Proprietary Procedure

Oxford BioDynamics' (OBD) EpiSwitchTM platform is the first industrial platform for the discovery, evaluation,validation and monitoring of a novel class of epigenetic biomarkers known as ‘chromosome conformationsignatures’ (“CCSs”). CCSs provide a robust, stable framework of regulatory genome architecture from whichchanges in the regulation of a genome can be analyzed, long before the results of these epigenetic changesmanifest themselves as obvious abnormalities.

Molecular biology offers several methods to detect long range chromosomal conformations. The underlyingprinciple allows for the capture of distant genomic juxtapositions by first stabilizing distant sites within intact cellnuclei, then isolating them and converting them into an artificial template with distant fragments of genome ligatedto each other in one DNA product and thereafter detecting the new products as evidence of long rangeinteractions. Early research protocols called chromosome conformation capture (3C) were developed in 2002.These were followed over the years by several other variants aimed at assessing different scopes and levels ofchromosome conformations. These protocols are used widely in academic settings today. Although all are veryuseful in providing insights and evidence for specific interactions, they remain time and labour consuming, oftenwith low resolution and frequent variability in readouts.

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Figure 1: A simplified workflow for detecting chromosomeconformations by microarray or PCR.

OBD, within its extensive research and developmentprogrammes has translated an academically variable assayinto a robust industrial platform to yield stable,reproducible and sensitive biomarkers.

The proprietary protocols used in processing and thedetection of long range interactions used by EpiSwitchTMin the analysis of clinical samples and other cellular inputshave taken screening and monitoring of chromosomeconformations to a different level in terms of industrystandards: speed, cost, sensitivity, resolution, accuracyand robustness.

Final quantitated readouts for individual markers andvalidated stratifying signatures are performed either inthe simple format of binary nested PCR or in the format ofMIQE-compliant qPCR with proprietary hydrolysableprobe design.

OBD has developed and achieved these standards byextensively modifying the protocol and procedures atevery stage of the process, right from identifying CCSmarkers within the genome to finalizing a classifier modelbased on CCS signature for stratifying groups of samples.

EpiSwitch Methodology

A robust and reliable methodology for personalised medicine

Figure 2: Oxford BioDynamics three-step biomarker discovery workflow using the EpiSwitchTM platform technology.

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Robustness built-in

Within the genome considering both cis (between genomic regions on the same chromosome) and transjuxtapositions (between genomic regions on different chromosomes), there are of the order of billions of potentialCCS interactions that can be experimentally measured. The majority of these interactions are riddled withstochastic noise and the experimental verification of any valid markers are overbearing. To screen from such a largepool of potential interactions in a high-throughput fashion to identify a subset of markers of interest for any givenstratification question, OBD has developed a proprietary Relevance Vector machine based algorithm with Bayesianstatistics. The software scans the genomic sequence and highlights interactions with a probability score for any locior gene that warrants experimental verification. This considerably reduces the downstream biochemical challengeand removes the stochastic noise from the interactions pool.

The shortlisted potential CCS interactions are experimentally evaluated and validated by OBD using a three-stageapproach detailed in our white paper (Figure 2). This ensures that the biomarker panels that emerge from thispipeline are biologically vetted and statistically robust to be used in pre-clinical and clinical practice.

Quality Control

The OBD reference laboratory is ISO-certified and regulated by the Human Tissue Authority, UK. All thebiospecimen samples are quarantined and quality checked before any laboratory usage. The company hasdeveloped a proprietary rapid and cost effective standard operating procedure to measure the quality of the inputblood samples for the EpiSwitchTM assay. This step immediately flags up any issue with the quality of patientsamples before any extensive studies.

In addition the EpiSwitchTM methodology uses standardized and normalized input for DNA extraction, PCR and qPCRassays, removing assay variability.

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Automation

OBD has automated every step in the EpiSwitchTM methodology. For the DNA extraction procedure from thebiospecimen samples, OBD uses Tecan Evo 150/200 machines, for high reproducibility and repeatability. Thestandard operating procedure uses proprietary reagents for high and consistent yield of chromatin templates fromthe samples. The processed DNA is quantified and normalized before PCR/qPCR readouts.

Automation has also increased the throughput of sample extraction, keeping the cost to a minimum. OBD has acapacity to process 384 extractions at the same time. Variation in yields between extractions for the same sampleextracted at different times are limited to 20%.

The nested PCR and qPCR set up are automated with Tecan Evo 200 and QIAgility machines. Nested PCR ampliconsare read automatically using Perkin Elmer LabChip Electrophoresis instruments.

Analytical sensitivity of qPCR

The minimum number of CCS copies EpiSwitchTMassays can measure is between 1-10 copies. Forexample, the FOXC1 chromosome conformationcopy number varies in the blood of patients withnon-malignant and advanced stage breast cancer.The atypical calibration curve with tissue matchednegative controls samples is shown in the figure 3.The amplification curves for the standards can beseen in pink (from a million copies, before cycle 20on the x-axis to 1 copy), and the patient samplesamplification curves are seen in blue. OBD’spatented probe technology specifically detects thechromosome conformation and no products aredetected in the non-template controls orconcentration matched negative controls. TheFOXC1 based signature assay significantlydiscriminated advanced stage breast cancer fromnon-malignant samples with the chromosomeconformation copy number.

Figure 3: The amplification curves for standards(pink) and patient samples (blue), for the FOXC1chromosome conformation qPCR assay.

Reproducibility and Repeatability

As part of our ISO standard requirements, the EpiSwitchTM methodology incorporates a number of measures tohave high repeatability and reproducibility. All the proprietary reagents used for DNA extraction are stability tested.Multiple extractions of the same patient sample are pooled and normalized before any downstream PCR ormicroarray read-out analysis. DNA templates are quantified and normalized before any PCR is set up. All PCRamplicons are readout in triplicates to avoid variability.

The EpiSwitchTM detection tested in parallel to a standard positive control of chromosome conformation at MMP1locus has shown >95% repeatability and reproducibility.

OBD has conducted multiple collaborative and commercial studies with clinical research institutions and industrypartners. Completed technology transfers of the EpiSwitchTM technology have demonstrated its robustness andreproducibility.

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Applications and References

OBD has built extensive expertise and knowledge of chromosome conformation signatures with a broad spectrumof successful biomarker developments and validations in a variety of therapeutic areas. This includes oncology(Melanoma, Non-Small Cell Lung Cancer, Breast Cancer, DLBCL, AML, CEL, Prostate Cancer, HCC, glioblastoma),immunology (Rheumatoid Arthritis, Multiple Sclerosis, Systemic Lupus Erythematosus, Ulcerative Colitis andFibrosis), immuno-oncology therapies (PD-1/PD-L1), neurology (Amyotrophic Lateral Sclerosis, Alzheimer’s Disease,Huntington’s Disease and Autism) and metabolic conditions (T2DM and pre-diabetic state).

OBD is a member of FNIH Biomarker Steering Committees (Washington, DC) in Oncology, Immunology andNeurodegeneration. As a valuable complimentary diagnostic biomarker tool for patient stratifications, several ofOBD applications have been integrated into prospective therapeutic trials in US and UK.

CASE STUDY: Predictive Biomarkers for Therapeutic Response

OBD has collaborated with a leading pharmaceutical company and a leading clinical consortium to solve one of themajor unmet clinical needs in rheumatoid disease. Rheumatoid arthritis (RA) is an autoimmune inflammatorydisorder that manifests clinically as painful, swollen, tender and stiff joints. For UK patients diagnosed with RA,methotrexate (MTX) is the first choice of disease modifying anti-rheumatic drug (DMARD) as recommended byEuropean League against Rheumatism (EULAR) and American College of Rheumatology (ACR). While MTX hastherapeutic benefit in some patients, approximately 35–59% of patients do not achieve clinically meaningfulresponses after starting therapy. Over the last several years, a range of newer targeted biologic drugs have beendeveloped and approved for RA, many of which show substantial clinical benefit. Thus, having a mechanism toidentify patients who are likely non-responders to MTX would facilitate earlier access to these more effectivetherapies, avoiding disease progression, unnecessary exposure to potentially toxic drugs and diminished quality oflife.

Using blood samples from the Scottish Early Rheumatoid Arthritis Inception cohort, we evaluated chromosomalinteractions at 123 genomic loci associated with RA. These loci were processed through the EpiSwitchTMannotations for the epigenetic landscape of 13,322 chromosomal interactions and were screened for associationwith response to MTX in RA. Quality controlled blood samples from 90 RA patients and control samples in a seriesof comparisons that used statistical filters reduced marker leads to five signature interactions (in IFNAR1, IL-21R, IL-23, IL-17A, and CXCL13 loci) that could accurately predict response toMTX. The 5-marker panel was validated on anindependent, blinded cohort of RA patients, with positive predictive value of over 90%, showing that the use of aCCS has practical utility as a predictive clinical tool.

CASE STUDY: Immuno-oncology

OBD is involved in multiple proprietary and commercial studies for development of predictive and responsebiomarkers for mono- and combination therapies based on checkpoint inhibitors PD-1 and PD-L1. Today OBD hascompleted 4 studies, identifying both common non-response profiles for patient exclusion, and asset specificresponse profiles that deliver high positive predictive value and discriminate against alternative competing assets.In its first study with Mayo Clinic, OBD has developed response biomarkers for late stage melanoma patients, whoresponded to Pembrolizumab (Keytruda). OBD has also applied its technology to patients with non-small cell lungcancer treated with various anti-PD1 and anti-PD-L1 assets.

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Copyright ©2018, Oxford BioDynamics, Plc. All rights reserved. Oxford BioDynamics® and EpiSwitch™ are registered trademarks of Oxford BioDynamics, Plc.

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Selected EpiSwitchTM Publications

Salter M, Powell R, Back J et al. Genomic architecture differences at the HTT locus underlie symptomatic and pre-symptomatic cases of Huntington’s

disease. [version 1; referees: awaiting peer review]. F1000Research 2018, 7:1757

Initial Identification of a Blood-Based Chromosome Conformation Signature for Aiding in the Diagnosis of Amyotrophic Lateral Sclerosis.

Salter M, Corfield E, Ramadass A, Grand F, Green J, Westra J, Lim CR, Farrimond L, Feneberg E, Scaber J, Thompson A, Ossher L, Turner M, Talbot K, Cudkowicz M, Berry J, Hunter E, Akoulitchev A.

EBioMedicine. 2018 Jul;33:169-184. doi: 10.1016/j.ebiom.2018.06.015. Epub 2018 Jun 23.

Chromosome conformation signatures define predictive markers of inadequate response to methotrexate in early rheumatoid arthritis.

Carini C, Hunter E; Scottish Early Rheumatoid Arthritis Inception cohort Investigators, Ramadass AS, Green J, Akoulitchev A, McInnes IB, Goodyear CS.J Transl Med. 2018 Jan 29;16(1):18. doi: 10.1186/s12967-018-1387-9.

Super-Enhancers and Broad H3K4me3 Domains Form Complex Gene Regulatory Circuits Involving Chromatin Interactions.

Cao F, Fang Y, Tan HK, Goh Y, Choy JYH, Koh BTH, Hao Tan J, Bertin N, Ramadass A, Hunter E, Green J, Salter M, Akoulitchev A, Wang

W, Chng WJ, Tenen DG, Fullwood MJ.Sci Rep. 2017 May 19;7(1):2186. doi: 10.1038/s41598-017-02257-3.

A pilot study of chromosomal aberrations and epigenetic changes in peripheral blood samples to identify patients with melanoma.

Jakub JW, Grotz TE, Jordan P, Hunter E, Pittelkow M, Ramadass A, Akoulitchev A, Markovic S.

Melanoma Res. 2015 Oct;25(5):406-11. doi: 10.1097/CMR.0000000000000182.

Chromatin barcodes as biomarkers for melanoma.Bastonini E, Jeznach M, Field M, Juszczyk K, Corfield E, Dezfouli M, Ahmat N, Smith A, Womersley H, Jordan P, Ramadass A, Akoulitchev A, Goding CR.

Pigment Cell Melanoma Res. 2014 Sep;27(5):788-800. doi: 10.1111/pcmr.12258. Epub 2014 May 27.

Formation of distinct chromatin conformation signatures epigenetically regulate macrophage activation.

Mukhopadhyay S, Ramadass AS, Akoulitchev A, Gordon S.Int Immunopharmacol. 2014 Jan;18(1):7-11. doi: 10.1016/j.intimp.2013.10.024. Epub 2013 Nov 6.

INNOVATION

SAVING LIVES

Version 1.0; Nov 2018; © Oxford BioDynamics Plc

Authors

Aroul Ramadass, PhDMatthew Salter, BScEwan Hunter, PhDWillem Westra, PhDAlexandre Akoulitchev, PhD

Regulated and Licensed by Human Tissue Authority