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

6

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).