Lessons learned from transgenic mouse models immune tolerant for interferon

Transcription

1 Lessons learned from transgenic mouse models immune tolerant for interferon Division of Drug Delivery Technology Leiden Academic Centre for Drug Research (LACDR) AAPS Annual Meeting San Diego November 2014

2 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)

3 Lesson #1: a transgenic mouse model is more than an animal Unfertilized ovum Fertilized ovum Microinjection of DNA fragment (encoding hifnβ) C57Bl/6 Reimplantation Transgenic C57Bl/6 x Wildtype FVB/N Wildtype and transgenic C57Bl/6 Hermeling et al. (2005) Pharm Res 22: Interferon β Wildtype and transgenic hybrid Van Beers et al. (2010) J Immunol Methods 352: 32-37

4 Lesson #2: wildtype mice are not discriminative Binding antibody titers (3-week injection protocol) Untreated Metal ox H 2 O 2 ox Guanidine Titers on day 21, wildtype mice Interferon β Van Beers et al., Pharm Res 28: (2011)

5 Lesson #3: transgenic mice are discriminative Binding antibody titers (3-week injection protocol) Untreated Metal ox H 2 O 2 ox Guanidine Titers on day 21, transgenic mice Interferon β Van Beers et al., Pharm Res 28: (2011)

6 Lesson #4: not all aggregates are equally immunogenic; combination of aggregation and oxidation increases risk Binding antibody titers (3-week injection protocol) Untreated Metal ox H 2 O 2 ox Guanidine Titers on day 21, transgenic mice Interferon β Van Beers et al., Pharm Res 28: (2011)

7 Lesson #4: not all aggregates are equally immunogenic; combination of aggregation and oxidation increases risk anti-rhifn 2b IgG titer Binding antibody titers (3-week injection protocol) t=14 days t=21 days 100 4/5 N= untreated M= metal-catalyzed oxidized G= glutaraldehyde treated H= oxidized by H 2 O 2 B= boiled 10 N M G H B Interferon α Hermeling et al., Pharm Res 22: (2005)

8 Lesson #5: aggregate dose affects immunogenicity IgG titer Binding antibody titers (3-week injection protocol) b 0 Wildtype Transgenic ** *** *** 4/ /5 10 ND A B C D E Increasing aggregate content Interferon α Hermeling et al., J Pharm Sci 95: (2006)

9 Lesson #5: aggregate dose affects immunogenicity Interferon β Kijanka et al., Pharm Res 30: (2013)

10 Lesson #6: product quality affects immunogenicity BABs: IgG titer BABs: IgG titer NABs: TRU/ml NABs: TRU/ml BABs: IgG titer NABs: TRU/ml Betaferon Avonex 0 BABs NABs BABs NABs 2/5 2/5 2/ Wildtype All < 20 Transgenic Wildtype Transgenic Rebif Betaferon contains highest aggregate levels 0 BABs NABs 4/4 4/ /4 Wildtype 0/5 All < 10 All < 20 Transgenic Van Beers et al., J Immunol Methods 352: (2010)

11 Lesson #7: subvisible particles may increase immunogenicity Particle count / mg protein (x 10 5 ) % Responders MFI analysis Immunogenicity in transgenic mice Bulk Reformulated Stressed Bulk Reformulated Stressed range (µm) > Size range (µm) >30 Hardly any particles in reformulated rhifnβ-1a Immunogenicity in transgenic immune tolerant mice correlates with subvisible particle counts (rather than total % aggregates) Interferon β van Beers et al., Pharm Res 27: (2010)

12 Lesson #7: subvisible particles may increase immunogenicity IgG titer Anti-rhIFNb antibody titers after a 3-week injection protocol 0 Metal Ø 14 μm 71% adsorbed Bulk Metal Glass Polystyrene Betaferon Glass Ø 1.1 μm 78% adsorbed Polystyrene Ø 0.21 μm 46 % adsorbed Interferon β Van Beers et al., J Pharm Sci 101: (2012)

13 Lesson #8: administration route affects immunogenicity Interferon α

14 Lesson #9: don t believe in dogmas Interferon β Kijanka et al., Pharm Res 30: (2013)

15 Lesson #10: immune mechanisms are not fully understood IgG titer Prime-boost experiment: Memory effect in wildtypes, not in transgenic mice Before After Before After Wildtype Transgenic Interferon β van Beers et al., Pharm Res 27: (2010)

16 The ultimate lesson 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 Johnson and Jiskoot, J Pharm Sci 101: (2012) Brinks et al., Pharm Res 30: (2013)

17 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 UIPS Utrecht Institute for Pharmaceutical Sciences University of Colorado Ted Randolph, John Carpenter, Jared Bee University Hospital San Luigi Gonzaga, Torino Francesca Gill Biogen Idec Kansas University Susan Goelz Christian Schöneich et al. Darren Baker Antitope Christine Bryson Matthew Baker

18 Thank you!