Challenges in Development and Validation of an Intracellular Cytokine Staining assay

Challenges in Development and Validation of an Intracellular Cytokine Staining assay Jenny Hendriks, Crucell Hatching @ EBF, Brussels, June 202 www.crucell.com

Vaccines vs Protein therapeutics Protein therapeutics Unwanted immunogenicity Adenovirus based vaccines Unwanted immunogenicity Responses against the vector Wanted immunogenicity Serology Cell Mediated Immunity

Unwanted immunogenicity may also be of a cellular nature While much attention is given to Anti-Drug-Antibodies (ADA), some ADA develop because of T cell help CD4 help to Neutralizing Abs against Factor VIII Delayed type hypersensitivity reactions are T cell mediated Committee for Medicinal Products for Human use (CHMP):GUIDELINE ON IMMUNOGENICITY ASSESSMENT OF BIOTECHNOLOGY-DERIVED THERAPEUTIC PROTEINS EMEA/CHMP/BMWP/4327/2006

Scatter Flow Cytometry basic principle Flow cytometry: principle First flow based cell counter in 953, Fluorescence was added in 968 Scatter Conventional fluochromes/flow cytometers stop at 8 Multiparameter data in one sample (one cell)

Intracellular Cytokine Staining Detecting Vaccine specific T cell responses Most, if not all, vaccines are developed on basis of protective Abs Vaccine development for : HIV, TB and Malaria In addition to antibodies, T cells are thought to play a pivotal role Aim Detect and characterize T cell responses to Vaccine/Pathogen CD3 APC-H7 CD8 PerCP-Cy5-5 CD4 Horizon V4 IFNγ - APC TNF - FITC IL-2 PE Live/dead cells Aqua Fluorescent Dye

Flow Cytometry towards a regulated environment Intracellular Cytokine Staining is an LBA both inside the cell and out Use adapted EMA guidance in absence of T cell/technology specific guidance Calibration of equipment Assay validation Assay run criteria

Intracellular Cytokine Staining Method in short T cells are incubated with Antigenic peptides in solution T cells recognize specific peptide antigen and are activated Activated antigen-specific T cells produce cytokines Cytokine secretion is blocked 0 4 0.44 0.94 0 3 cytokine 0 2 0 Cells are fixed and permeabilised Cells are stained with Cytokine- and phenotype- Specific antibodies 0 0 0 0 0 0 2 0 3 0 4 CD8 FACS analysis determines percentage of cytokine+ CD8+ or CD4+ Tcells

Intracellular Cytokine Staining Aim for standardization

ICS validation strategy CMV peptides CMV+ PBMC ICS development ICS Qualification ICS validation Basic ICS SOP Specific peptides Specific PBMC QC check QC check Bridging to Specific peptides Integrated ICS SOP

Development ICS Acquire material: Large batches PBMC isolated and frozen, test CMV reactivity Disposables, CMV peptides, SEB, Antibodies Establish separate cytokine staining first, then combine and test for interference Optimal staining; concentrations Ab, duration and temperature Optimal stimulation; concentrations stimuli and duration Selection FBS batch; test for lowest background and optimal antigen specific response Optimize Thaw protocol Number of stimulated cells Amount of acquired events Stability of stained samples Interference multi-cytokine staining Standardization of gating

Development ICS # of cells /well stimulation CD4 CD8 0 0 %IFN- γ + cells 0. % IFN- γ + cells 0. 0.0 0.0 Cells/well Cells/well # of events acquired CD8 CD8 0 CD8 % IFN- γ + cells 0 0. % TNF- α + cells 0 0. % IL-2 + cells 0. 0.0 0.0 0.0 Number of Eventts Number of Events Number of Events

Staining conditions determined in basic ICS are also applicable for multiple ICS CD4 responses CD4 responses CD4 responses % IFN γ + cells 0 0. % TNF α + cells 0 0. % IL-2+ cells 0 0. 0.0 0(single) 0(multiple) (single) (multiple) SEB 0(single) 0(multiple) (single) (multiple) CMV 0.0 0(single) 0(multiple) SEB (single) (multiple) 0(single) 0(multiple) (single) (multiple) CMV 0.0 0(single) 0(multiple) SEB (single) (multiple) 0(single) 0(multiple) (single) (multiple) CMV CD8 responses CD8 responses CD8 responses % IFN γ + cells 0 0. % TNF α + cells 0 0. % IL-2+ cells 0 0. 0.0 0(single) 0(multiple) (single) (multiple) SEB 0(single) 0(multiple) (single) (multiple) CMV 0.0 0(single) 0(multiple) (single) (multiple) SEB 0(single) 0(multiple) (single) (multiple) CMV 0.0 0(single) 0(multiple) (single) SEB (multiple) 0(single) 0(multiple) (single) (multiple) CMV

Development Linearity Decrease in cytokine response, increase in variation CD4 CD4 CD4 0 0 0 % IFN- γ + cells 0. % TNF- α + cells 0. % IL-2 + cells 0. 0.0 25 2.5 6.25 3.25.56 % CMV stimulated cells 0.8 0.0 25 2.5 6.25 3.25.56 % CMV stimulated cells 0.8 0.0 25 2.5 6.25 3.25.56 % CMV stimulated cells 0.8

Intracellular Staining (ICS) Assay IFNγ TNF IL-2

Reagent control mabs stability Increased background of IL-2 mabs 2.0 Lot: 82558.5 % Mock (IL-2).0 0.5 0.0 High CD4 Low CD4 High CD8 Low CD8 Expiry date may not reflect in use stability!

Development ICS Acquire material: Large batches PBMC isolated and frozen, test CMV reactivity Disposables, CMV peptides, SEB, Antibodies Establish separate cytokine staining first, then combine and test for interference Optimal staining; concentrations Ab, duration and temperature Optimal stimulation; concentrations stimuli and duration Selection FBS batch; test for lowest background and optimal antigen specific response Optimize Thaw protocol Number of stimulated cells Amount of acquired events Stability of stained samples Interference multi-cytokine staining Standardization of gating

Qualification ICS Linearity The slope should be close to and the 90%CI within the indifferent zone of [0.8-.2] 90% CI: 0.969-.04 90% CI: 0.970-.04

Qualification ICS Specificity mabs used for cytokine detection have been characterized before; use alternative method to establish specificity of entire procedure 3 2 r=0.75 ICS (%IFNg/CD4 and CD8.0 0.8 0.6 0.4 0.2 0.0 0 200 400 ELISPOT (IFNg SFU/0 6 ) 0 2000

Qualification ICS LLOQ IFNg/CD8 vs CV TNFa/CD8 vs CV IL-2/CD8 vs CV 200 200 200 CV% CV% CV% 0 0 0 20 30 0 0 0 20 30 0 0 0 20 30 %IFNg/CD8 %TNFa/CD8 %IL-2/CD8 IFNy/CD4 vs CV TNFa/CD4 vs CV IL-2/CD4 vs CV 200 200 200 CV% CV% CV% 0 0 0 20 30 0 0 0 20 30 0 0 0 20 30 %IFNg/CD8 %TNFa/CD4 %IL-2/CD4

Qualification ICS LLOQ LLOQ set at 0.05% for all cytokine/t cell, where 95% of values are <CV35% IFNg/CD8 vs CV TNFa/CD8 vs CV IL-2/CD8 vs CV 200 200 200 CV% CV% CV% 0 0.0 0.2 0.4 0.6 0.8.0 0 0.0 0.2 0.4 0.6 0.8.0 0 0.0 0.2 0.4 0.6 0.8.0 %IFNg/CD8 %TNFa/CD8 %IL-2/CD8 IFNy/CD4 vs CV TNFa/CD4 vs CV IL-2/CD4 vs CV 200 200 200 CV% CV% CV% 0 0.0 0.2 0.4 0.6 0.8.0 0 0.0 0.2 0.4 0.6 0.8.0 0 0.0 0.2 0.4 0.6 0.8.0 %IFNg/CD8 %TNFa/CD4 %IL-2/CD4

Qualification ICS Precision Variance component analysis; fully nested factorial model: insert your favorite parameter Assess what parameter that causes (most) variability operator day Plate* replicate *Plate, buffer, lotnr, Overall CV% CD4 IFNg 30.09 CD4 TNFa 37.76 CD4 IL-2 7.78 CD8 IFNg 34.9 CD8 TNFa 35.26 CD8 IL-2 37.7

Acceptance Criteria for Assay run Run Criteria Internal control: Viability > 85% >00 events in CD8 and CD4 gate SEB stimulation positive (>%) Mock stimulation: <0.6% (%IFNg/CD8) Control peptide response: (%IFNg/CD8) CV<35% %IFNg/CD8 within range of nominal response to control peptide Sample Criteria Viability > 85% >00 events in CD8 and CD4 gate SEB stimulation positive (>%) Mock stimulation<~0.% (cytokine specific) Peptide response that is greater than Mock: CV<35%

Internal controls trending Isolate PBMC from Buffycoat; freeze + aliquots; establish nominal response in at least 6 representative runs; ranges set at 2xSD+/-mean response 3.0000 B9 3.0000 B25 2.00 2.00 %IFNy+/CD8+ 2.0000.00.0000 %IFNy+/CD8+ 2.0000.00.0000 0.00 0.00 0.0000 27-Apr- 7-May- 06-Jun- 26-Jun- 6-Jul- 05-Aug- 0.0000 2-Jan-2 0-Feb-2 2-Feb-2 2-Mar-2 0-Apr-2 2-Apr-2 -May-2

Internal controls training Criteria for trainers as well as operators Job Training Guide Use Internal Controls for direct comparison Re-train if required 3 %IFNg+/CD8+ 2 0 Tech Tech 2 Tech 3

Major Challenges Standardization of assay execution and data analysis (training!) Comprehensive validation approach Inclusion of Assay Controls Data retention (def. raw data, readable format, CFR2part) Harmonization: need for gold standards, proficiency panels

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