BRM has experience developing preclinical models for systemic lupus erythematosus (SLE) including the NZB/W F1 and MRL/lpr mouse models. These mice develop anti-dsDNA antibodies and progressive glomerulonephritis similar to human SLE. BRM has validated methods for measuring disease outcomes like proteinuria, antibody levels, immune cell profiles, and histopathology in these mice. They have also demonstrated prevention and remission of lupus symptoms in NZB/W F1 mice using treatments like anti-CD40L monoclonal antibody.

1. Biomedical Research Models, Inc
Contract Discovery Research

2. Presentation Overview
• BRM Experience and Collaborations
• Background on Systemic Lupus Erythematosus
(SLE)
• BRM’s Models for SLE
• In Vivo and In Vitro Endpoints

3. 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.
3

4. Nat Rev Rheumatol 6:13-20, 2010
Pathogenesis of SLE

5. 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)

6. 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
6

7. Anti-dsDNA Ab Levels Increase at 16 Weeks of Age
Anti-dsDNA Ab ELISA
( SEM)
M
26A
(12
w
ks
0+)
M
26B
(12
w
ks
0+)
M
26C
(12
w
ks
0+)
M
26D
(16
w
ks
0+)
M
26E
(16
w
ks
0+)
M
26F
(16
w
ks
0+)
M
26I(20
w
ks
0+)
M
26S
(28
w
ks
1+)
M
26R
(40
w
ks
4+)
0.0
0.5
1.0
1.5
2.0
1:500
1:1000
OD405nm
Prevention vs.
remission:
Prevention = proteinuria
0+ with elevated
anti-dsDNAAbs
(start at 20 wks of age)
Remission = proteinuria
1 - 2+ with elevated auto
anti-dsDNAAbs (start at
28 wks of age)

8. 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

9. 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 reported
By Wang et al., Arthritis and
Rheumatism 48:495-506, 2003 and
Early et al., JI 157:3159-3164,
1996
0 5 10 15 20 25 30 35 40 45 50 55
0
20
40
60
80
100
No Rx
Control IgG
anti-CD40L
P = 0.0265
Weeks of Age
%<300mg/dLProteinuria

10. Decreased Anti-dsDNA Ab Levels in Mice
Treated with Anti-CD40L mAb
Results similar to those reported
By Wang et al., Arthritis and
Rheumatism 48:495-506, 2003
Anti-dsDNA Ab ELISA
(Sera from 28 weeks of age  SEM)
1:500
1:1000
0.0
0.5
1.0
1.5
No Rx)
Control IgG
anti-CD40L
Sera Dilution
OD405nm
Anti-dsDNA Ab ELISA
(Sera from Nx  SEM)
1:500
1:1000
0.0
0.5
1.0
1.5
No Rx
Control IgG
anti-CD40L
Sera Dilution
OD405nm

11. Reduced Immune Complex Deposition with anti-CD40L
Treatment: anti-CD40L
Proteinuria = 2+
at 52 weeks of age
Treatment: control IgG
Proteinuria = 3+
at 28 weeks of age
Results similar to those reported
By Wang et al., Arthritis and
Rheumatism 48:495-506, 2003
100x

12. Treatment of NZB/W F1 Mice with Moderate
Proteinuria (Remission Model)
NZB/W F1 female mice with
moderate proteinuria (1 - 2+)
were entered into groups at
28 weeks of age (n = 10/group)
and initiated treatment (bar).
Body weight was measured
once weekly and proteinuria
(Uristix) was measured once
every two weeks (the following
week to confirm a ≥ 3+ reading).
Humane survival end points:
≥ 3+ proteinuria on two
consecutive weeks; ≥ 20% body
weight loss; or prostration.
0
0
20
40
60
80
100
28 30 32 34 36 38 40 42 44 46
Control
Prednisolone
Weeks of Age
PercentDisease-freeSurvival

13. 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
CD21
CD23
Gated on CD19+ splenocytes
Prednisolone Effects on Splenic
B-cell Localization

14. ControlPrednisolone
CD4
CD69
Gated on CD3+ splenocytes
Prednisolone Effects on Splenic
T-cell Activation
CD8
CD4
CD8

15. 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
100x400x
3/1/1/3
3/1/1/3
1/0/0/2
1/0/0/2

16. MRL/lpr Lupus Mouse Model
0
0
20
40
60
80
100
8 12 16 20 24
Control
Weeks of Age
PercentDisease-freeSurvival
MRL/lpr female mice with
moderate proteinuria (1 - 2+)
were entered into groups at
6 weeks of age (n = 25/group)
and initiated treatment (PBS).
Body weight was measured
once weekly and proteinuria
(Uristix) was measured once
every 1 - 2 weeks (the following
week to confirm a ≥ 3+ reading).
Humane survival end points:
≥ 3+ proteinuria on two
consecutive weeks; ≥ 20% body
weight loss; or prostration.

17. 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.

18. Immune Complex Deposition in the
MRL/lpr Mouse
Proteinuria = 3+
at 17 weeks of age,
GN scores = 3/1/1/3
100x 400x

19. 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).