TREATMENT STRATIFICATION FOR AN EXACERBATION OF INFLAMMATION

Abstract

Provided are methods of analysing markers of eosinophil levels and/or markers of neutrophil levels in a blood sample from a patient suffering from an exacerbation of inflammation of a respiratory condition to determine the levels of eosinophils and/or neutrophils respectively. The methods may involve selecting an appropriate treatment. Systems and kits for performing the analysis are also provided.

Claims

1. A method for selecting a treatment to be administered to a patient suffering from an exacerbation of inflammation of a respiratory condition, the method comprising determining the levels of at least 3 markers of eosinophil levels and at least 3 markers of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition wherein: (i) perturbed levels of the at least 3 markers of eosinophil levels and no perturbation in the levels of the at least 3 markers of neutrophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation; (ii) perturbed levels of the at least 3 markers of neutrophil levels and no perturbation in the levels of the at least 3 markers of eosinophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation; or (iii) perturbed levels of the at least 3 markers of eosinophil levels and the at least 3 markers of neutrophil levels result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation; wherein determining the levels of the at least 3 markers of eosinophil levels comprises determining the levels of at least Eosinophil-derived neurotoxin (EDN), Myeloperoxidase (MPO) and Eosinophil cationic protein (RNASE3); and wherein determining the levels of the at least 3 markers of neutrophil levels comprises determining the levels of at least (i) Matrix metallopeptidase 9 (MMP9) and Eosinophil-derived neurotoxin (EDN); and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4.

2. The method according to claim 1 wherein determining the levels of the at least 3 markers of eosinophil levels further comprises determining the levels of at least 1 or 2 further markers selected from Human neutrophil elastase (HNE), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Calprotectin.

3. The method according to claim 1 wherein determining the levels of the at least 3 markers of eosinophil levels further comprises determining the levels of Soluble urokinase-type plasminogen activator receptor (SuPAR), and HNE.

4. The method according to claim 1 wherein determining the levels of the at least 3 markers of neutrophil levels comprises determining the levels of at least MMP9, EDN and LTB4.

5. The method according to claim 1 wherein determining the levels of the at least 3 markers of neutrophil levels further comprises determining the levels of MBP.

6. The method according to claim 1 wherein determining the levels of the at least 3 markers of neutrophil levels further comprises determining the levels of at least 1 or 2 further (different) marker(s) selected from CRP, SuPAR, A1AT and/or LTB4.

7. The method according to claim 1 wherein the levels of at least 5 markers of eosinophil levels and at least 5 markers of neutrophil levels are determined in the blood sample, wherein the markers of eosinophil levels are preferably EDN, RNASE3, SuPAR, HNE and MPO; and wherein the markers of neutrophil levels are preferably (i) MMP9 and EDN; and (ii) at least 3 markers selected from LTB4, A1AT, SuPAR and/or CRP; for example wherein the markers of neutrophil levels are MMP9, EDN, SuPAR, LTB4 and A1AT; or MMP9, C-reactive protein (CRP), EDN, A1AT and LTB4; or MMP9, EDN, CRP, SuPAR and A1AT.

8. A method for selecting corticosteroids to be administered as a treatment to a patient suffering from an exacerbation of inflammation of a respiratory condition, the method comprising determining the levels of at least 3 markers of eosinophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition, wherein perturbed levels of the at least 3 markers of eosinophil levels results in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation of a respiratory condition, wherein determining the levels of the at least 3 markers of eosinophil levels comprises determining the levels of EDN, MPO and RNASE3; optionally wherein determining the levels of the at least 3 markers of eosinophil levels further comprises determining the levels of HNE, SuPAR, and/or Calprotectin, preferably HNE and SuPAR.

9. A method for selecting antibiotics to be administered as a treatment to a patient suffering from an exacerbation of inflammation of a respiratory condition, the method comprising determining the levels of at least 3 markers of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition wherein perturbed levels of at least 3 markers of neutrophil levels results in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation, wherein determining the levels of the at least 3 markers of neutrophil levels comprises determining the levels of at least (i) Matrix metallopeptidase 9 (MMP9) and Eosinophil-derived neurotoxin (EDN); and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4, optionally at least 2 or 3 of these markers.

10. A method for selecting and monitoring treatment of a patient suffering from an exacerbation of inflammation of a respiratory condition, the method comprising: (i) selecting a treatment to be administered to the patient using a method as defined in claim 1; and (ii) with respect to the at least 3 markers for which levels were perturbed when determining the treatment to be administered of step (i), determining the levels of said at least 3 markers in a further blood sample taken from the patient at a later time point wherein: (a) perturbed levels of the at least 3 markers in the further sample indicate that the treatment should continue or be altered; or (b) a return to non-perturbed levels of the at least 3 markers in the further sample indicate or predict successful treatment of the exacerbation of inflammation; wherein optionally the method comprises a step of administering the selected treatment to the patient.

11. A system or test kit for selecting a treatment to be administered to a patient suffering from an exacerbation of inflammation of a respiratory condition, comprising: a. one or more testing devices for determining the levels of at least 3 markers of eosinophil levels and at least 3 markers of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition; b. a processor; and c. a storage medium comprising a computer application that, when executed by the processor, is configured to: i. Access and/or calculate the determined levels of the at least 3 markers of eosinophil levels and the at least 3 markers of neutrophil levels in a blood sample on the one or more testing devices; ii. Calculate whether there is a perturbed level of the at least 3 markers of eosinophil levels and the at least 3 markers of neutrophil levels in the blood sample; and iii. Output from the processor the treatment to be administered to the patient suffering from an exacerbation of inflammation, wherein: perturbed levels of the at least 3 markers of eosinophil levels and no perturbation in the levels of the at least 3 markers of neutrophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation; or perturbed levels of the at least 3 markers of neutrophil levels and no perturbation in the levels of the at least 3 markers of eosinophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation; or perturbed levels of the at least 3 markers of eosinophil levels and the at least 3 markers of neutrophil levels result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation; wherein the at least 3 markers of eosinophil levels comprise at least EDN, MPO and RNASE3; and wherein the at least 3 markers of neutrophil levels comprise at least (i) MMP9 and EDN; and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4.

12. A system or test kit for selecting corticosteroids to be administered as a treatment to a patient suffering from an exacerbation of inflammation of a respiratory condition, comprising: a. one or more testing devices for determining the levels of at least 3 markers of eosinophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition; b. a processor; and c. a storage medium comprising a computer application that, when executed by the processor, is configured to: i. Access and/or calculate the determined levels of the at least one marker of eosinophil levels in a blood sample on the one or more testing devices; ii. Calculate whether there is a perturbed level of the at least one marker of eosinophil levels in the blood sample; and iii. Output from the processor that corticosteroids are selected to be administered as the treatment for the exacerbation of inflammation if there is a perturbed level of the at least one marker of eosinophil levels in the blood sample, wherein the at least 3 markers of eosinophil levels comprise at least Eosinophil-derived neurotoxin (EDN), Myeloperoxidase (MPO) and Eosinophil cationic protein (RNASE3), and optionally the levels of HNE, SuPAR and/or Calprotecin are also determined.

13. A system or test kit for selecting antibiotics to be administered as a treatment to a patient suffering from an exacerbation of inflammation, comprising: a. one or more testing devices for determining the levels of at least one marker of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation; b. a processor; and c. a storage medium comprising a computer application that, when executed by the processor, is configured to: i. Access and/or calculate the determined levels of the at least one marker of neutrophil levels in a blood sample on the one or more testing devices; ii. Calculate whether there is a perturbed level of the at least one marker of neutrophil levels in the blood sample; and iii. Output from the processor that antibiotics are selected to be administered as the treatment for the exacerbation of inflammation if there is a perturbed level of the at least one marker of neutrophil levels in the blood sample; wherein the at least 3 markers of neutrophil levels comprise at least (i) MMP9 and EDN; and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4.

14. The system or test kit according to claim 11 further comprising a display for the output from the processor and/or wherein the one or more testing devices are disposable single use devices and/or wherein the one or more testing devices comprise lateral flow test strips, optionally comprising a lateral flow test strip for each marker that is determined.

15. A method for selecting initial treatment of a patient suffering from an exacerbation of inflammation of a respiratory condition as defined in claim 1, said method comprising (i) using antibodies to detect one or more or all of the markers; (ii) using a lateral flow assay to detect one or more or all of the markers; and/or (iii) using a system or test kit to detect one or more or all of the markers.

16. The method according to claim 1 wherein: (i) the treatment will be the first treatment to be administered to the patient suffering from an exacerbation of inflammation; and/or (iii) the subject is suffering from a respiratory disorder, optionally wherein the respiratory disorder is chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) or asthma, preferably COPD.

17. A computer application as defined in claim 11.

18. A method for selecting and monitoring initial treatment of a patient suffering from an exacerbation of inflammation of a respiratory condition as defined in claim 10, said method comprising (i) using antibodies to detect one or more or all of the markers; (ii) using a lateral flow assay to detect one or more or all of the markers; and/or (iii) using a system or test kit to detect one or more or all of the markers.

Description

DESCRIPTION OF THE FIGURES

[0405] The invention will now be described by way of example with respect to the accompanying drawings in which:

[0406] FIG. 1 is a schematic view of four different formats of the assay useful in the invention. Each format relies upon the same basic components of solid support (1), capture molecule (2), an indicator molecule containing a capture site (3) and a cleavage site (4) and a binding molecule (5) that binds to the indicator molecule only after cleavage (6) has occurred.

[0407] FIG. 2 is a schematic view of an enzyme detection device useful in the present invention and shows operation of the device in the absence (FIG. 2A) or presence (FIG. 2B) of enzyme cleavage activity.

[0408] FIG. 3 shows the visual read-out of the assay (shown in FIG. 2) as levels of MMP activity in the test sample are increased.

[0409] FIG. 4 is a schematic view of an enzyme detection device useful in the present invention. The figure specifies the exact longitudinal dimensions and position of each of the card components.

[0410] FIG. 5 shows an example of synthesis of a structurally constrained indicator molecule.

[0411] In FIG. 5A initially, a linear peptide (1) is synthesised, for example using solid phase Fmoc chemistry. The peptide may be purified for example by High Performance Liquid Chromatography (HPLC). The peptide is then constrained, or cyclised, by reaction between thiol groups on the peptide (2) and the scaffold molecule (3). This reaction produces a structurally constrained “clipped” peptide (4).

[0412] In FIG. 5B, the indicator molecule is synthesised to include the capture site (1), for example by synthesis of the linear peptide on a pre-loaded Biotin-PEG resin.

[0413] FIG. 6 shows schematically the ability of the binding molecules used in the invention to bind exclusively to the cleaved indicator molecule. In the absence of enzyme cleavage activity, the structurally constrained indicator molecule (1) is not bound by the antibody binding molecule (2). This antibody is generated using the cleaved indicator molecule (3) as antigen and thus only binds to this “open” form of the molecule.

[0414] FIG. 7 shows a number of scaffold molecules useful in the indicator molecules described herein.

[0415] FIG. 8 shows a number of scaffold molecules useful in the indicator molecules described herein, together with proposed nomenclature.

[0416] FIG. 9 shows some attachment options for scaffold molecules to the indicator molecules.

[0417] FIG. 9A shows products of cleavage at a single cleavage site and FIG. 9B shows products of cleavage at two separate cleavage sites.

[0418] FIG. 10—General algorithm for using the calculated marker levels to select treatment for an exacerbation of inflammation. BM=marker (any marker as described herein).

[0419] FIG. 11—Correlation of MBP with blood eosinophil levels (a) overall and (b) split between exacerbation and stable state.

[0420] FIG. 12—Correlation of Calprotectin with blood neutrophil levels (a) overall and (b) split between exacerbation and stable state.

[0421] FIG. 13—Correlation of MMP9 with blood neutrophil levels (a) overall and (b) split between exacerbation and stable state.

[0422] FIG. 14—Model 1: Correlations with eosinophil levels, the combination of 5 biomarkers produced an R.sup.2 of 0.752. The 5 biomarkers with significant levels in this model were: EDN (<0.001), MMP9 (<0.001), HNE ((<0.001), NGAL (0.001) and MBP (0.020) (in level of importance).

[0423] FIG. 15—Model 2: Correlations with neutrophil levels, the combination of 5 biomarkers produced an R.sup.2 of 0.489. The 5 biomarkers with significance levels in this model were: Calprotectin (0.00), MBP (0.003), MMP9 (0.012), CRP (0.043) and NGAL (0.057) (in level of importance).

[0424] FIG. 16A—Model 3: Correlation with neutrophil levels. The combination of 3 biomarkers produced an R.sup.2 of 0.514. The 3 biomarkers in this model were: MBP, Calprotectin and A1AT (as measured by Lateral flow).

[0425] FIG. 16B—Sensitivity and Specificity for Model 3 (MBP, Calprotectin and A1AT (LF). Optimal sensitivity of 90.91% and specificity of 92.11% with a cut-off of 8.66. a) scatter plot with median and interquartile ranges with Mann Whitney test p value b) Tukey's Box and whiskers plot c) ROC curve with AUC.

[0426] FIG. 17A—Model 4: Correlation with neutrophil levels. The combination of 5 biomarkers produced an R.sup.2 of 0.518. The 5 biomarkers in this model were: MBP, Calprotectin, A1AT (as measured by Lateral flow), MMP9 and CRP.

[0427] FIG. 17B—Sensitivity and Specificity for Model 4 (MBP, Calprotectin, A1AT (LF), MMP9 and CRP). Optimal sensitivity of 90.91% and specificity of 94.74% with a cut-off of 8.89. a) scatter plot with median and interquartile ranges with Mann Whitney test p value b) Tukey's Box and whiskers plot c) ROC curve with AUC.

[0428] FIG. 18A—Model 5: Correlation with neutrophil levels. The combination of 6 biomarkers produced an R.sup.2 of 0.514. The 6 biomarkers in this model were: MBP, Calprotectin, A1AT (as measured by Lateral flow), MMP9, CRP and NGAL.

[0429] FIG. 18B—Sensitivity and Specificity for Model 5 (MBP, Calprotectin, A1AT (LF), MMP9, CRP and NGAL). Optimal sensitivity of 90.91% and specificity of 92.11% with a cut-off of 8.695. a) scatter plot with median and interquartile ranges with Mann Whitney test p value b) Tukey's Box and whiskers plot c) ROC curve with AUC.

[0430] FIG. 19—Model 6: Correlations with neutrophil levels. The combination of 5 biomarkers produced an R.sup.2 of 0.308. The 5 biomarkers in this model were: MMP9, Calprotectin, HNE, CRP and A1AT (as measured by ELISA).

[0431] FIG. 20—Model 6 characterised on further defined group (removal of subgroup 2 and removal of grey zone samples) a) scatter plot with median and interquartile ranges with Mann Whitney test p value b) Tukey's Box and whiskers plot c) ROC curve with AUC.

[0432] FIG. 21—Model 7: Correlations with neutrophil levels. The combination of 4 biomarkers produced an R.sup.2 of 0.33. The 4 biomarkers in this model were: MMP9, Calprotectin, HNE and CRP.

[0433] FIG. 22—Model 8: Correlations with neutrophil levels. The combination of 6 biomarkers produced an R.sup.2 of 0.334. The 6 biomarkers in this model were: MMP9, Calprotectin, HNE, CRP, A1AT (as measured by LF) and NGAL.

[0434] FIG. 23—Model 9: Correlations with neutrophil levels. The combination of 5 biomarkers produced an R.sup.2 of 0.332. The 5 biomarkers in this model were: MMP9, Calprotectin, HNE, CRP and A1AT (as measured by LF).

[0435] FIG. 24A—Model 2: Correlations with neutrophil levels. The combination of 5 biomarkers produced the AUC for the training and validation sets of 0.9, and for the test set of 0.7. The 5 biomarkers in this model were: MMP9, LTB4, EDN, A1AT and SuPAR.

[0436] FIG. 24B—Model 4: Correlations with neutrophil levels. The combination of 5 biomarkers produced the AUC for the training and validation sets of 0.84, and for the test set of 0.87. The 5 biomarkers in this model were: MMP9, LTB4, EDN, A1AT and CRP.

[0437] FIG. 25—Model 1: Correlations with eosinophil levels. The combination of 5 biomarkers produced the AUC for the training and validation sets of 0.94 and 0.92, respectively, and for the test set of 0.81. The 5 biomarkers in this model were: EDN, MPO, RNASE3, HNE and SuPAR.

DETAILED DESCRIPTION

[0438] FIG. 1 is a schematic view of four different formats of an assay useful for performance of the invention, in particular for detecting molecules such as proteases (e.g. MMPs) in blood samples. Each format relies upon the same basic components of solid support (1), capture molecule (2), an indicator molecule containing a capture site (3) and a cleavage site (4) and a binding molecule (5) that binds to the indicator molecule only after cleavage (6) has occurred.

[0439] In formats 1 and 4, the capture molecule (2) is streptavidin. Here, the capture molecule (2) binds to a biotin capture site (3) within the indicator molecule. In formats 2 and 3, the capture molecule (2) is an antibody. Here, the capture molecule (2) binds to an epitope capture site (3) within the indicator molecule. The epitope is found in the alternative long peptide (ALP) which is derived from human chorionic gonadotropin (hCG).

[0440] Once the indicator molecule is added to a test sample, any enzyme specifically recognising the cleavage site (4) present, may cleave the indicator molecule (6). This cleavage event (6) produces a binding site for the specific antibody binding molecule (5). The binding molecule (5) is unable to bind to the indicator molecule until cleavage (6) has occurred. Thus, in formats 1 and 3 the antibody binding molecule (5) binds to the amino acid sequence GPQG produced as a result of cleavage of the GPQGIFGQ sequence. In formats 2 and 4, on the other hand, the antibody binding molecule (5) binds to the amino acid sequence QGFI, also produced as a result of cleavage of the GPQGIFGQ sequence.

[0441] In each format, the antibody binding molecule (5) does not bind to the GPQGIFGQ sequence prior to cleavage (not shown).

[0442] FIG. 2 is a schematic view of an enzyme detection device used in the present invention and shows operation of the device in the absence (FIG. 2A) or presence (FIG. 2B) of enzyme cleavage activity in the blood sample. The test strip includes an adhesive liner (1) upon which the other components of the device are assembled. From right to left, the sample application zone (2) is in the form of an absorbent pad. This is laid partially overlapping the conjugate pad (3), which is impregnated with the labelled binding molecules (7). In alternative embodiments, the labelled binding molecules may be impregnated in the sample application zone and this removes the need for a separate conjugate pad. The conjugate pad (3) is in fluid connection with a nitrocellulose membrane (4). The nitrocellulose membrane (4) contains immobilized streptavidin molecules (5) which define a capture zone. The membrane (4) further contains immobilized further binding molecules (6) downstream of the capture zone which bind to further labelled molecules (11) which pass through the device with the sample and form a separate control zone. Alternatively, the immobilised further binding molecules may bind to labelled binding molecules (7). The device optionally further comprises an absorbent pad (8) to absorb any test sample and reagents reaching the end of the device.

[0443] In use, the indicator molecule (9) is added to the test sample prior to bringing the test sample into contact with the sample application zone (8) of the device. As shown in FIG. 2A, in the absence of enzyme cleavage activity in the test sample, the indicator molecule (9) remains uncleaved at the cleavage site. Upon sample flow into the conjugate pad (3), the binding molecules (7) are unable to bind to the indicator molecule (9) because cleavage of the cleavage site has not occurred. The indicator molecules become bound at the capture zone via the interaction between streptavidin (5) and the biotin capture site (10) of the indicator molecule (9). The labelled binding molecules (7) are not immobilized at the capture zone because they cannot bind to the indicator molecules (9). Accordingly, the labelled binding molecules flow through to the control zone and beyond. Further labelled molecules (11) also pass through the device to the control zone where they are immobilized by binding to the immobilized further binding molecules (6). Thus, absence of enzyme cleavage activity is displayed as a signal only at the control zone, but not at the capture zone. Excess sample, potentially containing labelled binding molecules (7), flows into the absorbent pad (8).

[0444] As shown in FIG. 2B, in the presence of enzyme cleavage activity in the test sample, the indicator molecule (9) is cleaved at the cleavage site. Upon sample flow into the conjugate pad (3), the binding molecules (7) are able to bind to the indicator molecule (9) because cleavage of the cleavage site has occurred. The indicator molecules become bound at the capture zone via the interaction between streptavidin (5) and the biotin capture site (10) of the indicator molecule (9). The labelled binding molecules (7) are immobilized at the capture zone due to binding to the indicator molecules (9) at the cleavage site. Due to the relative excess of labelled binding molecule (7) to binding sites at the capture zone some labelled binding molecules (7) still flow through to the control zone and beyond. Further labelled molecules (11) also pass through the device to the control zone where they are immobilized by binding to the immobilized further binding molecules (6). Thus, level of enzyme cleavage activity may be measured via a signal at the capture zone (and a signal will also be present at the control zone). Excess sample, potentially containing cleavage products of the indicator molecule that do not contain the biotin capture site (10), flows into the absorbent pad (8).

[0445] It should be noted that the control zone is optional. The level of enzyme cleavage activity in the blood sample can be monitored based upon a measurement of the corresponding signal at the capture zone.

[0446] FIG. 3 shows the visual read-out of the assay (shown in FIG. 2) as levels of MMP activity in the test sample are increased. As can readily be seen, the signal at the control zone (1) is constant as MMP amounts increase. In contrast, as MMP amounts increase, the signal at the capture zone (2) also increases. This is due to cleavage of the indicator molecule at the cleavage site by MMP activity. This reveals a binding site, enabling binding of the binding molecules which is detected at the capture zone (2) via interaction between capture molecules defining the capture zone and the capture site of the indicator molecules. The intensity of the signal at the capture zone can be measured to provide the level of effector molecule in the blood sample. This may employ a suitable reader.

[0447] FIG. 4 is a schematic view of one specific enzyme detection device useful with the present invention. The table below provides a legend for the figure and specifies the longitudinal dimensions and position of each of the card components in this particular embodiment. Of course, the dimensions and positions may be varied as would be readily understood by one skilled in the art.

TABLE-US-00002 Position from Component Size Datum point Backing card (1) 60 mm 0 mm Nitrocellulose Membrane (2) 25 mm 20 mm Conjugate Pad (3) 17 mm 5 mm Sample Pad (4) 10 mm 0 mm Absorbent Pad (5) 22 mm 38 mm

[0448] FIG. 5 shows an example of synthesis of a structurally constrained indicator molecule. It should be noted that additional spacer or linker regions may be included between the cleavage region and the site of attachment of the scaffold molecule.

[0449] In FIG. 5A initially, a linear peptide (1) is synthesised, for example using solid phase Fmoc chemistry. The peptide may be purified for example by High Performance Liquid Chromatography (HPLC). The peptide is then constrained, or cyclised, by reaction between thiol groups on the peptide (2) and the scaffold molecule (3). This reaction produces a structurally constrained “clipped” peptide (4).

[0450] In FIG. 5B, the indicator molecule is synthesised to include the capture site (1), for example by synthesis of the linear peptide on a pre-loaded Biotin-PEG resin.

[0451] FIG. 6 shows schematically the ability of the binding molecules used in some embodiments of the invention to bind exclusively to the cleaved indicator molecule. In the absence of enzyme cleavage activity, the structurally constrained indicator molecule (1) is not bound by the antibody binding molecule (2). This antibody is generated using the cleaved indicator molecule (3) as antigen and thus only binds to this “open” form of the molecule.

[0452] FIGS. 7 and 8 show a range of suitable scaffold molecules for use in the invention.

[0453] FIG. 9 shows, in schematic form, some attachment options for scaffold molecules to the indicator molecules. FIG. 9A shows products of cleavage at a single cleavage site and FIG. 9B shows products of cleavage at two separate cleavage sites.

[0454] FIG. 10 presents an algorithm useful in the invention. This particular algorithm was designed based on the observed perturbations in marker levels in patients suffering from an exacerbation and the derived correlations between said perturbations and changes in eosinophil and/or neutrophil levels in the blood samples. The principles embodied in this algorithm are applicable to all of the markers and combinations described herein.

[0455] The invention may be further defined in the following set of numbered clauses, wherein any reference to “(the) at least one marker” in a dependent clause should be understood also to apply to “(the) at least 3 markers” of clause 1B or any other clause reciting “(the) at least 3 markers”.

[0456] For example, it should be understood that insofar as it is dependent on clause 1B, clause 5 is directed to the method according to any one of clauses 1-4 wherein at least one of the at least 3 markers of neutrophil levels is selected from: Calprotectin, C-reactive protein (CRP), Alpha-1-antitrypsin (A1AT), MBP, myeloperoxidase (MPO), Interleukin-8 (IL-8), Interleukin-6 (IL-6) and Interleukin-1β (IL-1β) (such that the at least 3 markers of neutrophil levels may, e.g., comprise or consist of MMP9, EDN, and A1AT; or comprise or consist of MMP9, EDN, LTB4 and IL-8)

wherein perturbed levels of the at least 3 markers result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation

[0457] Although some explicit references to clauses A and B are made, it should be understood that any reference to a numbered clause refers to embodiments A and B of that numbered clause (for example a clause dependent on clauses 1-18 depends on any of these clauses, including clause 1A, 1B, 17A and 17B):

[0458] 1A. A method for selecting a treatment to be administered to a patient suffering from an exacerbation of inflammation, the method comprising determining the levels of at least one marker of eosinophil levels and at least one marker of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation wherein: [0459] (i) perturbed levels of the at least one marker of eosinophil levels and no perturbation in the levels of the at least one marker of neutrophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation; [0460] (ii) perturbed levels of the at least one marker of neutrophil levels and no perturbation in the levels of the at least one marker of eosinophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation; or [0461] (iii) perturbed levels of the at least one marker of eosinophil levels and the at least one marker of neutrophil levels result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation.

[0462] 1B A method for selecting a treatment to be administered to a patient suffering from an exacerbation of inflammation of a respiratory condition, the method comprising determining the levels of at least 3 markers of eosinophil levels and at least 3 markers of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition wherein: [0463] (i) perturbed levels of the at least 3 markers of eosinophil levels and no perturbation in the levels of the at least 3 markers of neutrophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation; [0464] (ii) perturbed levels of the at least 3 markers of neutrophil levels and no perturbation in the levels of the at least 3 markers of eosinophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation; or [0465] (iii) perturbed levels of the at least 3 markers of eosinophil levels and the at least 3 markers of neutrophil levels result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation;
wherein determining the levels of the at least 3 markers of eosinophil levels comprises determining the levels of at least 3 markers selected from EDN, MPO, RNAse3, HNE, SuPAR and/or Calprotectin; preferably EDN, MPO and RNASE3 and optionally one or more further markers selected from HNE, SuPAR, and/or Calprotectin, preferably HNE and SuPAR;
and wherein determining the levels of the at least 3 markers of neutrophil levels comprises determining the levels of at least 3 markers selected from MMP9, EDN, LTB4, CRP, SuPAR and/or A1AT; preferably at least (i) Matrix metallopeptidase 9 (MMP9) and Eosinophil-derived neurotoxin (EDN); and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4.

[0466] 2. The method according to clause 1A or B wherein at least one marker of eosinophil levels is selected from: Eosinophil-derived neurotoxin (EDN), Major Basic Protein (MBP) and Eosinophil cationic protein (RNASE3);

wherein perturbed levels of the at least one marker result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0467] 3. The method according to clause 2 wherein increased levels of EDN, and/or MBP indicate increased levels of eosinophils and result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0468] 4. The method according to clause 2 or 3 wherein the at least one marker of eosinophil levels comprises EDN and MBP.

[0469] 5. The method according to any one of clauses 1(A or B)-4 wherein at least one marker of neutrophil levels is selected from: Calprotectin, C-reactive protein (CRP), Alpha-1-antitrypsin (A1AT), MBP, myeloperoxidase (MPO), Interleukin-8 (IL-8), Interleukin-6 (IL-6) and Interleukin-1β (IL-1β);

wherein perturbed levels of the at least one marker result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation.

[0470] 6. The method according to clause 5 wherein:

increased levels of Calprotectin and/or CRP;
decreased levels of MBP;
indicate increased levels of neutrophils and result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation.

[0471] 7. The method according to clause 5 or 6 wherein the at least one marker of neutrophil levels comprises: [0472] (i) MBP, Calprotectin and A1AT; or [0473] (ii) Calprotectin, IL-8, IL-6, CRP, MPO and IL-1β.

[0474] 8. The method according to any preceding clause further comprising at least one supporting (i.e. further) marker of eosinophil and neutrophil levels wherein perturbed levels of the at least one supporting marker: [0475] (i) in combination with perturbed levels of the at least one marker of eosinophil levels and no perturbation in the levels of the at least one marker of neutrophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation; [0476] (ii) in combination with perturbed levels of the at least one marker of neutrophil levels and no perturbation in the levels of the at least one marker of eosinophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation; or [0477] (iii) in combination with perturbed levels of the at least one marker of eosinophil levels and the at least one marker of neutrophil levels result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation.

[0478] 9. The method of clause 8 wherein the at least one supporting marker of eosinophil and neutrophil levels is selected from: Matrix metallopeptidase 9 (MMP9), Human neutrophil elastase (HNE) and neutrophil gelatinase-associated lipocalin (NGAL).

[0479] 10. The method according to clause 9 wherein:

increased levels of MMP9;
decreased levels of HNE;
indicate increased levels of neutrophils and eosinophils in combination with perturbed levels of at least one neutrophil and eosinophil marker respectively.

[0480] 11. The method of any preceding clause further comprising determining the levels of at least three markers, optionally at least four, five or six markers, in any combination of markers, provided that the at least three markers, optionally at least four, five or six markers, comprise at least one marker of eosinophil levels and at least one marker of neutrophil levels, preferably comprising determining at least 3, 4 or 5 markers of eosinophil levels and at least 3, 4 or 5 markers of neutrophil levels.

[0481] 12. The method according to any preceding clause wherein the markers comprise EDN, MMP9, HNE, NGAL and MBP.

[0482] 13. The method according to any preceding clause wherein the markers comprise MMP9, CRP and/or NGAL.

[0483] 14. The method according to any preceding clause wherein the markers comprise MMP9, Calprotectin, HNE and CRP.

[0484] 15. The method according to any preceding clause wherein the markers further comprise A1AT and/or NGAL.

[0485] 16. The method according to any preceding clause wherein: [0486] (i) increased levels of EDN; in combination with: [0487] (ii) increased levels of Calprotectin and/or CRP;
result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation.

[0488] 16B The method according to any preceding clause, preferably according to clause 1B, wherein the markers comprise

(i) EDN, MPO, RNAse, and MMP9; and

[0489] (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4.

[0490] 17A. A method for selecting corticosteroids to be administered as a treatment to a patient suffering from an exacerbation of inflammation, the method comprising determining the levels of at least one marker of eosinophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation wherein perturbed levels of the at least one marker of eosinophil levels results in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0491] 17B A method for selecting corticosteroids to be administered as a treatment to a patient suffering from an exacerbation of inflammation of a respiratory condition, the method comprising determining the levels of at least 3 markers of eosinophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition, wherein perturbed levels of the at least 3 markers of eosinophil levels results in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation of a respiratory condition, wherein determining the levels of the at least 3 markers of eosinophil levels comprises determining the levels of at least 3 markers selected from EDN, MPO, RNAse3, HNE, SuPAR and/or Calprotectin, preferably EDN, MPO and RNASE3 and optionally one or more further markers selected from HNE, SuPAR, and/or Calprotectin, preferably HNE and SuPAR.

[0492] 18. The method of clause 17A or B wherein at least one marker of eosinophil levels is selected from: Eosinophil-derived neurotoxin (EDN), Major Basic Protein (MBP) and Eosinophil cationic protein (RNASE3);

wherein perturbed levels of the at least one marker result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0493] 19. The method according to clause 18 wherein increased levels of EDN and/or MBP indicate increased levels of eosinophils and result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0494] 20. The method according to any one of clauses 17(A or B)-19 further comprising at least one supporting (i.e. further) marker of eosinophil levels wherein perturbed levels of the at least one supporting marker in combination with perturbed levels of the at least one marker of eosinophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0495] 21. The method according to clause 20 wherein the at least one supporting marker of eosinophil levels is selected from: MMP9, HNE and NGAL.

[0496] 22. The method according to clause 21 wherein:

increased levels of MMP9;
decreased levels of HNE and/or NGAL;
indicate increased levels of eosinophils in combination with perturbed levels of at least one eosinophil marker.

[0497] 24. The method according to any one of clauses 17(A or B)-23 comprising determining the levels of at least three, optionally at least four, five or six, markers in the blood sample.

[0498] 25. The method of clause 24 wherein perturbed levels of two, three or more of the at least one marker of eosinophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0499] 26. The method according to any one of clauses 17(A or B)-25 wherein the markers comprise EDN, MMP9, HNE, NGAL and MBP; or EDN, MPO, RNAse3, HNE and SuPAR.

[0500] 27A. A method for selecting antibiotics to be administered as a treatment to a patient suffering from an exacerbation of inflammation, the method comprising determining the levels of at least one marker of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation wherein perturbed levels of at least one marker of neutrophil levels results in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation, wherein the at least one marker of neutrophil levels comprises Calprotectin, A1AT, MBP, MPO, IL-8, IL-6 and/or IL-1β.

[0501] 27B A method for selecting antibiotics to be administered as a treatment to a patient suffering from an exacerbation of inflammation of a respiratory condition, the method comprising determining the levels of at least 3 markers of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition wherein perturbed levels of at least 3 markers of neutrophil levels results in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation,

wherein determining the levels of the at least 3 markers of neutrophil levels comprises determining the levels of at least 3 markers selected from MMP9, EDN, LTB4, CRP, SuPAR and/or A1AT; preferably at least (i) Matrix metallopeptidase 9 (MMP9) and Eosinophil-derived neurotoxin (EDN); and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4, optionally at least 2 or 3 of these markers.

[0502] 28. The method of clause 27A or B wherein the at least one marker further comprises CRP.

[0503] 29. The method according to clause 27(A or B) or 28 wherein:

[0504] 30. The method according to any of clauses 27(A or B)-29 wherein the at least one marker of neutrophil levels comprises: [0505] (i) MBP, Calprotectin and A1AT; or [0506] (ii) Calprotectin, IL-8, IL-6, CRP, MPO and IL-1β.

[0507] 31. The method according to any one of clauses 27(A or B)-30 further comprising at least one supporting (i.e. further) marker of neutrophil levels wherein perturbed levels of the at least one supporting marker in combination with perturbed levels of the at least one marker of neutrophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation.

[0508] 32. The method according to clause 31 wherein the at least one supporting marker of neutrophil levels is selected from: MMP9, HNE and NGAL.

[0509] 33. The method according to clause 32 wherein:

increased levels of MMP9;
decreased levels of HNE;
indicate increased levels of neutrophils in combination with perturbed levels of at least one neutrophil marker.

[0510] 34. The method according to any one of clauses 27(A or B)-33 comprising determining the levels of at least three, optionally at least four, five or six, markers in the blood sample.

[0511] 35. The method of clause 34 wherein perturbed levels of two, three or more markers of neutrophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation.

[0512] 36A. The method according to any one of clauses 27(A or B)-35 wherein the markers comprise (i) MMP9 and EDN and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4, optionally at least 2 or 3 of these markers.

[0513] 36B. The method according to any one of clauses 27(A or B)-35 or 36A wherein the markers comprise MBP, Calprotectin and A1AT.

[0514] 37. The method according to clause 36(A or B) wherein the markers further comprise MMP9, CRP and/or NGAL.

[0515] 38. The method according to any one of clauses 27(A or B)-35 or 36A or B wherein the markers comprise MMP9, Calprotectin, HNE and CRP.

[0516] 39. The method according to clause 38 wherein the markers further comprise A1AT and/or NGAL.

[0517] 40. The method according to any one of clauses 1(A or B)-39 wherein the treatment will be the first treatment to be administered to the patient suffering from an exacerbation of inflammation.

[0518] 41. A method for selecting and monitoring treatment of a patient suffering from an exacerbation of inflammation, the method comprising: [0519] (i) selecting a treatment to be administered to the patient using a method as defined in any one of clauses 1(A or B)-40; and [0520] (ii) with respect to the at least one marker for which levels were perturbed when determining the treatment to be administered of step (i), determining the levels of said at least one marker in a further blood sample taken from the patient at a later time point wherein: [0521] (a) perturbed levels of the at least one marker in the further sample indicate that the treatment should continue or be altered; or [0522] (b) a return to non-perturbed levels of the at least one marker in the further sample indicate or predict successful treatment of the exacerbation of inflammation.

[0523] 42. The method according to any one of clauses 1(A or B)-41 wherein the exacerbation of inflammation is exacerbation of lung inflammation.

[0524] 43. The method according to any one of clauses 1(A or B)-42 wherein the subject is suffering from a respiratory disorder.

[0525] 44. The method according to clause 43 wherein the respiratory disorder is chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) or asthma, preferably COPD.

[0526] 45A. A system or test kit for selecting a treatment to be administered to a patient suffering from an exacerbation of inflammation, comprising: [0527] a. one or more testing devices for determining the levels of at least one marker of eosinophil levels and at least one marker of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation; [0528] b. a processor; and [0529] c. a storage medium comprising a computer application that, when executed by the processor, is configured to: [0530] i. Access and/or calculate the determined levels of the at least one marker of eosinophil levels and the at least one marker of neutrophil levels in a blood sample on the one or more testing devices; [0531] ii. Calculate whether there is a perturbed level of the at least one marker of eosinophil levels and the at least one marker of neutrophil levels in the blood sample; and [0532] iii. Output from the processor the treatment to be administered to the patient suffering from an exacerbation of inflammation, wherein: [0533] perturbed levels of the at least one marker of eosinophil levels and no perturbation in the levels of the at least one marker of neutrophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation; or [0534] perturbed levels of the at least one marker of neutrophil levels and no perturbation in the levels of the at least one marker of eosinophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation; or [0535] perturbed levels of the at least one marker of eosinophil levels and the at least one marker of neutrophil levels result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation.

[0536] 45B. A system or test kit for selecting a treatment to be administered to a patient suffering from an exacerbation of inflammation of a respiratory condition, comprising:

[0537] a. one or more testing devices for determining the levels of at least 3 markers of eosinophil levels and at least 3 markers of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition;

[0538] b. a processor; and

[0539] c. a storage medium comprising a computer application that, when executed by the processor, is configured to: [0540] i. Access and/or calculate the determined levels of the at least 3 markers of eosinophil levels and the at least 3 markers of neutrophil levels in a blood sample on the one or more testing devices; [0541] ii. Calculate whether there is a perturbed level of the at least 3 markers of eosinophil levels and the at least 3 markers of neutrophil levels in the blood sample; and [0542] iii. Output from the processor the treatment to be administered to the patient suffering from an exacerbation of inflammation, wherein: [0543] perturbed levels of the at least 3 markers of eosinophil levels and no perturbation in the levels of the at least 3 markers of neutrophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation; or [0544] perturbed levels of the at least 3 markers of neutrophil levels and no perturbation in the levels of the at least 3 markers of eosinophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation; or [0545] perturbed levels of the at least 3 markers of eosinophil levels and the at least 3 markers of neutrophil levels result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation;
wherein the at least 3 markers of eosinophil levels comprise at least 3 markers selected from EDN, MPO, RNAse3, HNE, SuPAR and/or Calprotectin; preferably EDN, MPO and RNASE3 and optionally one or more further markers selected from HNE, SuPAR, and/or Calprotectin, preferably HNE and SuPAR;
and wherein the at least 3 markers of neutrophil levels comprise least 3 markers selected from MMP9, EDN, LTB4, CRP, SuPAR and/or A1AT; preferably at least (i) Matrix metallopeptidase 9 (MMP9) and Eosinophil-derived neurotoxin (EDN); and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4.

[0546] 46. The system or test kit according to clause 45(A or B) wherein at least one marker of eosinophil levels is selected from: Eosinophil-derived neurotoxin (EDN), Major Basic Protein (MBP) and Eosinophil cationic protein (RNASE3);

wherein perturbed levels of the at least one marker result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0547] 47. The system or test kit according to clause 46 wherein increased levels of EDN and/or MBP indicate increased levels of eosinophils and result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0548] 48. The system or test kit according to clause 46 or 47 wherein the at least one marker of eosinophil levels comprises EDN and MBP.

[0549] 49. The system or test kit according to any one of clauses 45(A or B)-48 wherein at least one marker of neutrophil levels is selected from: Calprotectin, C-reactive protein (CRP), Alpha-1-antitrypsin (A1AT), MBP, myeloperoxidase (MPO), Interleukin-8 (IL-8), Interleukin-6 (IL-6) and Interleukin-1β (IL-1β);

wherein perturbed levels of the at least one marker result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation.

[0550] 50. The system or test kit according to clause 49 wherein:

[0551] 51. The system or test kit according to clause 49 or 50 wherein the at least one marker of neutrophil levels comprises: [0552] (i) MBP, Calprotectin and A1AT; or [0553] (ii) Calprotectin, IL-8, IL-6, CRP, MPO and IL-1β.

[0554] 52. The system or test kit according to any one of clauses 45(A or B)-51 further comprising determining the levels of at least one supporting (i.e. further) marker of eosinophil and neutrophil levels in the sample, wherein:

[0555] i. the computer application, when executed by the processor, is configured to access and/or calculate the determined levels of the at least one supporting marker of eosinophil and neutrophil levels in the blood sample on the one or more testing devices;

[0556] ii. Calculate whether there is a perturbed level of the at least one supporting marker of eosinophil and neutrophil levels in the blood sample; and

[0557] iii. Output from the processor the treatment to be administered to the patient suffering from an exacerbation of inflammation, wherein perturbed levels of the at least one supporting marker: [0558] (a) in combination with perturbed levels of the at least one marker of eosinophil levels and no perturbation in the levels of the at least one marker of neutrophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation; [0559] (b) in combination with perturbed levels of the at least one marker of neutrophil levels and no perturbation in the levels of the at least one marker of eosinophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation; or [0560] (c) in combination with perturbed levels of the at least one marker of eosinophil levels and the at least one marker of neutrophil levels result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation.

[0561] 53. The system or test kit of clause 52 wherein the at least one supporting marker of eosinophil and neutrophil levels is selected from: Matrix metallopeptidase 9 (MMP9), Human neutrophil elastase (HNE) and neutrophil gelatinase-associated lipocalin (NGAL).

[0562] 54. The system or test kit according to clause 53 wherein:

[0563] 55. The system or test kit of any one of clauses 45-54 further comprising determining the levels of at least three markers, optionally at least four, five or six markers, in any combination of markers, provided that the at least three markers, optionally at least four, five or six markers, comprise at least one marker of eosinophil levels and at least one marker of neutrophil levels, preferably comprising determining at least 3, 4 or 5 markers of eosinophil levels and at least 3, 4 or 5 markers of neutrophil levels, more preferably wherein the at least 3 markers of eosinophil levels comprise at least EDN, MPO and RNASE3;

and preferably wherein the at least 3 markers of neutrophil levels comprise at least (i) MMP9 and EDN; and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4.

[0564] 56. The system or test kit according to any one of clauses 45-55 wherein the markers comprise EDN, MMP9, HNE, NGAL and MBP.

[0565] 57. The system or test kit according to any one of clauses 45-56 wherein the markers comprise MMP9, CRP and/or NGAL.

[0566] 58. The system or test kit according to any one of clauses 45-57 wherein the markers comprise MMP9, Calprotectin, HNE and CRP.

[0567] 59. The system or test kit according to any one of clauses 45-58 wherein the markers further comprise A1AT and/or NGAL.

[0568] 60. The system or test kit according to any one of clauses 45-59 wherein:

[0569] (i) increased levels of EDN; in combination with

[0570] (ii) increased levels of Calprotectin and/or CRP;

result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation.

[0571] 61A. A system or test kit for selecting corticosteroids to be administered as a treatment to a patient suffering from an exacerbation of inflammation, comprising: [0572] a. one or more testing devices for determining the levels of at least one marker of eosinophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation; [0573] b. a processor; and [0574] c. a storage medium comprising a computer application that, when executed by the processor, is configured to: [0575] i. Access and/or calculate the determined levels of the at least one marker of eosinophil levels in a blood sample on the one or more testing devices; [0576] ii. Calculate whether there is a perturbed level of the at least one marker of eosinophil levels in the blood sample; and [0577] iii. Output from the processor that corticosteroids are selected to be administered as the treatment for the exacerbation of inflammation if there is a perturbed level of the at least one marker of eosinophil levels in the blood sample.

[0578] 61B. A system or test kit for selecting corticosteroids to be administered as a treatment to a patient suffering from an exacerbation of inflammation of a respiratory condition, comprising:

[0579] a. one or more testing devices for determining the levels of at least 3 markers of eosinophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition;

[0580] b. a processor; and

[0581] c. a storage medium comprising a computer application that, when executed by the processor, is configured to: [0582] i. Access and/or calculate the determined levels of the at least one marker of eosinophil levels in a blood sample on the one or more testing devices; [0583] ii. Calculate whether there is a perturbed level of the at least one marker of eosinophil levels in the blood sample; and [0584] iii. Output from the processor that corticosteroids are selected to be administered as the treatment for the exacerbation of inflammation if there is a perturbed level of the at least one marker of eosinophil levels in the blood sample,
wherein the at least 3 markers of eosinophil levels comprise at least 3 markers selected from EDN, MPO, RNAse3, HNE, SuPAR and/or Calprotectin; preferably EDN, MPO and RNASE3 and optionally one or more further markers selected from HNE, SuPAR, and/or Calprotectin, preferably HNE and SuPAR.

[0585] 62. The system or test kit according to clause 61(A or B) wherein at least one marker of eosinophil levels is selected from: Eosinophil-derived neurotoxin (EDN), Major Basic Protein (MBP) and Eosinophil cationic protein (RNASE3);

wherein perturbed levels of the at least one marker result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0586] 63. The system or test kit according to clause 62 wherein increased levels of EDN and/or MBP indicate increased levels of eosinophils and result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0587] 64. The system or test kit according to any one of clauses 61(A or B)-63 further comprising determining the levels of at least one supporting (i.e. further) marker of eosinophil levels in the sample, wherein:

[0588] i. the computer application, when executed by the processor, is configured to access and/or calculate the determined levels of the at least one supporting marker of eosinophil levels in the blood sample on the one or more testing devices;

[0589] ii. Calculate whether there is a perturbed level of the at least one supporting marker of eosinophil levels in the blood sample; and

[0590] iii. Output from the processor the treatment to be administered to the patient suffering from an exacerbation of inflammation, wherein perturbed levels of the at least one supporting marker in combination with perturbed levels of the at least one marker of eosinophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0591] 65. The system or test kit according to clause 64 wherein the at least one supporting marker of eosinophil levels is selected from: MMP9, HNE and NGAL.

[0592] 66. The system or test kit according to clause 65 wherein:

increased levels of MMP9;
decreased levels of HNE and/or NGAL;
indicate increased levels of eosinophils in combination with perturbed levels of at least one eosinophil marker.

[0593] 67. The system or test kit according to any one of clauses 61-66 comprising determining the levels of at least three, optionally at least four, five or six, markers in the blood sample.

[0594] 68. The system or test kit of clause 67 wherein perturbed levels of two, three or more of the at least one marker of eosinophil levels result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation.

[0595] 69. The system or test kit according to any one of clauses 61-68 wherein the markers comprise EDN, MMP9, HNE, NGAL and MBP.

[0596] 70A. A system or test kit for selecting antibiotics to be administered as a treatment to a patient suffering from an exacerbation of inflammation, comprising: [0597] a. one or more testing devices for determining the levels of at least one marker of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation; [0598] b. a processor; and [0599] c. a storage medium comprising a computer application that, when executed by the processor, is configured to: [0600] i. Access and/or calculate the determined levels of the at least one marker of neutrophil levels in a blood sample on the one or more testing devices; [0601] ii. Calculate whether there is a perturbed level of the at least one marker of neutrophil levels in the blood sample; and [0602] iii. Output from the processor that antibiotics are selected to be administered as the treatment for the exacerbation of inflammation if there is a perturbed level of the at least one marker of neutrophil levels in the blood sample;
wherein the at least one marker of neutrophil levels comprises Calprotectin, A1AT, MBP, MPO, IL-8, IL-6 and/or IL-1β.

[0603] 70B A system or test kit for selecting antibiotics to be administered as a treatment to a patient suffering from an exacerbation of inflammation, comprising:

[0604] a. one or more testing devices for determining the levels of at least one marker of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation;

[0605] b. a processor; and

[0606] c. a storage medium comprising a computer application that, when executed by the processor, is configured to: [0607] i. Access and/or calculate the determined levels of the at least one marker of neutrophil levels in a blood sample on the one or more testing devices; [0608] ii. Calculate whether there is a perturbed level of the at least one marker of neutrophil levels in the blood sample; and [0609] iii. Output from the processor that antibiotics are selected to be administered as the treatment for the exacerbation of inflammation if there is a perturbed level of the at least one marker of neutrophil levels in the blood sample;
wherein the at least 3 markers of neutrophil levels comprise at least 3 markers selected from MMP9, EDN, LTB4, CRP, SuPAR and/or A1AT; preferably at least (i) Matrix metallopeptidase 9 (MMP9) and Eosinophil-derived neurotoxin (EDN); and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4.

[0610] 71. The system or test kit of clause 70(A or B) wherein the at least one marker further comprises CRP.

[0611] 72. The system or test kit according to clause 70(A or B) or 71 wherein:

[0612] 73. The system or test kit according to any of clauses 70(A or B)-72 wherein the at least one marker of neutrophil levels comprises: [0613] (i) MBP, Calprotectin and A1AT; or [0614] (ii) Calprotectin, IL-8, IL-6, CRP, MPO and IL-1β.

[0615] 74. The system or test kit according to any one of clauses 70(A or B)-73 further comprising determining the levels of at least one supporting marker of neutrophil levels in the sample, wherein:

[0616] i. the computer application, when executed by the processor, is configured to access and/or calculate the determined levels of the at least one supporting marker of neutrophil levels in the blood sample on the one or more testing devices;

[0617] ii. Calculate whether there is a perturbed level of the at least one supporting marker of neutrophil levels in the blood sample; and

[0618] iii. Output from the processor the treatment to be administered to the patient suffering from an exacerbation of inflammation, wherein perturbed levels of the at least one supporting marker in combination with perturbed levels of the at least one marker of neutrophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation.

[0619] 75. The system or test kit according to clause 74 wherein the at least one supporting marker of neutrophil levels is selected from: MMP9, HNE and NGAL.

[0620] 76. The system or test kit according to clause 75 wherein:

increased levels of MMP9;
decreased levels of HNE;
indicate increased levels of neutrophils in combination with perturbed levels of at least one neutrophil marker.

[0621] 77. The system or test kit according to any one of clauses 70(A or B)-76 comprising determining the levels of at least three, optionally at least four, five or six, markers in the blood sample.

[0622] 78. The system or test kit of clause 77 wherein perturbed levels of two, three or more markers of neutrophil levels result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation.

[0623] 79. The system or test kit according to any one of clauses 70(A or B)-78 wherein the markers comprise MBP, Calprotectin and A1AT.

[0624] 80. The system or test kit according to clause 79 wherein the markers further comprise MMP9, CRP and/or NGAL.

[0625] 81. The system or test kit according to any one of clauses 70(A or B)-78 wherein the markers comprise MMP9, Calprotectin, HNE and CRP.

[0626] 82. The system or test kit according to clause 81 wherein the markers further comprise A1AT and/or NGAL.

[0627] 83. The system or test kit according to any one of clauses 45(A or B)-82 wherein the treatment will be the first treatment to be administered to the patient suffering from an exacerbation of inflammation.

[0628] 84. The system or test kit according to any one of clauses 45(A or B)-83 further comprising a display for the output from the processor and/or wherein the one or more testing devices are disposable single use devices and/or wherein the one or more testing devices comprise lateral flow test strips, optionally comprising a lateral flow test strip for each marker that is determined.

[0629] 85. A method for selecting and monitoring initial treatment of a patient suffering from an exacerbation of inflammation as defined in any one of clauses 41-44 using a system or test kit as defined in one of clauses 45-84.

[0630] 86. The system or test kit according to any one of clauses 45(A or B)-85 wherein the exacerbation of inflammation is exacerbation of lung inflammation.

[0631] 87. The system or test kit according to any one of clauses 45(A or B)-86 wherein the subject is suffering from a respiratory disorder.

[0632] 88. The system or test kit according to clause 87 wherein the respiratory disorder is chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) or asthma.

[0633] 89. A computer application as defined in any one of clauses (A or B)- to 88.

[0634] 90. A method of analysis, the method comprising determining (detecting or measuring) the levels of at least 3 markers of eosinophil levels and/or at least 3 markers of neutrophil levels in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition

wherein
determining the levels of the at least 3 markers of eosinophil levels comprises determining the levels of at least 3 markers selected from EDN, MPO, RNAse3, HNE, SuPAR and/or Calprotectin; preferably EDN, MPO and RNASE3 and optionally one or more further markers selected from HNE, SuPAR, and/or Calprotectin, preferably HNE and SuPAR; and/or
wherein determining the levels of the at least 3 markers of neutrophil levels comprises determining the levels of at least 3 markers selected from MMP9, EDN, LTB4, CRP, SuPAR and/or A1AT; preferably at least (i) Matrix metallopeptidase 9 (MMP9) and Eosinophil-derived neurotoxin (EDN); and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4.

[0635] 91. The method of clause 90, wherein the method comprises

[0636] (i) providing a blood sample from a patient suffering from an exacerbation of inflammation of a respiratory condition;

[0637] (ii) determining one or more of all of the markers using marker-specific antibodies;

[0638] (iii) determining one or more of all of the markers using a lateral flow assay;

[0639] (iv) carrying out the determination on 2 or more blood samples obtained from the patient at different time points; and/or

[0640] (v) selecting and administering an appropriate treatment to the patient, wherein [0641] (a) perturbed levels of the at least 3 markers of eosinophil levels (and, if determined, no perturbation in the levels of the at least 3 markers of neutrophil levels) result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation; [0642] (b) perturbed levels of the at least 3 markers of neutrophil levels (and, if determined, no perturbation in the levels of the at least 3 markers of eosinophil levels) result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation; or [0643] (c) perturbed levels of the at least 3 markers of eosinophil levels and the at least 3 markers of neutrophil levels result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation;

[0644] 92. The method of clause 90 or 91, wherein any of the markers and/or the respiratory condition are as defined in any preceding clause.

[0645] 93. A method for selecting a treatment to be administered to a patient suffering from an exacerbation of inflammation of a respiratory condition, the method comprising determining in a blood sample taken from the patient suffering from an exacerbation of inflammation of a respiratory condition

[0646] (a) the levels of at least 3 markers selected from EDN, MPO, RNAse3, HNE, SuPAR and/or Calprotectin; preferably EDN, MPO and RNASE3 and optionally one or more further markers selected from HNE, SuPAR, and/or Calprotectin, preferably HNE and SuPAR to determine the levels of eosinophils; and/or

[0647] (b) determining the levels of at least 3 markers selected from MMP9, EDN, LTB4, CRP, SuPAR and/or A1AT; preferably at least (i) Matrix metallopeptidase 9 (MMP9) and Eosinophil-derived neurotoxin (EDN); and (ii) at least one of leukotriene B4 (LTB4), C-reactive protein (CRP), Soluble urokinase-type plasminogen activator receptor (SuPAR), and/or Alpha-1-antitrypsin (A1AT), preferably LTB4 to determine the levels of neutrophils, wherein: [0648] (i) perturbed eosinophil levels (as determined by determining the levels of at least 3 markers selected from EDN, MPO, RNAse3, HNE, SuPAR and/or Calprotectin) (and, if determined no perturbation in neutrophil levels (as determined by determining the levels of at least 3 markers selected from MMP9, EDN, LTB4, CRP, SuPAR and/or A1AT)) result in selection of corticosteroids to be administered as the treatment for the exacerbation of inflammation; [0649] (ii) perturbed neutrophil levels (as determined by determining the levels of at least 3 markers selected from MMP9, EDN, LTB4, CRP, SuPAR and/or A1AT) (and, if determined, no perturbation in the eosinophil levels (as determined by determining the levels of at least 3 markers selected from EDN, MPO, RNAse3, HNE, SuPAR and/or Calprotectin)) result in selection of antibiotics to be administered as the treatment for the exacerbation of inflammation; or [0650] (iii) perturbed eosinophil levels (as determined by determining the levels of at least 3 markers selected from EDN, MPO, RNAse3, HNE, SuPAR and/or Calprotectin) and perturbed neutrophil levels (as determined by determining the levels of at least 3 markers selected from MMP9, EDN, LTB4, CRP, SuPAR and/or A1AT) result in selection of corticosteroids and antibiotics to be co-administered as the treatment for the exacerbation of inflammation;

[0651] 94. The method of clause 93, wherein any of the markers and/or the respiratory condition are as defined in any preceding clause.

[0652] Reference to a “marker of eosinophil levels” as used above in the listed clauses may be replaced with a “marker of eosinophil activity”, as described herein. Reference to a “marker of neutrophil levels” as used above in the listed clauses may be replaced with a “marker of neutrophil activity”, as described herein. Reference to a “supporting marker of eosinophil levels” as used above in the listed clauses may be replaced with a “supporting marker of eosinophil activity”, as described herein. Reference to a “supporting marker of neutrophil levels” as used above in the listed clauses may be replaced with a “supporting marker of neutrophil activity”, as described herein. Reference to a “supporting marker of eosinophil and neutrophil levels” as used above in the listed clauses may be replaced with a “supporting marker of eosinophil and neutrophil activity”, as described herein. A marker of eosinophil levels may be used in conjunction with a marker of eosinophil activity. A marker of neutrophil levels may be used in conjunction with a marker of neutrophil activity. A supporting marker of eosinophil levels may be used in conjunction with a supporting marker of eosinophil activity. A supporting marker of neutrophil levels may be used in conjunction with a supporting marker of neutrophil activity. A supporting marker of eosinophil and neutrophil levels may be used in conjunction with a supporting marker of eosinophil and neutrophil activity.

[0653] The invention will be further understood with reference to the following experimental examples.

EXAMPLES

Example 1—Preliminary Experimentation Concerning Treatment Stratification for COPD Patients in Response to an Exacerbation

[0654] Introduction

[0655] COPD is a common disease that predominantly affects the elderly population. Its prevalence is increasing and it contributes to substantial morbidity and mortality. There are an estimated 80 million people that have moderate to severe COPD worldwide. COPD has an estimated annual death rate of over 4 million people globally. By 2020 it is predicted to be the 3rd leading cause of mortality worldwide. According to the World Health Organization (WHO) World Health Report 2007, the top five respiratory diseases, including COPD, account for 17% of all deaths and 13% of all Disability-Adjusted Life Years. The Global Strategy for the prevention and control of non-communicable diseases endorsed by the 53rd World Health Assembly cites chronic respiratory disease as one of the four priority disease groups to be addressed. In Europe, COPD annual expenditure is over €40 billion.

[0656] Patients with COPD have daily symptoms, a poorer health status, reduced exercise capacity, and impairment in lung function. The symptoms can deteriorate rapidly in response to infection or pollution. The acute and sustained worsening of the symptoms is termed a COPD exacerbation. COPD exacerbations account for 15% of all medical admissions, 1 million bed days and an annual UK NHS expenditure of £500 million. COPD and in particular COPD exacerbations are of a high public health and financial relevance associated with a significant negative impact on the quality of life.

[0657] The main therapeutic options are: antibiotics to tackle the infection (amoxicillin or doxycycline as first line, or co-amoxiclav); bronchodilators (beta-2-agonists, anticholinergics and theophylline) to make breathing easier; and corticosteroids (Prednisolone) to accelerate recovery by reducing inflammation. Bacterial and viral respiratory infections have an acknowledged association with exacerbations. Recent improvements in bacterial and moreover viral identification have increased the proportion of exacerbations that are associated with a pathogen. The proportion associated with bacteria alone is about 30%, virus alone about 25% and combined 25%. COPD exacerbations are also associated with an increase in airway inflammation. Both sputum neutrophil and eosinophil total cell counts and activation markers increase in COPD exacerbations.

[0658] Interestingly the neutrophil count increases irrespective of the associated pathogen, but the eosinophil count increases in viral infection either alone or in combination with bacteria.

[0659] Current guidelines advocate the use of oral corticosteroids for patients with a COPD exacerbation who have increased dyspnoea and antibiotics in those with a history of more purulent sputum. A Cochrane review of systemic corticosteroids that included 10 studies reported that corticosteroids increase the rate of recovery following a severe exacerbation, reduce the length of hospital admission by 1-2 days and reduce the proportion of patients that have treatment failure. This beneficial effect is similar for both oral and parenteral corticosteroids. In moderate exacerbations oral corticosteroids increase the time to next exacerbation. However, it is likely these small corticosteroid-related benefits are confined to a subgroup of patients. Likewise, antibiotic therapy in COPD exacerbations is beneficial. A recent Cochrane review that included 11 trials with a total of 917 patients found that antibiotics, regardless of choice, reduced the risk of short-term mortality by 77%, decreased the risk of treatment failure by 53% and the risk of sputum purulence by 44%. However, the range of response was large and it is estimated that antibiotics are of clinical benefit in only 25-50% of COPD exacerbations. Our inability to identify accurately which patients with a COPD exacerbation should receive antibiotics and or corticosteroids inevitably leads to inappropriate and excessive use of treatment.

[0660] The widespread use of antibiotics in the community has been implicated in the increase of antibiotic resistance, particularly MRSA, which now accounts for over 40% of Staphylococcus aureus blood isolates and the substantial increase of 17.2% in the number of cases of Clostridium difficile infection in patients aged 65 years and above in England during 2015. Systemic corticosteroids are also associated with well-established side-effects and in particular their use is complicated in patients with co-morbid diabetes mellitus and ischaemic heart disease. Indeed, the number needed to harm i.e. the number that need to receive systemic corticosteroids to observe an additional adverse reaction is only 6 patients. Therefore, there is a pressing need for either a single or more likely a composite of biomarkers to direct therapy in COPD exacerbations.

[0661] Targeted therapy for COPD exacerbations therefore requires reliable tests that can be performed in minutes without the need for laboratory support. Measuring biomarkers in blood samples provides a real opportunity to develop a near patient test for both secondary and primary care.

[0662] Solution

[0663] The Mologic multiplexed, blood biomarker diagnostic (Rightstart), with its integrated biomarker level interpretation algorithm, determines the levels of markers in a blood sample which have been found to correlate with levels of eosinophils and/or neutrophils. Thus, the levels of the markers can be used to determine whether an exacerbation is eosinophilic (i.e. high levels of eosinophils) or neutrophilic (i.e. high levels of neutrophils) in order to guide steroid or antibiotic treatment. Through the use of blood as the sample, the proposed point-of-care test has been designed to be minimally invasive, easy to use, rapid and simple to understand results, so that it can easily be integrated into a primary care setting (e.g. clinic).

[0664] Methods

[0665] Measurement of Blood (Serum) Biomarkers

[0666] Samples (banked, frozen) were provided from a Leicester study (MRC funded BEAT-COPD study ISRCTN2422949) Study details: From a two-staged single centre study, blood, sputum and urine samples from COPD subjects were longitudinally collected at four visit types: namely stable state (defined as being eight weeks free from an exacerbation visit), exacerbation (defined according to Anthonisen criteria and healthcare utilisation), two weeks post therapy and at recovery (six weeks post exacerbation visit). Exacerbations were treated with oral corticosteroids and antibiotics according to guidelines or trial study design. Clinical data including demographics, symptoms, lung function, inflammatory profiling in blood and sputum, bacteriology including standard culture, qPCR for common pathogens and microbiomics, viruses by PCR and fungal culture were undertaken.

[0667] Blood Biomarkers for Differentiating Eosinophil and Neutrophil Driven Exacerbations (to Guide Treatment)

[0668] 37 different biomarker assays were tested with serum.

[0669] Limited samples were available for testing (few exacerbations), therefore analysis focused only on which biomarkers correlated with blood eosinophils or neutrophils levels.

[0670] The most promising from a selection of 41 samples (where Leicester serum samples were also available) [0671] 24 stable samples [0672] 17 exacerbation samples

[0673] The biomarkers were selected on a rational basis and in the light of our increasing experience with urine samples from other clinical studies. Inflammatory leukocytes active in the lung cause a wide range of biomarkers to be released into lung fluid and blood, some originating from the leukocytes, some from the damage they cause to the surrounding tissue and some as a consequence of the signalling pathways that call them into the lung or control their activity.

[0674] The distribution of the continuous variables was studied using histograms, values of skewness and kurtosis, and normality was tested by the Kolmogorov-Smirnov test. Paired t test and Wilcoxon matched-pairs signed rank test were used to compare quantitative data in the two groups. Receiver operating characteristic (ROC) curve analysis was used to study the accuracy of the various diagnostic tests and logistic regression to find the best combination of biomarkers. P values<0.05 were considered to be statistically significant. Statistical analyses were carried out through the use of computer IBM software SPSS 21 (Chicago, Ill., USA), Graphpad Prism 5 and in R.

[0675] Results

[0676] Major Basic Protein correlated well with blood eosinophils. Calprotectin correlated well with blood neutrophils. MMP9 was tested by both Mologic and Leicester and correlated well with blood neutrophils in both cases. In general, the correlation was better with the exacerbation samples than stable (FIGS. 11-13).

[0677] Conclusion

[0678] As proof of principle, a panel of potential biomarkers had been selected based on blood neutrophil and eosinophil levels. Blood biomarkers may be used without the requirement of establishing a baseline value. As the test needs only to be used in the event of an exacerbation, a finger prick sample would be acceptable for less frequent testing.

Example 2—Biomarkers for Treatment Stratification for COPD Patients in Response to an Exacerbation

[0679] Samples

[0680] Samples (banked, frozen) were provided from University of Leicester study (MRC funded BEAT-COPD (Biomarkers to Target Antibiotic and Systemic Corticosteroid Therapy in COPD Exacerbations) study ISRCTN2422949).

[0681] Study details: From a two-staged single centre study, blood, sputum and urine samples from COPD subjects were longitudinally collected at four visit types: namely stable state (defined as being eight weeks free from an exacerbation visit), exacerbation (defined according to Anthonisen criteria [Anthonisen 2006] and healthcare utilisation), two weeks post therapy and at recovery (six weeks post exacerbation visit). Exacerbations were treated with oral corticosteroids and antibiotics according to guidelines or trial study design. Clinical data including demographics, symptoms, lung function, inflammatory profiling in blood and sputum, bacteriology including standard culture, qPCR for common pathogens and microbiomics, viruses by PCR and fungal culture were undertaken.

[0682] Laboratory methods: Blood samples were analysed for white cell count and C-reactive protein measurement as per usual care, and serum and plasma were isolated by centrifuge (10 minutes, 3000 rpm) before storage at −80° C. Sputum samples were sent for standard laboratory microscopy, culture and sensitivity analysis where patients were able to produce a sample.

[0683] Assays

[0684] Assays used for measuring the biomarkers in the samples were majority ELISAs (n=33) with some lateral flow assays (n=2) and a substrate assay for measurement of active Matrix metalloproteinase. Sample dilutions were optimised for each assay, as indicated by the table below.

TABLE-US-00003 Catalogue Assay Sample Assay Full Marker Name Supplier Number type dilution 1 CRP C reactive protein R&D DY1707 ELISA 1:100K systems 2 MPO Myeloperoxidase R&D DY3174 ELISA 1:750 systems 3 MMP9 Total Matrix R&D DY911 ELISA 1:1000 Metalloproteinase-9 systems 4 NGAL Neutrophil gelatinase- R&D DY1757 ELISA 1:100 associated lipocalin systems 5 Periostin Periostin R&D DY3548b ELISA 1:1000 systems 6 Calprotectin Calprotectin Biolegend 439707 ELISA 1:200 7 RNASE 3 Eosinophil cationic Cloud- SEB758Hu ELISA 1:200 protein clone 8 MBP Major Basic protein Cloud- SEB650Hu ELISA 1:10 clone 9 Active MMP Active protease ENZO BML-P276- Substrate 1:40 (Composite MMP 001 assay 2, 8, 9, 12, 13, 7) 10 HNE Human Neutrophil Mologic BHNEV1 ELISA 1:100 Elastase 11 Fibrinogen Fibrinogen Abcam 108841 ELISA 1:200 12 SLPI Secretory Mologic In-house ELISA 1:100 leukocyte developed protease inhibitor (see below) 13 IL-6 Interleukin-6 R&D DY206 ELISA 1:2 systems 14 Fibrinogen Fibrinogen Mologic In-house ELISA 1:2000 developed (see below) 15 fMLP N-Formylmethionine- Mologic BFMLPV1 Lateral 1:10 leucyl-phenylalanine Flow 16 Desmosine Desmosine Mologic BDESV1 ELISA 1:5 17 CC16 Club cell-16 R&D DY4218 ELISA 1:50 systems 18 TIMP1 Tissue inhibitor of R&D DY970 ELISA 1:600 metalloproteinase-1 systems 19 TIMP2 Tissue inhibitor of R&D DY971 ELISA 1:600 metalloproteinase-2 systems 20 CHI3L1 Chitinase 3 like 1 R&D DY2599 ELISA 1:500 protein systems 21 A1AT Alpha-1 antitrypsin Mologic BA1ATV1 ELISA 1:200K 22 Ac-PGP N-acetyl Proline- Mologic In-house ELISA 1:10 Glycine-Proline developed (see below) 23 B2M beta 2 Abcam 108885 ELISA 1:1000 Microglobulin 24 B2M beta 2 Mologic In-house ELISA 1:1000 Microglobulin developed (see below) 25 Cystatin C Cystatin C R&D DY1196 ELISA 1:1000 systems 26 MMP8 Total Matrix R&D DY908 ELISA 1:1000 Metalloproteinase-8 systems 27 RBP4 Retinol binding R&D DY3378 ELISA 1:100K protein-4 systems 28 HSA Human serum R&D DY1455 ELISA 1:100K Albumin systems 29 A1AT Alpha-1 antitrypsin Mologic BA1ATLF Lateral 1:200K FLow 30 IL-1b Interleukin-1β R&D DY201 ELISA 1:2 systems 31 IL-8 Interleukin-8 R&D DY208 ELISA 1:2 systems 32 Desmosine Desmosine Mologic In-house ELISA 1:5 Fragment Fragment developed (see below) 33 Large Large Elastin Mologic In-house ELISA 1:5 Elastin Fragment developed Fragment (see below) 34 Siglec 8 Siglec 8 Mologic In-house ELISA neat developed (see below) 35 sRAGE Soluble receptor Mologic In-house ELISA neat for advanced developed glycation end (see below) products 36 EDN Eosinophil-derived Alpco 30-EDNHU- ELISA 1:20 (RNASE2) neurotoxin E01

[0685] The units for each assay shown in the table above were ng/ml, with the exception of IL-6, IL-1β and IL-8 which were all pg/ml.

[0686] As shown above, the inventors have developed a number of enzyme immunoassays to detect marker levels. These are described in further detail below.

[0687] Secretory Leukocyte Protease Inhibitor (SLPI) Measurement

[0688] Disposable 96-well polystyrene plates were obtained from Fisher Scientific. The plate was sensitised with sheep anti SLPI (Mologic, CF 099 IgG cut) at 20 μg/ml in PBS overnight at ambient, 100 μl/well. After a wash step, the sensitised-well surfaces were blocked with buffer 1 (10 mM phosphate buffered saline pH7.5, supplemented with 1% (w/v) BSA) with 120 μl/well for 1 hour at room temperature.

[0689] Assay running procedure: recombinant SLPI (R&D systems cat. 1274-P1) was diluted in buffer 2 (10 mM phosphate buffered saline pH7.5, supplemented with 0.1% (v/v) Tween20 and 1% (w/v) BSA) to give concentrations between 0.781 and 50 ng/ml (1 in 2 serial dilution) to generate the standard curve. The standard and sample (diluted 1 in 100 in buffer 2) was added to the plate 100 μl/well after a wash step and incubated for 1.5 hours at room temperature with gentle agitation. After a further wash step, mouse anti-SLPI (Alere, 431) alkaline phosphatase conjugate at 1 in 2500 diluted in sample diluent were added 100 μl/well and incubated for 1 hour at room temperature with gentle agitation. After the final plate wash, the colour reaction was initiated with the addition of 100 μl of pNPP solution to each well. The absorbance was measured at 405 using an Omega plate reader and the standard curve was approximated in a sigmoid 4 parameter logistic model.

[0690] Fibrinogen Measurement

[0691] Disposable 96-well polystyrene plates were obtained from Fisher Scientific. The plate was sensitised with sheep anti Fibrinogen (Mologic, CF1765 affinity purified) at 2 μg/ml in PBS overnight at ambient, 100 μl/well. After a wash step, the sensitised-well surfaces were blocked with buffer 1 (10 mM phosphate buffered saline pH7.5, supplemented with 1% (w/v) BSA) with 120 μl/well for 1 hour at room temperature.

[0692] Assay running procedure: Fibrinogen (Scipac) was diluted in buffer 2 (10 mM phosphate buffered saline pH7.5, supplemented with 0.1% (v/v) Tween20 and 1% (w/v) BSA) to give concentrations between 0.625 and 40 ng/ml (1 in 2 serial dilution) to generate the standard curve. The standard and sample (diluted 1 in 2000 in buffer 2) was added to the plate 100 μl/well after a wash step and incubated for 1 hour at room temperature with gentle agitation. After a further wash step, sheep anti Fibrinogen (Mologic, CF1766) alkaline phosphatase conjugate at 1 in 4000 diluted in sample diluent were added 100 μl/well and incubated for 1 hour at room temperature with gentle agitation. After the final plate wash, the colour reaction was initiated with the addition of 100 μl of pNPP solution to each well. the absorbance was measured at 405 using an Omega plate reader and the standard curve was approximated in a sigmoid 4 parameter logistic model.

[0693] B2M Measurement

[0694] Disposable 96-well polystyrene plates were obtained from Fisher Scientific. The plate was sensitised with sheep anti-bet-2-Microglobulin (B2M) (Ig Innovations, NS15 affinity purified) at 1 μg/ml in PBS overnight at ambient, 100 μl/well. After a wash step, the sensitised-well surfaces were blocked with buffer 1 (10 mM phosphate buffered saline pH7.5, supplemented with 1% (w/v) BSA) with 120 μl/well for 1 hour at room temperature.

[0695] Assay running procedure: B2M (Scipac) was diluted in buffer 2 (10 mM phosphate buffered saline pH7.5, supplemented with 0.1% (v/v) Tween20 and 1% (w/v) BSA) to give concentrations between 0.01 and 50 ng/ml (1 in 4 serial dilution) to generate the standard curve. The standard and sample (diluted 1 in 1000 in buffer 2) was added to the plate 100 μl/well after a wash step and incubated for 1 hour at room temperature with gentle agitation. After a further wash step, sheep anti B2M (Ig Innovations, NS16) HRP conjugate at 1 in 20,000 diluted in sample diluent were added 100 μl/well and incubated for 1 hour at room temperature with gentle agitation. After the final plate wash, the colour reaction was initiated with the addition of 100 μl of OPD substrate to each well. the absorbance was measured at 450 using an Omega plate reader and the standard curve was approximated in a sigmoid 4 parameter logistic model.

[0696] Siglec 8 Measurement

[0697] Disposable 96-well polystyrene plates were obtained from Fisher Scientific. The plate was sensitised with Sheep anti Siglec 8 (Mologic, SA122 purified against peptide MOL624) at 2 μg/ml in PBS overnight at ambient, 120 μl/well. After a wash step, the sensitised-well surfaces were blocked (buffer 3) with 120 μl/well for 1 hour at room temperature.

[0698] Assay running procedure: Recombinant SIGLEC8 binding domain (Mologic, York) was diluted in buffer 3 to give concentrations between 7.81 and 500 ng/ml to generate the standard curve. The standard and serum sample (neat) were added to the plate 100 μl/well after a wash step and incubated for 1 hour at room temperature with gentle agitation. After a further wash step, sheep anti-siglec 8 (Mologic, SA122 purified against Siglec 8) alkaline phosphatase conjugate at 1 in 2000 were added 100 μl/well and incubated for 1 hour at room temperature with gentle agitation. After the final plate wash, the colour reaction was initiated with the addition of 100 μL of pNPP solution to each well. The absorbance was measured at 405 using an Omega plate reader and the standard curve was approximated in a sigmoid 4 parameter logistic model.

[0699] Soluble Receptor for Advanced Glycation End Products (sRAGE) Measurement

[0700] Disposable 96-well polystyrene plates were obtained from Fisher Scientific. The plate was sensitised with sheep anti sRAGE (Mologic, SA056 affinity purified) at 1 μg/ml in PBS overnight at ambient, 100 μl/well. After a wash step, the sensitised-well surfaces were blocked (buffer 2) with 120 μl/well for 1 hour at room temperature.

[0701] Assay running procedure: recombinant sRAGE (Novoprotein cat. C423) was diluted in buffer 2 to give concentrations between 0.02 and 5 ng/ml (1 in 2 serial dilution) to generate the standard curve. After a wash step, the standard and sample (neat) was added to the plate 50 μl/well with 50 μl/well of sample diluent and incubated for 1.5 hours at room temperature with gentle agitation. After a further wash step, rabbit anti-sRAGE (Mologic, RA040) alkaline phosphatase conjugate at 1 in 5000 diluted in sample diluent were added 100 μl/well and incubated for 1 hour at room temperature with gentle agitation. After the final plate wash, the colour reaction was initiated with the addition of 100 μl of pNPP solution to each well. The absorbance was measured at 405 using an Omega plate reader and the standard curve was approximated in a sigmoid 4 parameter logistic model.

[0702] Ac-PGP, Desmosine and LEF

[0703] Ac-PGP, Desmosine and LEF levels were measured using an in-house developed ELISA lateral flow assay based on a competition principle, where free marker in the sample competed with bound marker on a solid phase for a sheep polyclonal antibody conjugated to alkaline phosphatase. After washing, an alkaline phosphate enzyme substrate was added and subsequent colour was measured at 405 nm.

[0704] Results

[0705] Correlations with Eosinophil and Neutrophil Levels—High Eosinophil/Low Neutrophil Group

[0706] This subgroup of COPD samples consisted of samples collected from patients with high eosinophil and low neutrophil levels (1 sample with a neutrophil level of 10.85 which was just above the cut-off of 10 (×10.sup.9 cells/L)). The eosinophil cut-off level was 0.3 (×10.sup.9 cells/L) with levels of eosinophil ranging from 0.34 to 2.09. This is shown in the table below:

TABLE-US-00004 BEAT-COPD (SERUM) n status Range Neutrophil 38 Low 2.5-8.96 1 High 10.85 Eosinophil 0 Low — 39 High 0.34-2.09 Total 39

[0707] Multiple linear regression was performed using SPSS (version 21) for all analysis. The Automated Linear Modelling function in SPSS provides data transformation by trimming outliers. This was employed to generate models to predict neutrophil and eosinophil levels by combination of markers using a stepwise method with entry and removal of markers based on the information criterion (AICC).

[0708] Model 1: For the eosinophil correlation, a combination of 5 biomarkers produced an R.sup.2 of 0.752. The 5 biomarkers with significant levels in this model were: EDN (<0.001), MMP9 (<0.001), HNE ((<0.001), NGAL (0.001) and MBP (0.020) (in level of importance). Of note is that HNE and NGAL are negatively correlated with eosinophil levels. The results are also shown in FIG. 14.

[0709] Model 2: For the neutrophil correlations a combination of 5 biomarkers produced an R.sup.2 of 0.489. The 5 biomarkers with significance levels in this model were: Calprotectin (0.00), MBP (0.003), MMP9 (0.012), CRP (0.043) and NGAL (0.057) (in level of importance). Of note is that MBP, CRP and NGAL were negatively correlated with neutrophil levels. The results are also shown in FIG. 15.

[0710] Conclusion: The models generated significantly correlate with eosinophil and neutrophil levels. Thus, the models can be used to predict eosinophil and neutrophil levels.

[0711] Correlations with Neutrophil Levels—Mixed Levels of Low and High Eosinophil and Neutrophil Group

[0712] This subgroup of COPD samples consisted of samples collected from patients with mixed levels of high and low eosinophil and neutrophil levels. The neutrophil cut-off level was 10 (×10.sup.9 cells/L) and the neutrophil levels ranged from 2.5-18.96. The eosinophil biomarkers selected from example 1 were not available for all samples; therefore only neutrophil correlations were undertaken in this analysis. This is shown in the table below:

TABLE-US-00005 BEAT-COPD (SERUM) n status Range Neutrophil 38 Low 2.5-8.96 22 High 10-18.94 Eosinophil 17 Low 0.03-0.28 43 High 0.3-2.09 Total 60

[0713] Multiple linear regression was performed using a forward stepwise function using SPSS for all analysis. For each model developed the optimal sensitivity and Specificity was calculated based on the receiver operating characteristic (ROC) curve and area under the curve (AUC).

[0714] There were 3 models generated with a range of 3-6 biomarkers with similar R.sup.2 values: [0715] Model 3: MBP, Calprotectin and A1AT (as measured by Lateral flow)—producing an R.sup.2 of 0.514. Of note is that MBP, and A1AT were negatively correlated with neutrophil levels. [0716] Model 4: MBP, Calprotectin, A1AT (as measured by Lateral flow), MMP9 and CRP—producing an R.sup.2 of 0.518. Of note is that MBP, A1AT and CRP were negatively correlated with neutrophil levels. [0717] Model 5: MBP, Calprotectin. A1AT (as measured by Lateral flow), MMP9, CRP and NGAL—producing an R.sup.2 of 0.514. Of note is that MBP, A1AT and CRP were negatively correlated with neutrophil levels.

[0718] For all three models an AUC of approximately 0.93 was obtained. The sensitivity for all models using different cut off levels were 90.9% whereas the specificity varied slightly with the best result of 94.7% obtained from model 2 (slightly improved from the other two models). The results for models 3-5 are shown in FIGS. 16-18 respectively.

[0719] Conclusion: The models generated significantly correlate with neutrophil levels, with similar R.sup.2 values, AUC and sensitivity and specificity performance. The results indicate that the core biomarkers were MBP, Calprotectin and A1AT, with minor performance improvement with the addition of MMP9 and CRP. Thus, the models can be used to predict neutrophil levels.

[0720] Correlations with Neutrophil Levels—Mixed Levels of Low and High Eosinophil and Neutrophil Group—Larger Group Analysis and Sub Analysis

[0721] BEAT-COPD (n=601) samples were subdivided as shown in the table below according to eosinophil and neutrophil levels. The groups were defined using a cut-off of 0.3×10.sup.9 cells/L for eosinophils and 10×10.sup.9 cells/L for neutrophils.

TABLE-US-00006 BEAT-COPD (SERUM) n status Range Subgroup 1 402 Neutrophil Low 1.01-9.98 (L/L) Eosinophil low 0-0.29 Subgroup 2 172 Neutrophil Low 1.82-9.92 (L/H) Eosinophil High 0.3-2.09 Subgroup 3 17 Neutrophil High 10.66-18.94 (H/L) Eosinophil Low 0.03-0.28 Subgroup 4 5 Neutrophil High 10-10.99 (H/H) Eosinophil High 0.3-0.68 Total 601

[0722] Multiple linear regression was performed using a forward stepwise function using SPSS for all analysis. For each model developed the optimal sensitivity and specificity was calculated based on the ROC curve and AUC.

[0723] Multiple linear regression models were generated from data from all groups (n=601) and from a further stratified group—exclusion of sub-group 2 which contained all the samples with ‘high eosinophil’ levels (n=402). The developed models were then characterised on a further subgroup which removed all the grey zone samples—those samples with neutrophil levels between 5-10. This new group consisted of total number of 247 samples.

[0724] Model 6 was generated from all 601 samples and characterised on the other 2 subgroups described in the methods. [0725] Model 6: MMP9, Calprotectin, HNE, CRP and A1AT—producing an R.sup.2 of 0.308. Of note is that HNE and CRP were negatively correlated with neutrophil levels (see FIG. 19). [0726] N=601, ROC AUC of 0.8071, sensitivity 73%, specificity 74% [0727] N=402, ROC AUC of 0.8143, sensitivity 77%, specificity 75% [0728] N=247, ROC AUC of 0.9046, sensitivity 82%, specificity 80% (FIG. 20)

[0729] Models 7-9 were created on the newly defined group data (removed sub-group 2) and characterised on the further stratified group (removed grey-zone samples). [0730] Model 7: MMP9, Calprotectin, HNE and CRP— producing an R.sup.2 of 0.33. Of note is that HNE and CRP were negatively correlated with neutrophil levels (see FIG. 21). [0731] N=402, ROC AUC of 0.8263, sensitivity 77%, specificity 76% [0732] N=247, ROC AUC of 0.8948, sensitivity 82%, specificity 78% [0733] Model 8: MMP9, Calprotectin, HNE, CRP, A1AT (LF) and NGAL—producing an R.sup.2 of 0.334. Of note is that HNE, CRP and A1AT were negatively correlated with neutrophil levels (see FIG. 22). [0734] N=402, ROC AUC of 0.8341, sensitivity 77%, specificity 78% [0735] N=247, ROC AUC of 0.8945, sensitivity 82%, specificity 79% [0736] Model 9: MMP9, Calprotectin, HNE, CRP and A1AT (LF)—producing an R.sup.2 of 0.332. Of note is that HNE, CRP and A1AT were negatively correlated with neutrophil levels (see FIG. 23). [0737] N=402, ROC AUC of 0.8301, sensitivity 77%, specificity 78% [0738] N=247, ROC AUC of 0.8936, sensitivity 82%, specificity 79%

[0739] The results are summarised in the two following tables. The first table below describes the results of all 4 models tested on different stratified groups. The cohorts were split into 2 groups based on neutrophil levels with a cut-off of 10. Model 6 which was derived from all 601 samples was applied to all data (all 601) samples and a further stratified sample which had removed all high eosinophil samples in the low group.

TABLE-US-00007 Total Number Low Number High Mann Whitney Model Biomarkers Number Neutrophil Neutrophil test p value AUC Sens Spec Cut off Model 6 MMP9 Calprotectin 601 579 22 <0.0001 0.8071 72.73% 73.92% >6.33 HNE CRP A1AT Model 6 MMP9 Calprotectin 424 402 22 <0.0001 0.8143 77.27% 74.63 >6.305 tested on HNE CRP A1AT stratified cohort Model 7 MMP9 Calprotectin 424 402 22 <0.0001 0.8263 77.27% 75.87 >6.555 HNE CRP Model 8 MMP9 Calprotectin 424 402 22 <0.0001 0.8341 77.27% 77.61 >6.685 HNE CRP A1AT (LF) NGAL Model 9 MMP9 Calprotectin 424 402 22 <0.0001 0.8301 77.27% 78.11% >6.705 HNE CRP A1AT (LF)

[0740] The next table (below) describes the results of all 4 models as applied to a second stratified group which removed all grey zone samples (neutrophil levels 5-10) from the low group.

TABLE-US-00008 Total Number Low Number High Mann Whitney Model Biomarkers Number Neutrophil Neutrophil test p value AUC Sens Spec Cut off Model 6 MMP9 Calprotectin 247 225 22 <0.0001 0.9046 81.82% 80% >5.85 HNE CRP A1AT Model 7 MMP9 Calprotectin 247 225 22 <0.0001 0.8948 81.82% 77.78% >5.86 HNE CRP Model 8 MMP9 Calprotectin 247 225 22 <0.0001 0.8945 81.82% 78.67% >5.865 HNE CRP A1AT (LF) NGAL Model 9 MMP9 Calprotectin 247 225 22 <0.0001 0.8936 81.82% 78.67% >5.865 HNE CRP A1AT (LF)

[0741] Conclusion: There are clear linear regression models that can be used for neutrophil level prediction. From 33 biomarkers this is possible with a combination of key 4 biomarkers MMP9, Calprotectin, HNE and CRP, with some added value by the addition of A1AT and NGAL. When removing the ‘grey-zone’ samples i.e. those with neutrophil values between 5 and 10, this improves the performance as would be expected with standard variation around a single cut-off value.

Example 3—Eosinophil and Neutrophil Levels in Stable, Exacerbation and Mixed Samples and Marker Correlations

[0742] A total of 362 samples were analysed with a median and interquartile range of 5.18 (4.0-6.3) for blood neutrophil levels and 0.22 (0.1-0.4) for eosinophil levels, as shown in the table below.

TABLE-US-00009 Neutrophil Eosinophil Minimum 1.36 0 25% Percentile 4.048 0.13 Median 5.175 0.22 75% Percentile 6.268 0.3625 Maximum 12.46 1.13

[0743] Further sub group analysis was performed, stable group (n=322) exacerbation group (n=40) presented different neutrophil and eosinophil characteristics (see table below). Evaluation of neutrophil and eosinophil levels with discrimination between stable and exacerbation states produced a Mann Whitney test p value of 0.0002 and 0.5591 respectively. Thus, neutrophil levels significantly increased in exacerbation samples as compared with stable samples whereas eosinophil levels showed no significant change.

TABLE-US-00010 Neutrophil Eosinophil Stable Exacerbation Stable Exacerbation Minimum 1.36 2.63 0 0.03 25% Percentile 3.998 5.008 0.13 0.135 Median 4.99 6.08 0.225 0.22 75% Percentile 6.173 7.998 0.38 0.29 Maximum 10.85 12.46 1.13 0.88

[0744] Biomarker Data

[0745] Biomarkers that Correlate with Neutrophil Levels

[0746] A Spearman's r value >0.4 was deemed to be significant. To note, CRP levels measured by the hospital labs at time of collection correlated with the exacerbation samples only with an r-value of 0.55 with exacerbation samples and 0.20 with stable samples. Calprotectin was the only marker that correlated with neutrophil levels in both stable and exacerbation states.

TABLE-US-00011 Combined stable and exacerbation Stable Exacerbation Biomarker data only only IL-6 0.182 0.144 0.498 Calprotectin 0.587 0.566 0.578 MPO 0.325 0.294 0.957 MMP9 0.524 0.256 0.529 NGAL 0.359 0.327 0.452 IL1b 0.098 0.085 0.350 IL8 0.067 0.064 0.508 CRP 0.282 0.256 0.393

[0747] Biomarkers that Correlate with Eosinophil Levels

[0748] A Spearman's r value >0.4 was deemed to be significant. To note, other promising biomarkers i.e. EDN were not measured. MBP correlated with eosinophil levels in both stable and exacerbation states.

TABLE-US-00012 Combined stable and exacerbation Stable Exacerbation Biomarker data only only MBP 0.723 0.73 0.69

[0749] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Example 5—Biomarkers for Treatment Stratification for COPD Patients in Response to an Exacerbation—Large Study

[0750] Samples

[0751] A total of 292 patients were recruited into the study (44.7% Males). All samples were collected from blood of patients diagnosed with COPD and during an COPD exacerbation.

[0752] The data exists in a matrix with 16 biomarkers measurements. The frequency of measurements varied between the 2 classes for the Neutrophil model and the Eosinophil model. The Eosinophil model contained 70 high cases and 192 low cases, and the Neutrophil model contained 75 high and 187 low cases.

[0753] The cut-off used to discriminate between low and high eosinophil levels is 300 cells/μL and low and high neutrophil levels is 1000 cells/μL.

[0754] Results and Analysis

[0755] A. Preliminary Biomarker Selection

[0756] Neutrophil Subset

[0757] The levels of 16 biomarkers in blood samples from patients with low and high neutrophil levels were measured. The biomarker with the greatest significant difference between the low and high subgroups is MMP9 with a p value <0.0001 and an AUC of 0.75 (95% Cl 0.68-0.82 p value <0.0001). Additional biomarkers with a positive correlation are MMP8, MPO, PCT, CRP, HNE, NGAL and Calprotectin and with a negative correlation is LTB4 as determined by AUC values above or below 0.5.

TABLE-US-00013 Neutrophil < 1000 Neutrophil > 1000 t test ROC Biomarker Unit Median IQR Median IQR p value AUC 95 CI sig HNE ng/ml 10.8 5.8-17.0 13.8 8.1-28.9 0.000729544 0.59 0.51-0.67 0.01800398 RNASE3 ng/ml 5.1 2.3-15.7 5.1 2.0-9.6 0.775587778 0.48 0.40-0.56 0.568089722 Lactoferrin ng/ml 21.4 7.4-34.8 20.5 5.4-39.4 0.60217932 0.50 0.42-0.58 0.929574107 Calprotectin ng/ml 16239 7764-40000 26731 9553-44454 0.043347224 0.59 0.51-0.66 0.028613207 EDN ng/ml 15.5 9.5-25.1 13.6 10.9-21.7 0.292616197 0.47 0.40-0.55 0.520214613 LTB4 pg/ml 70.8 30.5-117.3 31.6 0-91.2 0.017534999 0.39 0.31-0.46 0.003685023 MBP ng/ml 1864 1150-2681 1648 927.7-3272 0.580089356 0.50 0.42-0.58 0.947509512 SuPAR ng/ml 2099 1672-2470 2275 1725-2616 0.282353394 0.56 0.48-0.64 0.162426795 CRP ng/ml 9820 3543-22614 16659 6461-41237 0.002670766 0.60 0.53-0.68 0.008101424 MMP8 pg/ml 16004 9420-30605 32699 14820-59998 2.40476E−05 0.67 0.59-0.74 2.13399E−05 MMP9 ng/ml 488 307.6-822.3 1229 572-1774 1.98043E−11 0.75 0.68-0.82 2.06812E−10 MPO pg/ml 74848 54287-109333 105585 78555-165884 0.00013088 0.66 0.58-0.73 7.75398E−05 NGAL ng/ml 286.8 193.2-391.2 355.1 272.3-451.7 0.005262488 0.63 0.56-0.71 0.000796647 PCT ng/ml 19.8 0-52.2 41.5 12.5-108 0.007506713 0.62 0.54-0.69 0.003059904 A1AT mg/ml 20.9 11.8-1254283 16.7 10.7-1161259 0.09441043 0.45 0.37-0.52 0.166257366 IgE ng/ml 299.2 0-833.6 490.2 189.7-962.3 0.906877455 0.57 0.50-0.64 0.078340166

[0758] The findings may be summarised as shown below.

TABLE-US-00014 ROC Biomarker p value AUC significance HNE 0.0007 0.59 0.018 RNASE3 0.7756 0.48 0.5681 Lactoferrin 0.6022 0.50 0.9296 Calprotectin 0.0434 0.59 0.0286 EDN 0.2926 0.47 0.5202 LTB4 0.0175 0.39 0.0037 MBP 0.5801 0.50 0.9475 SuPAR 0.2824 0.56 0.1624 CRP 0.0027 0.60 0.0081 MMP8 2.405E−05 0.67 2.134E−05 MMP9 1.980E−11 0.75 2.068E−10 MPO 0.0001 0.66 7.754E−05 NGAL 0.0053 0.63 0.0008 PCT 0.0075 0.62 0.0031 A1AT 0.0944 0.45 0.1663 IgE 0.9069 0.57 0.0783

[0759] Eosinophil Subset

[0760] The levels of 16 biomarkers in blood samples from patients with low and high eosinophil levels were measured. The biomarker with the greatest significant difference between the low and high subgroups is EDN with a p value of <0.0001 and an AUC of 0.76 (95% Cl 0.70-0.83, p value <0.0001). Additional biomarkers with a positive correlation are RNASE3, Lactoferrin, IgE and MBP and with negative correlations are CRP, MPO and PCT as determined by the AUC values above or below 0.5.

TABLE-US-00015 Eosinophil < 300 Eosinophil > 300 t test ROC Biomarker Unit Median IQR Median IQR p value AUC 95 CI sig HNE ng/ml 12.7 6.5-19.4 9.4 5.3-18.8 0.423346882 0.43 0.35-0.51 0.093421706 RNASE3 ng/ml 4.9 2.0-10.1 6.6 3.3-30.0 0.00384342 0.60 0.52-0.68 0.013102969 Lactoferrin ng/ml 18.2 5.6-34.7 24.8 13.5-39.1 0.029402338 0.58 0.50-0.68 0.053373548 Calprotectin ng/ml 18717 7993-40000 20657 8250-40000 0.96860856 0.50 0.4-0.58 0.93538545 EDN ng/ml 12.9 9.0-19.1 25.2 13.9-35.4 1.48929E−10 0.76 0.70-0.83 9.97967E−11 LTB4 pg/ml 53.5 10.7-112.0 76.6 28.8-126.7 0.621528688 0.57 0.49-0.64 0.099324444 MBP ng/ml 1689 1024-2653 1910 1247-2876 0.040618482 0.54 0.47-0.62 0.290654269 SuPAR ng/ml 2146 1717-2541 2105 1635-2446 0.757370831 0.47 0.39-0.55 0.489599202 CRP ng/ml 13527 5091-35845 7432 1794-13823 0.00102223 0.34 0.27-0.41 4.60655E−05 MMP8 pg/ml 18822 9918-41198 15658 9997-28049 0.176637158 0.44 0.36-0.51 0.113273563 MMP9 ng/ml 631 347.5-1218 499.5 327.3-803.5 0.112433516 0.43 0.35-0.50 0.072128963 MPO pg/ml 89457 59500-127266 69462 46198-102904 0.005925688 0.38 0.30-0.46 0.002862553 NGAL ng/ml 310.2 226.7-417.9 326 220.6-463.9 0.80965567 0.53 0.45-0.61 0.465047524 PCT ng/ml 25.5 4.9-73.3 20.1 3.0-60.8 0.044973392 0.45 0.37-0.53 0.227843769 A1AT mg/ml 18.8 11.6-1237821 20.8 10.6-1195161 0.487902417 0.50 0.42-0.58 0.986769514 IgE ng/ml 321.3 0-738.8 491.5 0-1242 0.001351863 0.57 0.49-0.66 0.063536059

[0761] The findings may be summarised as shown below.

TABLE-US-00016 ROC Biomarker p value AUC significance HNE 0.4233 0.43 0.0934 RNASE3 0.0038 0.60 0.0131 Lactoferrin 0.0294 0.58 0.0534 Calprotectin 0.9686 0.50 0.9354 EDN 1.489E−10 0.76 9.98E−11 LTB4 0.6215 0.57 0.0993 MBP 0.0406 0.54 0.2907 SuPAR 0.7573 0.47 0.4896 CRP 0.0010 0.34 4.607E−05 MMP8 0.1766 0.44 0.1133 MMP9 0.1124 0.43 0.0721 MPO 0.0059 0.38 0.0029 NGAL 0.8097 0.53 0.4650 PCT 0.045 0.45 0.2278 A1AT 0.4879 0.50 0.9868 IgE 0.0014 0.57 0.0635

[0762] Neural Network Analysis

[0763] A stepwise multilayer perceptron back propagation algorithm was applied to the data in order to determine the optimal panel of markers for inclusion into the final classification algorithm (Lancashire et al, 2010 A validated gene expression profile for detecting clinical outcome in breast cancer using artificial neural networks. Breast cancer research and treatment, 120 (1), pp. 83-93; and U.S. Pat. No. 8,788,444, both incorporated herein by reference).

[0764] To prevent overtraining of the model, the Monte-Carlo cross validation method was used as follows: the sample set was randomly divided into three groups: the training set, validation set and test set in a 60:20:20 ratio, respectively. The training set was used to train the model, the validation sets were used to stop training and prevent overfitting, while the test set was used to assess the accuracy of the trained model. After the model was trained once, the dataset was repeatedly divided again into the training, test and validation set, and this re-division process occurred 50 times, with new splits being randomly generated each time.

[0765] Stability Analysis

[0766] For both neutrophil and eosinophil biomarkers: 80 independent Artificial Neural Network cycles (loops) were performed each producing a diagnostic model to identify the most stable combination of features. The greater number of times the biomarkers appear in the top five the more stable the biomarkers are.

TABLE-US-00017 Number of Number of loops where loops where biomarker Sta- biomarker Sta- Neutrophil in the bility Eosinophil in the bility biomarker top 5 hits % biomarkers top 5 hits % MMP9 80 100 EDN 80 100 LTB4 48 60 MPO 66 83 MBP 39 49 SuPAR 41 51 EDN 37 46 RNASE3 30 38 A1AT 28 35 HNE 26 33 SuPAR 26 33 Calprotectin 23 29 HNE 23 29 A1AT 20 25 MMP8 18 23 PCT 18 23 CRP 15 19 MMP9 17 21 RNASE3 15 19 CRP 15 19 Lactoferrin 14 18 MBP 14 18 Calprotectin 14 18 LTB4 10 13 NGAL 14 18 Lactoferrin 8 10 MPO 10 13 MMP8 5 6 PCT 10 13 IgE 4 5 IgE 7 9 NGAL 2 3

[0767] Based on the stability analysis, the top five biomarkers of the neutrophil and eosinophil subset were carried forward for further optimisation (denoted as neutrophil model 1 and eosinophil model 1). In addition, alternative models exploring markers further down the list of stability were taken forward (denoted as neutrophil models 2-4 and eosinophil models 2 and 3).

[0768] Neutrophil Panel

TABLE-US-00018 Model 1 Model 2 Model 3 Model 4 MMP9 MMP9 MMP9 MMP9 LTB4 LTB4 LTB4 LTB4 EDN EDN EDN EDN A1AT A1AT A1AT A1AT MBP SuPAR HNE CRP

[0769] Eosinophil Panel

TABLE-US-00019 Model 1 Model 2 Model 3 EDN EDN EDN MPO MPO MPO RNASE3 RNASE3 RNASE3 HNE HNE SuPAR SuPAR Calprotectin Calprotectin

[0770] B. Preliminary Model Selection

[0771] Not all combinations of biomarkers were successful after further analysis and recreation of the models. For the neutrophil panel, models 2 and 4 were taken forward for optimisation and for the eosinophil panel, model 1 was taken forward for further optimisation. Criteria included an AUC>0.8, R.sup.2>0.3, sensitivity and specificity >70% (cut-off 0.5).

TABLE-US-00020 Neutrophil models Eosinophil models AUC Sensitivity Specificity AUC Sensitivity Specificity Model 1 0.72 85% 50% 0.92 88% 80% Model 2 0.89 87% 77% 0.69 57% 77% Model 3 0.69 54% 79% 0.80 72% 65% Model 4 0.84 76% 74%

[0772] C. Optimisation of Models

[0773] Multiple architectures of increasing complexity were assessed for performance. Once the most stable combination of features was identified further analysis was performed to recreate that model. The number of hidden nodes were optimised to maximise the predictive performance. Proprietary software developed in OpenCL, R python and Neurosolutions software was used to develop the algorithm.

[0774] Performance

[0775] The performance was assessed using the ROC curve and AUCs. In general, an AUC of 0.5 suggests no discrimination (i.e., ability to diagnose patients with and without the disease or condition based on the test), 0.7 to 0.8 is considered acceptable, 0.8 to 0.9 is considered excellent, and more than 0.9 is considered outstanding.

[0776] Two neutrophil models (model 2 and model 4) were developed, the results from the training, validation and test data are shown in FIG. 24.

[0777] Model 2 consists of 5 biomarkers: Matrix metalloproteinases 9 (MMP9), Eosinophil-derived neurotoxin (EDN), Soluble urokinase-type plasminogen activator receptor (SuPAR), Leukotriene B4 (LTB4) and Alpha-1 antitrypsin (A1AT). A significant p value <0.05 was obtained for all three data sets. The AUC for the training and validation sets was 0.9 and for the test set was 0.7. The combined AUC value was 0.84 (95% Cl 0.78-0.90) p<0.0001. At a cut off of 0.5 the sensitivity and specificity were 80% and 77%, respectively.

[0778] Model 4 consists of 5 biomarkers: Matrix metalloproteinases 9 (MMP9), C-reactive protein (CRP), EDN, A1AT and LTB4. A significant p value <0.05 was obtained for all three data sets. The AUC for the training and validation sets was 0.84 and for the test set was 0.87. The combined AUC value was 0.84 (95% Cl 0.78-0.90) p<0.0001. At a cut off of 0.4718 the sensitivity and specificity were 80% and 76%, respectively.

[0779] One eosinophil model (model 1) was developed, the results from the training, validation and test data are shown in FIG. 25.

[0780] Model 1 consists of 5 biomarkers: EDN, Eosinophil cationic protein (RNASE3), SuPAR, Human neutrophil elastase (HNE) and Myeloperoxidase (MPO). A significant p value <0.05 was obtained for all three data sets. The AUC for the training and validation sets was 0.94 and 0.92, respectively, and for the test set was 0.81. The combined AUC value was 0.9 (95% Cl 0.86-0.95) p<0.0001. At a cut off of 0.4843 the sensitivity and specificity were 90% and 84%, respectively.

[0781] Conclusion

[0782] The two neutrophil models and the eosinophil models how excellent performance.

[0783] D. Comparison of the Performance of Individual Biomarkers and the Optimised Neutrophil and Eosinophil Models

[0784] The performance of individual biomarkers from neutrophil models 2 and 4 and eosinophil model 1 was compared to the overall performance of neutrophil models 2 and 4 and eosinophil model 1 using the AUCs values.

TABLE-US-00021 Test Result Variable(s) AUC Std. Error.sup.a Neutrophil model 2 0.841 0.032 MMP9 0.755 0.042 LTB4 0.377 0.046 EDN 0.478 0.042 A1AT 0.456 0.046 SuPAR 0.586 0.046 Neutrophil model 4 0.843 0.030 MMP9 0.748 0.042 LTB4 0.388 0.046 EDN 0.485 0.042 A1AT 0.462 0.045 CRP 0.604 0.045 Eosinophil model 1 0.903 0.024 EDN 0.773 0.035 MPO 0.399 0.044 RNASE3 0.615 0.044 HNE 0.448 0.046 SuPAR 0.475 0.044 .sup.aunder the nonparametric assumption

[0785] The overall performance of a panel of biomarkers is considerably better than the performance of individual biomarkers. The results demonstrate that the performance is enhanced by the combination of the biomarkers.

[0786] The combination of stability assays and further optimisation experiments shows that an advantageous core set of biomarkers for the neutrophil panel includes MMP9 and EDN, and at least one of: LTB4, CRP, A1AT and SuPAR, whereas an advantageous core set of biomarkers for the eosinophil panel includes EDN, RNASE3 and MPO.

TREATMENT STRATIFICATION FOR AN EXACERBATION OF INFLAMMATION

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