ANTIBODIES BINDING SPECIFICALLY TO NT-PROBNP AND USE THEREOF

Abstract

Provided are an antibody that binds specifically to N-terminal pro-B-type natriuretic peptide (NT-proBNP) and use thereof. An antibody or antigen-binding fragment thereof, which binds specifically to an epitope of NT-proBNP including the amino acid sequence of HXLGXXX (SEQ ID NO: 2), may detect NT-proBNP, which is a heart disease biomarker, and heart disease can also be effectively diagnosed by using the same.

Claims

1. An antibody or antigen-binding fragment thereof, which specifically binds to an epitope of N-terminal pro-B-type natriuretic peptide (NT-proBNP) comprising the amino acid sequence of HXLGXXX.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the epitope comprises the amino acid sequence of SEQ ID NO: 3.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises an scFv fragment, an scFv-Fc fragment, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a mouse antibody, a goat antibody, a sheep antibody, a marmot antibody, a rat antibody, a rabbit antibody, or a humanized antibody.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 4, heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 5, and heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 7, light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 8, and light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 9.

5. A composition for detection of NT-proBNP, comprising the antibody or antigen-binding fragment thereof of claim 1.

6. The composition of claim 5, further comprising a second antibody or antigen-binding fragment thereof, which binds specifically to NT-proBNP comprising the amino acid sequence of SEQ ID NO: 1.

7. The composition of claim 5, wherein the composition is used to detect NT-proBNP of a canine.

8. A kit for detection of NT-proBNP, comprising the antibody or antigen-binding fragment thereof of claim 1.

9. A method of detecting N-terminal pro-B-type natriuretic peptide (NT-proBNP), the method comprising: contacting, with a biological sample isolated from a subject, an antibody or antigen-binding fragment thereof, which specifically binds to an epitope of NT-proBNP comprising the amino acid sequence of HXLGXXX; and detecting a complex formed by binding of the antibody or antigen-binding fragment thereof to the biological sample.

10. The method of claim 9, wherein the contacting further comprises contacting, with the biological sample isolated from a subject, a second antibody or antigen-binding fragment thereof, which binds specifically to NT-proBNP comprising the amino acid sequence of SEQ ID NO: 1.

11. An antibody or antigen-binding fragment thereof, which binds specifically to N-terminal pro-B-type natriuretic peptide (NT-proBNP), the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 4, heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 5, and heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 7, light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 8, and light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 9.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0080] FIG. 1 is a diagram schematically illustrating a method of manufacturing an NT-proBNP capture antibody and an NT-proBNP detector antibody and a method for detecting NT-proBNP by using the same;

[0081] FIG. 2 shows an image showing the results of primary epitope mapping of an NT-proBNP capture antibody;

[0082] FIG. 3 shows an image showing the results of secondary epitope mapping of an NT-proBNP capture antibody; and

[0083] FIGS. 4A and 4B show an image showing the results of quaternary epitope mapping of an NT-proBNP capture antibody.

DETAILED DESCRIPTION

[0084] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0085] Hereinafter, the disclosure will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the disclosure is not limited to these examples.

Example 1: Preparation of NT-proBNP Antibody—Preparation of Capture Antibody and Detector Antibody

[0086] The following experiment was performed to prepare a capture antibody and a detector antibody for detecting NT-proBNP derived from canines.

[0087] Specifically, the immunogen, which is NT-proBNP, and an antigen adjuvant were mixed to immunize a female 6-week-old mouse, and the spleen of the animal was isolated and the cells were separated therefrom. The antibody-producing splenocytes were fused with myeloma cells to prepare hybridomas. Then, from the hybridomas, a hybridoma that produces a monoclonal antibody that binds specifically to NT-proBNP was screened for. As the immunized animal, not only the mouse used in Examples, but also animals such as goats, sheep, marmots, rats or rabbits may be used, and monoclonal antibodies or polyclonal antibodies may be used.

Example 2: Epitope Mapping of NT-proBNP Capture Antibody

[0088] In order to confirm the NT-proBNP epitope region of the NT-proBNP capture antibody prepared in Example 1, the following experiment was performed.

2.1: Primary Epitope Mapping

[0089] For primary epitope mapping of the capture antibody, the entire sequence of NT-proBNP was divided into 15 regions to prepare a primary recombinant antigen (named CNT1 to CNT15), and a strip spot test was performed to identify whether the capture antibody and the recombinant antigen were bound to each other (Table 1).

TABLE-US-00001 TABLE 1 Reactivity with Character- Capture antibody Item Name istic Sequence Strip spot test Positive control Control Recombinant (Full length) O antigen HPLG to SPK Test  1 CNT1 Peptide HPLGGRSPASEASE O A  2 CNT2 Peptide RSPASEASEASEAS X G  3 CNT3 Peptide EASEASEASGLWA X VQ  4 CNT4 Peptide SEASGLWAVQELL X GR  5 CNT5 Peptide LWAVQELLGRLKD X AV  6 CNT6 Peptide ELLGRLKDAVSEL X QA  7 CNT7 Peptide LKDAVSELQAEQL X AL  8 CNT8 Peptide SELQAEQLALEPLH X R  9 CNT9 Peptide EQLALEPLHRSHSP X A 10 CNT10 Peptide EPLHRSHSPAEAPE X A 11 CNT11 Peptide SHSPAEAPEAGGTP X R 12 CNT12 Peptide EAPEAGGTPRGVL X AP 13 CNT13 Peptide GGTPRGVLAPHDS X VL 14 CNT14 Peptide GVLAPHDSVLQAL X RR 15 CNT15 Peptide HDSVLQALLRLRS X PK

[0090] In the strip spot test, the recombinant antigen was spotted on a nitrocellulose membrane, and then, the resultant structure was reacted by mixing a capture antibody, to which gold nanoparticles were conjugated, with a buffer, and binding can be confirmed by development of color.

[0091] As a result of the analysis, a weak reactivity occurred only with respect to the CNT1 recombinant antigen, indicating that an epitope region exists in CNT1 (FIG. 2).

2.2: Secondary Epitope Mapping

[0092] For secondary epitope mapping of the capture antibody, the C-terminal amino acid of the CNT1 recombinant antigen, which was confirmed to have reactivity in Example 2.1, was cleaved to prepare secondary recombinant antigens (named CNT1-1 to CNT1-5), and a strip spot test was performed to confirm whether the capture antibody and the recombinant antigen were bound to each other (Table 2).

TABLE-US-00002 TABLE 2 Reactivity with Character- Capture antibody Item Name istic Sequence Strip spot test Positive control Control Recombinant (Full length) O antigen HPLG to SPK Test 1 CNT1 Peptide HPLGGRSPASEASE O A 2 CNT1-1 Peptide HPLGGRSPASEAS O 3 CNT1-2 Peptide HPLGGRSPASE O 4 CNT1-3 Peptide HPLGGRSPA O 5 CNT1-4 Peptide HPLGGRS O 6 CNT1-5 Peptide HPLG X

[0093] As a result of the analysis, it was confirmed that there was the reactivity to CNT1-1 to CNT1-4 recombinant antigen, but not to CNT1-5 recombinant antigen. Based on this result, it can be seen that the amino acid sequence of CNT1-4 is an epitope region of the NT-proBNP capture antibody (FIG. 3).

2.3: Tertiary Epitope Mapping

[0094] In order to confirm that the amino acid sequence of CNT1-4 confirmed in the secondary epitope mapping is the minimum unit of the NT-proBNP capture antibody epitope, a recombinant antigen in which the C-terminal amino acid of the CNT1-4 recombinant antigen was additionally cleaved, was additionally prepared. (Named as CNT1-6 and CNT1-7), and a strip spot test was performed to confirm whether the capture antibody and the recombinant antigen were bound to each other (Table 3).

TABLE-US-00003 TABLE 3 Reactivity with Character- Capture antibody Item Name istic Sequence Strip spot test Positive control Control Recombinant (Full length) O antigen HPLG to SPK Test 1 CNT1 Peptide HPLGGRSPASEASE O A 2 CNT1-1 Peptide HPLGGRSPASEAS O 3 CNT1-2 Peptide HPLGGRSPASE O 4 CNT1-3 Peptide HPLGGRSPA O 5 CNT1-4 Peptide HPLGGRS O 6 CNT1-5 Peptide HPLG X 7 CNT1-6 Peptide HPLGGR X 8 CNT1-7 Peptide HPLGG X

[0095] As a result of the analysis, it was confirmed that there was the reactivity to the CNT1-4 recombinant antigen, but not to the recombinant antigen in which the C-terminal amino acid was additionally cleaved. Based on this, it may be seen that the amino acid sequence of CNT1-4 is the minimum reactivity epitope region of the NT-proBNP capture antibody.

2.4: Quaternary Epitope Mapping

[0096] To identify a key amino acid portion that plays a key role in the minimum unit epitope region of the NT-proBNP capture antibody identified in the tertiary epitope mapping, additional epitope mapping was performed through an alanine scanning mutagenesis test. First, the full-length canine NT-proBNP protein was expressed in the E. coli expression system, and the 1.sup.st to 8.sup.th amino acids were substituted with an alanine expression codon (GCT), respectively, and cloned to prepare an NT-proBNP recombinant antigen (Table 4).

TABLE-US-00004 TABLE 4 Canine NT- proBNP 1 2 3 4 5 6 7 8 up to 85 (aa) Origin. seq. H P L G G R S P . . . 1 A P L G G R S P . . . 2 H A L G G R S P . . . 3 H P A G G R S P . . . 4 H P L A G R S P . . . 5 H P L G A R S P . . . 6 H P L G G A S P . . . 7 H P L G G R A P . . . 8 H P L G G R S A . . .

[0097] Next, western blotting was performed to confirm the reactivity of the NT-proBNP capture antibody of the disclosure to the recombinant antigen. Specifically, the recombinant antigen (3 μg) was subjected to SDS-PAGE electrophoresis and blotted on a membrane, and then, reacted with the HRP-conjugated NT-proBNP capture antibody (10 μg/ml) and then, the substrate (CN/DAB) reactivity was identified. The results showed that when the 1.sup.st, 3.sup.rd and 4.sup.th amino acid sites were substituted with A, the band in western blotting was disappeared or weakened (where the band had been clearly identified in SDS-PAGE) (FIG. 4A).

[0098] Next, ELISA was performed to confirm the reactivity of the NT-proBNP capture antibody of the disclosure to the recombinant antigen. Specifically, the recombinant antigen (6 μg/ml) was coated on a plate, and the HRP-conjugated NT-proBNP capture antibody was treated at various concentrations, and a substrate (TMB) was added thereto to cause a reaction. Then, the absorbance of thereof was measured to identify the reactivity. As a result, like the results obtained from the western blotting, it was confirmed that when the 1.sup.st, 3.sup.rd and 4.sup.th amino acid sites were substituted with A, the reactivity was disappeared (FIG. 4B).

[0099] Accordingly, it can be seen that the 1.sup.st, 3.sup.rd and 4.sup.th amino acids of the amino acid sequence of CNT1-4, which is the minimum unit epitope region of the NT-proBNP capture antibody of the disclosure, are key epitopes.

Example 3: CDR Sequence Analysis of NT-proBNP Capture Antibody

[0100] The CDR sequence of the NT-proBNP capture antibody of the disclosure, for which the epitope was identified in Example 2, was analyzed, and the sequence information of the CDR regions in the light chain variable region and the heavy chain variable region is shown in Table 5.

TABLE-US-00005 TABLE 5 Amino acid sequence SEQ ID CDR regions (5′.fwdarw.3′) NO: Heavy CDR1 GFSLSTSGV 4 chain CDR2 WWDDD 5 CDR3 MDDYNYAFDY 6 Light CDR1 KSSQSLLYSNGKTYLN 7 chain CDR2 LVSKLDS 8 CDR3 VQGTHFPLT 9

Example 4: Evaluation of Clinical Performance of Diagnostic Kits Containing NT-proBNP Capture Antibody

[0101] The following experiment was performed to evaluate the diagnostic effect of the kit for diagnosis of the canine heart disease including the NT-proBNP capture antibody of the disclosure, for which epitope was identified in Example 2 and of which sequence was analyzed in Example 3.

4.1: Selection of Subjects for Clinical Evaluation and Collection of Samples

[0102] From Jul. 1, 2020 to Dec. 31, 2020, the subjects for clinical evaluation were selected from among the canines who visited veterinary hospitals of grade 2 or higher. Normal canines were selected from healthy subjects that do not have symptoms related to heart disease. Subjects diagnosed with heart disease through echocardiography were used as canines affected by heart disease (Subjects belonging to the group of canines affected by heart disease satisfied the NT pro-BNP range (250 pmol/L to 10,000 pmol/L)). The heart disease included myxomatous mitral valve degeneration.

[0103] Serum of blood samples collected from subjects for clinical evaluation was isolated and immediately tested. When the immediate test could not be performed, the serum was stored at −80° C. until the test.

4.2: Analysis of Characteristics of Subjects for Clinical Evaluation

[0104] The information about the subjects (n=100) for clinical evaluation, on which the NT pro-BNP test using the kit for diagnosis of canine animal heart disease containing the NT-proBNP capture antibody of the disclosure was performed, is as follows.

[0105] First, the heart disease group consisted of 50 subjects which all had myxomatous mitral valve degeneration, and the normal group consisted of 50 subjects which all were healthy subjects with no symptoms related to heart disease (Table 6).

TABLE-US-00006 TABLE 6 Name of Number of Ratio Group disease subjects (heads) (%) Heart disease Myxomatous mitral 50 50 (n = 50) valve degeneration Normal (n = 50) None 50 50 Total number of 100 100 subjects

[0106] As a result of echocardiography to evaluate the M-mode myocardial wall thickness and myocardial contractile function in the right parasternal short-axis papillary muscle view, in the case of the heart disease group, the fractional shortening (FS) value and the ejection fraction (EF) value were 52.90±9.02%, and 84.48±7.73%, respectively, and in the case of the normal group, the fractional shortening (FS) value and the ejection fraction (EF) value were 46.01±9.06%, and 78.43±9.57%, respectively (Table 7). For both items, the heart disease group showed significantly higher values than the normal group (p<0.001).

TABLE-US-00007 TABLE 7 Heart disease (n = 50, Normal (n = 50, average ± standard average ± standard Reference Values deviation) deviation) range P value IVSd (cm) 0.69 ± 0.18 0.68 ± 0.19 0.43-0.87 0.841 LVIDd (cm) 2.41 ± 0.56 2.39 ± 0.68 2.04-2.97 0.843 LVPWd (cm) 0.65 ± 0.21 0.65 ± 0.20 0.42-0.87 0.964 IVSs (cm) 1.08 ± 0.22 1.00 ± 0.25 0.63-1.16 0.076 LVIDs (cm) 1.15 ± 0.39 1.32 ± 0.50 1.18-2.09 0.061 LVPWs (cm) 1.22 ± 1.04 1.02 ± 0.27 0.69-1.24 0.180 FS (%) 52.90 ± 9.02  46.01 ± 9.06  33.4-45.9 <0.001** EF (%) 84.48 ± 7.73  78.43 ± 9.57  60.1-72.9 <0.001** IVSd: diastolic interventricular septum, LVIDd: diastolic left ventricular internal diameter, LVPWd: diastolic left ventricular posterior wall thickness, IVSs: systolic interventricular septum, LVIDs: systolic left ventricular internal diameter, LVPWs: systolic left ventricular posterior wall thickness, FS: fractional shortening, EF: ejection fraction. **P < 0.01

4.3: Analysis of NT Pro-BNP Test Results of Subjects for Clinical Evaluation

[0107] As a result of performing the NT pro-BNP test on the subject for clinical evaluation using the kit for diagnosis of canine heart disease including the NT-proBNP capture antibody of the disclosure, the heart disease group showed the value of 1815.14±1680.63 pmol/L and the normal group showed the value of 479.64±48.00 pmol/L. Accordingly, it can be seen that the heart disease group had a significantly greater value than the normal group (Table 8).

TABLE-US-00008 TABLE 8 Heart disease (n = 50, Normal (n = 50, average ± standard average ± standard Group deviation) deviation) P value BIONOTE 1815.14 ± 1680.63 479.64 ± 48.00 <0.001** (pmol/L)

[0108] Next, canines of the canine group affected by heart disease were classified according to the severity of heart disease using, in the case of myxomatous mitral valve degeneration, the ACVIM stage. The results obtained by comparing NT pro-BNP values according to the ACVIM stage showed that ACVIM Stage Cc showed the highest value of 2175.92±1733.52 pmol/L, followed by stage D (2035.23±1547.24 pmol/L), stage B2 (899.93±1341.01 pmol/L), and stage B1 (556.87±531.51 pmol/L)/L) and normal (479.64±48.00 pmol/L), and, that is, the normal group showed the lowest level (Table 9).

TABLE-US-00009 TABLE 9 Normal ACVIM B1 ACVIM B2 ACVIM Cc ACVIM D (n = 50, (n = 3, (n = 10, (n = 34, (n = 3, average ± average ± average ± average ± average ± standard standard standard standard standard Group deviation) deviation) deviation) deviation) deviation) P value BIONOTE 479.64 ± 556.87 ± 899.93 ± 2175.92 ± 2035.23 ± <0.001 (pmol/L) 48.00.sup.a 531.51.sup.ab 1341.01.sup.ab 1733.52.sup.b 1547.24.sup.ab [Different alphabets mean that there is a significant difference between groups (p < 0.05)]

[0109] Next, the positive predictive value, the negative predictive value, and the accuracy of the kit for diagnosis of canine heart disease containing the NT-proBNP capture antibody of the disclosure were analyzed. The results shows that the positive predictive value was 82%, the negative predictive value was 67%, and the accuracy was 72%. Accordingly, it was confirmed that the onset of heart disease was accurately diagnosed (Table 10).

TABLE-US-00010 TABLE 10 Disease diagnosis Heart Total Predictive disease Normal value value (%) BIONOTE Abnormal 28 6 34 Positive 82 range predictive (≥900 value pmol/L) Normal 22 44 66 Negative 67 range predictive (<900 value pmol/L) Total 50 50 100 Accuracy 72

[0110] Based on these results, it can be seen that the kit for diagnosis of canine heart disease including the NT-proBNP capture antibody of the disclosure can effectively detect NT-proBNP in canines with heart disease, and based on the detection result, heart disease can be accurately diagnosed.

[0111] The description of the disclosure described above is provided only for illustration, and those of ordinary skill in the art to which the disclosure pertains may understand that the embodiments presented herein may be easily modified into other specific forms without changing the technical spirit or essential features of the disclosure. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

[0112] An antibody or antigen-binding fragment thereof, which binds specifically to an epitope of NT-proBNP including the amino acid sequence of HXLGXXX (SEQ ID NO: 2) according to an aspect may detect NT-proBNP, a cardiac disease biomarker, and the heart disease can also be effectively diagnosed by using the same.

[0113] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.