MDC Connects: Biomarker identification - Assessing Immune Function
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This document discusses biomarkers for assessing immune function throughout the drug development process. It describes how various techniques can be used to identify, validate, and qualify biomarkers. These include flow cytometry to analyze cell populations and activation markers, Luminex to measure cytokine levels, and gene expression profiling using NanoString. Whole blood stimulation assays are discussed as a way to assess target engagement and immune responses ex vivo. The importance of assay validation and understanding sources of variation are also covered. Biomarkers can provide insights into mechanisms of action, safety, and efficacy to support clinical development.
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MDC Connects: Biomarker identification - Assessing Immune Function
- 1. BIOMARKER IDENTIFICATION: ASSESSING IMMUNE FUNCTION EVERY STEP OF THE WAY1 Russell Garland, Group Leader, Analytical Services Charles River Laboratories, Discovery Services, Portishead, UK
- 2. WHAT IS A BIOMARKER? 2 EVERY STEP OF THE WAY Source: IQVIA FDA-NIH Biomarker Working Group. BEST (Biomarkers, EndpointS, and other Tools) 2018. A defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions. Categories include: Susceptibility/risk Diagnostic / Monitoring Prognostic Predictive Pharmacodynamic/response Safety
- 3. Target Discovery and Validation Hit ID Lead-to-Candidate Preclinical Development Hit –to-Lead Clinical Development EVERY STEP OF THE WAY 3 EVERY STEP OF THE WAY Translationally-focused approach Target Expression Mechanism Of Action Pharmacology PK/PD Phase 0, 1, 2… cDNA, shRNA, CRISPR/Cas9, RNAi and genome editing Complex cell-based assays in primary human cells IN VIVO efficacy EX VIVO analyses Clinical Efficacy biomarker analyses
- 4. EVERY STEP OF THE WAY4 IMMUNOLOGY ACROSS THE THERAPEUTICS AREAS Immuno-Oncology, Autoimmune and Inflammatory Diseases, Infectious Diseases, Neuroinflammation Immuno-Oncology Autoimmune diseases Inflammatory conditions Infectious Diseases Neuroinflammation
- 5. CORE TECHNIQUES 5 Depth and breadth of data acquisition LuminexNanostring Flow cytometry: Analysis and Sorting Confocal microscopy qPCR IncuCyte In vivo Imaging, Disease Scoring Data: Quality Quantity Rapidity ELISpot Bacterial culture facilities Aerobic, microaerobic and anaerobicPHERAstar: TR-FRET, ELISA, Luminescence, HTS Isolator
- 7. NANOSTRING TECHNOLOGY 7 EVERY STEP OF THE WAY Summary • Each RNA molecule has two probes: a biotinylated capture probe, and a fluorescently-labelled barcode probe • Barcode probe contains string of 6 fluorochromes that are unique to each probe/RNA molecule • These are bound to a cartridge, aligned and then imaged by a high- resolution single-molecule CCD camera • Direct counting of the fluorescent barcodes enables direct counting of the RNA molecules • Up to 800 genes can be multiplexed. Fully automated system • Advantages include • No reverse-transcription or cDNA library generation, therefore less bias introduced • Reduced hands-on time • Fully automated system • Simple, fast data analysis • Can be informative for biomarker selection, for validation of a pre- selected biomarker, or if wider pathway analyses are required https://www.nanostring.com/scientific-content/technology-overview/ncounter-technology
- 8. N A N O S T R I N G A N A LY S I S O F G E N E E X P R E S S I O N P R O F I L E I N S T I M U L AT E D W H O L E B L O O D I D E N T I F I E S P O T E N T I A L C L I N I C A L B I O M A R K E R S 8 EVERY STEP OF THE WAY NanoString • LPS induces upregulation of many genes (left), inhibition of LPS-driven inflammation with dexamethasone results in downregulation of many of these genes (right). • Gene expression profiling (up to 800 targets) for biomarker selection. • Novel therapeutics screened to demonstrate target engagement and identify potential biomarkers. Upregulated genes Downregulated genes Significanceofchange Upregulated genes Downregulated genes Significanceofchange Healthy donor whole blood taken Stimulate with LPS or LPS + Dex Incubate 37°C, 24 h Take plasma supernatants for Luminex, extract RNA from cells for qPCR and Nanostring
- 9. U n s tim L P S L P S + D e x 0 2 0 4 0 6 0 8 0 IL -1 RelativeQuantity U n s tim L P S L P S + D e x 0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 IL -6 RelativeQuantity U n s tim L P S L P S + D e x 0 1 0 0 2 0 0 3 0 0 IFN RelativeQuantity U n s tim L P S L P S + D e x 0 2 4 6 8 T N F RelativeQuantity PRE- CL INICAL IN VIT RO ANALYSIS O F INF L AMMATORY CYTO KINE PRO DUCT IO N TO DEMO NST RAT E TARG ET ENG AG EMENT 9 EVERY STEP OF THE WAY qPCR and Luminex • Promising biomarkers - a more focused panel of analytes - are taken forward to confirm screening data. • Inflammatory cytokine production can be assessed at both the mRNA and protein levels by qPCR and Luminex, respectively. • The two techniques show good concordance for the cytokines tested. qPCR U n s tim L P S L P S + D e x 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 IL -1 pg/ml U n s tim L P S L P S + D e x 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 IL -6 pg/ml U n s tim L P S L P S + D e x 0 5 0 1 0 0 1 5 0 IFN pg/ml U n s tim L P S L P S + D e x 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 T N F pg/ml Luminex
- 10. Clinical biomarkers PD biomarkers – proof of mechanism - target engagement Cytokine analysis using Luminex (bead-based multiplex assays) Flow cytometry
- 11. BIOMARKER CATEGORIES 11 EVERY STEP OF THE WAY Assay validation guided by intended use of the data Source: IQVIA Type of biomarker Definition Comment Pharmacodynamic (PD) /response biomarker Used to show that a biological response has occurred in an individual who has been exposed to a medical product. Common exploratory endpoint in early phase trials. Proof of mechanism. Target engagement. Sponsor go/no-go decision. Predictive biomarker Used to identify individuals who are more likely (than similar individuals without the biomarker) to experience a favorable or unfavorable effect from exposure to a medical product. Used to select patients for treatment. Safety biomarker Measured before or after an exposure to a medical product to indicate the likelihood, presence, or extent of toxicity as an adverse effect. Safety endpoints. FDA-NIH Biomarker Working Group. BEST (Biomarkers, EndpointS, and other Tools) 2018.
- 12. Clinical Sample Analysis Fit for purpose Validation Phase Bespoke solutions driven by trial logistics WHAT IS THE INTENDED PURPOSE OF THE DATA? 12 EVERY STEP OF THE WAY • It is possible to measure the effects of immune modulating drugs and vaccines on the immune response in clinical trials – BUT only through the rigorous implementation of validated logistics and assay design. • Assays of soluble & cell based markers – most often functional assays for use in clinical trial settings. • Ex vivo activation is often required to elucidate immune modulation. • Pre-analytical factors need to be considered (blood anticoagulant, shipping & storage conditions). • Clinical Lab Manual sections generated accordingly. ‘Fit-for-Purpose’ validation & sample logistics
- 13. THE IMPORTANCE OF ASSAY VALIDATION Flow cytometry: Assay characterization Not all antibody clones give staining which withstands sample fixation (e.g. CD11b, CD80) 13 EVERY STEP OF THE WAY • Staining of some markers may be lost after fixation • During the validation phase, it is important to test antibody staining in samples exposed to conditions mimicking the clinical samples • Where issues are identified, different antibody clones may be employed to improve staining of phenotypic or activation markers
- 14. Cytokine analysis by Luminex Sample storage Plasma collection Stimulation and incubation Shipment to CRL Subject blood sample EX VIVO WHOLE BLOOD STIMULATION ASSAY 14 EVERY STEP OF THE WAY Sample Flow and Consideration Points Anti-coagulant Draw to process time Logistics Stimulation (e.g. LPS, CD3/CD28, SEB) Time-point Stability Recovery Assay panel Precision LLOQ/ULOQ Accuracy “Fit for purpose” validation
- 15. EX VIVO WHOLE BLOOD STIMULATION ASSAY 15 EVERY STEP OF THE WAY Luminex Validation Parameters Assay Panel • Upper and lower limits of quantitation (ULOQ/LLOQ) • Standard curve precision IL-2 IL-4 IL-6 IL-10 IL-13 IL-17A <10% <15% <10% <10% <10% <10% Observed 0.827 - 0.927 0.096 - 0.126 1.371 - 1.379 2.656 - 2.738 0.348 - 0.387 2.315 - 2.367 Expected 0.91 0.13 1.35 2.62 0.38 2.27 Observed 15126 - 15180 2266 - 2454 20449 - 20870 33732 - 34724 6120 - 6259 35420 - 35820 Expected 14842 2183 22147 42919 6178 37181 Standard curve Intra-assay precision LLOQ (pg/ml) ULOQ (pg/ml) Result Parameter Precision (% CV) • Intra-assay (within replicates) • Inter-assay (between plates) IL-2 IL-4 IL-6 IL-10 IL-13 IL-17A <10% <10% <10% <10% <10% <5% <15% <25% <10% <10% <20% <15% QC sample <5% <5% <5% <5% <5% <15% Samples analysis (stimulated Intra-assay precision Inter-assay precision Inter-assay precision Parameter Result Assay QC. Internally generated QC samples run on each plate. From FDA guidelines on Bioanalytical Method Validation: “The precision determined at each concentration level should not exceed 20% of the CV except for the LLOQ, where it should not exceed 25% of the CV”.
- 16. EX VIVO WHOLE BLOOD STIMULATION ASSAY 16 EVERY STEP OF THE WAY Understanding Assay Variation IL -2 IL -6 IL -8 IL -1 0 IF N - T N F - 0 5 0 1 0 0 1 5 0 A n a ly te R e c o v e ry a fte r F re e z in g Recovery(%) 1 week after storing at - 80°C IL -2 IL -6 IL -8 IL -1 0 IF N - T N F - 0 5 0 1 0 0 1 5 0 A n a ly te R e c o v e ry a fte r F re e z in g Recovery(%) 4 weeks after storing at -80°C Understanding: • Response variation between individuals (right) • Inter-operator variation • Intra-assay & Inter-assay precision • Cytokine stability during storage (below) • mimic study logistics - allows batching. • Activation of whole blood with appropriate stimuli & time-point • Cytokine panel selection • Reagent lot variability Stability following storage. Plasma isolated from 5 healthy individuals spiked with recombinant cytokine standard. Recovery assessed following 1 week and 4 weeks storage at -80°C, compared to fresh samples. Inter-donor variability. Responses in healthy individuals after 48 h +/- SEB activation.
- 17. EXPLORATORY BIOMARKERS OF SYSTEMIC IMMUNE MODULATION 17 EVERY STEP OF THE WAY Ex vivo whole blood stimulation assay (Luminex) Lead candidate results in reduced induction of IL-6 from human blood cells after 28 days upon agonist stimulation. Phase 1b clinical trial for inflammatory skin condition. • Blood samples from patients with mild to moderate psoriasis were taken at baseline and after 28 days of therapeutic (n=20) or placebo (n=18). Whole blood was incubated with an established TLR agonist. • Waterfall plots display the difference in cytokine induction from subject baseline across groups. The outcome: • Compared to placebo, a pronounced downward shift across subjects treated with therapeutic was observed in systemic markers of inflammation, notably IL-6. • Supportive evidence of therapeutic potential to treat inflammatory diseases. 5020 3011 5017 5012 5023 5005 5987 4007 3001 4005 3006 3008 5006 4001 4017 4010 5015 4015 −75% −50% −25% 0% 25% 50% 75% 100% 125% PercentagechangefrombaselineinIL6induction 4008 3002 3005 4011 4006 3012 4013 4003 3010 4018 3004 4004 3003 4012 4002 4014 4016 3007 4009 3009 −75% −50% −25% 0% 25% 50% 75% 100% 125% PercentagechangefrombaselineinIL6induction Placebo Treatment
- 18. FLOW CYTOMETRY Functional immune assays from whole blood / PBMCs
- 19. CONFIRMING MOA/ PREDICTING EFFICACY 19 EVERY STEP OF THE WAY Example: Novel peptide therapeutic for Graves disease (Phase 2a study) Sample logistics Clinical sites across EU Local PBMC prep and freeze Set up PBMC cultures with autoAg Flow cytometry panels: Treg panel: CD4, CD25, CD127, Tim3, LAG3, CD49b, FoxP3, Ki-67, IL-10 T effector panel: CD8, CD4, GATA-3, Tbet, CD71, CD40L, Ki-67, IFNγ, IL- 10, IL-17, IL-6 Luminex panel: IFN𝛾, IL-10, IL-6, IL-5 qPCR: IL-10 The problem: • The expectation is a shift in the nature of the T cell response to a single antigen. • Global changes are not expected. The solution: • Ex vivo characterisation of the T cell response to the pathogenic autoantigen tells you what is happening.
- 20. EXAMPLE: FLOW CYTOMETRY 20 EVERY STEP OF THE WAY Markers of rare T cell populations upon antigenic stimulation Gating strategy D0 Flow staining for proliferation /activation markers D6 Ex vivo restimulation with therapeutic peptide or positive control Thaw PBMC Ex vivo restimulation assay Patient (trial samples) Frozen PBMC positive control Triple antigen (Flu/TT/PPD) antigenic trigger Therapeutic peptide incubation 6 days readout Flow cytometry
- 21. SUMMARY
- 22. PUTTING PD/EFFICACY BIOMARKERS AT THE CENTER OF DRUG DISCOVERY 22 EVERY STEP OF THE WAY PD biomarkers demonstrate target engagement (proof of mechanism) & have a positive impact on project success Data generated from: Nature Reviews Drug Discovery 17, 171 (2018) 0 40 80 120 160 Costofdecision$M ≈ $15M ≈ $40M ≈ $150M • Clinical trials are hugely expensive. • High attrition rate at phase II in particular (insufficient efficacy, including poor target engagement). • Including PD biomarkers: increase project success rate or ‘fail quicker – fail cheaper’. Yes N o 0 20 40 60 80 100 Proof of mechanism demonstrated? Outcomeofprojects (%) Project Progressed Project Closed
- 23. Biomarkers of functional immune response Exploratory endpoints Early phase trials WHY INCLUDE PD BIOMARKERS? 23 EVERY STEP OF THE WAY • Inclusion of response biomarkers from early, pre- clinical stages of the drug development process can confirm target engagement. • Informative biomarker assays can be translated into clinical phase assays. • Traditionally, in First-in-Human clinical trials the primary/secondary endpoints are concerned with safety. • Inclusion of exploratory PD biomarkers can add value to early phase trials by providing proof of mechanism and inform go/no-go decisions. • Experimental approaches and solutions need to be designed around particular trial needs/ logistics. Validation phase is key!
- 24. CONTACT US Charles River Laboratories Discovery UK Charles River Laboratories Discovery House Quays Office Park Conference Avenue Portishead, UK BS20 7LZ POR-Client-Services@crl.com www.criver.com +44-1-275-460-903