COLLAGEN TYPE VII ALPHA 1 ASSAY

Abstract

A method of immunoassay for detecting in a biological sample a fragment of collagen type VII alpha 1 comprising an N- or C-terminal neo-epitope, the method comprising contacting the biological sample comprising the fragment of collagen type VII alpha 1 comprising the N- or C-terminal neo-epitope with an antibody of the invention, and determining the amount of binding of the antibody

Claims

1. An antibody reactive with a fragment of collagen type VII alpha 1 comprising an N- or C-terminal neo-epitope, wherein said antibody binds to the N- or C-terminal neo-epitope.

2. An antibody as claimed in claim 1, wherein the antibody is a monoclonal antibody.

3. An antibody as claimed in claim 1, wherein said N- or C-terminal neo-epitope is comprised in a non-collagenous amino-terminal domain of collagen type VII alpha 1 or is comprised in a central collagenous triple helical domain of collagen type VII alpha 1.

4. An antibody as claimed in claim 1, wherein said antibody binds to a C-terminal neo-epitope comprised in the amino acid sequence GPPGPPGRLV-COOH (SEQ ID NO: 1).

5. An antibody as claimed in claim 1, wherein said antibody binds to a C-terminal neo-epitope comprising the amino acid sequence PPGRLV-COOH (SEQ ID NO: 2).

6. An antibody as claimed in claim 4, wherein said antibody does not recognise or bind elongated amino acid sequence GPPGPPGRLVX-COOH (SEQ ID NO: 3), wherein X is one or more amino acids of the sequence of collagen type VII alpha 1.

7. An antibody as claimed in claim 1, wherein said antibody binds to an N-terminal neo-epitope comprised in the amino acid sequence H2N-EAPRVRAQHR (SEQ ID NO: 4).

8. An antibody as claimed in claim 1, wherein said antibody binds to an N-terminal neo-epitope comprising the amino acid sequence H2N-EAPRVR (SEQ ID NO: 5).

9. An antibody as claimed in claim 7, wherein said antibody does not recognise or bind elongated amino acid sequence H2N-XEAPRVRAQHR (SEQ ID NO: 6), wherein X is one or more amino acids of the sequence of collagen type VII alpha 1.

10. A method of immunoassay for detecting in a biological sample a fragment of collagen type VII alpha 1 comprising an N- or C-terminal neo-epitope, said method comprising contacting said biological sample comprising said fragment of collagen type VII alpha 1 comprising said N- or C-terminal neo-epitope with an antibody as claimed in claim 1, and determining the amount of binding of said antibody.

11. A method as claimed in claim 10, wherein said method is used to quantify the amount of the fragment of collagen type VII alpha 1 comprising said N- or C-terminal neo-epitope in biofluids.

12. A method as claimed in claim 11, wherein said biofluid is serum, plasma, bronchoalveolar lavage fluid, sputum, exhaled breath or urine.

13. A method as claimed in claim 10, wherein said immunoassay is a competition assay or a sandwich assay.

14. A method as claimed in claim 10, wherein said immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

15. A method as claimed in claim 11, further comprising correlating the quantity of the fragment of collagen type VII alpha 1 comprising an N- or C-terminal neo-epitope determined by said method with standard collagen type VII related disease samples of known disease severity to evaluate the severity of a collagen type VII related disease.

16. A method as claimed in claim 15, wherein the collagen type VII related disease is chronic obstructive pulmonary disease (COPD) or systemic sclerosis.

17. A peptide, wherein the peptide has an N-terminal amino acid sequence corresponding to an amino acid sequence of an N-terminal neo-epitope of a fragment of collagen type VII alpha 1 comprising said N-terminal neo-epitope, or wherein the peptide has a C-terminal amino acid sequence corresponding to an amino acid sequence of an C-terminal neo-epitope of a fragment of collagen type VII alpha 1 comprising said C-terminal neo-epitope.

18. A peptide as claimed in claim 17, wherein the N-terminal neo-epitope amino acid sequence is EAPRVRAQHR (SEQ ID NO: 4) or EAPRVR (SEQ ID NO: 5).

19. A peptide as claimed in claim 17, wherein the C-terminal neo-epitope amino acid sequence is GPPGPPGRLV (SEQ ID NO: 1) or PPGRLV (SEQ ID NO: 2).

20. A peptide as claimed in claim 17, wherein the peptide is conjugated to biotin.

21-23. (canceled)

Description

FIGURES

[0045] FIG. 1 shows a calibration curve for the “C7” assay.

[0046] FIG. 2 shows the correlation between “C7” and COPD.

[0047] FIG. 3 shows the correlation between “C7” and systemic sclerosis.

[0048] FIG. 4 shows a calibration curve for the “NB677” assay.

[0049] FIG. 5. Clinical evaluation of serum C7 in systemic sclerosis. Serum C7 levels were assessed in healthy donors (n=70) and a cohort of patients with systemic sclerosis (SSc; n=119). Data are presented as Tukey's box plots. Statistical significance was evaluated by Mann-Whitney test. ***p<0.0001.

EXAMPLES

Example 1—COPD Biomarker (“C7” Assay)

Rationale

[0050] Mass spectrometry was performed on serum samples from a patient with COPD, a patient with idiopathic pulmonary fibrosis (IPF), and a healthy donor.

[0051] The initial mass spectrometry analyses identified peptides derived from collagen type VII in serum. Peptides were isolated from serum using IMAC Cu beads. Identity significance threshold for individual peptides were 51. Serum samples were analyzed using an orbitrap (OrbiB) instrument.

[0052] Fragments of collagen type VII alpha 1 comprising the C-terminal neo-epitope GPPGPPGRLV-COOH (“C7”) (SEQ ID NO: 1) were found in the COPD sample but not in the IPF or healthy donor samples. The neo-epitope corresponds to the cleavage site located between amino acids Val-Asp at positions 1709-1710 of human collagen type VII. The protease responsible for this cleavage is as yet unknown. The sequence was analyzed using BLAST and was found to be unique for the collagen type VII alpha-1 chain.

Antibody

[0053] A monoclonal antibody was raised against the C-terminal neo-epitope amino acid sequence GPPGPPGRLV-COOH (SEQ ID NO: 1).

[0054] Briefly, four to six-week-old Balb/C mice were immunized subcutaneously with 200 μL emulsified antigen and 50 μg of a C7 synthetic peptide (KLH-CGG-GPPGPPGRLV, SEQ ID NO: 9) using Freund's incomplete adjuvant. Immunizations were performed every 2.sup.nd week until stable sera titer levels were reached. The mouse with highest serum titer was selected for fusion. The mouse was rested for one month and then boosted intravenously with 50 μg C7 peptide in 100 μL 0.9% sodium chloride solution three days before isolation of the spleen for cell fusion. Mouse spleen cells were fused with SP2/0 myeloma fusion partner cells. The resulting hybridoma cells were cloned using a semi-solid medium method, transferred into 96-well microtiter plates for further growth and incubated in a CO.sub.2 incubator. Standard limited dilution was used to promote monoclonal growth.

ELISA

[0055] A competitive ELISA using the monoclonal antibody raised against C7 was performed using the following procedure:

[0056] Streptavidin-coated plates were coated with 100 μL/well of 2.5 ng/mL biotin-labeled peptide (Biotin-KKGPPGPPGRLV, SEQ ID NO: 10) diluted in assay buffer (50 mM TBS-BTB, 2 g/L NaCl, pH 8.0) and incubated at 20° C., 300 rpm shaking for 30 minutes. Plates were washed five times in washing buffer (20 nM TRIS, 50 mM NaCl, pH 7.2). Sample or standard peptide (20 μL) was added in double determinations and followed immediately by addition of 100 μL/well of 200 ng/mL HRP-labeled monoclonal antibody diluted in assay buffer and plates were incubated at 20° C., 300 rpm shaking for 3 hours. The standard peptide was a synthetic peptide (GPPGPPGRLV, SEQ ID NO: 1) with a starting concentration of 125 ng/mL and diluted 2-fold to create an 11 points calibration curve (FIG. 1). After incubation, plates were washed five times in washing buffer. A volume of 100 μL 3,3′,5,5′-tetramethylbenzidine (TMB) was added and incubated for 15 min at 20° C. in the dark. To stop the enzyme reaction of TMB, 100 mL 0.1% sulphuric acid was added and the absorbance was measured at 450 nm with 650 nm as the reference using an ELISA reader. A calibration curve was plotted using a 4-parametric mathematical fit model. Each ELISA plate included both kit control and in-house quality control samples to monitor inter-assay variation. All samples were measured within the range of the assay. All samples below the lower limit of detection (LLOD) were assigned the value of LLOD.

[0057] The technical characteristics of the C7 ELISA are as follows:

TABLE-US-00001 Technical characteristics Results Biological matrix Human serum (undiluted measurements) Measurement range 1.5-105.6 ng/mL Normal range of healthy 3.5 ng/mL serum Inter-assay variation 13% (accepted if <15%) Intra-assay variation 9% (accepted if <10%) Dilution recovery Accepted (undiluted to 1:8) Spiking recovery 166% (peptide in serum) 131% (serum in serum) Analyte stability Accepted (freeze/thaw and storage)

[0058] The ELISA was shown to be specific for the cleavage site as reactivity was seen towards the standard peptide but not to an elongated peptide (GPPGPPGRLVD, SEQ ID NO: 11), indicating that the assay does not recognize intact collagen type VII protein (FIG. 1).

Clinical Utility

COPD

[0059] Serum levels of C7 were significantly elevated in a cohort of 68 patients with COPD when compared to healthy donors (FIG. 2).

[0060] These results show the utility of the C7 assay in identifying COPD, and may prove useful in evaluating COPD, for example as a diagnostic or prognostic tool.

Systemic Sclerosis

[0061] Serum levels of C7 were significantly elevated in 20 patients with early diffuse systemic sclerosis when compared to healthy control (p=0.022) (FIG. 3). The early stage of systemic sclerosis is associated with high disease activity, whereas the late stage patients are progressing slowly. In the group of patients with early diffuse disease, a subpopulation with intermediate progression rate (defined by the skin thickness progression rate) had significantly elevated levels when compared to controls (p=0.016).

[0062] The elevated level of C7 in early stage systemic sclerosis when compared to late stage systemic sclerosis suggests that the C7 assay may be capable of differentiating between early and late stages of systemic sclerosis, thereby providing a potentially useful diagnostic and/or prognostic tool for evaluating systemic sclerosis.

Example 2 (“NB677” Assay)

[0063] The signal peptide in collagen type VII alpha-1 is found at amino acids 1-16 [12]. The N-terminal neo-epitope sequence that is formed by cleavage of the signal peptide (17‘.EAPRVRAQHR’26) was analyzed using BLAST and was found to be unique for the collagen type VII alpha-1 chain.

[0064] Following the success of the “C7” assay for COPD, it is postulated that this unique collagen type VII alpha-1 neo-epitope may also be useful in the identification and/or evaluation of COPD and/or systemic sclerosis.

Antibody

[0065] Accordingly, a monoclonal antibody was raised against the N-terminal neo-epitope amino acid sequence H.sub.2N-EAPRVRAQHR (SEQ ID NO: 4).

[0066] Briefly, four to six-week-old Balb/C mice were immunized subcutaneously with 200 μL emulsified antigen and 50 μg of a NB677 synthetic peptide (EAPRVRAQHR-GGC-KLH, SEQ ID NO: 12) using Freund's incomplete adjuvant. Immunizations were performed every 2.sup.nd week until stable sera titer levels were reached. The mouse with highest serum titer was selected for fusion. The mouse was rested for one month and then boosted intravenously with 50 μg NB677 peptide in 100 μL 0.9% sodium chloride solution three days before isolation of the spleen for cell fusion. Mouse spleen cells were fused with SP2/0 myeloma fusion partner cells. The resulting hybridoma cells were cloned using a semi-solid medium method, transferred into 96-well microtiter plates for further growth and incubated in a CO.sub.2 incubator. Standard limited dilution was used to promote monoclonal growth.

ELISA

[0067] A competitive ELISA using the monoclonal antibody was performed using the following procedure:

[0068] Streptavidin-coated plates were coated with 100 μL/well of 2.0 ng/mL biotin-labeled peptide (EAPRVRAQHR-Lys-Biotin, SEQ ID NO: 13) diluted in coating buffer (50 mM PBS-BTE, 8 g/L NaCl, 10% sorbitol) and incubated at 20° C., 300 rpm shaking for 30 minutes. Plates were washed five times in washing buffer (20 nM TRIS, 50 mM NaCl, pH 7.2). Standard peptide (20 μL) was added in double determinations and followed immediately by addition of 100 μL/well of 120 ng/mL monoclonal antibody diluted in assay buffer (25 mM PBS-BTB, 8 g/L NaCl) and plates were incubated at 4° C., 300 rpm shaking for 20 hours. The standard peptide was a synthetic peptide (EAPRVRAQHR, SEQ ID NO: 4) with a starting concentration of 100 ng/mL and diluted 2-fold to create a calibration curve (FIG. 4). After incubation, plates were washed five times in washing buffer. 100 μL/well of secondary HRP-labeled antibody (rabbit anti-mouse IgG) was added diluted 1:3000 in assay buffer and plates were incubated at 20° C., 300 rpm shaking for 1 hour. A volume of 100 μL 3,3′,5,5′-tetramethylbenzidine (TMB) was added and incubated for 15 min at 20° C. in the dark. To stop the enzyme reaction of TMB, 100 mL 0.1% sulphuric acid was added and the absorbance was measured at 450 nm with 650 nm as the reference using an ELISA reader. A calibration curve was plotted using a 4-parametric mathematical fit model. The monoclonal antibody directed to the collagen type VII alpha 1 N-terminal neo-epitope has been confirmed to recognize the desired sequence, assessed by the reactivity to the standard peptide.

Example 3

[0069] The C7 ELISA was evaluated in a second, larger cohort of patients with systemic sclerosis (SSc). The C7 ELISA was re-calibrated (compared to the previous example) to improve accuracy of the assessments in human serum.

[0070] Results:

[0071] The biological relevance of the C7 ELISA was evaluated by comparing serum levels in healthy donors (n=70) with patients with SSc (n=119). Data are shown in FIG. 5. Median serum C7 level was significantly elevated in patients with SSc (9.3 ng/mL [IQR 6.7-13.2]) as compared with healthy donors (3.9 ng/mL [IQR 2.3-8.3 ng/mL]; p<0.0001).

[0072] The clinical data support that serum C7 levels are elevated in patients with SSc

[0073] In conclusion, the novel assays described herein utilise antibodies specific for an N- or C-terminal neo-epitope of collagen VII alpha 1. To the best of our knowledge, this is the first time that collagen type VII has been associated with COPD. Accordingly, it is envisaged that these assays may be used for assessing COPD as well as systemic sclerosis.

[0074] 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.

REFERENCES

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