Type XXIII Collagen Assay

Abstract

The present invention provides monoclonal antibodies that target collagen type XXIII, and to immunoassays and kits employing the antibodies for detecting and quantifying the epitope. The invention also provides a method for identifying and monitoring subjects with inflammatory bowel disease.

Claims

1: A monoclonal antibody that specifically recognises and binds to a peptide having the C-terminus amino acid sequence GLPVPGCWHK (SEQ ID NO: 1).

2: The monoclonal antibody of claim 1, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the C-terminus amino acid sequence GLPVPGCWHK (SEQ ID NO: 1).

3: The monoclonal antibody of claim 1, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPVPGCWHKX (SEQ ID NO: 2), wherein X represents any amino acid.

4: The monoclonal antibody of claim 1, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPVPGCWH (SEQ ID NO: 3).

5: The monoclonal antibody of claim 1, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPVQGCWNK (SEQ ID NO: 4).

6: The monoclonal antibody of claim 1, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPMPGCWQK (SEQ ID NO: 5).

7: The monoclonal antibody of claim 1, wherein the monoclonal antibody or fragment thereof comprises one or more complementarity-determining regions (CDRs): TABLE-US-00012 CDR-H1: (SEQ ID NO: 6) SYAMS, CDR-H2: (SEQ ID NO: 7) SISTAGRTYYPDTVR, CDR-H3: (SEQ ID NO: 8) PDYDYDGYIN, CDR-L1: (SEQ ID NO: 9) RSSKSLLHSNGVTYLY, CDR-L2: (SEQ ID NO: 10) QMSNLAS, or [[and]] CDR-L3: (SEQ ID NO: 11) AQNLELPLT.

8: The monoclonal antibody of claim 1, wherein the monoclonal antibody or fragment thereof has a light chain variable region comprising the CDR sequences: TABLE-US-00013 CDR-L1: (SEQ ID NO: 9) RSSKSLLHSNGVTYLY CDR-L2: (SEQ ID NO: 10) QMSNLAS and CDR-L3: (SEQ ID NO: 11) AQNLELPLT.

9: The monoclonal antibody of claim 1, wherein the monoclonal antibody or fragment thereof has a heavy chain variable region comprising the CDR sequences: TABLE-US-00014 CDR-H1: (SEQ ID NO: 6) SYAMS CDR-H2: (SEQ ID NO: 7) SISTAGRTYYPDTVR, and CDR-H3: (SEQ ID NO: 8) PDYDYDGYIN.

10: A method of immunoassay for detecting type XXIII collagen in a human biofluid sample, said method comprising contacting a human biofluid sample with a monoclonal antibody that specifically recognises and binds to a peptide having the C-terminus amino acid sequence GLPVPGCWHK (SEQ ID NO: 1), and detecting binding between the monoclonal antibody and peptides in the sample.

11: The method of claim 10, wherein the detection is quantitative.

12: The method of claim 10, wherein the immunoassay is a competitive immunoassay.

13: The method of claim 10, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the C-terminus amino acid sequence GLPVPGCWHK (SEQ ID NO: 1).

14: The method of claim 10, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPVPGCWHKX (SEQ ID NO: 2), wherein X represents any amino acid.

15: The method of claim 10, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPVPGCWH (SEQ ID NO: 3).

16: The method of claim 10, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPVQGCWNK (SEQ ID NO: 4).

17: The method of claim 10, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPMPGCWQK (SEQ ID NO: 5).

18: The method of claim 10, wherein the human biofluid sample is from a human patient having medical signs or symptoms indicative of inflammatory bowel disease.

19: The method of claim 10, wherein the method is an immunoassay method for diagnosing and/or monitoring and/or assessing the likelihood of inflammatory bowel disease in a patient, the method comprising contacting a biofluid sample obtained from said patient with the monoclonal antibody, detecting and determining the amount of binding between the monoclonal antibody and peptides in the sample, and correlating said amount of binding with values associated with normal healthy subjects and/or values associated with known disease severity and/or values obtained from said patient at a previous time point.

20: The method of claim 18, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

21: An assay kit comprising a monoclonal antibody that specifically recognises and binds to a peptide having the C-terminus amino acid sequence GLPVPGCWHK (SEQ ID NO: 1), and at least one of: a streptavidin coated well plate; a N-terminal biotinylated peptide having the C-terminus amino acid sequence GLPVPGCWHK (SEQ ID NO: 1); and a calibrator peptide having the C-terminus amino acid sequence GLPVPGCWHK (SEQ ID NO: 1).

22: The assay kit of claim 21, wherein the monoclonal antibody is a monoclonal antibody raised against a synthetic peptide having the C-terminus amino acid sequence GLPVPGCWHK (SEQ ID NO: 1).

23: The assay kit of claim 21, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPVPGCWHKX (SEQ ID NO: 2), wherein X represents any amino acid.

24: The assay kit of claim 21, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPVPGCWH (SEQ ID NO: 3).

25: The assay kit of claim 21, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPVQGCWNK (SEQ ID NO: 4).

26: The assay kit of claim 21, wherein the antibody does not specifically recognise or bind to a peptide having the C-terminus amino acid sequence GLPMPGCWQK (SEQ ID NO: 5).

Description

FIGURES

[0047] FIG. 1: PRO-C23 antibody 10F6 specificity. A) Sequence alignment for C-terminus of type XIII, XXIII and XXV collagens. The antibody recognizes the residues from 531 to 540 of type XXIII collagen. B) PRO-C23 antibody specificity towards different peptides. Reactivity to the selection peptide (GLPVPGCWHK (SEQ. ID No. 1)), the elongated peptide (GLPVPGCWHKA (SEQ. ID No. 16)), the truncated peptide (GLPVPGCWH), peptide from type XIII collagen (GLPVQGCWNK (SEQ. ID. No. 4)) and peptide from type XXV collagen (GLPMPGCWQK (SEQ. ID No. 5)) was tested in the PRO-C23 assay. C) Western blot results of recombinant type XXIII collagen using 10F6 as primary antibody.

[0048] FIG. 2: Correlation of PRO-C23 levels in three kinds of plasma and serum (N=16)

[0049] FIG. 3: PRO-C23 levels in rat DSS model. A) PRO-C23 serum levels in control rats (n=8) and DSS rats with DAI≥5 (n=8). B) The serum levels of PRO-C23 increased upon induction of colitis and returned towards baseline at reversal of the intestinal inflammation. Data are presented as median±95% confidence interval. * Asterisk (*) represent statistical difference. * P<0.05.

[0050] FIG. 4: PRO-C23 levels in human cohort 1. Data are presented as median±95% confidence interval. * Asterisk (*) represent statistical difference. * P<0.05.

[0051] FIG. 5: PRO-C23 levels in human cohort 2 (CD) and 3 (UC). Data are presented as median±95% confidence interval. * Asterisks (*) represent statistical differences. *(p<0.05) ** (p<0.01)*** (p<0.001)

EXAMPLES

[0052] Various embodiments are described and disclosed in the following Examples, which are set forth to aid in the understanding of the present disclosure and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Methods

Antibody Development for PRO-C23

[0053] The last 10 amino acids of the type XXIII collagen al chain (.sup.531‘GLPVPGCWHK’.sup.540, (SEQ. ID No. 1) Genscript, USA) were used as the immunogenic peptide to generate specific monoclonal antibodies. 4-6-week-old Balb/C mice were immunized subcutaneously with 100 μg of the immunogen (KLH-CGG-GLPVPGCWHK (SEQ. ID No. 1) emulsified with Stimmune adjuvant (Thermo Fisher, USA). Consecutive immunizations were performed at 2-week intervals. The mouse with highest antiserum titer and the best peptide reactivity was selected for fusion. Mouse spleen cells were fused with SP2/0 myeloma cells. The fusion cells were raised in 96-well plates and incubated in the CO2-incubator. Cell lines specific to the selection peptide and without cross-reactivity to neither elongated peptide (GLPVPGCWHKA (SEQ. ID No. 16)), truncated peptide (GLPVPGCWH) nor deselection peptides (GLPVQGCWNK, (SEQ. ID. No. 4) type XIII collagen, GLPMPGCWQK (SEQ. ID No. 5), type XXV collagen) (Genscript, USA) were selected and subcloned. At last, the antibodies were purified using an IgG column (GE health, USA).

[0054] The antibodies generated were sequenced and the CDRs determined.

[0055] The sequence of the chains are as follows:

[0056] CDRs underlined and in bold

Constant Region Italic:

[0057]

TABLE-US-00008 Heavy chain: Amino acid sequence (454 aa) (SEQ. ID No. 17) EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLAW VASISTAGRTYYPDTVRGRITISRDNARNILYLQMSSLRSEDTAIYY CARPDYDYDGYINWGQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSV TLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTV PSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHK CPAPNIEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQI SWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCK VNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLV VGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSK WEKTDSFSCNVRHEGLKNYYLKKTISRSPGK Light chain: Amino acid sequence (219 aa) (SEQ. ID No. 18) DIVMTQAAFSNPVTLGTSAYISCRSSKSLLHSNGVTYLYWYLQKPGQ SPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCA QNLELPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFL NNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTK DEYERHNSYTCEATHKTSTSPIVKSFNRNEC

PRO-C23 Assay and Technical Evaluation

[0058] ELISA-plates used for the assay development were Streptavidin-coated from Roche (cat.: 11940279). All ELISA plates were analyzed with the ELISA reader from Molecular Devices, SpectraMax M, (CA, USA). The selected monoclonal antibody was labelled with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit according to the instructions of the manufacturer (Innovabioscience, Babraham, Cambridge, UK). A 96-well streptavidin plate was coated with biotin-GLPVPGCWHK (SEQ. ID No. 1) (Genscript, USA) and incubated 30 minutes at 20° C. 20 μL of standard peptide or samples were added to appropriate wells, followed by 100 μL of HRP conjugated monoclonal antibody 10F6, and incubated 20 hour at 4° C. Finally, 100 μL tetramethylbenzidine (TMB) (Kem-En-Tec cat.4380H) was added and the plate was incubated 15 minutes at ° C. in the dark. All the above incubation steps included shaking at 300 rpm. After each incubation step, the plate was washed five times. The TMB reaction was stopped by adding 100 μL of stopping solution (1% H.sub.2SO.sub.4) and measured at 450 nm with 650 nm as the reference.

[0059] The lower limit of detection (LLOD) was determined from 21 zero samples (i.e. buffer) and calculated as the mean+3×standard deviation. Upper limit of detection (ULOD) was determined as the mean−3×SD of 10 measurements of Standard A. The intra-assay and inter-assay variation was the mean variations of 10 QC samples run 10 independent times in duplicate. Dilution recovery was determined in 4 serum samples and 4 plasma samples and was calculated as a percentage of recovery of diluted samples from the 100% sample. Correlation between serum and plasma was determined in serum and the matched heparin plasma, citrate plasma, EDTA plasma from 16 individuals (Innovative Research, USA).

Western Blotting with Recombinant Human Type XXIII Collagen

[0060] Recombinant human type XXIII collagen (R&D system, 4165-CL) was diluted in sample buffer containing 80 mM DTT and run on a 10% SDS-PAGE gel, and subsequently transferred onto a nitrocellulose membrane. The nitrocellulose membranes were then blocked for non-specific binding by incubation for 1 hour at room temperature in TBS-T containing 5% skim milk powder. This was followed by incubation with 1 μg/ml 10F6 or commercial type XXIII collagen antibody (R&D system, MAB4165) diluted in TBS-T milk for overnight. Then the membranes were washed in TBS-T three times, followed by incubation in the secondary peroxidase conjugated antibody. Finally, the membranes were washed in TBS-T three times, and then the results were visualized using the ECL system (GE healthcare, cat #RPN2109).

DSS Rat Model

[0061] Male Sprague-Dawley rats, 12 weeks of age, were divided into 2 groups: 6% DSS group (12 rats) and a control group (9 rats). Acute colitis was induced in the DSS group by adding 6% of DSS to the drinking water for 5 days. After day 5, DSS was withdrawn from drinking water. Half of the rats in 6% DSS group and 3 control rats were sacrificed at day 6 to remove colon. The remaining the rats were allowed to recover from the DSS induced colitis until sacrifice at day 16. Blood samples were taken at baseline, day 6, 7 and 16. Disease activity index (DAI) was scored every day to evaluate the progression of colitis. It was based on the following parameters: weight loss, stool consistency and blood in feces or rectal bleeding. The weight loss score in DSS rats was compared to the mean weight of age-matched controls: score 0=0-4% weight loss; score 1=5-10.9% weight loss; score 2=11-15.9% weight loss; score 3=16-20% weight loss. Stool consistency: score 0=normal and well formed; score 1=soft and sticky stool visible at base of tail; score 2=very soft and unformed; score 4=diarrhoea and watery stool. Blood in feces or rectal bleeding: score 0=normal color stool; score 2=reddish color stool; score 4=bloody stool or bleeding from rectum. The score for each parameter resulted in a daily total DAI score ranging between 0 and 12. A total score of 5 or above was considered as high disease activity.

IBD Cohorts

[0062] Three different cohorts were measured to evaluate biological relevance of PRO-C23 assay. Serum samples were collected after informed consent and approval by the local Ethics Committee. In cohort 1, serum from CD and UC patients were obtained from commercial vendor Reprocell (USA), whereas as the serum from healthy donors were obtained from Valley Biomedical (USA) (table 1). In cohort 2 and cohort 3, serum from patients with CD and UC were obtained, respectively (table 2) For any of the cohorts, there were no significant statistical differences between the patient demographics (gender and age) of healthy donors, CD and UC patients.

TABLE-US-00009 TABLE 1 Patient demographics of cohort 1 Healthy controls CD UC (N = 10) (N = 10) (N = 10) P value Age 43.5 40.5 45.5 0.57 Male, n (%) 7 (70%) 8 (80%) 7 (70%) 0.85 Comparison of age and gender was performed using Kruskal-Wallis test. P-values below 0.05 were considered significant. Abbreviations: CD: Crohn's disease, UC: Ulcerative colitis.

TABLE-US-00010 TABLE 2 Patient demographics of cohort 2 and 3 Crohn's Ulcerative disease colitis Healthy P- General (cohort 2) (cohort 3) donors value Total samples 44 29 29 Gender: n (%) 16 (37%) 14 (41%) 12 (41%) 0.599 female Age (years, 36 [19-73] 39 [17-62) 38 [22-58] 0.392 mean [range]) Crohn's Disease 24 (54%) NA Activity Index (CDAI) > 150 Partiel mayo NA 14 (48%) score (>1) Age at diagnosis NA (n(%)) A1 (<16) 0 (0%) A2 (17-40) 31 (71%) A3 (>40) 13 (29%) Disease location NA start IFx (n(%)) L1 (n, %) 5 (11%) L2 (n, %) 14 (32%) L3 (n, %) 25 (57%) Disease behavior NA start IFx (n(%)) B1: Luminal 20 (45%) disease B2: Stricturing 11 (25%) B3: Penetrating 13 (30%) Disease extension NA E1: proctitis 2 (7%) E2: Left-sided 8 (28%) E3: Pan-colitis 19 (66%) Disease severity NA S1: Mild 15 (52%) S2: Moderate 13 (45%) S3: Severe 1 (3%) Peri-anal disease  0  0 NA Comparison of age and gender was performed using a Mann-Whitney U test or fisher's exact test. P-values below 0.05 were considered significant. Abbreviations: CD: Crohn's disease.

Statistics

[0063] Statistical analysis was performed using MedCalc version 14 and GraphPad Prism version 7. The biomarker levels were presented as median±95% confidence interval. The differences of PRO-C23 between DSS rats and controls were determined by unpaired t-test. In human cohorts, comparison of age and gender was performed using a Kruskal-Wallis test. The differences of PRO-C23 between patients and healthy controls were determined by Mann-Whitney t-test. The diagnostic power of biomarkers was investigated by the area under the receiver-operating characteristics (ROC) curve (AUC) with 95% confidence interval (CI). Sensitivity and specificity were determined for appropriate cut-off values based on the ROC curves. Significance threshold was set at P<0.05.

Results

Characterization of PRO-C23 Assay

[0064] Like type XXIII collagen, type XIII and XXV collagens are also transmembrane collagens and share highly similar sequences in their C-terminus (FIG. 1A). To fully investigate the specificity of the antibody, a series of peptides were synthesized and included in the inhibition test. The chosen antibody 10F6 specifically recognized the last 10 amino acids of C-terminus of type XXIII collagen .sup.531‘GLPVPGCWHK’.sup.540, (SEQ. ID No. 1) but did not recognize elongated peptide GLPVPGCWHKA (SEQ. ID No. 16), truncated peptide GLPVPGCWH(SEQ. ID No. 3), type XIII collagen C-terminal peptide GLPVQGCWNK (SEQ. ID. No. 4), or type XXV collagen C-terminal peptide GLPMPGCWQK. (SEQ. ID No. 5) (FIG. 1B). Western blot of recombinant type XXIII collagen ectodomain (4165-CL, R&D system) showed that the chosen antibody 10F6 recognized type XXIII collagen ectodomain around 60 kD, while reference commercial antibody (MAB4165, R&D system) was also shown (FIG. 1C).

[0065] PRO-C23 competitive ELISA provided a measurement range from 0.38 ng/ml (LLOD) to 18.73 ng/ml (ULOD). The inter- and intra-assay variability were 8.1% and 3.5%, respectively. The dilution recovery and spiking recovery in human serum was shown in Table 3. The correlations between values in human serum and three kinds of plasmas were relatively high (FIG. 2, P<0.0001), showing that PRO-C23 levels were independent of blood preparation method.

TABLE-US-00011 TABLE 3 Technical performance of PRO-C23 assay Detection range (LLOD-ULOD) 0.38 ng/mL-18.73 ng/mL Intra-assay variability 3.5% Inter-assay variability 8.1% Dilution recovery in serum Within 100 ± 20% Spiking recovery in serum 75.8%  Interference No HAMA, biotin, intralipid and hemoglobin interference

PRO-C23 Biomarker in DSS Rat Model

[0066] To investigate the biological relevance of PRO-C23 fragment, a rat model of DSS induced colitis was used. PRO-C23 biomarker was measured in serum samples. DSS rats with high disease activity index (DAI≥5) at day 6 and 7 had significantly higher PRO-C23 serum levels (p>0.05, FIG. 3A) compared to controls. The serum levels of PRO-C23 increased upon induction of colitis and returned towards baseline at reversal of the intestinal inflammation (FIG. 3B).

PRO-C23 Biomarker in Human IBD Cohorts

[0067] PRO-C23 was measured in serum from three independent human cohorts. In cohort 1, PRO-C23 were quantified in 10 CD and 10 UC patients, together with 10 age-matched healthy donors. Results showed that CD and UC patients have significantly higher levels of PRO-C23 (p<0.05, FIG. 4) compared to healthy donors. No information of the activity of the disease in these patients was given. In cohort 2 and 3, 44 CD patients and 29 UC patients were included, together with 29 age-matched healthy donors. PRO-C23 levels were elevated in active CD patients and active UC patients compared to healthy donors (CD: p<0.05, UC: p<0.001 FIG. 5).

DISCUSSION

[0068] It was hypothesized that type XXIII collagen could be cleaved from cell surface during epithelial damage. Moreover, the loss of type XXIII collagen could contribute to the epithelial adhesion change in IBD and so used as a biomarker for IBD. Therefore, a PRO-C23 ELISA measuring the ectodomain of type XXIII collagen was developed.

[0069] The antibody only recognized the C-terminus sequence of type XXIII collagen and had no cross-reaction with C-terminus of type XIII and XXV collagen which showed similar sequences. This data clearly confirmed the specificity of the antibody. Thereafter, the antibody was applied in a competitive ELISA and optimized for human serum and plasma measurement. The data demonstrated that the ectodomain of type XXIII could be detected in circulation by the competitive ELISA independent of blood preparation method.

[0070] In order to further validate the biomarker in in vivo studies, the PRO-C23 biomarker was measured in a DSS induced colitis rat model. DSS can cause intestinal epithelial cell injury. The animals exhibit IBD-like symptoms, such as diarrhoea, rectal bleeding and weight loss [25,26]. One study also showed that DSS can induce altered tight junction protein expression [27]. Therefore, DSS rat model could be an appropriate animal model to validate PRO-C23 biomarker. Type XXIII collagen was found to be elevated in rats with active disease and weakly correlated with disease activity. This finding indicated the ectodomain of type XXIII collagen found in circulation related with disease activity of DSS rats.

[0071] To further validate the PRO-C23 biomarker, it was measured in two human cohorts. PRO-C23 was elevated in human CD patients (cohort 2) and in UC patients (cohort 3) with active disease. These data suggested the release of ectodomain of type XXIII collagen was reinforced in the active intestinal damage, which was consistent with the results in animal model.

[0072] Type XXIII collagen has been suggested to be a potential biomarker for prostate cancer recurrence [18], non-small cell lung cancer [17] and clear cell renal cell carcinoma [14]. It showed significantly higher expression in those cancer tissues, especially in the metastatic tissue. It is believed that type XXIII collagen facilitates cell-cell adhesion and cell-matrix adhesion [13]. Silencing type XXIII collagen in lung cancer and clear cell renal cell lines showed altered adhesion protein expressions and less ability on cell adhesion and migration [13,14]. However, type XXIII collagen is also present in other tissue, and the function and the use in other diseases are yet unknown. To the inventors' knowledge, this is the first study that showed type XXIII collagen level was modulated in IBD. The results indicate that type XXIII collagen may also play an important role in cell adhesion in intestines and contribute to the pathologies of IBD.

CONCLUSION

[0073] The data indicates that the biomarkers of epithelium PRO-C23 can be used as non-invasive surrogates of disease activity in CD patients and thus aid in monitoring patients. Higher levels of PRO-C23 were measured in serum of CD and UC patients with active disease compared to inactive disease.

[0074] All prior teachings acknowledged in this specification 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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