Division of Drug Delivery Technology Leiden Academic Centre for Drug Research (LACDR) AAPS

Annual Meeting San Diego

November 2014

Lessons learned from transgenic mouse models immune tolerant for interferon

Lesson #1: a transgenic mouse model is more than an animal

Some of the crucial factors:

• The animal (sub)species

• The right transgene

• Animal facilities

• Time

• Patience

• Perseverence

• The protein

• The dose

• The injection schedule

• The sampling schedule

• The right assay(s)

• …

Approximate development time for transgenic mouse models in our hands (plus resulting papers):

• Interferon-alfa: 2 years (terminated)

(3 publications)

• Interferon-beta: 11 years (ongoing)

(10 publications)

• IgG: 5 years (unfinished)

(1 publication)

• Insulin: 5 years (unfinished)

(1 publication)

Lesson #1: a transgenic mouse model is more than an animal

Wildtype and transgenic C57Bl/6

Unfertilized ovum

Fertilized ovum

Reimplantation

Microinjection of DNA fragment

(encoding hIFNβ)

C57Bl/6

Hermeling et al. (2005) Pharm Res 22: 847-51

x

Wildtype FVB/N

Transgenic C57Bl/6

Wildtype and transgenic hybrid

Van Beers et al. (2010) J Immunol Methods 352: 32-37 Interferon β

Lesson #2: wildtype mice are not discriminative

Binding antibody titers (3-week injection protocol)

Van Beers et al., Pharm Res 28: 2393–2402 (2011)

H2O2 ox

Guanidine

Metal ox

Untreated

H2O2 ox

Guanidine

Metal ox

Untreated

Titers on day 21, wildtype mice

Interferon β

Lesson #3: transgenic mice are discriminative

Binding antibody titers (3-week injection protocol)

Van Beers et al., Pharm Res 28: 2393–2402 (2011)

H2O2 ox

Guanidine

Metal ox

Untreated

H2O2 ox

Guanidine

Metal ox

Untreated

Titers on day 21, transgenic mice

Interferon β

Lesson #4: not all aggregates are equally immunogenic; combination of aggregation and oxidation increases risk

Binding antibody titers (3-week injection protocol)

Van Beers et al., Pharm Res 28: 2393–2402 (2011)

H2O2 ox

Guanidine

Metal ox

Untreated

H2O2 ox

Guanidine

Metal ox

Untreated

Titers on day 21, transgenic mice

Interferon β

Lesson #4: not all aggregates are equally immunogenic; combination of aggregation and oxidation increases risk

Binding antibody titers (3-week injection protocol)

Hermeling et al., Pharm Res 22: 1997-2006 (2005)

N M G H B10

100

1000

t=14 days t=21 days

5/5

4/5

an

ti-r

hIF

N

2b

Ig

G t

iter

N= untreated

M= metal-catalyzed oxidized

G= glutaraldehyde treated

H= oxidized by H2O2

B= boiled

Interferon α

Lesson #5: aggregate dose affects immunogenicity

Binding antibody titers (3-week injection protocol)

Hermeling et al., J Pharm Sci 95: 1084-1096 (2006)

A B C D E10

100

1000

10000

Wildtype

***** ***

b Transgenic

2/5

4/5

5/5

5/5

IgG

tit

er

ND

Increasing aggregate content

Interferon α

Kijanka et al., Pharm Res 30:1553–1560 (2013)

Lesson #5: aggregate dose affects immunogenicity

Interferon β

Rebif

Van Beers et al., J Immunol Methods 352: 32-37 (2010)

Wildtype Transgenic10

100

1000

10000

0

200

400

600

800

1000BABs NABs

5/5

5/5

5/5

All< 20

BA

Bs

: Ig

G

tite

r NA

Bs

: TR

U/m

l

Betaferon

Wildtype Transgenic10

100

1000

10000

0

200

400

600

800

1000BABs NABs

5/55/5

2/5

2/5

BA

Bs

: Ig

G

tite

r NA

Bs

: TR

U/m

l

Avonex

Wildtype Transgenic10

100

1000

10000

0

200

400

600

800

1000BABs NABs

4/4

4/4All

< 20All

< 10

BA

Bs

: Ig

G

tite

r NA

Bs

: TR

U/m

l

5/5 5/5

4/4

5/5

2/5

0/5

Betaferon contains highest aggregate levels

Lesson #6: product quality affects immunogenicity

0.75

-1 1-2

2-4

4-6

6-8

8-10

10-1

5

15-2

0

20-3

0>3

0

0

2

4

6

8

10

12

14Bulk

Reformulated

Stressed

Size range (µm)

Part

icle

co

un

t /

mg

pro

tein

(x 1

05)

• Hardly any particles in reformulated rhIFNβ-1a

• Immunogenicity in transgenic immune tolerant mice correlates with subvisible particle counts (rather than total % aggregates)

van Beers et al., Pharm Res 27: 1812-1824 (2010)

0.75

-1 1-2

2-4

4-6

6-8

8-10

10-1

5

15-2

0

20-3

0>3

0

0

2

4

6

8

10

12

14Bulk

Reformulated

Stressed

Size range (µm)

Part

icle

co

un

t /

mg

pro

tein

(x 1

05) Immunogenicity in transgenic mice

% R

esp

on

der

s

MFI analysis

Lesson #7: subvisible particles may increase immunogenicity

Interferon β

Bulk

Met

al

Glass

Polys

tyre

ne

Bet

afer

on

10

100

1000

10000

IgG

tit

er

Van Beers et al., J Pharm Sci 101: 187-199 (2012)

Anti-rhIFNb antibody titers after a 3-week injection protocol

Metal Ø 14 μm 71% adsorbed

Glass Ø 1.1 μm 78% adsorbed

Polystyrene Ø 0.21 μm 46 % adsorbed

Lesson #7: subvisible particles may increase immunogenicity

Interferon β

Lesson #8: administration route affects immunogenicity

Interferon α

Kijanka et al., Pharm Res 30:1553–1560 (2013)

Lesson #9: don’t believe in dogmas

Interferon β

Prime-boost experiment: Memory effect in wildtypes, not in transgenic mice

Before After Before After10

100

1000

10000

100000

Wildtype Transgenic

IgG

tit

er

Lesson #10: immune mechanisms are not fully understood

van Beers et al., Pharm Res 27: 1812-1824 (2010) Interferon β

Immune tolerant animal models (and any other currently available so-called ‘predictive’ models) do not predict clinical immunogenicity Rather, they can be used to identify product-related factors contributing to immunogenicity risk

The ultimate lesson

Johnson and Jiskoot, J Pharm Sci 101: 3586-3592 (2012)

Brinks et al., Pharm Res 30: 1719-1728 (2013)

Acknowledgements

Leiden University

Miranda Van Beers, Vasco Filipe,

Riccardo Torosantucci, Andrea Hawe

Utrecht University

Huub Schellekens, Daan Crommelin, Vera Brinks,

Suzanne Hermeling, Melody Sauerborn,

Grzegorz Kijanka, Andhyk Halim

University of Colorado

Ted Randolph, John Carpenter,

Jared Bee

University Hospital San Luigi Gonzaga, Torino

Francesca Gill

Kansas University

Christian Schöneich et al.

UIPS Utrecht Institute for Pharmaceutical Sciences

Antitope

Christine Bryson

Matthew Baker

Biogen Idec

Susan Goelz

Darren Baker

Thank you!