Biochemical Markers for Pulmonary and Other Diseases

Abstract

A method of bioassay for the quantification of peptide fragments elevated in lung diseases such as COPD, SCC, or IPF, comprising a neo-epitope formed at a cleavage site by cleavage in vivo of elastin by a proteinase by contacting a sample with an antibody having specific binding affinity for a the neo-epitope amino acid sequence and determining the level of binding, where the antibody binds one of the following terminal sequences:

TABLE-US-00001 ...FGPGVV ...VPGLGV IKAPKL...
and antibodies and immunoassay kits for use in such methods.

Claims

1. An immunological binding partner specifically reactive with one of the following C-terminal sequences derived from elastin: TABLE-US-00005 (SEQIDNO:2) FGPGVV||'334 (SEQIDNO:3) VPGLGV||'602 or reactive with the following N-terminal sequence derived from elastin: TABLE-US-00006 (SEQIDNO:4) 208'||IKAPKL wherein the immunological binding partner not specifically reactive with the sequence ... ... ...FGPGVVG (SEQ ID NO:5) if it is reactive with ... ... ...FGPGVV (SEQ ID NO: 2), and is not specifically reactive with ... ... ...VPGLGVG (SEQ ID NO: 6) if it is reactive with ... ... ...VPGLGV (SEQ ID NO: 3), and is not specifically reactive with PIKAPKL... ... (SEQ ID NO: 7) if it is reactive with IKAPKL... ... (SEQ ID NO: 4).

2. An immunological binding partner as claimed in claim 1, which is a monoclonal antibody or a binding fragment thereof.

3. A cell line producing a monoclonal antibody as claimed in claim 1.

4. An immunoassay kit comprising an immunological binding partner as claimed in claim 1, and a competition agent which binds said immunological binding partner, and optionally one or more of a wash reagent, a buffer, a stopping reagent, an enzyme label, an enzyme label substrate, calibration standards, an anti-mouse antibody and instructions for conducting an assay using said kit.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0036] FIG. 1 shows results obtained in Example 3 of an ELISA and shows a measured B/B.sub.0 ratio compared to the concentration of free peptide (SEQ ID NO: 8) and elongated peptide (NB590) (GGPGFGPGVVG, SEQ ID NO: 16) after incubation with the NB590 monoclonal antibody. There is almost no reactivity towards the elongated peptide.

[0037] FIG. 2 shows results characterising the NB590 monoclonal antibody obtained in Example 3.

[0038] FIG. 3 shows concentrations of the NB590 free peptides in serum from COPD, IPF and squamous cell carcinoma (SCC) of patients as well as controls obtained in Example 3. * denotes a p-value 0.05 and ** denotes a p-value 0.05. The standard error of the mean is also shown.

[0039] FIG. 4 shows results obtained in Example 4 of an ELISA and shows a measured B/B.sub.0 ratio compared to the concentration of free peptide (SEQ ID NO: 9) and elongated peptide (NB592) (VGAGVPGLGVG, SEQ ID NO: 17) after incubation with NB592 monoclonal antibody. There is almost no reactivity towards the elongated peptide.

[0040] FIG. 5 shows results characterising the NB592 monoclonal antibody obtained in Example 4.

[0041] FIG. 6 shows concentrations of the NB592 free peptides in serum from COPD, IPF and squamous cell carcinoma (SCC) of patients as well as controls obtained in Example 4. The standard error of the mean is shown. Free peptide concentrations were applied as reference.

[0042] FIG. 7 shows results obtained in Example 5 of an ELISA and shows a measured B/B.sub.0 ratio compared to the concentration of free peptide (NB593) (SEQ ID NO: 11) and elongated peptide (AGLGGVLGGA, SEQ ID NO: 13) after incubation with the NB593 monoclonal antibody. There is almost no reactivity towards the elongated peptide.

[0043] FIG. 8 shows results characterising the NB593 monoclonal antibody obtained in Example 5.

[0044] FIG. 9 shows the results of testing NB593 antibody on various disease samples from patients as well as healthy controls. The NB593 fragments were undetectable in serum.

[0045] FIG. 10 shows the results of an ELISA conducted in Example 6 and shows the percent inhibition of the signal by free peptide (NB595) (SEQ ID NO: 12) and elongated peptide (GVFYPGAGLGA, SEQ ID NO: 14) of the NB595 monoclonal antibody. There is almost no inhibition by the elongated peptide.

[0046] FIG. 11 shows results characterising the NB595 monoclonal antibody obtained in Example 6.

[0047] FIG. 12 shows concentrations of the NB595 fragments in serum from COPD patients and healthy controls. The NB595 fragment is not elevated in COPD.

[0048] FIG. 13 shows the results of an ELISA conducted in Example 7 and shows the percent inhibition of signal by free peptide (NB599) (SEQ ID NO: 10) and elongated peptide (PIKAPKLPGGY, SEQ ID NO: 15) of the NB599 monoclonal antibody. There is a low reactivity with the antibody towards the elongated peptide compared to the free peptide.

[0049] FIG. 14 shows results characterising the NB599 monoclonal antibody which were obtained in Example 7.

[0050] FIG. 15 shows the concentration of the NB599 elastin fragments in serum from controls, squamous cell carcinoma (SCC), COPD and IPF patients. There is a significant difference between control and SCC and between COPD and controls. There is also a statistical difference between IPF and control.

DETAILED DESCRIPTION OF THE INVENTION

[0051] The invention will be further described and illustrated with reference to the following examples illustrating the principles and practice of preferred embodiments. The Examples make reference to the results presented in the accompanying drawings.

EXAMPLE 1

Selection of Peptides for Immunizations

[0052] The following peptides were selected for immunisation:

TABLE-US-00004 NB590: (SEQIDNO:8) AminoAcid#325'GGPGFGPGVV||'334(HNEderived) NB592: (SEQIDNO:9) AminoAcid#593'VGAGVPGLGV||'602(HNEderived) NB599: (SEQIDNO:10) AminoAcid#208'||IKAPKLPGGY'217(MMP7derived) NB593: (SEQIDNO:11) AminoAcid#743'||GLGGVLGGA'752(HNEderived) NB595: (SEQIDNO:12) AminoAcid#41'||VFYPGAGLGA'50 (MMP7derived)
The cleavage sites involved are amongst those previously determined by others (He, Turino, & Lin 2010d; Heinz et al. 2011a). We chose the immunisation peptides as the first 10 amino acids either downstream or upstream of the cleavage sites.

EXAMPLE 2

Development of Monoclonal Antibodies

[0053] Six 4-6 week old Balb/C mice were immunized subcutaneously in the abdomen with 200 L emulsified antigen (50 g per immunization) using mentioned immunogen peptide conjugated to KLH at its N-terminus for C-terminal epitopes and at its C-terminus for N-terminal epitopes according to a standard procedure. Immunizations were continued until stable titer levels were obtained.

[0054] The mouse with the highest titer was selected for fusion and boosted intravenously with 50 g immunogen in 100 L 0.9% sodium chloride solution three days before isolation of the spleen for cell fusion.

[0055] The fusion procedure was followed by known procedures (Gefter, Margulies, & Scharff 1977). Supernatants were collected and monoclonal antibodies were purified using Protein G columns according to manufacturer's instructions (GE Healthcare Life Science).

Selection of Neo-Epitope Specific Monoclonal Antibodies

[0056] Antibodies were selected upon their peptide-specificity using the homologous peptide used for immunisation (selection peptide) in non-conjugated form and de-selected against an elongated version of the peptide extended by one amino acid of the elastin sequence beyond the cleavage site thereby ensuring that only antibodies with a neo-epitope specificity was selected for further analysis.

[0057] Subsequently, the reactivity of the antibodies against intact, native and cleaved elastin was tested.

[0058] And finally, serum samples from healthy and diseased subjects were tested in the above mentioned assay protocol.

Assay Protocol

[0059] A 96-well streptavidin plate was coated with screening peptide dissolved in coater buffer and incubated for 30 minutes at 20 C. After incubation, the plate was washed five times in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2). 20 L of peptide (selection, de-selection or nonsense) or human sample were added in duplicate to appropriate wells, followed by 100 L of POD-conjugated monoclonal antibody, and then the plate was incubated for 1 hour on a shaker. After washing, 100 L tetramethylbenzidine (TMB) (Kem-En-Tec) was added and the plate was incubated for 15 minutes at 20 C. in the dark.

[0060] All the above incubation steps included shaking at 300 rpm.

[0061] The TMB reaction was stopped by adding 100 L of stopping solution (1% H.sub.2SO.sub.4). Absorbance was measured at 450 nm with 650 nm as the reference. A master calibrator prepared from the selection peptide was used as a calibration curve and plotted using a 4-parametric mathematical fit model.

[0062] All serum samples were diluted 1:2 in incubation buffer prior to measurements.

EXAMPLE 3

Monoclonal Antibody NB590

[0063] FIG. 1 shows the reactivity of the free and elongated peptide towards the NB590 antibody. A decrease in absorbance is seen together with an increase in free peptide concentration. This is due an increased reaction with the free peptide instead of the screening peptide in the wells. This was not the case for the elongated peptide as the stable B/B.sub.0 levels indicated a very low or no reaction between the NB590-01 antibody and the elongated peptide.

[0064] Subsequently, the NB590 antibody was tested towards elastin (ELN), elastin cleaved in vitro with MMP2, MMP7, MMP9 and HNE and the proteases in their respective buffers (FIG. 2). The reactivity of the selected proteases in their buffers only, is also included. The NB590 monoclonal antibody clearly measures the elastin fragments derived by HNE. Free peptide concentrations were applied as reference. The NB590 antibody has a very high reactivity towards elastin cleaved with HNE. The reactivity of the NB590 antibody and intact elastin or elastin cleaved with other proteases is close to the lower limit of detection of the assay.

[0065] Finally, the NB590 antibody was tested in serum samples from patients with COPD, IPF or squamous cell carcinoma and compared to healthy controls (FIG. 3). There is a significant difference in the concentration of the NB590 fragments in COPD and squamous cell carcinoma patients compared to the healthy controls. Although a tendency for higher levels was observed, there was no statistically significant difference between controls and patients with IPF. This might be explained by the fact that this was a cross-sectional study whereas a longitudinal study of IPF patients may show a more significant difference.

EXAMPLE 4

Monoclonal Antibody NB592

[0066] FIG. 4 shows the reactivity of the free and elongated peptide towards the NB592 antibody. A decrease in absorbance is seen together with an increase in free peptide concentration. This is due an increased reaction with the free peptide instead of the screening peptide in the wells. This was not the case for the elongated peptide as the stable B/B.sub.0 levels indicated a very low or no reaction between the elongated peptide and the NB592 antibody.

[0067] Subsequently, the reactivity of the NB592 antibody was tested towards elastin, elastin cleaved in vitro with MMP2, MMP7, MMP9 and HNE and the proteases in their respective buffers (FIG. 5). The reactivity of the selected proteases in their buffers only, is also included. The NB592 monoclonal antibody clearly measures the elastin fragments derived by HNE. Free peptide concentrations were applied as reference. The NB592 antibody has a very high reactivity towards elastin cleaved with HNE. The reactivity of the NB592 antibody and intact elastin or elastin cleaved with other proteases is close to the lower limit of detection of the assay. A minor signal is shown for the HNE and its buffer alone but the signal is over 100 times smaller than the signal for elastin cleaved with HNE.

[0068] Finally, the NB592 antibody was tested in serum samples from patients with COPD, IPF or squamous cell carcinoma and compared to healthy controls (FIG. 6). Although there was a high tendency for increases in levels there were no significant differences (p<0.05) in the concentration of the NB592 fragments in COPD and squamous cell carcinoma patients compared to the healthy controls. There was no difference between controls and patients with IPF. This might be explained by the fact that this was a cross-sectional study whereas a longitudinal study of IPF patients may show a difference.

EXAMPLE 5 (COMPARATIVE)

Monoclonal Antibody NB593

[0069] FIG. 7 shows the reactivity of the free and elongated peptide towards the NB593 antibody. A decrease in absorbance is seen together with an increase in free peptide concentration. This is due an increased reaction with the free peptide instead of the screening peptide in the wells. This was not the case for the elongated peptide as the stable B/B.sub.0 levels indicated a very low or no reaction between the NB593 antibody.

[0070] Subsequently (FIG. 8), the reactivity of the NB593 antibody was tested towards elastin, elastin cleaved in vitro with HNE and the HNE buffer. The 593 monoclonal antibody clearly has a very high reactivity towards elastin fragments derived by HNE compared to the intact elastin and measures such fragments. Free peptide concentrations were applied as reference. The reactivity of the NB593 antibody towards intact elastin or the HNE buffer is very low.

[0071] And finally, FIG. 9 shows the reactivity of the NB593 antibody and human serum samples from COPD patients and healthy controls. The data shows that the fragment recognised by antibody NB593 is absent, or only present in undetectable amounts, in the serum of healthy controls and diseased.

EXAMPLE 6 (COMPARATIVE)

Monoclonal Antibody NB 595

[0072] FIG. 10 shows the reactivity of the free and elongated peptide towards the NB595 antibody. A decrease in absorbance is seen together with an increase in free peptide concentration. This is due to an increased reaction with the free peptide instead of the screening peptide in the wells. This was not the case for the elongated peptide as the stable B/B.sub.0 levels indicated a very low or no reaction with the NB595 antibody.

[0073] Subsequently (FIG. 11), the reactivity of the NB595 antibody was tested towards elastin, elastin cleaved in vitro with MMP2, MMP7, MMP9 and HNE and the proteases in their respective buffers. The 595 monoclonal antibody clearly measures the elastin fragments derived by MMP7 and has a high reactivity towards elastin cleaved with MMP7. Free peptide concentrations were applied as reference. The reactivity of the NB595 antibody towards intact elastin or elastin cleaved with other proteases is close to the lower limit of detection of the assay.

[0074] Finally, FIG. 12 shows the reactivity of the NB595 antibodies and pooled human serum samples from COPD patients and healthy controls. The data show that the NB595 fragment is not elevated in COPD patients. There is therefore limited biological relevance for this antibody.

EXAMPLE 7

NB599

[0075] FIG. 13 shows the reactivity of the free and elongated peptide towards the NB599 antibody. A decrease in absorbance is seen together with an increase in free peptide concentration. This is due an increased reaction with the free peptide instead of the screening peptide in the wells. The decrease in absorbance was significantly lower for elongated peptide as the B/B.sub.0 levels indicated weak reactions between the peptide and the NB599 antibody.

[0076] Subsequently, the reactivity of the NB599 antibody towards elastin, elastin cleaved in vitro with MMP2, MMP7, MMP9 and HNE and the proteases in their respective buffers (FIG. 14) was determined. The NB599 monoclonal antibody clearly measures the elastin fragments derived by MMP7. The NB599 antibody has a much higher reactivity towards elastin cleaved with MMP7 than elastin cleaved with other proteases. There is some reactivity towards the HNE+HNE buffer but this is still much lower than elastin cleaved with HNE.

[0077] And finally, the presence of Elastin fragments was tested in serum samples from patients with COPD, IPF or squamous cell carcinoma and compared to healthy controls (FIG. 15) using the NB599 test. There is a significant difference in the concentration of the NB599 fragments in COPD, IPF and squamous cell carcinoma patients compared to the healthy controls. Especially the squamous cell carcinoma and COPD patients had increased levels of the NB599 elastin fragment.

[0078] In this specification, unless expressly otherwise indicated, the word or is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator exclusive or which requires that only one of the conditions is met. The word comprising is used in the sense of including rather than in to mean consisting of. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

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