Human TNFα Transgenic Mouse
Model of Spontaneous Arthritis

HEIDELBERG PHARMA AT A GLANCEl CRO situated in Ladenburg, near Heidelberg, Germany
l 45 employees, 2000 m2 of lab spacel Core competence: pre-clinical profiling of small molecules
and biologicals
l Focus: cancer, inflammatory & autoimmune diseasesl Services: Explorative pharmacology, drug-metabolism andpharmacokinetics (DMPK), molecular biologyl Standard models, customized experimental design, newsolutions

HEIDELBERG PHARMAINFLAMMATION & AUTOIMMUNITY
Mechanistic modelsl Thioglycolate induced peritonitisl LPS-induced cytokine release (IL-2; -4; -5; -6; -10, MCP-1; IL-12p70; IFNγ andTNFα)l Anti-CD3- induced cytokine release (Il-2; -4; -5; -6; -10, MCP-1; IL-12p70;
IFNγ and TNFα)l DTH (delayed type hypersensitivity) model with KLH (keyhole limpethemocyanin)
Autoimmune disease models
l Experimental Autoimmune Encephalitis (EAE, Multiple Sclerosis) in SJL/J micel Collagen - Induced Arthritis (CIA) in DBA/1 micel Diabetes (DIO model)

HEIDELBERG PHARMAONCOLOGY
Syngenic models• Syngenic models using s.c., i.p. or i.v. application:
Leukemia, lung, colon, testicular teratoma and melanoma
Standard xenograft models• Several subcutaneous xenograft models are established:
Glioma, Stomach, Cervix, Ovary, Pancreas, Colon, Kidney, Lung, Breast,Prostate, Bladder
Orthotopic xenograft models• Luciferase transfected cell lines suitable for Bioimaging. Implantation sites:
Caecum, Pancreas, Prostate, Kidney in development
Metastasis models using human cell lines• Left-ventricular inoculation of luciferase transfected cell lines suitable forBioimaging.
Models to evaluate bispecific Antibodies• Implantation of a mixture of cancer cell lines with human peripheral bloodmononuclear cells (hPBMCs)
4

Rheumatoid Arthritis

Rheumatoid Arthritis : Pathogenesis

PRECLINICAL MODELS FOR RA

SIMILARITIES & DIFFERENCES

Origin of the TNFα transgenic model• The TNFα transgenic mice were generated using a constructthat contains a 2.8 kb fragment of the human TNFα gene,including the entire coding region and promoter, fused tothe human β-globin 3' untranslated region (UTR) thatreplaces the endogenous 3'UTR of the human TNFα gene– Designed to model dysregulated human TNFα expression• This transgenic line was produced by pronuclear
microinjection of B6SJLF2 hybrid zygotes• The animals have been backcrossed for over 21 generations
onto the C57BL6/NTac genetic backgroundProgressive arthritis in the TNFα mice
• The TNFα mice develop inflammatory arthritis
spontaneously• Ideal for screening new small molecules and biologics forthe treatment of arthritis

Experimental procedures• Treatment was initiated when mice were 5 weeks oldfollowing the randomization of the experimental mice into
groups of 10 mice based on their body weights• Treatment was given through i.p. injection of 100 µl ofworking concentration of Humira freshly prepared justbefore each dosing• Doses of 0.25, 1, 10 and 25 mg/kg Humira were used
• The arthritis disease progression in the experimental
animals was monitored by clinical scoring twice weekly.
• After giving total 22 doses to each animal, the study wasterminated when the animals reached 15 weeks old– Paws were fixed in 10% buffered formalin for histology analysis

Arthritis clinical assessment criteria
Maximum 24 scores were given to each mouse.
The sum score of all 4 paws from each mouse will be used for graphing and statistical analysis
ü 20 digits: score 0 or 0.2 for each digit (maximum 4 scores)

ü0 = normal
ü0.2 = one or more swollen joints

ü 4 paws: score 0 or1 or 2 (maximum 8 scores)

ü0 = normal
ü1 = noticeable swollen
ü2 = severe swollen

ü 2 wrists: score 0 or 1 or 2 (maximum 4 scores)

ü0 = normal
ü1 = noticeable swollen
ü2 = severe swollenü 2 ankles: score 0 or 2 or 4 (maximum 8 scores)

ü0 = normal
ü2 = noticeable swollen
ü4 = severe swollen with stiffness of ankle joint

Dose dependent effect of treatment
on clinical progression of arthritis
Clinical Score

10.0
8.0Vehicle (n=10)
Humira, 25mg/kg (n=9)

RA Score

6.0

Humira, 10mg/kg (n=10)
Humira, 1mg/kg (n=9)
Humira, 0.25mg/kg (n=9)

4.0
2.0

Arthritis Clinical Scores

1.5

Two-Way ANOVA

1.0
0.5
0.0TimeP<0.0001

Time/Treatment

P<0.0001

Treatment

P<0.0001

Post-hoc LSD Test-0.5047.07.5

8.0

8.5

9.0

Week post Treatment Initiation

9.5

G1 vs G2

P<0.0001

G1 vs G3

P<0.0001

G1 vs G4

P=0.559

G1 vs G5

P=0.709

Clinical manifestation of arthritis
in treated and untreated animals

VehicleHumira10 mg/kg

Humira25 mg/kg

Histopathology gradingof joint lesions• Grade 0: no lesions• Grade 1: minimal to mild leukocyte infiltration• Grade 2: moderate leukocyte infiltration• Grade 3: severe leukocyte infiltration, often muchof the joints spaces were filled with abundant
exudate, inflammatory lesions

Histopathology scores of front
and rear paw joints
3.5
3.0
G1: Vehicle (n=10)
G2: HUMIRA, 25mg/kg (n=9)

Histo Score

2.5

G3: HUMIRA, 10mg/kg (n=10)
G4: HUMIRA, 1mg/kg (n=9)

2.0

G5: HUMIRA, 0.25mg/kg (n=9)

1.5
***

1.0

* = P<0.05** = P<0.01*** = P<0.001

0.5
***
0.0

Treatment groups
One way ANOVA
P<0.0001••••

Post Hoc Analysis:
Dunnett's Multiple Comparison Test
G1 vs G2: P<0.001
G1 vs G3: P<0.001
G1 vs G4: ns
G1 vs G5: ns

Grade 0: no lesions
Grade 1: minimal to mild leukocyte infiltration
Grade 2: moderate leukocyte infiltration
Grade 3: severe leukocyte infiltration, often much of the joints spaces were filledwith abundant exudate, inflammatory lesions

Representative histopathologyof ankles from experimental micemuscleFat in bonemarrow

JtS
InflamJtS

Bone
Inflamed ankle joint , 100x ,# 560 (non-treated)

Normal ankle joint, 100x, # 561(25mg/kg HUMIRA treated)

Paw tissue pro-inflammatory
cytokines: IL-1β and mKC

IL-1β Levels in the Joints

mKC Levels in the Joints

8000.0800.0

G1: Vehicle (n=4)

6000.0
G1: Vehicle (n=4)

G2: Humira, 25mg/kg (n=4)

600.0

G3: Humira, 10mg/kg (n=4)
G4: Humira, 1mg/kg (n=3)

G3: Humira, 10mg/kg (n=4)

4000.0

G4: Humira, 1mg/kg (n=3)
G5: Humira, 0.25 mg/kg (n=4)

pg/ml

pg/ml

G2: Humira, 25mg/kg (n=4)

G5: Humira, 0.25mg/kg (n=4)

400.0

2000.0
200.0

***

0.0

***

Treatment groups
One way ANOVA
P<0.0001

Post Hoc Analysis:
Dunnett's Multiple Comparison Test
G1 vs G2: P<0.001
G1 vs G3: P<0.001
G1 vs G4: ns
G1 vs G5: ns

***
***

0.0

Treatment groups
One way ANOVA
P<0.0001

Post Hoc Analysis:
Dunnett's Multiple Comparison Test
G1 vs G2: P<0.001
G1 vs G3: P<0.001
G1 vs G4: ns
G1 vs G5: ns* = P<0.05** = P<0.01*** = P<0.001

Study at Heidelberg Pharma
Animalsl Age at delivery

5 weeksl Age at start of experiment

6 weeks

Treatment Groups (n=8)
l Vehiclel Humira 5 mg/kg ip
l Humira 10 mg/kg ipl Schedule 2x weekly
Readouts
l Clinical Scorel Paw swelling
l Histology : Paws fixed and embedded

Body Weight• Steady increase in body weight• No effect on body weight by treatment with HumiraMedian body weight (g)33Group 1PBS

31292725

Group 2Humira®
5 mg/ml

23211917
1507

14 21 28 35 42 49 56 63 70 77 84 91
Days after start of treatment

Group 3
Humira®
10 mg/mlPaw swelling

Mean left hind paw (mm) +SD

1.8
Group 1PBS

1.6
1.4
1.2
1.0Group 2
Humira®
5 mg/ml

0.80.60.40.20.02734414855
6269
Days after start of treatment

768390Group 3
Humira®
10 mg/ml

Mean right hind paw (mm) +SD

1.8
Group 1
PBS

1.6
1.4
1.2
1.0Group 2
Humira®
5 mg/ml

0.8
0.6
0.4
0.2
0.0
27

34414855
6269
Days after start of treatment

768390Group 3
Humira®
10 mg/ml• Only minor swellingin hind paws
• Humira effectively
inhibited pawswellingPaw swelling

Mean left fore paw (mm) +SD

3.5
Group 1PBS

3.0
2.5
2.0Group 2
Humira®
5 mg/ml

1.5
1.00.50.02734414855
62
69
Days after start of treatment

7683

90

Group 3
Humira®
10 mg/ml

Mean right fore paw (mm) +SD

3.5
Group 1PBS

3.0
2.5
2.0

Group 2Humira®
5 mg/ml

1.5
1.0
0.5
0.0
27

34414855
6269
Days after start of treatment

768390Group 3
Humira®
10 mg/ml• Similar swelling inboth fore paws
• Humira effectively
inhibited pawswellingClinical Score

Mean fore left
visual CIA scoring + SD

4.0
Group 1
PBS

3.5
3.0
2.5
2.0

Group 2Humira®
5 mg/ml

1.5
1.0
0.5
0.02734414855
6269
Days after start of treatment

768390Group 3
Humira®
10 mg/ml• Similar development
of disease in bothfore paws
• Humira effectively
inhibited
progression ofdisease

Mean fore rightvisual CIA scoring + SD

4.0
Group 1
PBS

3.5
3.0
2.5
2.0

Group 2Humira®
5 mg/ml

1.5
1.0
0.5
0.0
2734
41
48
55
6269
Days after start of treatment

7683

90

Group 3
Humira®
10 mg/ml

Scores:0: No evidence of erythema and swelling
1: Erythema and mild swelling confined to the midfoot (tarsals) and ankle joint
2: Erythema and mild swelling extending from theankle to the mid-foot
3: Erythema and moderate swelling extending fromthe ankle to the metatarsal joints
4: Erythema and severe swelling encompass theankle, foot, and digits(acc. to Current Protocols in Immunology, 15.5.11)

Experimental considerations• Males often preferred– Males have earlier onset and more severe disease phenotype• Age at study initiation––––

To see best therapeutic effect, start study with young mice
If wish to see efficacy against advanced disease, start with older miceInflammation seen first; this can be reversed
As the disease progresses, bone and tissue remodeling occurs, which
may not be reversible• Readouts– Clinical score, histopathology and cytokine measurements all relevant– Understand time course of cytokine induction and pick relevant
timepoints

Summary• Model– Spontaneous, no immunization– Paw swelling, Clinical score, histopathology and cytokine measurements– Similar results at two different sites• Advantages– Highly reproducible– 100% incidence of disease– Highly similar to human RA• Suitable for– Anti-TNF compounds– Biologic drugs & small molecules in relevant pathway

Contact

Prof. Andreas Pahl
Heidelberg Pharma GmbH
Schriesheimer Straße 101
68526 Ladenburg, Germany
Phone:
Fax:+49 (0)6203 1009-45+49 (0)6203 1009-19

E-Mail:Web:a.pahl@hdpharma.com
www.heidelberg-pharma.com

BackupINVOLVEMENT OF IMMUNE SYSTEMINVOLVEMENT OF CYTOKINES

Other physiological consequences of
constitutive human TNFα expression
no visible
signs

mild

moderatesevereArthritis
normaldecreased

Fertilitynone or minor

pronounced

Metabolic AbnormalitiesAge(weeks)

510

15

202530TNFα: overview
Cell Source

Inducers

Inhibitors

Cell Target

Primary Effects on
Each Target

Mononuclear
phagocytes, Tcells, B cells,
NK cells,
vascularendothelialcells,
keratinocytes,
smoothmuscle cells,
mast cells,
neutrophils,
astrocytes,
glial cells.

Lipopolysaccharide,zymosan, phorbolesters, ultraviolet light,
viral infection,
allogenic B cells,
protozoa, and othermicroorganisms.

Prostaglandins,corticosteroids,
IL-4, IL-6,TGF-b

Mononuclear
phagocytes

Activation (Inflammationand Infection)Neutrophils,
eosinophils

Activation (Inflammation)

Endothelialcells

Activation (Inflammation,coagulation)

Hypothalamus

FeverLiver

Acute phase reactants(serum ameloid A protein)

Muscle, fat

Catabolism (cachexia)

Thymocyte

Costimulator

Cytokines and otherendogenous mediators(TNF-a, IL-1, IFN-g,
IFN-a, GM-CSF, IL-2,TGF-b, substance P,
platelet activatingfactor).

Humira levels in mouse serum
during the study
HUMIRA Levels in the Serum
10000
1000
100

G2: HUM IRA, 25mg/kg
G3: HUM IRA, 10mg/kgµg/ml

10G4: HUM IRA, 1mg/kg
G5: HUM IRA, 0.25mg/kg

10.1
0.010.001

0.000107142128

Days post Treatment Initiation

35

Proof of concept study with Humira• Humira (adalimumab) is a biologic drug approved for thetreatment of arthritis– Recombinant human IgG1 monoclonal antibody– Mechanism of action involves binding to TNFα to block signalingExperimental groups

Group #Treatment

Dosing Schedule Route/Volume

Concentration

1Placebo

Twice weekly

i.p./100µl

1:10 dilution in PBS

2HUMIRA-022512E

Twice weekly

i.p./100µl

25mg/kg

3

HUMIRA-022512ETwice weekly

i.p./100µl10mg/kg

4HUMIRA-022512E

Twice weekly

i.p./100µl1mg/kg

5HUMIRA-022512E

Twice weekly

i.p./100µl0.25mg/kg

Experimental considerations• Group size
– Minimum group size = 8. Recommended group size = 10.

• Readouts– Clinical score, histopathology and cytokine measurements all relevant– Understand time course of cytokine induction and pick relevant
timepoints• Immunogenicity and efficacy– Biologic drugs can induce an immune response in mice
– Important to monitor drug concentrations over course of study– May need to use progressively higher concentrations to preserve
efficacy– Humira has low immunogenicity and thus decrease in effective
concentrations is not a big concern over typical study