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Sebastian Spindeldreher

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1 Sebastian Spindeldreher
Aggregation of Human Recombinant Monoclonal Antibodies Enhances Their Presentation by Dendritic Cells In Vitro Sebastian Spindeldreher Novartis Institutes for Biomedical Research / DMPK-Biologics 7th Open Scientific EIP Symposium, Lisbon, February 24, 2015

2 Aims of this study Do proteinaceous SVP have increased immunogenic potential? Why (mechanism)? Which factors influence immunogenicity: Size? Type of stress/structure? | EIP Symposium | S. Spindeldreher | Feb 24, 2015

3 Acknowledgements MAPPs data Anette Karle Sascha Gottlieb
Stephan Koepke Laetitia Sordé Xavier Charles Leber Steffen Hartmann DC data: Andrea Kiessling Babette Wolf Jennifer Krieg* Stewart Jones* T cell data Aida Baban Aggregates generation and phys/chem characterization: Verena Rombach-Riegraf Atanas Koulov* Bahman Ossuli Dolores Tinhan Philippe Simeoni Marie-Claude Djidja Kamal Egodage Markus Blümel Margit Jeschke Maria Brauchle | EIP Symposium | S. Spindeldreher | Feb 24, 2015 * Left Novartis

4 Selected stress conditions
2 model antibodies: mAb1 and mAb2 (terminated projects) Both IgG1 subclass targeting soluble plasma proteins Very different biophysical properties from one another with regard to isoelectric point, melting temperature, surface hydrophobicity, colloidal stability Three stress conditions Heat/shake (HS) Unstressed Stress level 1 (sl1) 10 min at 65°C and 1400 rpm Stress level 2 (sl2) 6 min at 80°C and 1400 rpm Shear stress (S) Stress level 1 (sl1) draw/empty syringe once Stress level 2 (sl2) draw/empty syringe four times Freeze Thaw (FT) Stress level 1 (sl1) 3 FT cycles Stress level 2 (sl2) 10 FT cycles | EIP Symposium | S. Spindeldreher | Feb 24, 2015

5 Applied technologies Phys/Chem analytics Biological assays:
Microflow Imaging Size Exclusion Chromatography Dynamic Light Scattering Capillary Electrophoresis Sodium Dodecyl Sulfate LC/MS peptide mapping Biological assays: DC activation assay MAPPs assay T cell assay | EIP Symposium | S. Spindeldreher | Feb 24, 2015

6 Particle size distribution
Determined via Microflow imaging unstressed FT stress level 1 FT stress level 2 mAb1 mAb2 mAb1 mAb2 HS FT S | EIP Symposium | S. Spindeldreher | Feb 24, 2015

7 Immune cell interplay T-cell assay MAPPs DC-assay
| EIP Symposium | S. Spindeldreher | Feb 24, 2015

8 DC maturation assay Assay procedure
CD83 CD80 CD14 isolation with magnetic beads GM-CSF IL-4 Ficoll Maturation 3d CD86 5d CD11c Healthy Blood Donor Buffy Coat PBMC Monocyte Immature Dendritic Cell drug stain cytokines 72h maturation cocktail 72h medium control 72h DPBS | EIP Symposium | S. Spindeldreher | Feb 24, 2015

9 Induction of DC maturation by stressed mAb materials
Response index: response relative to unstressed condition | EIP Symposium | S. Spindeldreher | Feb 24, 2015

10 Relative number of positive responder with response index at least 1.5
| EIP Symposium | S. Spindeldreher | Feb 24, 2015

11 Identification of naturally processed HLA peptides MHC-associated Peptide Proteomics (MAPPs)
Cell isolation Differentiation Maturation Immuno-precipitation Washing and peptide elution Nano LC-MS and database search Buffy Coat mass spectra Monocyte peptide mAb Immature DC Mature DC | EIP Symposium | S. Spindeldreher | Feb 24, 2015

12 MAPPs example: clusters in human interferon beta
One Donor Different Peptides Heat map of different peptides | EIP Symposium | S. Spindeldreher | Feb 24, 2015

13 Peptide clusters presented on DCs from heat stressed mAb1 and mAb2
| EIP Symposium | S. Spindeldreher | Feb 24, 2015

14 Naturally processed peptides: MAPPs assay
Number of different peptides and clusters mAb1 mAb2 | EIP Symposium | S. Spindeldreher | Feb 24, 2015

15 Correlation of peptide presentation and protein amount in particles
Linear regression analyses of increase of HLA-DR associated peptides/clusters as functions of the calculated amount of protein present in subvisible particles. | EIP Symposium | S. Spindeldreher | Feb 24, 2015

16 T cell assay Aim: Link to other studies that show enhanced T cell activation PBMCs prepared from buffy coats of healthy human donors by density gradient centrifugation. On day 0, challenge with different preparations of biotherapeutics at 200 mg/mL. IFN-y ELISpot after 7 days in culture Limited no. of donors tested (8 blood donors tested for FT and 13 different for HS) MAb2 not tested due to direct interference in the assay Shear stress not tested Naïve CD4+ T cell APC T cell proliferation T cell expansion (7 days) Activated CD4+ T cell Activated CD4+ T cell Activated CD4+ T cell | EIP Symposium | S. Spindeldreher | Feb 24, 2015

17 T cell assay results mAb1 only due to direct interference of mAb2 in T cell assay | EIP Symposium | S. Spindeldreher | Feb 24, 2015

18 Summary Aggregates can induce DC maturation. More particles induce stronger DC maturation in more donors Aggregates lead to increased HLA-restricted antigen presentation Extent of presentation correlates exceptionally well with calculated amount of protein contained within the subvisible particles Aggregates can lead to increased T cell responses Thus, highly aggregated proteins are able to induce adaptive immune responses | EIP Symposium | S. Spindeldreher | Feb 24, 2015

19 Summary cont’d Aggregates and subvisible particles are present, to a limited extent, in every biopharmaceutical product sold on the market today. The aggregation levels typically observed for marketed therapeutic antibodies are much lower than the levels generated for this study. To shed further light on the mechanistic involvement of aggregates during induction of immunogenicity additional investigations have been initiated as part of ABIRISK: Additional aggregated monoclonal antibodies (marketed formulations) which are also part of clinical investigations in ABIRISK Advanced assays performed by multiple labs | EIP Symposium | S. Spindeldreher | Feb 24, 2015

20 Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the RISK

21 WP 3 Evaluation and development of technologies for predicting immunogenicity Aim 1: Evaluate clinical relevance and gain a greater understanding of technologies of prediction of immunogenicity Aim 2: Develop and assess novel prediction methods Aim 3: Assess effects of aggregation on immunogenicity Co-leaders Bernard Maillere, CEA Christian Pedersen Ross, Novo Nordisk Sebastian Spindeldreher, Novartis Pharma

22 Aim 3: Effect of aggregates A sneak preview
Aggregate generation and characterization Focus on antibodies (rituximab, infliximab, natalizumab, adalimumab) Syringe stress: SS-L1: 3x "up and down“ SS-L2: 10x "up and down“ Heat stress: HS-L1: 24 °C HS-L2: 72 Physicochemical analytics : SEC, DLS, MFI and Turbidity Applied methods MAPPs DC activation assays T cell assay

23 Natalizumab: Changes in peptide presentation

24 Infliximab: Changes in peptide presentation

25 Novartis internal study
Increased stress leads to increased peptide presentation rituximab adalimumab infliximab natalizumab mAb 1 mAb 2 New IMI study Novartis internal study

26 Conclusion MAPPs on 4 marketed antibodies confirmed the findings of the Novartis study The stronger the stress, the more peptides are being presented on HLA class II The baseline presentation as well as the degree of change in peptide presentation depends on the molecule. Ongoing: DC activation assays with multiple endpoints: Cell surface activation markers Cytokine secretion Chemokine and cytokine transcription Cell signaling (phosphorylation) Selection of T cell assay setup for aggregate study

27 WP3 partners INSERM U996 Marc Pallardy Sophie Tourdot Isabelle Turbica
Yu-Chun Lone Novartis Sebastian Spindeldreher Anette Karle Andrea Kiessling Anja Matter Hannah Morgan Sascha Gottlieb Stephan Koepke Verena Rombach-Riegraf IRB Antonio Lanzavecchia Federica Sallusto INSERM U872 Sebastien Lacroix-Desmazes Anastas Pashov Sandrine Delignatat M. Zhu Novo Nordisk Christian Ross Pedersen Thomas Bäckström Anne Månsson Kvarnhammar Bayer Hans-Werner Vohr Jeannette Lo Margret Leineweber Fred Aswad Pedro Paz SANOFI Vincent Mikol Catherine Prades Daniel Kramer Laurent Duhau Magali Agnel Pierre Lafarguette Stephane Coren DRK-BSD Peter Milanov Stefanie Roth CEA Bernard Maillere Moustafa Hamze Sylvain Meunier SciCross Pierre Dönnes

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