human tnfα transgenic mouse model of …...a transgenic model • the tnfα transgenic mice were...
TRANSCRIPT
![Page 1: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/1.jpg)
Human TNFα Transgenic Mouse Model of Spontaneous Arthritis
![Page 2: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/2.jpg)
l CRO situated in Ladenburg, near Heidelberg, Germany
l 45 employees, 2000 m2 of lab space
l Core competence: pre-clinical profiling of small molecules and biologicals
l Focus: cancer, inflammatory & autoimmune diseases
l Services: Explorative pharmacology, drug-metabolism and pharmacokinetics (DMPK), molecular biology
l Standard models, customized experimental design, new solutions
HEIDELBERG PHARMA AT A GLANCE
![Page 3: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/3.jpg)
Mechanistic models
l Thioglycolate induced peritonitis
l LPS-induced cytokine release (IL-2; -4; -5; -6; -10, MCP-1; IL-12p70; IFNg and TNFa)
l Anti-CD3- induced cytokine release (Il-2; -4; -5; -6; -10, MCP-1; IL-12p70; IFNg and TNFa)
l DTH (delayed type hypersensitivity) model with KLH (keyhole limpet hemocyanin)
Autoimmune disease models
l Experimental Autoimmune Encephalitis (EAE, Multiple Sclerosis) in SJL/J mice
l Collagen - Induced Arthritis (CIA) in DBA/1 mice
l Diabetes (DIO model)
HEIDELBERG PHARMA INFLAMMATION & AUTOIMMUNITY
![Page 4: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/4.jpg)
4
HEIDELBERG PHARMA ONCOLOGY
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 for
Bioimaging.
Models to evaluate bispecific Antibodies • Implantation of a mixture of cancer cell lines with human peripheral blood
mononuclear cells (hPBMCs)
![Page 5: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/5.jpg)
Rheumatoid Arthritis
![Page 6: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/6.jpg)
Rheumatoid Arthritis : Pathogenesis
![Page 7: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/7.jpg)
PRECLINICAL MODELS FOR RA
![Page 8: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/8.jpg)
SIMILARITIES & DIFFERENCES
![Page 9: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/9.jpg)
Origin of the TNFa transgenic model
• The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene, including the entire coding region and promoter, fused to the human β-globin 3' untranslated region (UTR) that replaces the endogenous 3'UTR of the human TNFα gene – Designed to model dysregulated human TNFa 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
![Page 10: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/10.jpg)
Progressive arthritis in the TNFa mice
• The TNFa mice develop inflammatory arthritis spontaneously
• Ideal for screening new small molecules and biologics for the treatment of arthritis
![Page 11: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/11.jpg)
Experimental procedures
• Treatment was initiated when mice were 5 weeks old following 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 of working concentration of Humira freshly prepared just before 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 was terminated when the animals reached 15 weeks old – Paws were fixed in 10% buffered formalin for histology analysis
![Page 12: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/12.jpg)
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 = normalü0.2 = one or more swollen joints
ü 4 paws: score 0 or1 or 2 (maximum 8 scores)
ü0 = normalü1 = noticeable swollenü2 = severe swollen
ü 2 wrists: score 0 or 1 or 2 (maximum 4 scores)
ü0 = normalü1 = noticeable swollenü2 = severe swollen
ü 2 ankles: score 0 or 2 or 4 (maximum 8 scores)
ü0 = normalü2 = noticeable swollenü4 = severe swollen with stiffness of ankle joint
![Page 13: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/13.jpg)
Clinical Score
0 4-0.5
0.0
0.5
1.0
1.5
7.0 7.5 8.0 8.5 9.0 9.5
2.0
4.0
6.0
8.0
10.0
Week post Treatment Initiation
RA S
core
Humira, 1mg/kg (n=9)Humira, 0.25mg/kg (n=9)
Humira, 25mg/kg (n=9)Vehicle (n=10)
Humira, 10mg/kg (n=10)
Arthritis Clinical Scores Two-Way ANOVA
Time P<0.0001
Time/Treatment P<0.0001
Treatment P<0.0001
Post-hoc LSD Test
G1 vs G2 P<0.0001
G1 vs G3 P<0.0001
G1 vs G4 P=0.559
G1 vs G5 P=0.709
Dose dependent effect of treatment on clinical progression of arthritis
![Page 14: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/14.jpg)
Vehicle
Humira 10 mg/kg
Humira 25 mg/kg
Clinical manifestation of arthritis in treated and untreated animals
![Page 15: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/15.jpg)
Histopathology grading of joint lesions
• 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 filled with abundant exudate, inflammatory lesions
![Page 16: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/16.jpg)
Histopathology scores of front and rear paw joints
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
G1: Vehicle (n=10)Hi
sto
Scor
eG2: HUMIRA, 25mg/kg (n=9)G3: HUMIRA, 10mg/kg (n=10)G4: HUMIRA, 1mg/kg (n=9)G5: HUMIRA, 0.25mg/kg (n=9)
One way ANOVAP<0.0001
***
Post Hoc Analysis:Dunnett's Multiple Comparison TestG1 vs G2: P<0.001G1 vs G3: P<0.001G1 vs G4: nsG1 vs G5: ns
***
Treatment groups
* = P<0.05 ** = P<0.01
*** = P<0.001
• 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 filled
with abundant exudate, inflammatory lesions
![Page 17: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/17.jpg)
Representative histopathology of ankles from experimental mice
Inflamed ankle joint , 100x , # 560 (non-treated)
Normal ankle joint, 100x, # 561 (25mg/kg HUMIRA treated)
Inflam
muscle
Bone
Fat in bone marrow
JtS
JtS
![Page 18: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/18.jpg)
Paw tissue pro-inflammatory cytokines: IL-1β and mKC
* = P<0.05 ** = P<0.01
*** = P<0.001
IL-1b Levels in the Joints
0.0
2000.0
4000.0
6000.0
8000.0
G1: Vehicle (n=4)
pg/m
l
G2: Humira, 25mg/kg (n=4)G3: Humira, 10mg/kg (n=4)G4: Humira, 1mg/kg (n=3)G5: Humira, 0.25 mg/kg (n=4)
Post Hoc Analysis:Dunnett's Multiple Comparison TestG1 vs G2: P<0.001G1 vs G3: P<0.001G1 vs G4: nsG1 vs G5: ns
One way ANOVAP<0.0001
*** ***
Treatment groups
mKC Levels in the Joints
0.0
200.0
400.0
600.0
800.0
G1: Vehicle (n=4)
pg/m
l
G2: Humira, 25mg/kg (n=4)G3: Humira, 10mg/kg (n=4)G4: Humira, 1mg/kg (n=3)G5: Humira, 0.25mg/kg (n=4)
***
Post Hoc Analysis:Dunnett's Multiple Comparison TestG1 vs G2: P<0.001G1 vs G3: P<0.001G1 vs G4: nsG1 vs G5: ns
One way ANOVAP<0.0001
***
Treatment groups
![Page 19: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/19.jpg)
Study at Heidelberg Pharma
Animals
l Age at delivery 5 weeks
l Age at start of experiment 6 weeks
Treatment Groups (n=8)
l Vehicle
l Humira 5 mg/kg ip
l Humira 10 mg/kg ip
l Schedule 2x weekly
Readouts
l Clinical Score
l Paw swelling
l Histology : Paws fixed and embedded
![Page 20: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/20.jpg)
Body Weight
15
17
19
21
23
25
27
29
31
33
0 7 14 21 28 35 42 49 56 63 70 77 84 91
Med
ian
bod
y w
eigh
t (g)
Days after start of treatment
Group 1PBS
Group 2Humira®5 mg/ml
Group 3Humira®10 mg/ml
• Steady increase in body weight • No effect on body weight by treatment with Humira
![Page 21: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/21.jpg)
Paw swelling
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
27 34 41 48 55 62 69 76 83 90
Mea
n le
ft hi
nd p
aw (m
m) +
SD
Days after start of treatment
Group 1PBS
Group 2Humira®5 mg/ml
Group 3Humira®10 mg/ml
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
27 34 41 48 55 62 69 76 83 90
Mea
n rig
ht h
ind
paw
(mm
) +SD
Days after start of treatment
Group 1PBS
Group 2Humira®5 mg/ml
Group 3Humira®10 mg/ml
• Only minor swelling in hind paws
• Humira effectively inhibited paw swelling
![Page 22: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/22.jpg)
Paw swelling
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
27 34 41 48 55 62 69 76 83 90
Mea
n le
ft fo
re p
aw (m
m) +
SD
Days after start of treatment
Group 1PBS
Group 2Humira®5 mg/ml
Group 3Humira®10 mg/ml
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
27 34 41 48 55 62 69 76 83 90
Mea
n rig
ht fo
re p
aw (m
m) +
SD
Days after start of treatment
Group 1PBS
Group 2Humira®5 mg/ml
Group 3Humira®10 mg/ml
• Similar swelling in both fore paws
• Humira effectively inhibited paw swelling
![Page 23: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/23.jpg)
Clinical Score
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
27 34 41 48 55 62 69 76 83 90
Mea
n fo
re le
ft vi
sual
CIA
sco
ring
+ SD
Days after start of treatment
Group 1PBS
Group 2Humira®5 mg/ml
Group 3Humira®10 mg/ml
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
27 34 41 48 55 62 69 76 83 90
Mea
n fo
re ri
ght
visu
al C
IA s
corin
g +
SD
Days after start of treatment
Group 1PBS
Group 2Humira®5 mg/ml
Group 3Humira®10 mg/ml
• Similar development of disease in both fore paws
• Humira effectively inhibited progression of disease
Scores: 0: No evidence of erythema and swelling 1: Erythema and mild swelling confined to the mid-foot (tarsals) and ankle joint 2: Erythema and mild swelling extending from the ankle to the mid-foot 3: Erythema and moderate swelling extending from the ankle to the metatarsal joints 4: Erythema and severe swelling encompass the ankle, foot, and digits (acc. to Current Protocols in Immunology, 15.5.11)
![Page 24: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/24.jpg)
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 mice – Inflammation 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
![Page 25: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/25.jpg)
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
![Page 26: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/26.jpg)
Contact
Prof. Andreas Pahl Heidelberg Pharma GmbH Schriesheimer Straße 101 68526 Ladenburg, Germany Phone: +49 (0)6203 1009-45 Fax: +49 (0)6203 1009-19 E-Mail: [email protected] Web: www.heidelberg-pharma.com
![Page 27: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/27.jpg)
Backup
![Page 28: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/28.jpg)
INVOLVEMENT OF IMMUNE SYSTEM
![Page 29: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/29.jpg)
INVOLVEMENT OF CYTOKINES
![Page 30: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/30.jpg)
Arthritis
Fertility
Metabolic Abnormalities
5 10 15 20 25 30 Age (weeks)
no visible signs mild moderate severe
normal decreased
none or minor pronounced
Other physiological consequences of constitutive human TNFa expression
![Page 31: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/31.jpg)
TNFa: overview
Cell Source Inducers Inhibitors Cell Target Primary Effects on Each Target
Mononuclear phagocytes, T cells, B cells, NK cells, vascular endothelial cells, keratinocytes, smooth muscle cells, mast cells, neutrophils, astrocytes, glial cells.
Lipopolysaccharide, zymosan, phorbol esters, ultraviolet light, viral infection, allogenic B cells, protozoa, and other microorganisms. Cytokines and other endogenous mediators (TNF-a, IL-1, IFN-g, IFN-a, GM-CSF, IL-2, TGF-b, substance P, platelet activating factor).
Prostaglandins, corticosteroids, IL-4, IL-6, TGF-b
Mononuclear phagocytes Neutrophils, eosinophils Endothelial cells Hypothalamus Liver Muscle, fat Thymocyte
Activation (Inflammation and Infection) Activation (Inflammation) Activation (Inflammation, coagulation) Fever Acute phase reactants (serum ameloid A protein) Catabolism (cachexia) Costimulator
![Page 32: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/32.jpg)
Humira levels in mouse serum during the study
Days post Treatment Initiation
µg/m
l
0 7 14 21 28 350.0001
0.001
0.01
0.1
1
10
100
1000
10000
G2: HUMIRA, 25mg/kgG3: HUMIRA, 10mg/kgG4: HUMIRA, 1mg/kgG5: HUMIRA, 0.25mg/kg
HUMIRA Levels in the Serum
![Page 33: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/33.jpg)
Proof of concept study with Humira
• Humira (adalimumab) is a biologic drug approved for the treatment of arthritis – Recombinant human IgG1 monoclonal antibody – Mechanism of action involves binding to TNFa to block signaling
![Page 34: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/34.jpg)
Experimental groups
Group # Treatment Dosing Schedule Route/Volume Concentration
1 Placebo Twice weekly i.p./100µl 1:10 dilution in PBS
2 HUMIRA-022512E Twice weekly i.p./100µl 25mg/kg
3 HUMIRA-022512E Twice weekly i.p./100µl 10mg/kg
4 HUMIRA-022512E Twice weekly i.p./100µl 1mg/kg
5 HUMIRA-022512E Twice weekly i.p./100µl 0.25mg/kg
![Page 35: Human TNFα Transgenic Mouse Model of …...a transgenic model • The TNFα transgenic mice were generated using a construct that contains a 2.8 kb fragment of the human TNFα gene,](https://reader033.vdocuments.mx/reader033/viewer/2022041807/5e54f919dbbbb229f72cc2f6/html5/thumbnails/35.jpg)
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