histopathological scoring
(0 - 5 scale) for: • glomerulonephropathy• dilated tubules• degenerate tubules• lymphocyte aggregates
(arrows)
Control Prednisolone
Renal Histopathology
Performed by an independent pathologist blinded to treatment group and disease status
100x
400x
3/1/1/3
3/1/1/3
1/0/0/2
1/0/0/2
MRL/lpr Lupus Mouse Model
00
20
40
60
80
100
8 12 16 20 24
Control
Weeks of Age
Per
cen
t D
isea
se-f
ree
Su
rviv
al
MRL/lpr female mice withmoderate proteinuria (1 - 2+)were entered into groups at 6 weeks of age (n = 25/group)and initiated treatment (PBS).
Body weight was measuredonce weekly and proteinuria(Uristix) was measured onceevery 1 - 2 weeks (the followingweek to confirm a ≥ 3+ reading).
Humane survival end points:≥ 3+ proteinuria on twoconsecutive weeks; ≥ 20% bodyweight loss; or prostration.
MRL/lpr Lupus Mouse
• ♀/♂ disease incidence similar, though ♀ are preferred• Rapid, severe onset of immune complex-mediated
glomerulonephritis (compared to NZB/W F1 ♀ mice)◦ Lymphadenopathy (up to ~100x normal size)◦ Splenomegaly (up to ~3x normal size)◦ Arthritis (no physical disability from our experience)◦ Dermatitis (areas of the tail, back, and ears)
• Disease dependent on Faslpr mutation, with MRL background playing a minor role.
• ~2007, the MRL/lpr line lost the lupus phenotype, but no genetic drift was found. The line was recovered from cryopreserved embyos. Unknown whether this event could happen again.
Immune Complex Deposition in the MRL/lpr Mouse
Proteinuria = 3+at 17 weeks of age,GN scores = 3/1/1/3
100x 400x
Comparative Renal Histopathology in the NZB/W F1 and MRL/lpr Models
Proteinuria = 3+at 46 weeks of age
NZB/W F1 MRL/lpr
Proteinuria = 3+at 15 weeks of age
Both mice reached the same humane endpoint, but lymphocyte aggregates were noticeably more severe in MRL/lpr mice (mean score = 3) compared to NZB/W F1 mice (mean score = 2).