Method for determining responsiveness of CHB patient to IFN-alpha treatment

Abstract

The invention generally relates to the field of diagnostics and prognostics. In particular, the invention relates to a method for determining the responsiveness of a chronic hepatitis B (CHB) patient to IFN-alpha treatment. In an aspect of the invention, there is provided a method for determining the responsiveness of a chronic hepatitis B (CHB) patient to IFN-alpha treatment, comprising: (a) measuring an expression level of at least one biomarker of a prognostic biomarker panel selected from the group consisting of NQO2, MBL2, IGKV3D-20, PKHD1L1, PGLYRP1, KHSRP, LCN2, and ROBO1 in a biological sample obtained from the patient, and (b) comparing the expression level of the at least one biomarker to corresponding expression level of a known non-responder, wherein an expression level higher than the corresponding expression level is indicative of a likelihood of a positive response to IFN-alpha treatment.

Description

DESCRIPTIONTITLE OF THE INVENTION: METHOD FOR DETERMINING RESPONSIVENESS OF CHB PATIENT TO IFN-ALPHA TREATMENTFIELD OF THE INVENTION

[0001] The invention generally relates to the field of diagnostics and prognostics. In particular, the invention relates to a method for determining the responsiveness of a chronic hepatitis B (CHB) patient to IFN-alpha treatment.BACKGROUND OF THE INVENTION

[0002] Treatment of chronic Hepatitis B virus (CHB) infection remains challenging. Current CHB treatment strategies involve mainly treating CHB patients with either Nucleoside Analogues (NAs) or immunomodulatory drug such as Interferon-alpha (IFN-alpha). Despite availability of these treatments, functional cure from CHB infection is still rare, with only 10% to 20% of treated CHB patients responding to the treatment and achieving functional cure. Given the low treatment success rate and associated adverse side effects, administration of treatments should be prudent and selective. Currently, low blood quanti-HBsAg (HBV S antigens) level is used as a prognostic marker for identifying responders. However, this approach has its limitations, with a substantial number of low qHBsAg CHB patients still not responding to treatment.

[0003] There is thus a need for a method for stratifying IFN-alpha treatment responders and non-responders among CHB patients that overcome the drawbacks of the prior art. Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the accompanying drawings and this background of the disclosure.SUMMARY OF THE INVENTION

[0004] In an aspect of the invention, there is provided a method for determining the responsiveness of a chronic hepatitis B (CHB) patient to IFN-alpha treatment, comprising: (a) measuring an expression level of at least one biomarker of a prognostic biomarker panel selected from the group consisting of NQO2, MBL2, IGKV3D-20, PKHD1 L1, PGLYRP1 , KHSRP, LCN2, and ROBO1 in a biological sample obtained from the patient, and (b)comparing the expression level of the at least one biomarker to corresponding expression level of a known non-responder, wherein an expression level higher than the corresponding expression level is indicative of a likelihood of a positive response to IFN-alpha treatment.

[0005] In various embodiments, the prognostic biomarker panel consists of NQO2 and one or more biomarkers selected from the group consisting of MBL2, IGKV3D-20, PKHD1 L1 , PGLYRP1 , KHSRP, LCN2, and ROBO1.

[0006] In various embodiments, the prognostic biomarker panel consists of MBL2 and one or more biomarkers selected from the group consisting of NQO2, IGKV3D-20, PKHD1 L1, PGLYRP1 , KHSRP, LCN2, and ROBO1.

[0007] In various embodiments, the prognostic biomarker panel consists of NQO2 and MBL2.

[0008] In various embodiments, the prognostic biomarker panel consists of NQO2, MBL2, IGKV3D-20, PKHD1 L1 , PGLYRP1 , KHSRP, LCN2, and ROBO1.

[0009] In various embodiments, an expression level of at least 20% higher than the corresponding expression level is indicative of the likelihood of a positive response to IFN- alpha treatment.

[0010] In various embodiments, the biological sample is selected from the group consisting of a blood sample and a plasma sample.

[0011] In various embodiments, an immunoassay is used to measure the expression level of the at least one biomarker of the prognostic biomarker panel. The immunoassay may be selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), lateral Flow Immunoassay (LFIA), Chemiluminescent Immunoassay (CLIA), Radioimmunoassay (RIA), Fluorescence Immunoassay (FIA), Western Blotting, Multiplex Immunoassay selected from the group consisting of bead-based arrays and microarrays, Electrochemiluminescent Immunoassay (ECLIA), Dot Blot Immunoassay, Surface Plasmon Resonance (SPR)-Based Immunoassay, targeted mass spectrometry assay selected from the group consisting of Multiple Reaction Monitoring (MRM) assay and Parallel Reaction Monitoring (PRM) assay, and Microsphere-Based Immunoassay.

[0012] In various embodiments, the immunoassay comprises at least one antibody or antigen binding fragment thereof that binds specifically to the at least one biomarker.

[0013] In various embodiments, the method further comprises a step for calculating a cumulative expression score for a plurality of the biomarkers, wherein the cumulative score is used to predict the response to IFN-alpha treatment.

[0014] In another aspect of the invention, there is provided the use of IFN-alpha in the manufacture of a medicament for treating CHB, wherein the medicament is to be administeredto a patient who has been determined to be likely to have a positive response to IFN-alpha treatment, and wherein the determination of the likelihood of a positive response to IFN-alpha treatment is made using a method of this invention.

[0015] In another aspect of the invention, there is provided the use of an alternative therapeutic agent which is not IFN-alpha in the manufacture of a medicament for treating CHB, wherein the medicament is to be administered to a patient who has been determined to be not likely to have a positive response to IFN-alpha treatment, and wherein the determination of the likelihood of a positive response to IFN-alpha treatment is made using a method of this invention.

[0016] In another aspect of the invention, there is provided a kit for determining the responsiveness of a chronic hepatitis B (CHB) patient to IFN-alpha treatment using the method of any one of claims 1 to 11, the kit comprising: (a) at least one antibody or antigen binding fragment thereof that binds specifically to the at least one biomarker in the biological sample that allows for quantifying the expression level of the at least one biomarker in the biological sample; and (b) at least one reference standard indicating a corresponding expression level of the at least one biomarker in a known non-responder.

[0017] In various embodiments of the kit, the at least one antibody or antigen binding fragment thereof and / or the at least one reference standard is freeze-dried.

[0018] In another aspect of the invention, there is provided a method of treating a CHB patient with IFN-alpha, comprising: (a) determining the patient’s responsiveness to IFN-alpha treatment using a method of the invention; and (b) administering IFN-alpha to the patient if the patient is determined to be likely to have a positive response to the IFN-alpha treatment.

[0019] In various embodiments, the method further comprises the step of administering a nucleoside analogue or a nucleotide analogue to the patient.

[0020] In another aspect of the invention, there is provided a method of treating a CHB patient, comprising: (a) determining the patient’s responsiveness to IFN-alpha treatment using a method of this invention; and (b) administering an alternative therapeutic agent which is not IFN-alpha to the patient if the patient is determined to be not likely to have a positive response to the IFN-alpha treatment.

[0021] In various embodiments, the alternative therapeutic agent may be selected from the group consisting of: a nucleoside analogue or nucleotide analogue selected from the group consisting of Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide (TAF), Entecavir (ETV), Lamivudine (LAM), Adefovirdipivoxil (ADV), and Telbivudine (LdT); a Capsid Assembly Modulator (CAM) selected from the group consisting of JNJ-56136379, AB-506, and GLS4; a RNA Interference (RNAi) Therapeutics selected from the group consisting of Janssen’s JNJ-3989 and Arrowhead’s ARO-HBV; an Entry Inhibitor such as Bulevirtide; and an HBsAg Inhibitor selected from the group consisting of ALG-010133 and REP 2139.In various embodiments, the patient is one that had previously received IFN-alpha treatment, and that has discontinued administration of IFN-alpha after being determined to be not likely to have a positive response to IFN-alpha treatment.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0023] Fig. 1 shows a workflow of Plasma Proteome Profiling of INACTIVE patient cohort using TMT-based MS quantification approach, followed by computational biology to identify prognostic biomarkers for singling out treatment responders.

[0024] Fig. 2 shows (A) Boxplots of putative prognostic biomarkers for identification of treatment responders among Chronic HBV patients. (B) Receiver operating characteristic (ROC) analysis of qHBsAg and putative prognostic biomarkers identified in the present study.

[0025] Fig. 3 shows (A) ROC of top 7 putative prognostic biomarkers, in comparison with qHBsAg. (B) ROC of NQO2 and MBL2 as panel.

[0026] Fig. 4 shows the validation of NQO2 as a prognostic biomarker in the INACTIVE cohort. (A) NQO2 band intensity determined through Western blot, expressed as a z-score. (B) ROC analysis of NQO2 revealed strong prognostic biomarker performance.

[0027] Fig. 5 shows the validation of MBL2 as a prognostic biomarker in the INACTIVE cohort. (A) MBL2 levels in plasma determined by ELISA using a standard curve approach, expressed in ng / mL. (B) ROC analysis of MBL2 showed strong biomarker performance.DETAILED DESCRIPTION OF THE INVENTION

[0028] Interferon (IFN-a) treatment is one of the main strategies for treating chronic hepatitis B infection (CHB). However, treatment does not always lead to a hepatitis B functional cure. Globally, the rate of hepatitis B functional cure from IFN-a stands at 10%. Hence, most CHB patients undergo unnecessary treatment, enduring avoidable side effects and financial burden. Currently, in the prior art, low circulating viral antigen (HBsAg) levels serve as prognostic biomarkers for IFN-a treatment responders. However, this has been proven to be inadequate, as a number of CHB patients with low qHBsAg still do not respondto IFN-a treatment. Hence, to improve stratification and clinical management of CHB patients, treatment prognostic biomarkers with better predictive power and accuracy are needed to identify potential IFN-a treatment responders. To identify IFN-a treatment prognostic biomarkers, the present inventors performed mass spectrometry-based plasma proteome profiling on the INACTIVE patient cohort. The INACTIVE cohort consists of CHB patients responding to IFN-a treatment (Responder group), CHB patients not responding to IFN-a treatment (Non-responder group), and CHB patients who did not undergo IFN-a treatment (Control group). From this screening, the present inventors identified eight putative treatment prognostic biomarkers (AUC > 0.78) that perform better than qHBsAg (AUC = 0.5), and when combined, this biomarker panel achieved an AUC > 0.8. Further longitudinal analysis of the INACTIVE cohort revealed that the prognostic biomarker NQO2 exhibited an expression profile consistent with a protein mediating treatment response. Through pathway analysis, the present inventors hypothesized that NQO2’s role in suppressing elevated oxidative stress during IFN-a treatment is critical for CHB patient response to IFN-a. To further study these biomarkers, the present inventors aimed to validate these biomarkers in a larger clinical cohort.

[0029] While nucleoside / nucleotide analogues (NAs) are becoming increasingly popular for CHB (Chronic Hepatitis B) treatment and direct-acting antivirals (DAAs) are slowly replacing IFN in hepatitis C virus (HCV) treatment, it is important to note that NA treatment does not always lead to a functional cure. Hence, combination treatments, involving IFN and NA, for CHB are still in practice, underscoring IFN’s continued relevance.

[0030] Furthermore, IFN was reported as a potential immune modulator for treatment of other diseases, including multiple sclerosis and cancers such as lymphoma, melanoma, and Kaposi sarcoma 1-5. Hence, identifying prognostic biomarkers for IFN treatment remains clinically and scientifically important.

[0031] Existing biomarkers for IFN response among CHB patients, such as low HBsAg levels, are insufficient predictors of treatment success. The present disclosure’s predictive biomarker panel aims to: reduce side effects and improve patients’ quality of life (QoL) and lower the economic burden associated with ineffective treatments.

[0032] The key technical features of the present disclosure include the following proteins as treatment prognostic biomarkers for Chronic Hepatitis B infection, which can be utilised individually or in combination as biomarkers panel.:1. NQO2 (Uniprot: P16083), AUC: 0.8912. MBL2 (Uniprot: P11226), AUC: 0.7823. IGKV3D-20 (Uniprot: A0A0C4DH25), AUC: 0.76364. PKHD1 L1 (Uniprot: Q86W11), AUC: 0.8365. PGLYRP1 (Uniprot: 075594), AUC: 0.9276. KHSRP (Uniprot: M0R0C6), AUC: 0.8737. LCN2 (Uniprot: X6R8F3), AUC: 0.8188. ROBO1 (Uniprot: Q9Y6N7), AUC: 0.7829. Biomarker Panel A: (NQO2 and MBL2), AUC: 0.84610. Biomarker Panel B: (NQO2, MBL2, IGKV3D-20, PKHD1 L1 , PGLYRP1 , KHSRP, LCN2 and ROBO1), AUC: 0.851

[0033] These treatment prognostic biomarkers, when used individually or as in a panel, offer superior performance over existing qHBsAg (AUC=0.582) biomarker in identifying potential IFN-a treatment responder among CHB patients.

[0034] As used herein, the term “IFN-alpha” is used interchangeably with “interferon-alpha” and “I FN- a”.

[0035] As used herein, the term “responsiveness” in the context of a method for determining the responsiveness of a CHB patient to IFN-alpha treatment is meant to include the likelihood or potential for a positive therapeutic outcome in a CHB patient following administration of the IFN-alpha treatment. The positive therapeutic outcome includes a clinically significant improvement in the patient’s disease state, which may be measured by markers such as virological, biochemical or histological markers.

[0036] In various embodiments, the method for determining the responsiveness of a CHB patient to IFN-alpha treatment as described herein involves obtaining a biological sample from the patient. The measurement of an expression level of the at least one biomarker is carried out on the biological sample, and not on the human or animal body.

[0037] As used herein, the term “at least one biomarker” in the phrase “measuring an expression level of at least one biomarker of a prognostic biomarker panel selected from the group consisting of NQO2, MBL2, IGKV3D-20, PKHD1L1, PGLYRP1 , KHSRP, LCN2, and ROBOT’ is meant to include one biomarker (e.g. only NQO2, only MBL2 etc.) or more than one biomarker (e g. a combination of two or more biomarkers out of all possible combinations in the list).

[0038] As used herein, the term “expression level” refers to the quantity or amount of a gene product transcribed or translated from a specific gene sequence. This quantity may be measured in terms of its concentration, mass, activity, or abundance within a biological sample.

[0039] As used herein, the term “corresponding expression level of a known nonresponder” is meant to refer to the expression level of the relevant biomarker(s) in a non-responder. A non-responder refers to a CHB patient that did not respond to IFN-alpha treatment. In various embodiments, a non-responder may have a higher level of viral antigen expression, such as a HBsAg level of more than III of 0.05.

[0040] As used herein, the phrase “likelihood of a positive response to IFN-alpha treatment” refers to an assessment that the patient’s prognosis is significantly higher than the average positive response rate observed in an unselected population of CHB patients. As the average positive response rate observed in an unselected population of CHB patients is 10% to 20%, the phrase “likelihood of a positive response to IFN-alpha treatment” refers to a likelihood of a positive response rate of greater than 20%. In other words, it refers to an assessment that the patient has a greater than 20% chance of achieving a positive response to IFN-alpha treatment. In one embodiment, the prognostic biomarker identifies CHB patients with high innate tendency to respond to IFN-alpha treatment, instead of altering the likelihood of outcome.Non-Responder expression Responder expressionUniprot_GN (meanistdev) (meanistdev)IFN-alpha treatment, estimated to be 80% of CHB patient treated with IFN-alpha as shown in Yalcin et. al, Comparison of 12-Month Courses of lnterferon-a-2b-Lamivudine Combination Therapy and lnterferon-a-2b Monotherapy among Patients with Untreated Chronic Hepatitis B, Clinical Infectious Diseases, Volume 36, Issue 12, 15 June 2003, Pages 1516-

[0043] As used herein, the term “treat” or “treating” in the context of treating a disease such as CHB is meant to include improving clinical condition of patients having the disease. This includes reducing the severity and preventing or slowing the progression of the disease.

[0044] As used herein, the term “positive therapeutic response” is intended to include an improvement in the disease or condition, and / or an improvement in the symptoms associated with the disease or condition, and / or prevent the worsening of symptoms associated with the disease or condition. Positive therapeutic responses in any given disease or condition can be determined by standardized response criteria specific to that disease or condition. In addition to these positive therapeutic responses, the subject undergoing therapy may experience the beneficial effect of an improvement in the symptoms associated with the disease.

[0045] Combination therapy with an additional therapeutic agent may also be contemplated by the disclosure. In one embodiment, the method of treating a CHB patient as described herein may include administering IFN-alpha and a nucleotide / nucleoside analogue. The term “combination” or “combination therapy” as used throughout the specification, is meant to encompass the administration of the referred therapeutic agents to a subject suffering from a disease, disorder or pathological condition, in the same or separate pharmaceutical formulations, and at the same time or at different times. If the therapeutic agents are administered at different times they should be administered sufficiently close in time to provide for the potentiating or synergistic response to occur. In such instances, it is contemplated that one would typically administer both therapeutic agents within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1 , 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. In other situations, it might be desirable to reduce the time between administration, administering both therapeutic agents within seconds or minutes to hours, preferably within about 6 hours from each other, more preferably within about 1 or 3 hours.

[0046] Advantageously, the methods of the present disclosure, by measuring the expression level of the at least one biomarker of the prognostic biomarker panel, allow for improved stratification and clinical management of CHB patients.

[0047] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in theuse of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0048] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0049] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0050] MATERIALS AND METHODS

[0051] High abundance proteins depletion and protein extraction

[0052] To deplete High abundance proteins in the plasma, 100 pL of plasma was applied to High-Select Top14 abundant protein depletion midi spin column containing 1000 pL of depletion resin reconstituted with 10 mM PBS, 0.02% sodium azide, at 50% slurry (ThermoFischer Scientific). The column was incubated at room temperature and mix gently end-over-end on rotator set at 40 rpm for 10 mins. After incubation, the depleted plasma was collected into a new 15 mL falcon tube by centrifuge at 1000g for 2 mins. Depleted plasma was then concentrated using Amicon Ultra 4 with 3k MWCO (Millipore). After which, around 100 pL of concentrated depleted plasma was collected. To linearised the plasma proteins, 9 M urea buffer was added to the concentrate to final concentration of 6 M urea. Protein concentration was then determined using 660nm protein assay (Pierce).

[0053] In solution tryptic digestion of depleted plasma proteins

[0054] Tryptic peptides were prepared from 200 pg of depleted plasma proteins. Briefly, proteins in 6M urea were first reduced with 5 mM DTT for 30 mins, followed by alkylation with 10 mM lAA for 30 mins. After reduction and alkylation, proteins were digested with lysC (1 :100 ratio) overnight in 37 oC. Next, the lysate was dilute down to 1 M urea with 50 mM TEABbefore digesting with Trypsin (Promega) at 1 :100 ration for 8 hours in 37°C. After digestion trypsin was inactivated by adding 20% (v / v) TFA to final concentration of 0.5%. Peptides cleanup was subsequently carried out using 1 mL C18 desalting column (Oasis). C18 column was preconditioned with 500 pL of Methanol, 250 pL of 80% (v / v) Acetonitrile in 0.1% TFA and 200 pL of 0.1% TFA. Peptides were added and bind to the C18 column before washing with 0.1% TFA and eluted with 80% AON in 0.1% TFA. The eluted peptides were then lyophilised using a speed vacuum.

[0055] Tandem-Mass Tagging of tryptic peptides and Basic reverse phase fractionation

[0056] Lyophilised peptides were reconstituted with 50 mM TEAB and quantified using Nandrop spectrometer before labelling with 10-plex TMT reagents. 100 pg of peptides was labelled separately with 0.8mg TMT reagents dissolved in 100% anhydrous ACN for 1 hour before quenching with 5% HA for 15 mins in the dark. After quenching, the labelled peptides were pooled with 1 :1:1 :1:1 :1 :1:1 :1 :1 ratio. The labelled peptide mixture was then lyophilised using a speed vacuum. The labelled peptide mixture was then reconstituted in 20 mM Ammonium Formate pH 10 and quantitated using nandrop spectrometer. 1 mg of labelled peptides were subsequently basic reverse phase fractionated using HPLC (Simazdu), with 80% ACN in 20 mM Ammonia formate, pH 10 as buffer B and 20 mM Ammonia Formate as buffer A. Fractionation was carried out over 80 mins with ACN gradient starting from 5 to 40 %. In total, 50 fractions were collected and orthogonally concatenate to 16 fractions before lyophilising using speed vacuum. Lyophilised peptides were stored at -80°C until ready to be analysed by Mass Spectrometry.

[0057] Mass Spectrometry acquisition

[0058] Before MS analysis, the lyophilised peptides were reconstituted in 0.1% FA and quantitated using nanodrop spectrometer. Peptides were adjusted to 0.5 pg / pLwith 0.1 % FA, and 2 pg were injected into EASY-nanoLC 1200 UPLC (n1200) for online MS analysis. Injected peptides were fractionated and eluted from C18 EASY-Spray 50 cm column (Thermo Fisher Scientific) with 205 mins gradient configured at 200 nL / min flow rate. For the nLC buffer, mobile phase A was 0.1% formic acid in 2% acetonitrile, and mobile phase B was 0.1 % formic acid in 80% Acetonitrile. The nLC gradient was configured to ramp from 5% to 50% B over 180 mins, followed by 50% to 100% B for 10 mins and lastly hold at 100% B for 15 mins. Eluted peptides were analysed using high resolution Orbitrap Explorise 480 Mass spectrometer (Thermo Fischer Scientific). MS acquisition was carried out in DDA mode with the following parameters. For full MS scan, the resolution was set at MOK, with ACG target of 3e6, maximum injection time of 60 ms and scan range of 380 to 1600 m / z. For MS2, the resolution was kept at 35k with AGC target of 1e5 and maximum injection time of 105 ms. Loop count was set at 20 foranalysis of top 20 precursors. Isolation window was set at 1.2 m / z and normalised collision energy was set at 30. TurboTMT mode was activated for analysis of TMT labelled peptides.

[0059] MS data processing

[0060] Raw data generated from MS was processed using Proteome Discoverer Software 3.0 (Thermo Fischer Scientific). Peptide identification was performed using the sequest HT and Perlocator node with. Database used for the search are Human reference proteome database obtained from Uniprot (June 2017 release, with 9277 entries including isoforms). Standard static modifications included for cysteine carbamidomethylation (+57,021 Da) and TMT16plex (+229.163 Da) modification on lysin and N-terminal. As for dynamic modifications, methionine oxidation (+15.995 Da) and N-terminal acetylation (+42.011 Da) were included. The precusor and fragment mass tolerance was set at 10 ppm and 0.02 Da respectively. For reporter ion quantification, unique and razor peptides were taken into consideration. The coisolation threshold was set at 50% and signal to noise ratio cut-off of 1.5 was used.

[0061] Bioinformatic analysis

[0062] Protein abundance derived from TMT intensity was median normalised across different TMT set, using abundance of pooled samples at channel 126 as reference. Differentially Expressed Proteins (DEP) were identified using FLEXSTAT advance statistic analytic software. Gene Ontology, pathway analysis and network analysis were carried out using Stringdb, DAVID, GSEA and ClueGO plugin in Cytoscape 3.0. Data visualisation was performed using Perseus and GraphPad PRISM software.

[0063] Biomarker validation

[0064] Biomarker validation was performed on existing INACTIVE cohort samples (nonresponder n=6, responder n=8). NQO2 (Uniprot: P16083) and MBL2 (Uniprot: P11226) biomarkers were prioritised for validation based on their predictive power and potential functional role in mediating IFN-a treatment response. For MBL2, biomarker validation was performed using ELISA. For Elisa, plasma was diluted by 400x before 20 uL of the diluted plasma was assayed with MBL2 specific Elisa kit. MBL2 quantification was determined using standard curve approach. For NQO2, biomarker validation was carried out using western blot assay with NQO2 specific antibody. For this assay, the plasma was diluted 16x before 4ul was used for analysis. NQO2 plasma level was quantitate through Imaged and standard curve approach.

[0065] Composite z-score calculation for multiple biomarkersZ-score is a normalisation calculation based on measure value, eg “peak intensity” or “protein / expression level”. It indicates how far the value deviate from the mean value. Fordiagnosis with multiple biomarkers, composite z-score will be calculated based on “peak intensity” obtained from MS analysis or plasma “protein level” obtained from ELISA assay. For each biomarker XBM, the usual z-score is calculated using:For multiple Biomarkers, unweighted composite z-score is calculated usingwhere p is the number of biomarkers.

[0066] EXAMPLES

[0067] Non-limiting examples of the invention and comparative examples will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

[0068] Example 1 : Discovery of IFN-alpha prognostic biomarkers using deep plasma proteome profiling

[0069] The eight prognostic biomarkers were identified through in-depth comparison of plasma proteome profile between IFN-a treatment responders against non-responders at baseline or pretreatment (Fig. 1). Briefly, plasma proteome profiling for biomarker discovery was first performed on the INACTIVE cohort using TMT-based Mass Spectrometry approach.

[0070] The selection criteria of the putative prognostic biomarker for responder are as followed: (1) The protein is upregulated in responder group at baseline or pretreatment, (2) the difference in expression when compared to non-responder should be significant with p- value<0.05. (3) AUC (Area Under Curve) of ROC is more than 0.75 in the discovery cohort. Taking these selection criteria into consideration, eight putative prognostic biomarkers for responder were identified. They are NQO2 (Uniprot: P16083), MBL2 (Uniprot: P11226), IGKV3D-20 (Uniprot: A0A0C4DH25), PKHD1 L1 (Uniprot: Q86WI1), PGLYRP1 (Uniprot: 075594), KHSRP (Uniprot: M0R0C6), LCN2 (Uniprot: X6R8F3) and ROBO1 (Uniprot: Q9Y6N7) (Fig. 2A). These prognostic biomarkers perform better than qHBsAg level in term of stratification of treatment responders, with AUC (Area Under Curve) ranging from 0.78 to 0.927 (Fig. 2B).

[0071] Example 2: MBL2 identified as high predictive functional prognostic biomarker for IFN-alpha treatment

[0072] In addition, GSEA analysis was also performed. Through this ranked based analysis, several relevant pathways and GO terms, such as creation of C4 and C2 activators (R-H SA- 166786), initial triggering of complement system (R-H SA- 166663), innate immune response activating cell surface receptor signaling pathway (G0:0002429) and acute phase response (G0:0006953), were found to be associated with IFN-a treatment responder (Fig. 3). Consistently, and MBL2 (Uniprot: P11226) (a member of innate immune system) was found to be involved in these pathways. Further analysis revealed that MBL2 level of treatment responders remains high, throughout the treatment course, when compared to both control and non-responder group (Fig. 2A and 2B). As prognostic biomarkers, the performance of MBL2 is decent with AUG 0.782, outperforming qHBsAg (Fig. 2B).

[0073] Example 3: Biomarker panel outperform HBsAq as prognostic biomarker

[0074] Evidently, when considered as a biomarker panel, the top 8 prognostic biomarkers outperformed qHBsAg with combined AUC of 0.851 , comparing with 0.582 of qHBsAg (Fig. 3A). Based on the known functions of these prognostic biomarkers, NQO2 and MBL2 were deemed to have characteristic and functions relevant to CHB infection. Thus, the present inventors also proposed the inclusion of NQO2 and MBL2 as a panel of functional treatment prognostic biomarkers, with decent AUC of 0.846 observed (Figure 3B). In conclusion, the present study provides several putative prognostic biomarkers that can be used to better stratify Chronic HBV patients to those who will or will not respond to IFN-a treatment.

[0075] Example 4: Validation of biomarkers using ortholoqous approach

[0076] To validate NQO2 and MBL2 as prognostic biomarkers of IFN-a treatment, the plasma levels of NQO2 and MBL2 were tested in the INACTIVE cohort using Western blot and ELISA, respectively, on existing INACTIVE cohort samples (non-responder n=6, responder n=8). Through Western blot quantification, a significant elevation of NQO2 in the plasma of responders was observed, with a fold change of 1.2 (p=0.0169 and AUC=0.8704) (Figure 4a and b). Through ELISA, MBL2 levels in responders were elevated, with a fold change of 1.9 (p=0.002 and AUC=0.9583) (Figure 5a and b). These observations are consistent with the present study’s MS-based plasma proteome profiling of the INACTIVE cohort, validating NQO2 and MBL2 as prognostic biomarkers for IFN-a treatment.

[0077] While embodiments of the invention have been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

Claims

CLAIMS1 . A method for determining the responsiveness of a chronic hepatitis B (CHB) patient to IFN-alpha treatment, comprising:(a) measuring an expression level of at least one biomarker of a prognostic biomarker panel selected from the group consisting of NQO2, MBL2, IGKV3D-20, PKHD1L1, PGLYRP1, KHSRP, LCN2, and ROBO1 in a biological sample obtained from the patient, and(b) comparing the expression level of the at least one biomarker to a corresponding expression level of a known non-responder, wherein an expression level higher than the corresponding expression level is indicative of a likelihood of a positive response to IFN-alpha treatment.

2. The method of claim 1 , wherein the prognostic biomarker panel consists of NQO2 and one or more biomarkers selected from the group consisting of MBL2, IGKV3D-20, PKHD1L1, PGLYRP1 , KHSRP, LCN2, and ROBO1.

3. The method of claim 1, wherein the prognostic biomarker panel consists of MBL2 and one or more biomarkers selected from the group consisting of NQO2, IGKV3D-20, PKHD1 L1 , PGLYRP1 , KHSRP, LCN2, and ROBO1.

4. The method of claim 1 , wherein the prognostic biomarker panel consists of NQO2 and MBL2.

5. The method of claim 1 , wherein the prognostic biomarker panel consists of NQO2, MBL2, IGKV3D-20, PKHD1 L1 , PGLYRP1 , KHSRP, LCN2, and ROBO1.

6. The method of claim 1 , wherein an expression level of the at least one biomarker of at least 20% higher than the corresponding expression level in the known non-responder is indicative of the likelihood of a positive response to IFN-alpha treatment.

7. The method of claim 1, wherein the biological sample is selected from the group consisting of a blood sample and a plasma sample.

8. The method of claim 1 , wherein an immunoassay is used to measure the expression level of the at least one biomarker of the prognostic biomarker panel.

9. The method of claim 8, wherein the immunoassay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), lateral Flow Immunoassay (LFIA), Chemiluminescent Immunoassay (CLIA), Radioimmunoassay (RIA), Fluorescence Immunoassay (FIA), Western Blotting, Multiplex Immunoassay selected from the group consisting of bead-based arrays and microarrays, Electrochemiluminescent Immunoassay (ECLIA), Dot Blot Immunoassay, Surface Plasmon Resonance (SPR)- Based Immunoassay, targeted mass spectrometry assay selected from the group consisting of Multiple Reaction Monitoring (MRM) assay and Parallel Reaction Monitoring (PRM) assay, and Microsphere-Based Immunoassay.

10. The method of claim 9, wherein the immunoassay comprises at least one antibody or antigen binding fragment thereof that binds specifically to the at least one biomarker.

11. The method of claim 1, further comprising a step for calculating a composite z-score for a plurality of the biomarkers, wherein the cumulative z-score is used to predict the response to IFN-alpha treatment.

12. Use of IFN-alpha in the manufacture of a medicament for treating CHB, wherein the medicament is to be administered to a patient who has been determined to be likely to have a positive response to IFN-alpha treatment, and wherein the determination of the likelihood of a positive response to IFN-alpha treatment is made using the method of any one of claims 1 to 11 .

13. Use of an alternative therapeutic agent which is not IFN-alpha in the manufacture of a medicament for treating CHB, wherein the medicament is to be administered to a patient who has been determined to be not likely to have a positive response to IFN- alpha treatment, and wherein the determination of the likelihood of a positive response to IFN-alpha treatment is made using the method of any one of claims 1 to 11.

14. A kit for determining the responsiveness of a chronic hepatitis B (CHB) patient to IFN- alpha treatment using the method of any one of claims 1 to 11, the kit comprising:(a) at least one antibody or antigen binding fragment thereof that binds specifically to the at least one biomarker in the biological sample that allows for quantifying the expression level of the at least one biomarker in the biological sample; and(b) at least one reference standard indicating a corresponding expression level of the at least one biomarker in a known non-responder.

15. The kit of claim 14, wherein the at least one antibody or antigen binding fragment thereof and / or the at least one reference standard is freeze-dried.

16. A method of treating a CHB patient with IFN-alpha, comprising:(a) determining the patient’s responsiveness to IFN-alpha treatment using the method of any one of claims 1 to 11 ; and(b) administering IFN-alpha to the patient if the patient is determined to be likely to have a positive response to the IFN-alpha treatment.

17. The method of claim 16, further comprising the step of administering a nucleoside analogue or a nucleotide analogue to the patient.

18. A method of treating a CHB patient, comprising:(a) determining the patient’s responsiveness to IFN-alpha treatment using the method of any one of claims 1 to 11 ; and(b) administering an alternative therapeutic agent which is not IFN-alpha to the patient if the patient is determined to be not likely to have a positive response to the IFN-alpha treatment. The use of claim 13 or the method of claim 18, wherein the alternative therapeutic agent is selected from the group consisting of: a nucleoside analogue or nucleotide analogue selected from the group consisting of Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide (TAF), Entecavir (ETV), Lamivudine (LAM), Adefovir dipivoxil (ADV), and Telbivudine (LdT); a Capsid Assembly Modulator (CAM) selected from the group consisting of JNJ-56136379, AB-506, and GLS4; a RNA Interference (RNAi) Therapeutics selected from the group consisting of Janssen’s JNJ-3989 and Arrowhead’s ARO-HBV; an Entry Inhibitor such as Bulevirtide; and an HBsAg Inhibitor selected from the group consisting of ALG-010133 and REP 2139.

19. The use of claim 13 or the method of claim 18, wherein the patient is one that had previously received IFN-alpha treatment, and that has discontinued administration of IFN-alpha after being determined to be not likely to have a positive response to IFN- alpha treatment.

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