LSHB-CT

Transcription

1 Implementation of the results from the EU-FP6 project Sens-it-iv Integrated Data Analysis of In Vitro Testing Approaches Advancing BioTech Product Development and Safety ELRo_Sens-it-iv_PATC 1

2 Overall objective of the Sens-it-iv project Development of tools that are ready for prevalidation. Physiologically relevant Cell culture conditions supporting relevant cell phenotype, cell-cell interactions and cell-compound interactions Mechanism of action -omics Addressing specific questions Solid knowledge about in vivo processes required SOP(s) Including detailed performance criteria Tools that can be incorporated in ITS ELRo_Sens-it-iv_PATC 2

3 Overview of the available toolbox ELRo_Sens-it-iv_PATC 3

4 Toolbox content LSHB-CT Protein binding N TSF SOPs Status Keratinocytes NCTC2544 test 30 X X PV Human reconstituted skin 30 X X PV Lung EC Precision cut lung slices 12 X CT Human reconstituted alveolar epithelium 12 X X E Human reconstituted bronchial epithelium 12 X X E Specific sensitizer profil 12 X pv DC Xenobiotic sensing (genomic profile) 49 X X pv Maturation #1 (CD86, CD54, IL-8, ) Stoppe d PV: prevalidation; CT: confirmation test; E: evaluation; pv: prevalidation light X Maturation #2 (DotSCan) 20 X X pv Migration 12 X X pv T-cells Primary T-cell stimulation 6 X X E Other Neutrophil - THP-1 metabolization tests 12 X E Proteomics marker profile (combined list) 12 X E ELRo_Sens-it-iv_PATC 4

5 Implementation of selected tests from the Sens-it-iv toolbox ELRo_Sens-it-iv_PATC 5

6 Challenges related to implementation LSHB-CT Formulating the right question Establishing a testing strategy that provides the proper answer Getting around the econ-omics Full ITS parallel testing 10x AM* Full ITS sequencial testing 5x AM The minimal ITS providing the necessary information 0.8-2x AM * AM: animal model ELRo_Sens-it-iv_PATC 6

7 A proposed complete sequencial testing strategy for chemcial sensitizers. LSHB-CT Binding to protein (haptenization) - Direct Peptide Reactivity Binding Assay - 85% accuracy - Protein Binding Assay (Gerberick et al) - Respiratory Sensitization Testing 3a. Xenobiotic sensing by keratinocytes -KeratinoSens (Nrf2) - 85% accuracy - - IL-18 expression - 98% accuracy - (Nash et al) 3b. Xenobiotic sensing by DC (GARD) - 98% accuracy Dendritic cell (DC) activation - HCLAT (Sakaguchi & Ashikaya),MUSST (Ade & Python) (maturation) - 85% accuracy - DotScan (maturation) - DC migration - no misclassification yet - 5. T cell priming assay - 1. Penetration and absorption - accuracy: 79% for potency ranking, 92% for classifying compounds into the correct potency groups - ELRo_Sens-it-iv_PATC 7

8 A proposed need-to-know testing strategy for chemcial sensitizers. LSHB-CT Binding to protein (haptenization) - Direct Peptide Reactivity Binding Assay - 85% accuracy - Protein Binding Assay (Gerberick et al) - 3a. Xenobiotic sensing by keratinocytes -KeratinoSens (Nrf2) - 85% accuracy - (Nash et al) Respiratory Sensitization Testing - IL-18 expression - 98% accuracy - - 3b. Xenobiotic sensing by DC (GARD) - 98% accuracy - 1. Penetration and absorption - accuracy: 79% for potency ranking, 92% for classifying compounds into the correct potency groups - ELRo_Sens-it-iv_PATC 8

9 Animal-free assessment of the sensitizing properties of proteins In vitro and computational (B T cell epitopes) ELRo_Sens-it-iv_PATC 9

10 Measuring the impact of a protein on cells and cellular processes in vitro INNATE RESPONSES PROTEIN-SPECIFIC IMMUNE RESPONSES RECONSTITUTED TISSUE MODELS DC-based* DC-based* T CELL-based SKIN** EYE** LUNG INTESTINAL MATURATION MIGRATION PRIMING ASSAY CD86 DOTScan IRRITATION (IL-8, IL-1) GENOMIC PROFILE CD profile -ACTIVATION - EPITOPES * : dendritic cell **: proteases only - BARRIER FUNCTION - CILIA ACTIVITY -RECOVERY - CYTOKINE PROFILING - GENOMIC PROFILING UPREGULATION (FACS) NUMBER OF CELLS ELRo_Sens-it-iv_PATC 10

11 Testing strategy for determining the lung sensitizing capacity and relative potency of proteins. 1a. Sensing by lung epithelial cells a. Alveolar model b. Bronchial model Identification of marker profile for proteins in progress Implementation by Novozymes in progress 2. Relative potency Based upon dose-dependent changes in TEER (lung epitheial cell models (1a)) and DC profile (1b) 3. Dendritic cell (DC) activation - DotScan (maturation) - DC migration Assessed on proteins identification of a predictive marker profile in progress 1b. Sensing by DC (GARD) Assessed on proteins identification of a predictive marker profile in progress 4. T cell priming assay Assessed on proteins ready for use Lung Sensitizer ELRo_Sens-it-iv_PATC 11

12 In Vitro: Air/liquid phase model using primary human lung EC (Epithelix) After 1-2 months: 1. Appearing of ciliated, mucus and basal cells 2. Establishment of absorption / secretion properties 3. Stabilisation of electrophysiological properties Exposure regimes: Basal: the cells can be incubated during 24, 48 or 72 h Apical: only a short time exposure immersion (> 1 hr) induced inflammatory response chronic exposure (10x 1hr/day) Read-outs: viability cytokine profiles (IL1, IL-6 and IL-8) TEER ELRo_Sens-it-iv_PATC 12

13 TEER [ohm.cm2] % of Cytotoxicity TEER [ohm.cm2] % of Cytotoxicity and Viability Cilia Beating Frequency [Hz] TEER [ohm.cm2] % of Cytotoxicity TEER [ohm.cm2] % of Cytotoxicity and Viability Cilia Beating Frequency [Hz] LSHB-CT Reconstitiuted human bronchial tissue: Barrier function, cilia beating and recovery 1200 Effect of Amylase on Tissue Integrity and Cytotoxicity - after 24h exposure - 180% Effect of Amylase on Tissue Integrity, Cytotoxicity and Cell Viability - after 6 days recovery % 12 Effect of Amylase on Cilia Beating Frequency - after 24h exposure % % % % % % % % % % Amylase [mg/ml] 0% TEER Cytotoxicity Amylase [mg/ml] 0% TEER Cytotoxicity Viability Amylase [mg/ml] 1200 Effect of Protease on Tissue Integrity and Cytotoxicity - after 24h exposure - 180% Effect of Protease on Tissue Integrity, Cytotoxicity and Cell Viability - after 6 days recovery % 12 Effect of Protease on Cilia Beating Frequency - after 6 days recovery % % % % % % % % % % Protease [mg/ml] 0% TEER Cytotoxicity Protease [mg/ml] 0% TEER Cytotoxicity Viability Protease [mg/ml] Collaboration with Epithelix ELRo_Sens-it-iv_PATC 13

14 Reconstitiuted human alveolar tissue: Cytokine profiling PROTEASE NCI-H441Cell line ISO-HAS-1 Cell line upper well lower well TNF-ɑ CXCL12 IL-16 IL-8 IL-1ß M-CSF strem-1 PAI-1 MIF-1 CXCL11 IL-1ɑ IFN-γ CD54 CD154 CD5a CCL-5 CCL-2 IL-23 IL-17E IL-6 G-CSF LIPASE AMYLASE TNF-ɑ; CXCL12; IL-32a; IL-16; IL-12p70; IL-10; IL-8; IL-1ß Collaboration with Mainz University ELRo_Sens-it-iv_PATC 14

15 ng M-CSF/ml LSHB-CT Ranking of enzymes according to sensitizing potency: an example Protease 1 Protease 2 Protease ,001 0,01 0, mg protease/ml ELRo_Sens-it-iv_PATC 15

16 Testing strategy for determining the lung sensitizing capacity and relative potency of proteins. 1a. Sensing by lung epithelial cells a. Alveolar model b. Bronchial model Identification of marker profile for proteins in progress Implementation by Novozymes in progress 2. Relative potency Based upon dose-dependent changes in TEER (lung epitheial cell models (1a)) and DC profile (1b) 3. Dendritic cell (DC) activation - DotScan (maturation) - DC migration Assessed on proteins identification of a predictive marker profile in progress 1b. Sensing by DC (GARD) Assessed on proteins identification of a predictive marker profile in progress 4 T cell priming assay Assessed on proteins ready for use Lung Sensitizer ELRo_Sens-it-iv_PATC 16

17 Genomic Analysis of MUTZ-3 Cells after treatment C. Borrebaeck et al. Lund University (S) ELRo_Sens-it-iv_PATC 17

18 Testing strategy for determining the lung sensitizing capacity and relative potency of proteins. 1a. Sensing by lung epithelial cells a. Alveolar model b. Bronchial model Identification of marker profile for proteins in progress Implementation by Novozymes in progress 2. Relative potency Based upon dose-dependent changes in TEER (lung epitheial cell models (1a)) and DC profile (1b) 3. Dendritic cell (DC) activation - DotScan (maturation) - DC migration Assessed on proteins identification of a predictive marker profile in progress 1b. Sensing by DC (GARD) Assessed on proteins identification of a predictive marker profile in progress 4 T cell priming assay Assessed on proteins ready for use Lung Sensitizer ELRo_Sens-it-iv_PATC 18

19 Maturation marker induction after treatment: confounded by irritation Allergens Potency (in vivo data) Non-allergens Irritation (TEER, IL-8) C. Borrebaeck et al. Lund University (S) ELRo_Sens-it-iv_PATC 19

20 Integration of data read across computational in vitro ELRo_Sens-it-iv_PATC 20

21 Integration of the data LSHB-CT Enzyme activity Variant / class In vivo data Computational Innate immune responses Adaptive immunc responses Animal Human B cell T cell Barrier function (mg/ml) Cilia beating (mg/ml) Recovery Cytokine profile (mg/ml) Genomic profile DC maturation DC migration T cell priming Protease 1 Animals Epitope lists no Analysis available in 2 Guinea pig, progress Rat, Overlaps and no 0.1 Mouse differences 3 identified Serological data yes 10 Analysis in progress Epitopes identified Amylase A Immunochemcial Epitope lists characterization available yes Others (Lipase) B Overlaps and No effect No effect -- Humans differences identifed Clinical studies, Occupational data Serological data Epitope lists available No effect No effect -- ELRo_Sens-it-iv_PATC 21

22 Log2 Titre Log2 Titre pg/ml M-CSF LSHB-CT Proof of principle (1): Selection based on in vitro data only Alcalase Savinase (PS0000) Subtilisin Novo Protease X PS398 Selected proteases were evaluated in a MINT mouse model for respiratory sensitisation. 250 Alcalase - day 31, IgE Protease X - day 31, IgE µg/ml enzyme ug enzyme protein per animal ug enzyme protein per animal ELRo_Sens-it-iv_PATC 22

23 Log2 Titre Log2 Titre Log2 Titre pg/ml M-CSF LSHB-CT Proof of principle (2): Selection based on in vitro, computational data and read-across Selected protease was evaluated in a MINT mouse model for respiratory sensitisation. 250 Protease X Variant 58 Variant 63 Variant µg/ml enzyme Alcalase - day 31, IgE Protease X - day 31, IgE Variant 58 - day 31, IgE ug enzyme protein per animal ug enzyme protein per animal ug enzyme protein per animal ELRo_Sens-it-iv_PATC 23

24 Summary Set-up still under development Sufficient/adequate testing? Avoid overkill for the sake of time and money Integration of historical data, computational data and in vitro data to be optimised The weight of each input still to be established Ambition to be able to perform relative hazard identification and risk assessment Learnings from industrial enzymes should help to establish testing strategies for BioPharmaceuticals ELRo_Sens-it-iv_PATC 24