Antibodies that recognize genetic variants

Abstract

The present invention relates to antibodies that specifically bind to mutated NT-proBNP, said mutated NT-proBNP comprising i) a mutation in which arginine at position 46 is replaced with histidine, or ii) a mutation in which glutamate at position 43 is replaced with aspartic acid. In addition, the present invention relates to mutated NT-proBNP or fragments thereof. Further contemplated by the present invention are kits comprising the antibodies of the present invention or the mutated NT-proBNP of the present invention. The present invention also relates to methods for diagnosing heart failure.

Description

[0001] The present invention relates to i) an antibody that specifically binds a mutated NT-proBNP, said mutated NT-proBNP comprising a mutation in which arginine at position 46 is replaced by histidine, and ii) an antibody that specifically binds a mutated NT-proBNP, said mutated NT-proBNP comprising a mutation in which glutamate at position 43 is replaced by aspartic acid. In addition, the present invention relates to the mutated NT-proBNP. Also contemplated by the present invention are kits comprising the antibodies of the present invention or the mutated NT-proBNP of the present invention. The present invention also relates to methods for diagnosing heart failure.

[0002] Heart failure (HF) is one of the leading causes of morbidity and mortality in many countries worldwide. Measurement of natriuretic peptide markers such as B-type natriuretic peptide (BNP) or its amino-terminal fragment N-terminal proBNP (NT-proBNP) has emerged as an important tool for the diagnosis and risk stratification of patients with HF.

[0003] Brain natriuretic peptide (BNP) is a 32-amino acid polypeptide. BNP is synthesized as a 134-amino acid preprohormone ("preproBNP"). Removal of the N-terminal signal peptide, which has a length of 26 amino acids, generates the prohormone ("proBNP", 108 aa in length). Subsequently, the prohormone is cleaved into NT-proBNP (the N-terminal of prohormone brain natriuretic peptide, 76 aa in length) and bioactive brain natriuretic peptide (BNP). NT-proBNP and BNP are produced in equimolar amounts. Several studies have shown that the determination of BNP and NT-proBNP can be reliably used for the diagnosis of heart failure (see, for example, Prontera et al., Clinica Chimica Acta 400 (2009) 70–73).

[0004] BNP is metabolized in the blood. Due to its rapid degradation rate both in vivo and in vitro, it has a short half-life. NT-proBNP circulates in the blood as an intact molecule and is eliminated by the kidneys as such. It has a longer half-life than the active peptide BNP and is more stable in vitro. The pre-analytical handling of NT-proBNP is thus more robust, which allows for easy transport of samples to a central laboratory (Mueller 2004, Clin Chem Lab Med 42: 942-4). Blood samples can be stored at room temperature for several days or can be mailed or transported without loss of recovery. In contrast, storage of BNP at room temperature or 4 °C for 48 hours results in at least a 20% loss of concentration (Mueller loc.cit.; Wu 2004, Clin Chem 50: 867-73).

[0005] Due to the advantages of NT-proBNP, the peptide NT-proBNP, which has no biological activity, is currently the preferred biomarker for the diagnosis of heart failure. This biomarker is routinely used in laboratory tests but also in the context of point-of-care medicine.

[0006] All NT-proBNP assays currently on the market are sandwich immunoassays using capture and signal antibodies. Some NT-proBNP assays currently on the market contain antibodies that bind to epitopes encompassing amino acids 42 to 46 of NT-proBNP (see Saenger et al., Clinical Chemistry 63:1 351–358 (2017), for example Supplementary Table 2, or Clerico et al., Crit Rev Clin Lab Sci, 2015; 52(2): 56–69).

[0007] In the basic research of the present invention, point mutations (Arg46His, R46H) were identified in the amino acid sequence of NT-proBNP. This point mutation is within the epitope of the signal antibody of various NT-proBNP assays. The point mutation can be found in the Ensembl database (Yates et al. Ensembl 2016, Nucleic Acids Res. 2016 44 Database issue:D710-6, 87th edition, December 2016, dbSNP Cluster ID: rs61761991. Search of the database revealed a second point mutation Glu43Asp (E43D) within amino acids 42 to 46 of NT-proBNP (dbSNP Cluster ID:rs74613227).

[0008] An object of the present invention is to provide means and methods for diagnosing heart failure in subjects having a mutation in the amino acid sequence of NT-proBNP. This technical problem is solved by the claims and the embodiments characterized hereinafter.

[0009] Accordingly, the present invention relates to antibodies that specifically bind to mutant NT-proBNP, said mutant NT-proBNP comprising a mutation in which arginine at position 46 is replaced by histidine. In addition, the present invention also relates to antibodies that specifically bind to mutant NT-proBNP, said mutant NT-proBNP comprising a mutation in which glutamate at position 43 is replaced by aspartic acid.

[0010] The term "NT-proBNP" (the N-terminal fragment of pro-brain natriuretic peptide) is well known in the art. As used herein, the term refers to the 76-amino acid N-terminal fragment of pro-brain natriuretic peptide (proBNP), a secreted protein that functions as a cardiac hormone after cleavage. Preferably, NT-proBNP is human NT-proBNP. Thus, the mutated NT-proBNP should be mutated human NT-proBNP.

[0011] The sequence of wild-type human NT-proBNP (also referred to herein as "unmutated" NT-proBNP) is well known in the art and has been described in detail in the prior art, such as WO 02 / 089657, WO 02 / 083913, Bonow 1996, New Insights into the cardiac natriuretic peptides. Circulation 93:1946-1950. Preferably, wild-type NT-proBNP has the amino acid sequence shown in SEQ ID NO: 3.

[0012] The antibodies of the present invention should specifically bind to the mutated NT-proBNP. Thus, compared to wild-type NT-proBNP, the NT-proBNP specifically bound by the antibodies of the present invention should contain a mutation.

[0013] In one embodiment, the antibodies of the present invention should specifically bind to a mutated NT-proBNP that contains a mutation in which arginine at position 46 is replaced with histidine. Thus, arginine at position 46 in wild-type NT-proBNP should be replaced with histidine (in other words, the mutated NT-proBNP contains histidine at position 46). This mutation is also referred to herein as the "Arg46His" or "R46H" mutation.

[0014] In another embodiment, the antibodies of the present invention specifically bind to a mutated NT-proBNP that contains a mutation in which glutamate at position 43 is replaced with aspartic acid. Thus, glutamate at position 43 in wild-type NT-proBNP should be replaced with aspartic acid (in other words, the mutated NT-proBNP contains aspartic acid at position 43). This mutation is also referred to herein as the "Glu43Asp" or "E43D" mutation.

[0015] The positions of the mutations given herein are amino acid positions in the NT-proBNP sequence, specifically the wild-type NT-proBNP having the sequence shown in SEQ ID NO: 3. Thus, the R46H mutation is at position 46 of NT-proBNP. In the longer precursor polypeptide pre-proBNP, the corresponding mutation is at position 72. Thus, the R46H mutation is also referred to as the R72H mutation herein, for example in the Examples section, and the E43D mutation is also referred to as the E69D mutation.

[0016] The mutations mentioned above are substitutions. It is to be understood that the mutations / substitutions are with respect to the wild-type NT-proBNP, specifically the wild-type NT-proBNP having the sequence shown in SEQ ID NO: 3. For example, wild-type human NT-proBNP contains an arginine residue at position 46, while the mutated NT-proBNP containing the mutation of substituting the arginine at position 46 with histidine contains a histidine residue at that position.

[0017] One or more further mutations may be present in the mutated NT-proBNP. However, these one or more mutations should not be located in the epitope specifically bound by the antibodies of the present invention.

[0018] In one embodiment of the present invention, the mutated NT-proBNP containing the mutation of substituting the arginine at position 46 with histidine contains the amino acid sequence shown in SEQ ID NO: 1. This sequence is also shown in Table A. The histidine residue at position 46 is indicated in bold.

[0019] In one embodiment, the mutated NT-proBNP containing the mutation of substituting the glutamate at position 43 with aspartate contains the amino acid sequence shown in SEQ ID NO: 2. This sequence is also shown in Table A. The aspartate residue at position 43 is indicated in bold.

[0020] The term "antibody" is known in the art. As used herein, the term refers to any immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains. As used herein, the term "antibody" also includes antigen-binding fragments of antibodies. The term "antigen-binding fragment" is explained elsewhere herein. This definition applies accordingly.

[0021] Antibodies according to the invention can be polyclonal or monoclonal antibodies. In a preferred embodiment, the antibody is a monoclonal antibody. The term "monoclonal antibody" is well known in the art. As used herein, the term preferably refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the population comprises individual antibodies that are identical and / or bind the same epitope, except for possible variants that may occur during the production of the monoclonal antibody (such variants generally occur in small amounts). Monoclonal antibodies of the invention can be prepared by the well-known hybridoma method described by Kohler and Milstein, Nature, 256:495 (1975), or can be prepared by recombinant DNA methods.

[0022] In a preferred embodiment of the invention, antibodies are prepared by applying the mutated NT-proBNP or fragment of the invention (described in more detail elsewhere herein) to a mammal, preferably a mouse, more preferably a sheep. In particular, it is contemplated that the NT-proBNP or its fragment comprises (and is thus operably linked to) a carrier protein (for the definition of the term "carrier protein", see elsewhere herein). Depending on the host species, various adjuvants can be used to increase the immune response. Such adjuvants preferably include Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, complex polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Monoclonal antibodies according to the invention can then be prepared using well-known hybridoma techniques. Further details regarding the preparation of the antibodies of the invention are described in the accompanying examples below.

[0023] Preferred antibodies of the invention are IgG antibodies.

[0024] In one embodiment, the antibody of the invention is an isolated antibody. Thus, the antibody should be a purified antibody. Purification of the antibody can be achieved by methods well known in the art, such as size exclusion chromatography (SEC). Accordingly, the antibody should be isolated from the cells in which it is produced. In some embodiments, the isolated antibody is purified to greater than 70% by weight of the antibody, as determined, for example, by the Lowry method, and in some embodiments, purified to greater than 80%, 90%, 95%, 96%, 97%, 98% or 99% by weight. In a preferred embodiment, the isolated antibody according to the invention is purified to greater than 90% purity, as determined by SDS-PAGE under reducing conditions using Coomassie blue staining for protein detection.

[0025] Preferably, the monoclonal antibodies as described herein are selected from ovine monoclonal antibodies, murine monoclonal antibodies, rabbit monoclonal antibodies, goat monoclonal antibodies, equine monoclonal antibodies, avian monoclonal antibodies. More preferably, the monoclonal antibody is a murine monoclonal antibody. Most preferably, the monoclonal antibody is an ovine antibody.

[0026] The antibodies of the invention can be used as capture or detection (signal) antibodies in a sandwich assay, in combination with at least one other antibody that binds a different second NT-proBNP epitope. Preferably, the secondary antibody is derived from a species different from the species from which the antibody of the invention has been obtained. For example, if the antibody of the invention is an ovine antibody, the secondary antibody can be a murine antibody.

[0027] The signal antibody and the capture antibody can be used in a sandwich assay. Sandwich assays are among the most useful and common assays and encompass many variations of sandwich assay techniques. For example, in a typical assay, an unlabeled (capture) binder is immobilized or can be immobilized on a solid matrix, and the sample to be tested is contacted with the capture binder. After a suitable incubation period, for a time period sufficient to allow the formation of a binder-biolinker complex, a second (detection) binder labeled with a reporter molecule capable of generating a detectable signal is then added and incubated for a time sufficient to form another complex of binder-biolinker-labeled binder. Any unreacted material can be washed away, and the presence of the biolinker is determined by observing the signal generated by the reporter molecule that is bound to the detection binder. The results can be qualitative by simple observation of a visible signal or can be quantitative by comparison with a control sample containing a known amount of mutated NT-proBNP (described elsewhere herein as a standard or calibrator).

[0028] The incubation steps of a typical sandwich assay can be varied as needed and appropriately. Such variations include, for example, simultaneous incubation, in which two or more binders and the biolinker are co-incubated. For example, both the sample to be analyzed and the labeled binder are added simultaneously to the immobilized capture binder. It is also possible to first incubate the sample to be analyzed and the labeled binder and then add an antibody that binds to the solid phase or is capable of binding to the solid phase.

[0029] The complex formed between the specific binder and the biolinker should be proportional to the amount of biolinker present in the sample. It should be understood that the specificity and / or sensitivity of the binder to be applied defines the degree of proportion of at least one label present in the sample that can bind specifically. Further details on how measurements can be made are also found elsewhere herein. The amount of the complex formed should be converted into an amount of biolinker that reflects the amount that is actually present in the sample.

[0030] The antibodies as described herein can be included in a test strip.

[0031] The antibody (or antigen-binding fragment thereof) of the present invention should be able to specifically bind to the mutated NT-proBNP (N-terminal fragment of the precursor of brain natriuretic peptide). Thus, the antibody (or antigen-binding fragment thereof) of the present invention should be able to specifically bind to the epitope contained in the mutated NT-proBNP. The term "epitope" is well known in the art. As used herein, the term preferably refers to the portion of the mutated NT-proBNP that can be specifically bound by the antibody of the present invention. However, an epitope according to the present invention can also be formed by a certain three-dimensional structure, and such conformational epitopes are also contemplated herein. In one embodiment, the epitope has a length of at least 5 but no more than 50 amino acids, particularly no more than 20 amino acids.

[0032] It should be understood that the epitope should contain the mutations as mentioned herein, i.e., the mutated amino acid residues. Thus, the antibody of the present invention should bind to the epitope in the mutated NT-proBNP, which contains the mutations (and thus the histidine residue at position 46 of NT-proBNP or the aspartic acid residue at position 43 of NT-proBNP).

[0033] The antibody or antigen-binding fragment thereof as described herein should specifically bind to the corresponding antigen (e.g., the mutated NT-proBNP or its fragment as defined elsewhere herein). Thus, an antibody that "binds" or "specifically binds" to an antigen is expected to refer to an antibody (or its antigen-binding fragment) that specifically binds to the antigen. The expressions "specific binding" or "specifically binding" are well understood and are used to indicate that the antibody (or its antigen-binding fragment) does not significantly bind to other biomolecules. In particular, an antibody that specifically binds to the mutated NT-proBNP (or its fragment) as described herein preferably does not bind to wild-type NT-proBNP (and thus does not bind to NT-proBNP that does not contain the mutation of replacing arginine at position 46 with histidine and / or the mutation of replacing glutamate at position 43 with aspartic acid). The expression "the antibody does not bind to wild-type NT-proBNP" or "the antibody fragment does not bind to wild-type NT-proBNP" means that the antibody (or its fragment) does not significantly bind to wild-type NT-proBNP. For example, as described hereinafter, it is contemplated that the antibody (or its fragment) of the present invention has a K D for wild-type NT-proBNP that is D at least a hundred-fold lower than its K for the mutated NT-proBNP as mentioned herein.

[0034] Accordingly, binding levels to wild-type NT-proBNP, as determined by ELISA or affinity assays using, for example, a Biacore T200 instrument, result in negligible binding affinities. As shown in the Examples, even at high analyte concentrations, kinetic measurements did not show any determinable binding rate constant ka (1 Ms) for such antibodies relative to wild-type NT-proBNP.

[0035] K D is the dissociation constant that can be determined by a binding assay such as surface plasmon resonance technology (BIAcore ® , GE-Healthcare Uppsala, Sweden). As described in the Examples section, two monoclonal antibodies designated S-22.2.195 and S-23.4.66 were generated and tested in the basic research of the present invention. Antibody S-22.2.195 binds to NT-proBNP with the R46H mutation. Antibody S-23.4.66 binds to NT-proBNP with the E43D mutation. The K D , k a and k d values for these antibodies against wild-type NT-proBNP, NT-proBNP with the R46H mutation, and NT-proBNP with the E43D mutation were determined in Example 3 of the Examples section.

[0036] In particular, an antibody (or antigen-binding fragment thereof) that specifically binds to the mutated NT-proBNP R46H as mentioned herein preferably has a K D value of no more than 0.05 nM at 13 °C, particularly no more than 0.08 nM at 25 °C and / or no more than 0.15 nM at 37 °C for the antigen (i.e., NT-proBNP containing a mutation with histidine substituted for arginine at position 46).

[0037] In particular, the antibody of the present invention (or antigen-binding fragment thereof) that specifically binds to the mutated NT-proBNP E43D as mentioned herein preferably has a K D value of no more than 0.4 nM at 13 °C, particularly no more than 3 nM at 25 °C and / or no more than 20 nM at 37 °C for the antigen (i.e., NT-proBNP containing a mutation with aspartic acid substituted for glutamic acid at position 43).

[0038] It is particularly contemplated that the antibody (or antigen-binding fragment) of the present invention specifically binds to its true antigen and does not show detectable off-target interactions as investigated by SPR (surface plasmon resonance).

[0039] Preferably, the antibody (or antigen-binding fragment thereof) of the present invention shows a lower affinity interaction relative to the recognition of wild-type NT-proBNP (compared to the interaction with its antigen, i.e., the mutated NT-proBNP). More preferably, the KD of the antibody or its fragment for wild-type NT-proBNP is at least a hundred-fold lower, especially at least three hundred-fold lower, than its binding to the antigen (i.e., the mutated NT-proBNP).

[0040] Another means of describing the kinetic binding characteristics of an antibody to its antigen is to resolve the dissociation constant into its kinetic rate contributions, since there is an association rate constant k a and a dissociation rate constant k d . The association rate constant k a characterizes the rate of antibody / antigen complex formation and is time- and concentration-dependent.

[0041] Preferably, as mentioned herein, the association rate constant of an antibody (or antigen-binding fragment thereof) that specifically binds to mutated NT-proBNP R46H preferably has a k a value exceeding 4.0E+05 1 / Ms at 13 °C, especially exceeding 7.5E+05 1 / Ms at 25 °C and / or exceeding 1.0E+06 1 / Ms at 37 °C (for its antigen).

[0042] Preferably, as mentioned herein, the association rate constant of an antibody (or antigen-binding fragment thereof) that specifically binds to mutated NT-proBNP E43D preferably has a k a value exceeding 6.0E+04 1 / Ms at 13 °C, especially exceeding 5.5E+04 1 / Ms at 25 °C and exceeding 1.0E+05 1 / Ms at 37 °C.

[0043] As mentioned herein, an antibody that specifically binds to mutated NT-proBNP preferably shows an association rate constant that is at least 1.6-fold slower than that of wild-type NT-proBNP at 25 °C.

[0044] The dissociation rate constant indicates the dissociation rate of an antibody from its antigen. Thus, the dissociation rate constant indicates the probability of the complex decomposing per unit time. The lower the dissociation rate constant, the tighter the binding of the antibody to its antigen.

[0045] Preferably, as mentioned herein, the dissociation rate constant of an antibody (or antigen-binding fragment thereof) that specifically binds to mutated NT-proBNP R46H preferably has a k d value less than 1.0E-05 1 / s at 13 °C, especially less than 3E-05 1 / s at 25 °C and less than 9E-05 1 / s at 37 °C.

[0046] Preferably, as mentioned herein, the dissociation rate constant of an antibody (or an antigen-binding fragment thereof) that specifically binds to the mutated NT-proBNP E43D preferably has a k value of less than 1.0E-05 1 / s at 13 °C, particularly less than 1.5E-04 1 / s at 25 °C and / or less than 2.0E-03 1 / s at 37 °C. d value.

[0047] As mentioned herein, an antibody that specifically binds to the mutated NT-proBNP preferably exhibits a dissociation rate that is at least a hundred-fold faster at 25 °C relative to wild-type NT-proBNP (compared to the dissociation rate constant with respect to the mutated NT-proBNP).

[0048] Kinetic rate constants change continuously in a temperature gradient. The temperature-dependent binding kinetics at 13 °C and 37 °C can be used to characterize antibody-antigen interactions. The quotient of the binding rate velocities at k a 37 °C and k a 13 °C (the velocity factor, US20140256915) is a means of characterizing antibody interactions. If the quotient has a value below 10, the antibody-antigen binding is enthalpy-driven, while a velocity factor VF > 10 increases the likelihood of entropy-driven kinetics.

[0049] Preferably, the quotient of an antibody (or an antigen-binding fragment thereof) that specifically binds to wild-type NT-proBNP or its fragment or mutant protein is below 10.

[0050] The antibodies of the present invention should comprise at least one light chain, particularly two light chains, and at least one heavy chain, particularly two heavy chains.

[0051] In a preferred embodiment of the present invention, the antibody binds to a mutated NT-proBNP containing a mutation in which arginine at position 46 is replaced by histidine. Preferably, the light chain (particularly two light chains) of the antibody comprises the amino acid sequence of SEQ ID NO: 4, and the heavy chain (particularly two heavy chains) of the antibody comprises the amino acid sequence of SEQ ID NO: 5.

[0052] The light chain comprising the amino acid sequence as shown in SEQ ID NO: 4 is preferably encoded by a polynucleotide comprising the nucleic acid sequence as shown in SEQ ID NO: 10. The heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 5 is preferably encoded by a polynucleotide comprising the nucleic acid sequence as shown in SEQ ID NO: 11.

[0053] In another preferred embodiment of the present invention, the antibody binds to a mutated NT-proBNP comprising a mutation in which glutamic acid at position 43 is replaced with aspartic acid. Preferably, the light chain (especially both light chains) of the antibody comprises the amino acid sequence of SEQ ID NO: 12, and the heavy chain (especially both heavy chains) of the antibody comprises the amino acid sequence of SEQ ID NO: 13.

[0054] The light chain comprising the amino acid sequence as shown in SEQ ID NO: 12 is preferably encoded by a polynucleotide comprising the nucleic acid sequence as shown in SEQ ID NO: 14. The heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 13 is preferably encoded by a polynucleotide comprising the nucleic acid sequence as shown in SEQ ID NO: 15.

[0055] In the basic research of the present invention, an antibody comprising the above light chain and heavy chain was generated:

[0056] Antibody S-22.2.195 specifically binds to NT-proBNP with the R46H mutation. This antibody comprises a light chain having the sequence as shown in SEQ ID NO: 4, and a heavy chain having the sequence as shown in SEQ ID NO: 5.

[0057] Antibody S-23.4.66 specifically binds to NT-proBNP with the E43D mutation. This antibody comprises a light chain having the sequence as shown in SEQ ID NO: 12, and a heavy chain having the sequence as shown in SEQ ID NO: 13.

[0058] The amino acid sequences of the heavy chain and light chain of the generated antibody are also shown in Table A.

[0059] Table A further shows the complementarity-determining regions (CDRs) that are mainly responsible for the binding affinity of the antibody. Each light chain and heavy chain has three CDRs (CDR1, CDR2, and CDR3). Thus, each antibody has a total of six different CDRs.

[0060] The CDRs of the heavy chain and light chain of the two antibodies generated in the research of the present invention are indicated in bold in Table A below (in the order from the N-terminus to the C-terminus). The CDRs were annotated on a computer (Lefranc, M.-P., IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc. June 1, 2011; 2011(6)).

[0061] In a preferred embodiment, the antibody (or antigen-binding fragment thereof) of the present invention comprises six CDRs contained in S-22.2.195 or six CDRs contained in S-23.4.66 (preferably in the same order as in the light chain / heavy chain of the respective antibody).

[0062] Accordingly, it is contemplated that the antibody (or antigen-binding fragment thereof) comprises in the light chain (preferably in both light chains):

[0063] CDR1 having the sequence LLDDAY (as shown in SEQ ID NO: 16),

[0064] CDR2 having the sequence KDS, and

[0065] CDR3 having the sequence LSVDSSEYSV (as shown in SEQ ID NO: 17),

[0066] and

[0067] in the heavy chain (preferably in both heavy chains) comprises:

[0068] CDR1 having the sequence GFSLIGEY (as shown in SEQ ID NO: 18),

[0069] CDR2 having the sequence MASGGTI (as shown in SEQ ID NO: 19), and

[0070] CDR3 having the sequence VRSSVSPGDDRDV (as shown in SEQ ID NO: 20).

[0071] The antibody described above specifically binds to NT-proBNP with the R46H mutation. Thus, an antibody that specifically binds to NT-proBNP with the R46H mutation is characterized in that the light chain variable domain comprises CDR1 having the sequence LLDDAY (SEQ ID NO: 16), CDR2 having the sequence KDS, and CDR3 having the sequence LSVDSSEYSV (SEQ ID NO: 17), and the heavy chain variable domain comprises CDR1 having the sequence GFSLIGEY (SEQ ID NO: 18), CDR2 having the sequence MASGGTI (SEQ ID NO: 19), and CDR3 having the sequence VRSSVSPGDDRDV (SEQ ID NO: 20).

[0072] Furthermore, it is contemplated that the antibody (or antigen-binding fragment thereof) of the present invention comprises in the light chain (preferably in both light chains):

[0073] CDR1 having the sequence SSNVGYGNY (as shown in SEQ ID NO: 21),

[0074] A CDR2 having the sequence SAT, and

[0075] a CDR3 having the sequence VSYDSSSKFGV (as shown in SEQ ID NO: 22),

[0076] and

[0077] comprising, in the heavy chain (preferably in both heavy chains):

[0078] a CDR1 having the sequence GFSVTNSG (as shown in SEQ ID NO: 23),

[0079] a CDR2 having the sequence INNDGVA (as shown in SEQ ID NO: 24), and

[0080] a CDR3 having the sequence GTRDLPSDVRYGNMYINY (as shown in SEQ ID NO: 25).

[0081] The above antibody specifically binds to NT-proBNP having the E43D mutation. Thus, the antibody that specifically binds to NT-proBNP having the E43D mutation is characterized in that the light chain variable domain comprises a CDR1 having the sequence SSNVGYGNY (SEQ ID NO: 21), a CDR2 having the sequence SAT, and a CDR3 having the sequence VSYDSSSKFGV (SEQ ID NO: 22), and the heavy chain variable domain comprises a CDR1 having the sequence GFSVTNSG (SEQ ID NO: 23), a CDR2 having the sequence INNDGVA (SEQ ID NO: 24), and a CDR3 having the sequence GTRDLPSDVRYGNMYINY (SEQ ID NO: 25).

[0082] The CDRs mentioned above should consist of the variable regions of the long or short chains respectively, preferably in the order shown in Table A.

[0083] The present invention also relates to antigen-binding fragments of the antibodies of the present invention (also referred to herein as "antibody fragments"). As used herein, an antigen-binding fragment of an antibody should be capable of specifically binding to an antigen (particularly the mutated NT-proBNP as described above, i.e., the mutated NT-proBNP comprising a mutation in which histidine replaces arginine at position 46, or the mutated NT-proBNP comprising a mutation in which aspartic acid replaces glutamic acid at position 43). Thus, an antigen-binding fragment of an antibody is a fragment that retains the ability of the (full-length) antibody to specifically bind to an antigen (such as the mutated NT-proBNP).

[0084] An antibody fragment preferably comprises a part of a full-length antibody, preferably its variable domain, or at least its antigen-binding site. In one embodiment, the antigen-binding fragment is selected from Fab fragment, Fab' fragment, Facb fragment, F(ab')2 fragment, scFv fragment, and Fv fragment. For example, the antigen-binding fragment is an F(ab')2 fragment.

[0085] How to generate antigen-binding fragments is well known in the art. For example, the fragments can be generated by enzymatic cleavage of the antibodies of the present invention. Additionally, the fragments can be generated by synthetic or recombinant techniques. Fab fragments are preferably generated by papain digestion of the antibody, Fab' fragments are generated by pepsin digestion and partial reduction, F(ab')2 fragments are generated by pepsin digestion, and Facb fragments are generated by plasmin digestion. Fv or scFv fragments are preferably generated by molecular biology techniques.

[0086] The antigen-binding fragment of an antibody can also be a diabody, which is a small antibody fragment having two antigen-binding sites. The diabody preferably comprises a heavy-chain variable domain linked to a light-chain variable domain in the same polypeptide chain.

[0087] In one embodiment of the present invention, the antibody or antigen-binding fragment of the present invention is used as a signal antibody or signal fragment (also referred to herein as a detection antibody or detection fragment). The term signal antibody or signal fragment refers to an antibody or fragment that can be directly detected or detected by a label amplified by a detection means. In an alternative embodiment of the present invention, the antibody or antigen-binding fragment of the present invention is used as a capture antibody or capture fragment.

[0088] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention is linked to a detectable label. As described herein, the detectable label is preferably a label that is not naturally linked to the antibody or its antigen-binding fragment. Thus, the detectable label is preferably heterologous to the antibody. Suitable labels are any labels that can be detected by an appropriate detection method. In one embodiment, the detectable label is an enzyme, biotin, a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a gold label, or a magnetic label. In a preferred embodiment, the label is an electrochemiluminescent label.

[0089] Enzymatic labels include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and luciferase. Substrates for these enzymes are well known in the art. Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-chloronitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate). Suitable enzyme-substrate combinations can result in colored reaction products, fluorescence, or chemiluminescence, which can be measured according to methods known in the art. Fluorescent labels include, for example, 5-carboxyfluorescein, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, Cy2, Cy3, and Cy5, fluorescent proteins such as GFP (green fluorescent protein), Texas Red, and Alexa dyes. Radioactive labels include, for example, radioactive isotopes of iodine, cobalt, selenium, tritium, carbon, sulfur, and phosphorus. Radioactive labels can be detected by any known and appropriate method, such as photographic film or a phosphor imager. Magnetic tags include, for example, paramagnetic and superparamagnetic labels. Chemiluminescent labels used can include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, or oxalates.

[0090] In particular, it is contemplated that the antibodies described herein comprise electrochemiluminescent labels (especially signal antibodies).

[0091] The most commonly used electrochemiluminescent compound is ruthenium. Thus, the electrochemiluminescent label preferably comprises ruthenium. In particular, the electrochemiluminescent label should comprise a bipyridine-ruthenium (II) complex. Thus, it is particularly contemplated that the antibody is a ruthenated antibody. How to ruthenate an antibody is described, for example, in the Examples section.

[0092] The present invention also relates to host cells that produce the antibodies or antigen-binding fragments thereof of the present invention. In a preferred embodiment, the host for producing the antibodies of the present invention is a hybridoma cell. In addition, the host cell can be any type of cell system that can be engineered to generate the antibodies according to the present invention. For example, the host cell can be an animal cell, particularly a mammalian cell. In one embodiment, HEK293 cells or CHO cells are used as host cells. In another embodiment, the host cell is a non-human animal or mammalian cell.

[0093] For example, the antibody S-23.4.66 generated in the basic research of the present invention was produced by a hybridoma. The antibody rS-22.2.195 was produced by a hybridoma and a library of CHO cells (Chinese hamster ovary cells) in which the antibody was recombinantly expressed. Subsequently, stable CHO lines were generated for large-scale production of this antibody.

[0094] The host cell according to the present invention should be an isolated cell. Thus, the host cell should be present in cell culture, in other words, outside the organism.

[0095] The host cell preferably comprises at least one polynucleotide encoding the light chain of the antibody of the present invention and at least one polynucleotide encoding the heavy chain of the antibody of the present invention. The polynucleotide should be operably linked to a suitable promoter.

[0096] According to the present invention, it is further contemplated to use an antibody (or an antigen-binding fragment thereof) that specifically binds a mutated NT-proBNP containing a mutation in which arginine at position 46 is replaced by histidine, in combination with an antibody (or an antigen-binding fragment thereof) that specifically binds a mutated NT-proBNP containing a mutation in which glutamate at position 43 is replaced by aspartic acid. The combined use of these antibodies allows the detection of NT-proBNP in subjects containing the R46H mutation and subjects containing the E43D mutation.

[0097] Furthermore, it is contemplated to use the R46H antibody (or an antigen-binding fragment thereof), or the E43D antibody (or an antigen-binding fragment thereof), or both the R46H and E43D antibodies (or antigen-binding fragments thereof), in combination with an antibody (or an antigen-binding fragment thereof) that specifically binds wild-type NT-proBNP. As used herein, an "antibody that specifically binds wild-type NT-proBNP" preferably specifically binds to a region of wild-type NT-proBNP comprising amino acid residues 42 to 46 of wild-type NT-proBNP. Accordingly, the antibody should bind to an epitope contained within amino acids 42 to 46 of wild-type NT-proBNP. The epitope of such an antibody should therefore not contain the E43D and R46H mutations. Preferably, an antibody that specifically binds wild-type NT-proBNP does not significantly bind to the mutated NT-proBNP having the E43D or R46H mutation.

[0098] Accordingly, the present invention also relates to a kit or composition comprising:

[0099] a) i) an antibody or an antigen-binding fragment thereof that specifically binds a mutated NT-proBNP containing a mutation in which arginine at position 46 is replaced by histidine, and ii) an antibody or an antigen-binding fragment thereof that specifically binds a mutated NT-proBNP containing a mutation in which glutamate at position 43 is replaced by aspartic acid, or

[0100] b) i) An antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP contains a mutation in which arginine at position 46 is replaced by histidine, and ii) an antibody or an antigen-binding fragment thereof that specifically binds to wild-type NT-proBNP, wherein the antibody specifically binds to a region of wild-type NT-proBNP that contains amino acid residues 42 to 46 of wild-type NT-proBNP, or

[0101] c) i) An antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP contains a mutation in which glutamate at position 43 is replaced by aspartic acid, and ii) an antibody or an antigen-binding fragment thereof that specifically binds to wild-type NT-proBNP, wherein the antibody specifically binds to a region of wild-type NT-proBNP that contains amino acid residues 42 to 46 of wild-type NT-proBNP, or

[0102] d) i) An antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP contains a mutation in which arginine at position 46 is replaced by histidine, and ii) an antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP contains a mutation in which glutamate at position 43 is replaced by aspartic acid, and iii) an antibody or an antigen-binding fragment thereof that specifically binds to wild-type NT-proBNP, wherein the antibody specifically binds to a region of wild-type NT-proBNP that contains amino acid residues 42 to 46 of wild-type NT-proBNP, or

[0103] e) An antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP contains a mutation in which arginine at position 46 is replaced by histidine, or

[0104] f) An antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP contains a mutation in which glutamate at position 43 is replaced by aspartic acid.

[0105] Preferably, the antibody comprised in the kit is a monoclonal antibody. In one embodiment, the antibody is a monoclonal sheep antibody.

[0106] Preferably, the antibody (or antigen-binding fragment thereof) mentioned under a), b), c), d), e) or f) of the kit or composition should comprise a detectable label (for the definition of this term, see elsewhere in this text). Thus, each antibody (or antigen-binding fragment thereof) as described under a), b), c), d), e) or f) should be operably linked to a detectable label. In particular, it is contemplated that the antibodies bind to the same label. Preferably, each antibody (or antigen-binding fragment thereof) is ruthenated.

[0107] Preferably, the kit or composition of the present invention comprises a further antibody or antigen-binding fragment thereof that binds both the mutated NT-proBNP and the wild-type NT-proBNP. Thus, the antibody (or antigen-binding fragment thereof) should specifically bind to different regions in NT-proBNP, particularly to a region that does not include amino acids 42 to 46 of NT-proBNP. Preferably, the further antibody (or antigen-binding fragment thereof) specifically binds to an epitope present in amino acids 1 to 35 of NT-proBNP (particularly NT-proBNP having the sequence shown in SEQ ID NO: 3). More preferably, the further antibody (or antigen-binding fragment thereof) binds to a specific epitope within amino acids 27 to 31 of NT-proBNP (particularly NT-proBNP having the sequence shown in SEQ ID NO: 3). The antibody can be a monoclonal antibody or a polyclonal antibody. However, preferably, the antibody is a monoclonal antibody. In one embodiment, the further antibody described in this paragraph is a monoclonal murine antibody.

[0108] The further antibody (or antigen-binding fragment thereof) described above is preferably used as a capture antibody (fragment). The antibody (or antigen-binding fragment thereof) should be capable of being immobilized on a solid support (such as a plate, bead or tube). Preferably, the further antibody should capture the mutated NT-proBNP (as described elsewhere in this text) and the wild-type NT-proBNP. In a preferred embodiment, the further antibody (or fragment thereof) is biotinylated. Thus, the antibody (or fragment thereof) can bind to a solid support comprising avidin or streptavidin.

[0109] Unless otherwise stated, the definitions and explanations given above apply mutatis mutandis to the following text.

[0110] Furthermore, the present invention relates to a mutated NT-proBNP or fragment thereof as described above. The mutated NT-proBNP has been defined above in the context of binding to the antibodies of the present invention.

[0111] In one embodiment, the present invention relates to a mutated NT-proBNP or a fragment thereof comprising a mutation in which arginine at position 46 is replaced by histidine. The fragment should comprise the replacement of arginine by histidine at position 46. Accordingly, the fragment should comprise histidine at the position corresponding to position 46 of SEQ ID NO: 1 (or position 46 of SEQ ID NO: 3). Preferably, the mutated NT-proBNP mentioned in this paragraph comprises the amino acid sequence as shown in SEQ ID NO: 1.

[0112] In an alternative embodiment, the present invention relates to a mutated NT-proBNP or a fragment thereof comprising a mutation in which glutamate at position 43 is replaced by aspartic acid. The fragment should comprise the replacement of glutamate by aspartic acid at position 43. Accordingly, the fragment should comprise aspartic acid at the position corresponding to position 43 of SEQ ID NO: 2 (or position 43 of SEQ ID NO: 3). Preferably, the mutated NT-proBNP mentioned in this paragraph comprises the amino acid sequence as shown in SEQ ID NO: 2.

[0113] The mutated NT-proBNP or a fragment thereof of the present invention can be, for example, used to generate an antibody of the present invention. Further, the mutated NT-proBNP or a fragment thereof can be used as a standard or calibrator for an assay in which an antibody (or a fragment thereof) of the present invention is used to determine the amount of the mutated NT-proBNP as mentioned herein. Accordingly, the above-mentioned kit of the present invention can further comprise a) a mutated NT-proBNP or a fragment thereof comprising a mutation in which arginine at position 46 is replaced by histidine, or b) a mutated NT-proBNP or a fragment thereof comprising a mutation in which glutamate at position 43 is replaced by aspartic acid.

[0114] It should be understood that the mutations comprised by the fragment of the present invention are generally not at positions 43 or 46 of the fragment, since the fragment is shorter than the mutated NT-proBNP. Instead, the mutations comprised by the fragment should be the mutation corresponding to the replacement of arginine by histidine at position 46, or the replacement of glutamate by aspartic acid at position 43. Therefore, the fragment should comprise a histidine residue at the position corresponding to position 46 of the wild-type NT-proBNP, or an aspartic acid residue at the position corresponding to position 43 of the wild-type NT-proBNP.

[0115] Preferably, the fragment of the present invention has a length of at least 7 amino acids, more preferably at least 10 amino acids, and most preferably at least 15 amino acids. It is also preferred that the fragment should have a length of at least 20 amino acids.

[0116] The fragment should be shorter than the mutated NT-proBNP mentioned above, i.e., shorter than 76 amino acids.

[0117] In a preferred embodiment, the fragment of the mutated NT-proBNP has a length of no more than 75 amino acids, or in particular no more than 70 amino acids. In another preferred embodiment, the fragment has a length of no more than 50 amino acids. It is further contemplated that the fragment has a length of no more than 30 amino acids. Thus, the fragment preferably has a length of 7 to 70 amino acids (or 7 to 75 amino acids), 10 to 70 amino acids (or 10 to 75 amino acids), or 20 to 70 amino acids (or 20 to 75 amino acids). Also preferably, the fragment has a length of 10 to 30 amino acids.

[0118] The mutated NT-proBNP or its fragment can advantageously be used as an antigen for generating the antibodies of the present invention. In addition, the mutated NT-proBNP or fragment can be used as a positive control in the kit of the present invention (see above).

[0119] In a preferred embodiment, the fragment containing the mutation of substituting arginine at position 46 with histidine contains the sequence shown in SEQ ID NO: 6, or consists of the sequence shown in SEQ ID NO: 6. In another preferred embodiment, the fragment containing the mutation of substituting glutamate at position 43 with aspartic acid contains the sequence shown in SEQ ID NO: 7, or consists of the sequence shown in SEQ ID NO: 7.

[0120] In a preferred embodiment, the fragment containing the mutation of substituting glutamate at position 43 with aspartic acid contains the sequence shown in SEQ ID NO: 8, or consists of the sequence shown in SEQ ID NO: 8. In another preferred embodiment, the fragment containing the mutation of substituting glutamate at position 43 with aspartic acid contains the sequence shown in SEQ ID NO: 9, or consists of the sequence shown in SEQ ID NO: 9.

[0121] In one embodiment of the present invention, the mutated NT-proBNP or its fragment further comprises a purification tag. The tag should be operably linked to the mutated NT-proBNP or its fragment.

[0122] The tag should allow for the purification of NT-proBNP or a fragment thereof. Such tags are well known in the art. As used herein, the term "purification tag" preferably refers to an additional amino acid sequence (a peptide of the polypeptide) that allows for the purification of the mutated NT-proBNP or a fragment thereof of the present invention. In one embodiment, the purification tag is a peptide or polypeptide that is not naturally linked to the mutated NT-proBNP or a fragment thereof. Thus, the purification tag should be heterologous to the mutated NT-proBNP or a fragment thereof.

[0123] Preferably, the purification tag is selected from the group consisting of polyhistidine tags, polyarginine tags, glutathione-S-transferase (GST), maltose binding protein (MBP), influenza virus HA tag, thioredoxin, staphylococcal protein A tag, FLAG™ epitope, and c-myc epitope. In a preferred embodiment, the purification tag is a polyhistidine tag. Preferably, the polyhistidine tag comprises at least 6 consecutive histidine residues.

[0124] In one embodiment of the present invention, the mutated NT-proBNP or a fragment thereof further comprises a carrier protein. The carrier protein should be operably linked to the mutated NT-proBNP or a fragment thereof.

[0125] The carrier protein is preferably heterologous to the mutated NT-proBNP or a fragment thereof. The carrier protein is desirably a protein that elicits an immune response. Thus, the carrier protein should have a high degree of immunogenicity. Such carrier proteins are well known in the art. Preferably, the carrier protein is selected from keyhole limpet hemocyanin (KLH), tetanus toxoid, and diphtheria toxoid, bovine serum albumin (BSA), ovalbumin, and thyroglobulin, especially KLH.

[0126] The present invention also relates to a composition comprising i) the mutated NT-proBNP of the present invention or ii) a fragment of the present invention, and further comprising an immunoadjuvant. As used herein, the term "immunoadjuvant" refers to a substance or composition that, when administered to an organism together with an antigenic substance, can enhance the immune response against the antigenic substance. Preferred adjuvants are described above. In one embodiment, the immunoadjuvant is selected from alum, Freund's incomplete adjuvant, and especially Freund's complete adjuvant.

[0127] Furthermore, the present invention relates to the use of the mutated NT-proBNP or a fragment thereof of the present invention for the production of an antibody of the present invention. Preferably, the antibody should specifically bind to the mutated NT-proBNP as defined elsewhere herein. In one embodiment, the antibody is a monoclonal antibody. Alternatively, the composition defined in the previous paragraph is used for the generation of antibodies.

[0128] The present invention further relates to a polynucleotide encoding the mutated NT-proBNP of the present invention or a fragment of the present invention.

[0129] As used herein, the term "polynucleotide" refers to a linear or circular nucleic acid molecule. It encompasses DNA as well as RNA molecules. Preferably, the polynucleotide of the present invention should be provided as an isolated polynucleotide (i.e., isolated from its natural environment) or in a genetically modified form. The term encompasses single-stranded as well as double-stranded polynucleotides. In addition, also included are chemically modified polynucleotides, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides, such as biotinylated polynucleotides. The polynucleotide of the present invention is characterized in that it should encode a polypeptide as mentioned above. Due to the degeneracy of the genetic code, polynucleotides encoding specific amino acid sequences as described above are encompassed. In one embodiment, the polynucleotide does not comprise intron sequences.

[0130] In one embodiment, the polynucleotide of the present invention is operably linked to a promoter. The promoter should allow expression of the polynucleotide. In one embodiment, the promoter is a heterologous promoter. In addition, the polynucleotide of the present invention can be linked to a terminator.

[0131] The present invention also contemplates a vector comprising one of the aforementioned polynucleotides of the present invention. In one embodiment, the vector is an expression vector, wherein the polynucleotide of the present invention is operably linked to a promoter, in particular to a heterologous promoter.

[0132] The term "vector" preferably encompasses phage, plasmid, viral or retroviral vectors, as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. More preferably, the vector of the present invention is an expression vector. In such expression vectors, the polynucleotide contains an expression cassette as specified above, allowing expression in eukaryotic cells or their isolated fractions. In addition to the polynucleotide of the present invention, the expression vector may further contain additional regulatory elements, including transcriptional and translational enhancers. Preferably, the expression vector is also a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses or bovine papillomaviruses can be used to deliver the polynucleotide of the present invention into a targeted cell population. Methods well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y., and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994).

[0133] The present invention also relates to host cells comprising the mutated NT-proBNP or a fragment thereof, the polynucleotide or the vector of the present invention.

[0134] The term "host cell" mentioned in the previous paragraph should be a prokaryotic or eukaryotic cell. Eukaryotic cells include protists, fungi, plant and animal cells. In another embodiment, the host cells include, but are not limited to, the prokaryotic cell line Escherichia coli (E. Coli); the mammalian cell lines CHO, HEK293, COS, NSO, SP2 and PER.C6; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae. In combination with the present invention, it is also contemplated that the host cell is a non-human host cell. In one embodiment, the mutated NT-proBNP, fragment, polynucleotide or vector comprised by the host cell of the present invention is heterologous to the host cell. For example, it is contemplated that the host cell has been transfected with the polynucleotide of the present invention.

[0135] Furthermore, the present invention relates to a kit or composition comprising:

[0136] (i) a mutated NT-proBNP or a fragment thereof comprising a mutation in which arginine at position 46 is replaced by histidine, or

[0137] (ii) a mutated NT-proBNP or a fragment thereof that contains a mutation substituting aspartic acid for glutamic acid at position 43, or

[0138] (iii) a mutated NT-proBNP or a fragment thereof that contains a mutation substituting histidine for arginine at position 46, and a mutated NT-proBNP or a fragment thereof that contains a mutation substituting aspartic acid for glutamic acid at position 43.

[0139] The term "fragment" has been explained elsewhere herein. The definitions and explanations apply accordingly.

[0140] In one embodiment of the foregoing kit (or composition), the kit (or composition) further comprises wild-type NT-proBNP or a fragment thereof. Preferably, the fragment comprises at least amino acid residues 42 to 46 of wild-type NT-proBNP. In one embodiment, the fragment has a length of at least 10 amino acids.

[0141] In addition, the present invention relates to a method for diagnosing heart failure in a subject, which comprises the following steps:

[0142] a) determining the amount of NT-proBNP in a sample from the subject, and

[0143] b) comparing the amount of NT-proBNP determined in step a) with a reference amount, thereby diagnosing heart failure.

[0144] In a preferred embodiment of the foregoing method, determining the amount of NT-proBNP comprises the step of contacting the sample with at least one antibody (or at least one antigen-binding fragment of the antibody) of the present invention. In particular, the determination of NT-proBNP comprises the step of contacting the sample with at least the following:

[0145] (i) an antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP that contains a mutation substituting histidine for arginine at position 46, or

[0146] (ii) an antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP that contains a mutation substituting aspartic acid for glutamic acid at position 43, or

[0147] (iii) an antibody or antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP comprises a mutation in which arginine at position 46 is replaced by histidine, and an antibody or antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP comprises a mutation in which glutamate at position 43 is replaced by aspartic acid.

[0148] In one embodiment of step a) of the foregoing method, the sample is further contacted with an antibody or an antigen-binding fragment of the antibody that specifically binds to wild-type NT-proBNP, wherein the antibody specifically binds to a region of wild-type NT-proBNP comprising amino acid residues 42 to 46 of wild-type NT-proBNP.

[0149] Thus, the determination of NT-proBNP comprises the step of contacting a sample with at least the following:

[0150] (i) an antibody or antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP comprises a mutation in which arginine at position 46 is replaced by histidine, and an antibody or antigen-binding fragment thereof that specifically binds to wild-type NT-proBNP, wherein the antibody specifically binds to a region of wild-type NT-proBNP comprising amino acid residues 42 to 46 of wild-type NT-proBNP, or

[0151] (ii) an antibody or antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP comprises a mutation in which glutamate at position 43 is replaced by aspartic acid, and an antibody or antigen-binding fragment thereof that specifically binds to wild-type NT-proBNP, wherein the antibody specifically binds to a region of wild-type NT-proBNP comprising amino acid residues 42 to 46 of wild-type NT-proBNP, or

[0152] (iii) an antibody or antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP comprises a mutation in which arginine at position 46 is replaced by histidine, and an antibody or antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP comprises a mutation in which glutamate at position 43 is replaced by aspartic acid, and an antibody or antigen-binding fragment thereof that specifically binds to wild-type NT-proBNP, wherein the antibody specifically binds to a region of wild-type NT-proBNP comprising amino acid residues 42 to 46 of wild-type NT-proBNP.

[0153] The subject of the method according to the invention is preferably a human.

[0154] In one embodiment, the subject is heterozygous, or particularly homozygous, for a mutation in NT-proBNP, in which arginine at position 46 is replaced by histidine. Preferably, the antibody / antigen-binding fragment in i) or iii) as described above is used.

[0155] In an alternative embodiment, the subject is heterozygous, or particularly homozygous, for a mutation in NT-proBNP, in which glutamate at position 43 is replaced by aspartic acid. Preferably, the antibody / antigen-binding fragment in ii) or iii) as described above is used.

[0156] In one embodiment of the invention, the sample is a blood, serum or plasma sample. For example, a blood sample, i.e., a whole blood sample, can be used for NT-proBNP determination in a point-of-care medical context. The most preferred samples are plasma and serum. Samples of this type are routinely used in laboratory tests.

[0157] Furthermore, the invention relates to the use of at least one antibody (or at least one antigen-binding fragment thereof) of the invention for diagnosing heart failure in a sample of a subject (as defined hereinabove). Preferably, the antibody (or antibodies) as described in i), ii), iii) is used in combination with step a) of the method for diagnosing heart failure. In addition to the antibody (or antibodies) or its antigen-binding fragment, it is contemplated to use an antibody or an antigen-binding fragment of the antibody that specifically binds wild-type NT-proBNP, wherein the antibody specifically binds to a region of wild-type NT-proBNP comprising amino acid residues 42 to 46 of wild-type NT-proBNP.

[0158] The antibody or antigen-binding fragment applied in the use or method of the invention may comprise a detectable label as described elsewhere.

[0159] Hereinafter, the preferred embodiments of the invention are summarized. The definitions given in the above description and claims are accordingly applied.

[0160] 1. An antibody that specifically binds mutant NT-proBNP, wherein the antibody is selected from

[0161] a) an antibody that specifically binds mutant NT-proBNP, the mutant NT-proBNP comprising a mutation in which arginine at position 46 is replaced by histidine, and

[0162] (b) An antibody that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP contains a mutation in which glutamic acid at position 43 is replaced with aspartic acid.

[0163] 2. The antibody of embodiment 1, wherein the antibody is a monoclonal antibody.

[0164] 3. The antibody of embodiment 1 or 2, wherein the antibody is an ovine antibody.

[0165] 4. The antibody of any one of embodiments 1 to 3, wherein the NT-proBNP is a mutated human NT-proBNP.

[0166] 5. The antibody of any one of embodiments 1 to 4, wherein

[0167] (i) the mutated NT-proBNP containing a mutation in which arginine at position 46 is replaced with histidine contains the amino acid sequence as shown in SEQ ID NO: 1, or

[0168] (ii) the mutated NT-proBNP containing a mutation in which glutamic acid at position 43 is replaced with aspartic acid contains the amino acid sequence as shown in SEQ ID NO: 2.

[0169] 6. The antibody of any one of embodiments 1 to 5, wherein the antibody does not significantly bind to wild-type NT-proBNP.

[0170] 7. The antibody of any one of embodiments 1 to 6, wherein

[0171] i) the antibody specifically binds to a mutated NT-proBNP that contains a mutation in which arginine at position 46 is replaced with histidine, and wherein the light chain of the antibody contains the amino acid sequence of SEQ ID NO: 4, and wherein the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO: 5, or

[0172] ii) the antibody specifically binds to a mutated NT-proBNP that contains a mutation in which glutamic acid at position 43 is replaced with aspartic acid, and wherein the light chain of the antibody contains the amino acid sequence of SEQ ID NO: 12, and wherein the heavy chain of the antibody contains the amino acid sequence of SEQ ID NO: 13.

[0173] 8. An antigen-binding fragment of the antibody of any one of embodiments 1 to 7.

[0174] 9. The antigen-binding fragment of embodiment 8, wherein the antigen-binding fragment is selected from Fab fragment, Fab' fragment, Facb fragment, F(ab')2 fragment, scFv fragment and Fv fragment.

[0175] 10. The antibody of any one of embodiments 1 to 7, or the antigen-binding fragment of embodiment 8 or 9, wherein the antibody or the antigen-binding fragment is linked to a detectable label.

[0176] 11. The antibody or antigen-binding fragment of embodiment 10, wherein the detectable label is an enzyme, biotin, a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a gold label or a magnetic label, particularly wherein the detectable label is an electrochemiluminescent label.

[0177] 12. The antibody or antigen-binding fragment of embodiment 10 or 11, wherein the detectable label comprises ruthenium.

[0178] 13. A host cell that produces the antibody of any one of embodiments 1 to 7, or the antigen-binding fragments of embodiments 8 and 9.

[0179] 14. A kit or composition comprising the following:

[0180] a) i) An antibody or its antigen-binding fragment that specifically binds to mutant NT-proBNP, the mutant NT-proBNP comprising a mutation in which arginine at position 46 is replaced with histidine, and ii) An antibody or its antigen-binding fragment that specifically binds to mutant NT-proBNP, the mutant NT-proBNP comprising a mutation in which glutamate at position 43 is replaced with aspartic acid, or

[0181] b) i) An antibody or its antigen-binding fragment that specifically binds to mutant NT-proBNP, the mutant NT-proBNP comprising a mutation in which arginine at position 46 is replaced with histidine, and ii) An antibody or its antigen-binding fragment that specifically binds to wild-type NT-proBNP, wherein the antibody specifically binds to the region of wild-type NT-proBNP comprising amino acid residues 42 to 46 of wild-type NT-proBNP, or

[0182] c) i) An antibody or its antigen-binding fragment that specifically binds to mutant NT-proBNP, the mutant NT-proBNP comprising a mutation in which glutamate at position 43 is replaced with aspartic acid, and ii) An antibody or its antigen-binding fragment that specifically binds to wild-type NT-proBNP, wherein the antibody specifically binds to the region of wild-type NT-proBNP comprising amino acid residues 42 to 46 of wild-type NT-proBNP, or

[0183] d) i) An antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP contains a mutation in which arginine at position 46 is replaced with histidine, and ii) An antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, wherein the mutated NT-proBNP contains a mutation in which glutamate at position 43 is replaced with aspartic acid, and iii) An antibody or an antigen-binding fragment thereof that specifically binds to wild-type NT-proBNP, wherein the antibody specifically binds to a region of wild-type NT-proBNP that contains amino acid residues 42 to 46 of wild-type NT-proBNP.

[0184] 15. The kit or composition of embodiment 14, wherein the antibody mentioned in a), b), c) or d) is conjugated to a detectable label, preferably conjugated to an electrochemiluminescent label, particularly wherein the antibody is ruthenated.

[0185] 16. The kit or composition of any one of embodiments 13 to 15, which further comprises an antibody that binds to a different epitope in NT-proBNP, particularly wherein the antibody binds to an epitope present in amino acids 1 to 35 of NT-proBNP.

[0186] 17. A mutated NT-proBNP, which comprises

[0187] i) A mutation in which arginine at position 46 is replaced with histidine, or

[0188] ii) A mutation in which glutamate at position 43 is replaced with aspartic acid,

[0189] or a fragment of the mutated NT-proBNP, wherein the fragment comprises

[0190] i) A mutation in which arginine at position 46 is replaced with histidine, or

[0191] ii) A mutation in which glutamate at position 43 is replaced with aspartic acid.

[0192] 18. The mutated NT-proBNP or its fragment of embodiment 17, wherein the mutated NT-proBNP comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0193] 19. The mutated NT-proBNP or its fragment of embodiment 17 or 18, wherein the mutated NT-proBNP or fragment is operably linked to a purification tag or a carrier protein.

[0194] 20. A polynucleotide encoding the mutated NT-proBNP or a fragment thereof according to any one of embodiments 17 to 19.

[0195] 21. An expression vector comprising the polynucleotide of embodiment 20.

[0196] 22. A host cell comprising the mutated NT-proBNP or a fragment thereof according to any one of embodiments 17 to 19, the polynucleotide of embodiment 20, or the expression vector of embodiment 21.

[0197] 23. A composition comprising an immune adjuvant and the mutated NT-proBNP or a fragment thereof according to any one of embodiments 17 to 19.

[0198] 24. Use of the mutated NT-proBNP or a fragment thereof according to any one of embodiments 17 to 19, or the composition of embodiment 23 for the production of antibodies.

[0199] 25. A kit or composition comprising:

[0200] (i) a mutated NT-proBNP or a fragment thereof comprising a mutation replacing arginine at position 46 with histidine, or

[0201] (ii) a mutated NT-proBNP or a fragment thereof comprising a mutation replacing glutamate at position 43 with aspartic acid, or

[0202] (iii) a mutated NT-proBNP or a fragment thereof comprising a mutation replacing arginine at position 46 with histidine and a mutated NT-proBNP or a fragment thereof comprising a mutation replacing glutamate at position 43 with aspartic acid.

[0203] 26. The kit or composition of embodiment 25, further comprising wild-type NT-proBNP or a fragment thereof, wherein the fragment comprises at least amino acid residues 42 to 46 of wild-type NT-proBNP.

[0204] 27. A method for diagnosing heart failure in a subject, comprising the steps of:

[0205] a) determining the amount of NT-proBNP in a sample from the subject, and

[0206] b) comparing the amount of NT-proBNP determined in step a) with a reference amount to thereby diagnose heart failure.

[0207] wherein determining the amount of NT-proBNP comprises the step of contacting the sample with at least the following:

[0208] (i) an antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, said mutated NT-proBNP comprising a mutation in which arginine at position 46 is replaced by histidine, or

[0209] (ii) an antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, said mutated NT-proBNP comprising a mutation in which glutamate at position 43 is replaced by aspartic acid, or

[0210] (iii) an antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, said mutated NT-proBNP comprising a mutation in which arginine at position 46 is replaced by histidine, and an antibody or an antigen-binding fragment thereof that specifically binds to a mutated NT-proBNP, said mutated NT-proBNP comprising a mutation in which glutamate at position 43 is replaced by aspartic acid.

[0211] 28. The method of embodiment 27, wherein the sample is further contacted with an antibody or an antigen-binding fragment of said antibody that specifically binds to wild-type NT-proBNP, wherein said antibody specifically binds to a region of wild-type NT-proBNP comprising amino acid residues 42 to 46 of wild-type NT-proBNP.

[0212] All of the references mentioned above are incorporated by reference in their entirety as to their entire disclosures and as to the specific disclosures specifically mentioned in the foregoing description.

[0213] In Figure 1 and 2 and in the following Examples section, the mutations R72H and E69D in NT-proBNP are mentioned. As described elsewhere herein, the R72H mutation is the same mutation as the R46H mutation, and the E69D mutation is the same mutation as the E46D mutation.

[0214] In the drawings:

[0215] Figure 1 : Concentration-dependent antibody kinetics for antibodies S-23.4.66 and rS-22.2.195 (see Example 3)

[0216] Figure 2 : Antibody sandwich experiments using recombinant proBNP and M-18.4.34-IgG as the primary antibody, and S-23.4.66-IgG or rS-22.2.195-IgG as the secondary antibody. M-18.4.34-IgG was used as a homologous secondary antibody control (see Example 4).

[0217] Figure 3 : Specificity of antibody rS-22.2.195 for NT-proBNP-R46H

[0218] Figure 4 : Specificity of antibody S-23.4.66 for NT-proBNP-E43D. Example

[0219] The following examples will illustrate the invention. However, they should not be construed as limiting the scope of the invention.

[0220] Example 1: Production of recombinant N-terminal proBNP (1-76) mutant proteins NTproBNP (1-76) mutant protein [R72H] (i.e., the mutant protein containing the R46H substitution) and NTproBNP (1-76) mutant protein [E69D] (i.e., the mutant protein containing the E43D substitution).

[0221] The nucleotide sequence of N-terminal proBNP (amino acid sequence 1-76) was generated by genetic synthesis via PCR, and the amplified gene was cloned into a suitable expression vector (pQE80), allowing expression with a C-terminal histidine tag. To obtain optimal expression of the gene, the DNA sequence was adapted to the most frequently used codons in Escherichia coli. The translated protein sequence consists of 92 amino acids, containing an N-terminal tetra-amino acid sequence MRGS (SEQ ID NO: 29), followed by NTproBNP (1-76), amino acids GGGS (SEQ ID NO: 30), and 8 histidines:

[0222] NTproBNP (1-76) mutant protein [R72H]: MRGS-NTproBNP (1-76) [R72H]-GGGSHHHHHHHH (see SEQ ID NO: 27)

[0223] NTproBNP (1-76) mutant protein [E69D]: MRGS-NTproBNP (1-76) [E69D]-GGGSHHHHHHHH (see SEQ ID NO: 28)

[0224] The plasmid was transformed into Escherichia coli. The recombinant E. coli clone was inoculated in Super Broth (with 100 μg / ml ampicillin) at an OD600 of 0.2, and induced with IPTG (isopropyl β-D-thiogalactopyranoside; final concentration 0.5 mM) at an OD600 of 1.0 – 1.5. After induction, the culture was further incubated at 37 °C for 3 h. The culture was then centrifuged, and the cell pellet was collected in 20 mM sodium phosphate buffer, pH 7.5, 500 mM NaCl. After disrupting the cell suspension by high pressure, the suspension was centrifuged, and the supernatant was applied to a Ni-NTA (nitrilotriacetic acid) column. After a washing step with 50 mM sodium phosphate buffer, pH 7.5, 500 mM NaCl, 20 mM imidazole, the histidine-tagged NT-pro BNP protein was eluted using a linear gradient of increasing concentrations of imidazole (from 20 mM to 500 mM) in 50 mM sodium phosphate buffer, pH 7.5, 500 mM NaCl buffer.

[0225] Example 2: Generation and screening of monoclonal antibodies against NTproBNP(1-76) mutant protein [R72H] and NTproBNP(1-76) mutant protein [E69D]

[0226] Sheep were immunized with recombinant NTproBNP(1-76) mutant protein [R72H] and recombinant NTproBNP(1-76) mutant protein [E69D]. Freund's complete was used as adjuvant. After four primary immunizations, the immunizations were repeated at monthly intervals. One sheep lymph node with a positive reaction titer was excised under general anesthesia and pain medication. After surgical excision, a single cell preparation was made from the lymph node tissue. The lymph node lymphocytes and the permanent myeloma cell line 1C10 (Bioventix) were fused at a ratio of 2:1 with PEG and cultured in Dulbecco's modified Eagle's medium supplemented with 4.5 mg / ml glucose, 10% fetal bovine serum (Hyclone), 100 μmol / L non-essential amino acids (Gibco), 5.7 μM azaserine / 100 μM hypoxanthine (Sigma), 50 U / ml human interleukin-6 (Roche), 100 IU / ml penicillin / 100 μg / ml streptomycin (Sigma). Fusion was carried out according to the well-known method of Köhler and Milstein (Nature 256, 1975, pages 495-497). Hybridomas secreting antibodies specific for their respective immunogen sequences were finally cloned by single cell deposition using a FACSAria III.

[0227] To identify the presence and specificity of antibodies against the NTproBNP (1-76) mutant protein [R72H] or the NTproBNP (1-76) mutant protein [E69D] in the culture supernatant of hybridoma cells, the clones were evaluated by ELISA according to the following test principle:

[0228] a) Reactivity with NTproBNP(1 - 76) mutant protein [R72H]

[0229] A streptavidin-coated 384-well microtiter plate (Microcoat) was used with 1 μg / ml biotinylated MAK <ntprobnp>M-18.4.34 IgG-Bi mono (Roche) was coated for 1 hour at room temperature in incubation buffer (PBS buffer + 0.5% Byco C). M-18.4.34 binds to the region encompassing amino acids 27 to 31 of SEQ ID NO: 3. For example, the antibody binds to the mutated NT-proBNP and wild-type as described herein. After washing with the washing solution (0.9% NaCl + 0.05% Tween 20), further incubation with the antigen NTproBNP(1-76) mutant protein [R72H] diluted to 100 ng / ml in the incubation buffer was carried out for 1 hour at room temperature. After a further washing step with the washing solution, antibody sample incubation (hybridoma supernatant) was carried out at 50 μl / well for 1 hour at room temperature. After a further washing step, incubation with AffiniPure Donkey Anti-Sheep-IgG (H+L) detection antibody (Jackson Imm.Research) diluted 1:15,000 in the incubation buffer was carried out for 1 hour at room temperature. After a further washing step with the washing buffer, peroxidase activity was detected by incubation with ABTS (ready-to-use solution, Roche) for 20 minutes at room temperature, and the absorbance difference was read in mU at 405 nm with the aid of an ELISA plate reader.

[0230] The positive-reacting hybridoma supernatants were then similarly screened for cross-reactivity against the NTproBNP(1-76) mutant protein [E69D] and wild-type NTproBNP(1-76).

[0231] b) Reactivity with NTproBNP(1 - 76) mutant protein [E69D]

[0232] Using the NTproBNP(1-76) mutant protein [E69D] as the antigen, the initial ELISA screening was performed in a similar manner as described above. The positive-reacting hybridoma supernatants were then screened for cross-reactivity against the NTproBNP(1-76) mutant protein [R72H] and wild-type NTproBNP(1-76).

[0233] Example 3: Characterization of the kinetic properties of monoclonal antibodies

[0234] The T200 instrument is equipped with a Biacore Series S Sensor Chip CM5. The system buffer is 1 mM KH2PO4, 10 mM Na2HPO4 pH 7.4, 500 mM NaCl, 2.7 mM KCl, 0.05 % Tween 20. To determine the rate factor, the system was incubated at 13 °C and 37 °C. The sample buffer is the system buffer supplemented with 1 mg / ml CMD (carboxymethyl dextran, Fluka). An antibody capture system was established. As described by the manufacturer, 5000 RU of rabbit anti-sheep polyclonal antibody (ibid: 313-005-045, Dianova) was immobilized at 30 μg / ml in 10 mM sodium acetate buffer pH 5.0 at 25 °C by EDC / NHS coupling. The capture system was regenerated by washing with concentrated HBS buffer (100 mM HEPES pH 7.4, 1.5 M NaCl, 0.05% (w / v) Tween 20) for 15 seconds at 20 μl / min, and injection of 100 mM HCl for 30 seconds at 20 μl / min, followed by injection of 10 mM glycine buffer pH 1.5 for 1 minute at 20 μl / min. The antibody to be captured was injected for 1 minute at 10 μl / min at a concentration of 40 nM diluted in separate system buffer pH 7.4. After antibody capture, the system was washed with 2.5-fold concentrated system buffer at 60 μl / min for 30 seconds, followed by 2 minutes of baseline stabilization. A concentration-dependent analyte series was injected in 1:3 dilution steps, from 0.4 nM, 1.1 nM, 3.3 nM, in two injections at 10 nM, 30 nM and 90 nM. In another embodiment, the analyte NTproBNP E69D was injected in a concentration series from 1.1 nM, 3.3 nM, 10 nM, in two injections at 30 nM, 90 nM and 270 nM. The analyte contact time was 5 minutes, and the dissociation time was 10 minutes. Analyte kinetics were performed at 60 μl / min. Sheep antibodies were captured as ligands on the sensor surface: mAb<NTproBNP(E69D)>S-23.4.66-IgG, mAb<NTproBNP(R72H)>rS-22.2.195-IgG. Analytes manufactured by Roche were injected into the solution: NT-proBNP (aa 1-76) (MW 8.5 kDa); proBNP (aa 27-102) (MW 9.9 kDa); NT-proBNP E69D (MW 10.2 kDa); NT-proBNP R72H (MW 10.2 kDa); system buffer as control; 0 - serum SB150610-001.Use the Biaevaluation software V.3.0 according to the instructions of the manufacturer GEHC. Apply a local 1:1 binding model to determine the kinetic rate. MAX The local 1:1 binding model was used to determine the kinetic rate.

[0235] Figure 1 The concentration-dependent antibody kinetics performed at 37 °C are shown. Antibody S-23.4.66 binds to NT-proBNP E69D, but does not show any detectable interaction with full-length wild-type NT-proBNP (aa 1-76), wild-type proBNP (aa27-102), and NT-proBNP R72H. The recombinant sheep antibody rS-22.2.195 does not show measurable interaction with respect to NT-proBNP (aa 1-76), proBNP (aa27-102), and NT-proBNP E69D, but specifically binds to NT-proBNP R72H. The kinetic data are summarized in Table 1.

[0236]

[0237] ka: Association rate constant [M-1s-1]; kd: Dissociation rate constant [s-1]; KD: Dissociation constant [M]; t / 2diss: Half-life of complex dissociation [minutes] (ln(2) / (kd * 60)) [minutes]; Rmax: Maximum analyte binding capacity [RU], Chi2: Statistical fit model quality; MR: Molar ratio, binding ratio of analyte to antibody, MW(antibody) / MW(antigen) * response (analyte-antibody binding (RU) / antibody capture (RU)). n.d. means not detectable.

[0238] To evaluate the thermodynamic binding properties of the sheep antibodies, temperature-dependent binding kinetic data at 13 °C and 37 °C were used to calculate the velocity factor (US20140256915, Table 2). Briefly, the velocity factor is the quotient of the binding rate velocities at ka 37°C and ka 13°C. If the quotient is below a value of 10, VF < 10, then the antibody-antigen binding is enthalpy-driven, while a velocity factor VF > 10 increases the likelihood of entropy-driven kinetics. For issues of specificity and biophysical stability, preferably, enthalpy-driven antibody binding interactions are used to detect NT-proBNP wild-type and NT-proBNP mutants in immunoassays.

[0239] Table 2:

[0240]

[0241] Example 4: Antibody Sandwich Characterization

[0242] Antibody sandwich SPR measurements were performed at 25 °C. Using the T200 instrument under the same buffer conditions and sensor device as described in Example 3. As described by the manufacturer, 12,000 RU of RbAMFcg (rabbit anti-mouse Fcγ, PAK <m-fcg>Rb-IgG (IS), code: 315-005-046, Jackson Immuno Research) was immobilized at 40 μg / ml in 10 mM sodium acetate buffer pH 5.0 at 25 °C. The capture system was regenerated by washing with concentrated HBS buffer (100 mM HEPES pH 7.4, 1.5 M NaCl, 0.05% (w / v) Tween 20) for 15 s at 20 μl / min, injection of 10 mM glycine buffer pH 1.5 for 1 min at 20 μl / min, followed by two injections of 10 mM glycine buffer pH 1.7 for 1 min each at 20 μl / min. 150 nM primary murine anti-proBNP antibody M-18.4.34 (27-31) was captured for 1 min at 10 μl / min. The free binding capacity of the capture system was saturated with the blocking solution. The blocking solution was injected at 30 μl / min for 3 min, followed by 2 min baseline stabilization. Analytes NT-proBNP (aa 1-76) (MW 8.5 kDa); proBNP (aa 27-102) (MW 9.9 kDa) and NT-proBNP R72H (MW 10.2 kDa) were injected at 90 μM at 30 μL / min for 3 min. Analyte NT-proBNP E69D (MW 10.2 kDa) was injected at 180 μM at 30 μL / min for 3 min. In another embodiment, instead of the recombinant proBNP analyte, human serum was applied as the analyte in solution. The serum was diluted 1:5 with sample buffer and injected at 5 μl / min for 20 min such that the displayed primary antibody M-18.4.34 (27-31) was saturated with the native proBNP antigen derivative derived from patient serum. Serum analytes were: 0 - serum SB150610-001; NT-proBNP R72H serum; NT-proBNP wild type serum SE 1053 DI 0580 with 27.39 ng / mL proBNP; To evaluate the sandwich performance, each of 250 nM of the secondary antibodies mAb<NTproBNP(E69D)>S-23.4.66-IgG and mAb<NTproBNP(R72H)>rS-22.2.195-IgG SP / Q) was injected at 30 μl / min for 3 min binding time and 3 min dissociation time. Biaevaluation software V.3.0 was used according to the instructions of the manufacturer GEHC.

[0243] Antibody sandwich experiments using recombinant proBNP and M-18.4.34-IgG as the primary antibody and S-23.4.66-IgG or rS-22.2.195-IgG as the secondary antibody are shown in Figure 2 In this case, M-18.4.34-IgG was applied as a homologous secondary antibody control. All sensorgrams showed analyte saturation for the primary antibody M-18.4.34-IgG, followed by injection of the secondary antibody. The primary antibody binds to the antigen with high complex stability. rS-22.2.195-IgG showed specific complex formation with NT-proBNP R72H. Antibody S-23.4.66-IgG specific complexes were formed on NT-proBNP E69D.

[0244] Similar experiments were performed using human serum instead of the recombinant analyte. Figure 2 Attention was paid to the injection of the secondary antibody as overlaid data. Negative serum (0-serum SB150610-001) was referenced. Antibody rS-22.2.195-IgG specifically formed an antibody sandwich complex with NT-proBNP R72H serum and did not form a complex with wild-type NT-proBNP serum. S-23.4.66-IgG did not show complex formation in the serum sample (NT-proBNP E69D serum was not available).

[0245] Example 5: Purification and ruthenation of monoclonal antibodies rS-22.2.195 and S-23.4.66

[0246] MAK <ntprobnp>Purification of S - 23.4.66 - IgG< / ntprobnp>

[0247] The cell-free ovine hybridoma culture supernatant (CELLine, INTEGRA Biosciences AG) was adjusted to pH 4.75 and incubated at RT for 30 minutes. After centrifugation, 0.5 M ammonium sulfate and 150 mM NaCl were added to the supernatant containing IgG, and the pH was raised to 8.5. The solution was applied to a protein A column (ProSep® Ultra Plus, MerckMillipore), and the bound IgG was eluted with 100 mM citrate, 100 mM NaCl, pH 5.5. After dialysis against 50 mM K-phosphate, 150 mM NaCl pH 7.5, trace amounts of bovine IgG from FCS in the medium were removed using an immunoadsorbent resin that specifically binds bovine IgG. The final product was concentrated (Amicon Ultra-30, Merck Millipore), filtered (0.22 μm) and stored at -80 °C.

[0248] MAK <ntprobnp>Purification of rS - 22.2.195 - IgG< / ntprobnp>

[0249] The cell-free culture supernatant of the CHO cell line expressing the recombinant antibody was adjusted to pH 4.75 and incubated at RT for 30 minutes. After centrifugation, 0.5 M ammonium sulfate and 150 mM NaCl were added to the supernatant containing IgG, and the pH was raised to 8.5. The solution was applied to a Protein A column (ProSep® Ultra Plus, Merck Millipore), and the bound IgG was eluted with 100 mM citrate, 100 mM NaCl, pH 5.5. After dialysis against 50 mM K-phosphate, 150 mM NaCl pH 7.5, the final product was concentrated (Amicon Ultra-30, Merck Millipore), filtered (0.22 μm) and stored at -80 °C.

[0250] MAK <ntprobnp>S-23.4.66-IgG and MAK <ntprobnp>Ruthenation of rS - 22.2.195< / ntprobnp> < / ntprobnp>

[0251] The Protein A-purified antibody was dialyzed against ruthenation buffer (100 mM K-phosphate pH 8.5), and then the solution was adjusted to a protein concentration of 1 mg / ml. Ruthenium (II) tris(bipyridyl)-UEEK-N-hydroxysuccinimide ester was dissolved in DMSO at a concentration > 5 mg / ml. 15-fold molar excess of BPRu-UEEK-DDS was added to the IgG solution, and the reaction was carried out at 25 °C for 45 minutes while mixing vigorously. The reaction was terminated by adding L-lysin to a final concentration of 10 mM. Using 100 mM K-phosphate, 150 mM KCl pH 7.5 as the running buffer, the excess labeling reagent was removed by gel permeation chromatography on Superdex 200 (GE Healthcare Life Sciences). The fractions containing the conjugated IgG were pooled, and the final product was stabilized with 6.5% sucrose and stored at -80 °C.

[0252] Example 6: Method for detecting mutated NT-proBNP

[0253] One method for detecting NT-proBNP is the well-established Roche Elecsys ® assay technology, which is based on electrochemiluminescence. The Elecsys ® assay is a heterogeneous immunoassay and functions according to the sandwich principle: an antibody-antigen-antibody complex is formed by capture and detection (signal) antibodies that are generated against different epitopes of the analyte. The Elecsys ® assay for detecting mutated NT-proBNP comprises the following steps and components:

[0254] a) Incubate the sample together with a monoclonal capture antibody such as M-18.4.34, and either rS-22.2.195 or S-23.4.66 or both as monoclonal signal antibodies, in a phosphate buffer (100 mM, pH 5.8) matrix containing preservatives (Oxy-Pyrion 0.1%, Methylisothiazolon 0.1%), detergents (0.1%), and stabilizing and interference-avoiding proteins (such as albumin, avidin).

[0255] b) A biotinylated capture antibody (such as M-18.4.34), and either a ruthenium-labeled detection antibody rS-22.2.195 specific for NT-proBNP-R46H or S-23.4.66 specific for NT-proBNP-E43D forms a sandwich complex with the analyte.

[0256] c) The sandwich complex binds to streptavidin-coated microparticles, which are then magnetically captured onto the surface of the electrode.

[0257] d) Use ProCell to remove unbound substances.

[0258] e) Chemiluminescent emission is induced by applying a voltage to the electrode and measured by a photomultiplier tube.

[0259] f) Calculate the NT-proBNP value via the calibration curve specified by the instrument.

[0260] If the Elecsys assay is to detect mutant and wild-type NT-proBNP, an additional signal antibody specific for wild-type NT-proBNP, such as S-1.21.3, can be included in the assay mixture.

[0261] Example 7: Antibodies rS-22.2.195 and S-23.4.66 are highly specific for mutant NT-proBNP

[0262] As described in Example 5, evaluate the specificity of two newly generated antibodies rS-22.2.195 and S-23.4.66 using an NT-proBNP-specific Elecsys ® assay. A series of recombinant wild-type and mutant peptide spiked sera without human NT-proBNP are used such that they cover the NT-proBNP concentration range observed in heart failure patients. Then use rS-22.2.195 specific for NT-proBNP-R46H ( Figure 3 ) or S-23.4.66 specific for NT-proBNP-E43D ( Figure 4 ) Detect NT-proBNP in the sample.

[0263] Figure 3 and 4 The results in and showed that both antibodies rS-22.2.195 and S-23.4.66 did not bind to wild-type NT-proBNP at all, but recognized the mutant peptides with high intensity. In addition, both antibodies showed a wide range of linear correlation of the signal with respect to the analyte concentration and thus allowed quantification of NT-proBNP across the entire concentration range typically found in heart failure patients.

[0264] Example 8: Reactivity with Mutated NT-proBNP (R46H) in Patient Samples

[0265] In serum or plasma samples from 8 patients (Table 4, Patients 1 - 8) known to show distinct symptoms of heart failure, the reactivity of the new antibodies with native mutated NT-proBNP was evaluated. Only patients with the R46H mutation were available. For three patients, the presence of the R46H mutation was also confirmed by sequencing. The Elecsys ® assay system described in Example 5 was used to quantify NT-proBNP, using rS-22.2.195 specific for NT-proBNP-R46H as the signal antibody and M-18.4.34 as the capture antibody. For comparison, the same patient samples were measured with the Elecsys ® kit, which contains S-23.4.66 specific for NT-proBNP-E43D as the signal antibody and M-18.4.34 as the capture antibody. Calibration of both assay systems was completed using calibrators based on recombinant mutant NT-proBNP (R46H or E43D, respectively).

[0266] Table 3:

[0267]

[0268] * The R46H mutation was confirmed by sequencing.

[0269] The assay kit containing the rS-22.2.195 antibody clearly detected NT-proBNP-R46H in all patient samples. The control kit using S-23.4.66 as the signal antibody did not detect a significant amount of NT-proBNP (≤7 pg / mL). This result again shows the high specificity of rS-22.2.195 and S-23.4.66 for the two point mutations R46H and E43D, respectively, but now is also consistent with the native samples. The NT-proBNP concentration quantified with rS-22.2.195 ranged from 202 to 22321 pg / mL. In the past, the determination threshold for diagnosing cardiac dysfunction was determined to be 125 pg / mL (wild type) NT-proBNP (Roche Elecsys ® proBNP II assay). Therefore, all measured concentrations of the mutated NT-proBNP > 125 pg / mL may reflect the heart failure symptoms of the patients.

[0270] The sequences mentioned in this article are shown in the sequence listing and the following table.

[0271]

[0272]

[0273]

[0274]

[0275] < / ntprobnp>

Claims

1. An antibody that specifically binds to mutant human NT-proBNP, wherein the antibody is selected from (a) an antibody that specifically binds to mutant human NT-proBNP, the mutant human NT-proBNP comprising a mutation in which arginine at position 46 is replaced with histidine, and (b) an antibody that specifically binds to mutant human NT-proBNP, the mutant human NT-proBNP comprising a mutation in which glutamate at position 43 is replaced with aspartic acid, wherein i) the antibody specifically binds to mutant NT-proBNP, the mutant NT-proBNP comprising a mutation in which arginine at position 46 is replaced with histidine, and wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising CDR1 consisting of the sequence LLDDAY (SEQ ID NO: 16), CDR2 consisting of the sequence KDS, and CDR3 consisting of the sequence LSVDSSEYSV (SEQ ID NO: 17), and the heavy chain variable domain comprising CDR1 consisting of the sequence GFSLIGEY (SEQ ID NO: 18), CDR2 consisting of the sequence MASGGTI (SEQ ID NO: 19), and CDR3 consisting of the sequence VRSSVSPGDDRDV (SEQ ID NO: 20), or ii) the antibody specifically binds to mutant NT-proBNP, the mutant NT-proBNP comprising a mutation in which glutamate at position 43 is replaced with aspartic acid, and wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising CDR1 consisting of the sequence SSNVGYGNY (SEQ ID NO: 21), CDR2 consisting of the sequence SAT, and CDR3 consisting of the sequence VSYDSSSKFGV (SEQ ID NO: 22), and the heavy chain variable domain comprising CDR1 consisting of the sequence GFSVTNSG (SEQ ID NO: 23), CDR2 consisting of the sequence INNDGVA (SEQ ID NO: 24), and CDR3 consisting of the sequence GTRDLPSDVRYGNMYINY (SEQ ID NO: 25), and wherein the positions of the mutations are the amino acid positions in the wild-type NT-proBNP as shown in SEQ ID NO:

3.

2. The antibody of claim 1, wherein the antibody is a monoclonal antibody.

3. The antibody of claim 1, wherein the antibody is a monoclonal ovine antibody.

4. The antibody of any one of claims 1 to 3, wherein (i) The mutated NT-proBNP containing a mutation of substituting arginine at position 46 with histidine consists of the amino acid sequence shown in SEQ ID NO: 1, or (ii) The mutated NT-proBNP containing a mutation of substituting glutamate at position 43 with aspartic acid consists of the amino acid sequence shown in SEQ ID NO:

2.

5. An antibody according to any one of claims 1 to 3, wherein the antibody does not bind to wild-type NT-proBNP.

6. An antigen-binding fragment of an antibody according to any one of claims 1 to 3, wherein the antigen-binding fragment is selected from Fab fragment, Fab' fragment, F(ab′)2 fragment, scFv fragment, and Fv fragment.

7. An antibody according to any one of claims 1 to 3, wherein the antibody is linked to a detectable label.

8. An antigen-binding fragment according to claim 6, wherein the antigen-binding fragment is linked to a detectable label.

9. An antibody according to claim 7, wherein the detectable label is an enzyme, biotin, a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a gold label, or a magnetic label.

10. An antigen-binding fragment according to claim 8, wherein the detectable label is an enzyme, biotin, a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a gold label, or a magnetic label.

11. An antibody according to claim 7, wherein the detectable label is an electrochemiluminescent label containing ruthenium.

12. An antigen-binding fragment according to claim 8, wherein the detectable label is an electrochemiluminescent label containing ruthenium.

13. A kit or composition comprising the following: i) An antibody or its antigen-binding fragment that specifically binds to mutated human NT-proBNP, the mutated human NT-proBNP containing a mutation of substituting arginine at position 46 with histidine, and ii) An antibody or its antigen-binding fragment that specifically binds to mutated NT-proBNP, the mutated NT-proBNP containing a mutation of substituting glutamate at position 43 with aspartic acid, wherein i) An antibody or an antigen-binding fragment thereof specifically binds to a mutated NT-proBNP, the mutated NT-proBNP comprising a mutation in which arginine at position 46 is replaced by histidine, and wherein the antibody or the antigen-binding fragment thereof comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising CDR1 consisting of the sequence LLDDAY (SEQ ID NO: 16), CDR2 consisting of the sequence KDS, and CDR3 consisting of the sequence LSVDSSEYSV (SEQ ID NO: 17), and the heavy chain variable domain comprising CDR1 consisting of the sequence GFSLIGEY (SEQ ID NO: 18), CDR2 consisting of the sequence MASGGTI (SEQ ID NO: 19), and CDR3 consisting of the sequence VRSSVSPGDDRDV (SEQ ID NO: 20), or ii) An antibody or an antigen-binding fragment thereof specifically binds to a mutated NT-proBNP, the mutated NT-proBNP comprising a mutation in which glutamate at position 43 is replaced by aspartic acid, and wherein the antibody or the antigen-binding fragment thereof comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising CDR1 consisting of the sequence SSNVGYGNY (SEQ ID NO: 21), CDR2 consisting of the sequence SAT, and CDR3 consisting of the sequence VSYDSSSKFGV (SEQ ID NO: 22), and the heavy chain variable domain comprising CDR1 consisting of the sequence GFSVTNSG (SEQ ID NO: 23), CDR2 consisting of the sequence INNDGVA (SEQ ID NO: 24), and CDR3 consisting of the sequence GTRDLPSDVRYGNMYINY (SEQ ID NO: 25), and wherein the position of the mutation is the amino acid position in the wild-type NT-proBNP as shown in SEQ ID NO:

3.

14. The kit or composition of claim 13, wherein the kit or composition further comprises an antibody that binds to a different epitope in NT-proBNP.

15. The kit or composition of claim 14, wherein the antibody binds to an epitope present in amino acids 1 to 35 of NT-proBNP, and wherein the position of the amino acids is the amino acid position in the wild-type NT-proBNP as shown in SEQ ID NO: 3.

Images