Human BNP Immunospecific Antibodies

FIELD OF THE INVENTION

The present invention relates to antibodies that immunospecifically bind to human brain natriuretic peptide or a human brain natriuretic peptide fragment with a high binding affinity, methods for producing and selecting said antibodies, immunoassays for human brain natriuretic peptide or a human brain natriuretic peptide fragment that employ said antibodies and therapeutic compositions containing said antibodies.

BACKGROUND OF THE INVENTION

Atrial natriuretic peptide (hereinafter referred to as “ANP”), brain natriuretic peptide (hereinafter referred to as “BNP”), C-type natriuretic peptide (hereinafter referred to as “CNP”) and Dendroaspis natriuretic peptide (hereinafter referred to as “DNP”) are each members of a family of hormones known as “natriuretic peptides”. ANP and BNP share a wide spectrum of biological properties and belong to the cardiac natriuretic system. Both ANP and BNP are of myocardial cell origin while CNP is of endothelial cell origin. DNP was isolated from the venom of the green mamba snake and possesses structural similarity to ANP, BNP and CNP.

BNP received its name because it was first isolated from porcine brain, thus “BNP” stood for “brain natriuretic peptide”. However, because BNP belongs to the cardiac natriuretic system, “brain” has been changed to “B-type”. Therefore, “BNP” now refers to “B-type natriuretic peptide”.

ANP is secreted by the heart in the atria. BNP is secreted by the heart through the coronary sinus, predominantly from the cardiac ventricles. BNP is secreted as a 108 amino acid polypeptide precursor (See Valli et al., J. Lab. Clin. Med., 134(5):437-444 (November 1999)). The mature form of BNP is made up of 32 amino acids (representing amino acids 77-108 of the 108 amino acid polypeptide precursor) with a 17 amino acid ring closed by a disulfide bond between two cysteine residues, an amino-terminal tail of 9 amino acids, and a carboxyl-terminal tail of 6 amino acids. ANP and CNP also have a 17 amino acid ring closed by a disulfide bond between two cysteine residues. Eleven of the seventeen amino acids in the ring are conserved between the three molecules. In addition to the 17 amino acid ring structure, ANP has an amino-terminal tail of 6 amino acids and a carboxy-terminal tail of 5 amino acids. ANP is produced as a 126 amino acid pro-ANP form that is the major storage form of ANP. After proteolytic cleavage between amino acids 98 and 99, the mature 28 amino acid peptide ANP is found in coronary sinus plasma (See Yandle, J. Internal Med., 235:561-576 (1994)).

CNP is found in the brain and cerebral spinal fluid and is the most prevalent of the three peptides in the central nervous system. Little if any CNP is present in the heart. Pro-CNP is a 103 amino acid peptide that is processed into either CNP-53 (amino acids 51 to 103) or CNP-22 (amino acids 82 to 103) that are the active peptides. In addition the 17 amino acid ring structure, CNP-22 has an amino-terminal tail of 5 amino acids and contains no carboxy-terminal tail. CNP-53 is identical to CNP-22 except for a 31 amino acid extension at the amino terminal end.

As mentioned previously, DNP was isolated from the venom of the green mamba snake. The mature form of DNP is made up of 38 amino acids. DNP-like immunoreactivity (DNP-L1) has been reported in human plasma and the plasma concentration of DNP-L1 has been found to be elevated in patients with congestive heart failure (See, Cataliotti, et al., Mayo Clin. Proc., 76:111-1119 (2001)). Additionally, it is also known that the infusion of synthetic DNP results in marked natriuresis and diuresis in association with increased plasma and urinary cyclic guanosine monophosphate. Id.

One of the problems with natural human natriuretic peptides is that they are unstable in plasma and serum. Specifically, enzymes, such as proteases, cleave these peptides. For example, proteases cleave BNP (natural and synthetic) at various locations along its amino acid chain. For example, protease cleavage is known to occur at the amino terminus of BNP between amino acids 2-3 (Shimizu et al., Clinica Chimica Acta, 316:129-135 (2002)) and at its carboxy terminus between amino acids 30-32. Moreover, endopeptidase cleavage of BNP is also known in the art (Davidson and Struthers, J. Hypertension, 12:329-336 (1994)).

The measurement of mature BNP (i.e., the 32 amino acid molecule (amino acids 77-108 of the precursor polypeptide of BNP)) in humans (hereinafter referred to has “hBNP”), in the general population has been found to reflect cardiac diseases, such as congestive heart failure, ischemic heart diseases, atrial fibrillation and renal dysfunction. In fact, elevated levels of BNP in human plasma have been reported in heart disease, following acute myocardial infarction and during symptomless or subclinical ventricular dysfunction (See Mukoyama et al., J. Clin. Invest., 87:11402-11412 (1991), Motwani et al., Lancet, 341:1109-1113 (1993), Yoshibayashi et al., New Eng. J. Med., 327:434 (1992)). Increased circulating levels of ANP are seen in congestive heart failure, chronic renal failure and in severe hypertension. The presence of CNP in human plasma remains controversial with reports of its absence or presence as CNP-22 (See Yandle, J. Internal Med., 235:561-576 (1994)).

A ligand binding assay is an analytical technique for measuring concentrations of substances commonly referred to as ligands that react selectively with specific binding proteins. Immunoassays that measure the concentrations of antigens that react selectively with specific antibodies are an example of a class of ligand binding assays.

Ligand binding assays, such as immunoassays, for measuring human natriuretic peptides in plasma, particularly hBNP, are well-known in the art and are commercially available. These immunoassays require the use of at least one or two specific antibodies as well as at least one calibrator and, ideally, at least one control. In addition to the calibrators and controls, immunoassays require the use of at least one test sample. Test samples are normally biological samples derived from serum, plasma, whole blood or other bodily fluids (normally from a human patient). The levels of at least one human natriuretic peptide in the test sample is quantified in the immunoassay.

For example, U.S. Pat. No. 6,162,902 (hereinafter referred to as the “'902 patent”) discloses isolated antibodies that are monospecifically reactive to epitopes 1-10, 5-13 and 15-25 of hBNP. More particularly, the '902 patent describes two isolated monoclonal antibodies. The first monoclonal antibody is produced by hybridoma cell line 106.3 (A.T.C.C. Accession No. HB-12044) and is monospecifically reactive to epitopes 5-13 of hBNP. The second monoclonal antibody is produced by hybridoma cell line 201.3 (A.T.C.C. Accession No. HB 12045) and is monospecifically reactive to epitopes 1-10 of hBNP. The '902 patent also describes the use of the above antibodies in immunoassays for the purpose of quantifying the amount of hBNP in a biological sample. U.S. Pat. No. 6,677,124 (hereinafter referred to as the “'124 patent”) discloses a monoclonal antibody that binds to an epitope having the amino acid sequence of LYS-VAL-LEU-ARG-ARG-HIS that is found in the C-terminal region of hBNP, namely epitopes 27-32. More particularly, the '124 patent describes a monoclonal antibody produced by hybridoma cell line BC203 (FERM BP-3515). The '124 patent also describes immunoassays for hBNP using this monoclonal antibody.

It is generally known in the art that the specificity and sensitivity of the antibodies used in immunoassays, such as hBNP immunoassays, are very important. One way in which to increase both the specificity and sensitivity of one or more antibodies is to improve the binding affinity of an antibody for its intended target (i.e., an antigen). Antibodies having an improved binding affinity for their intended targets should exhibit increased specificity and sensitivity. Therefore, there is a need in the art for new antibodies that specifically bind to human BNP with a high binding affinity and thus exhibit high specificity and sensitivity when used in said hBNP immunoassays.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an isolated antibody which immunospecifically binds to an epitope comprising amino acid residues 5 through 13 of human brain natriuretic peptide (“hBNP”) with at least about a two fold improvement in its equilibrium dissociation constant (K_D) when compared with an antibody produced by hybridoma cell line 106.3, said cell line having A.T.C.C. Accession No. HB-12044. More specifically, the antibody of the present invention exhibits at least about a three fold improvement, at least about a five fold improvement, at least about a ten fold improvement, at least about a fifteen fold improvement, at least about a twenty fold improvement or at least about a twenty-five fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 106.3. The isolated antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof.

In another aspect, the present invention relates to an isolated antibody which immunospecifically binds to hBNP, wherein said antibody has an association rate (k_a) of between about 5.0×10⁴and about 1.0×10⁸M⁻¹s⁻¹More specifically, the antibody of the present invention has an association rate of between about 3.3×10⁴and about 1.0×10⁹M⁻¹s⁻¹, between about 2.5×10⁴and about 1.0×10⁸M⁻¹s⁻¹or between about 2.4×10⁴and about 1.35×10⁷M⁻¹s⁻¹The isolated antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof. Additionally, this isolated antibody immunospecifically binds to an epitope comprising amino acid residues 5 through 13 of hBNP.

In another aspect, the present invention relates to an isolated antibody which immunospecifically binds to hBNP, wherein said antibody has a dissociation rate (k_d) of between about 1.0×10⁻³and about 1.0×10⁻⁶·s⁻¹. More specifically, the antibody of the present invention has a dissociation rate of between about 1.0×10⁻³and about 1.0×10⁻⁵s⁻¹or between about 1.0×10⁻³and about 1.0×10⁻⁴·s⁻¹. The isolated antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof. Additionally, this isolated antibody immunospecifically binds to an epitope comprising amino acid residues 5 through 13 of hBNP.

In another aspect, the present invention relates to an isolated antibody which immunospecifically binds to hBNP wherein said antibody has an equilibrium dissociation constant (K_D) of between about 4.2×10⁻¹¹M and about 1×10⁻¹⁵M. More specifically, the antibody of the present invention has an equilibrium dissociation constant of between about 4.0×10⁻¹¹M and about 1.0×10⁻¹⁴M, between about 3.0×10⁻¹¹M and about 1.0×10⁻¹³M between about 2.0×10⁻¹¹M and about 8.0×10⁻¹³M, or between about 1.0×10⁻¹²M and about 7.4×10⁻¹³M. The isolated antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof. Additionally, this isolated antibody immunospecifically binds to an epitope comprising amino acid residues 5 through 13 of hBNP.

In still another aspect, the present invention relates to a Chinese hamster ovary (“CHO”) cell line 106.3 AM1 having A.T.C.C. Accession No. PTA-6987.

In still yet another aspect, the present invention relates to an antibody made from DNA extracted from CHO cell line 106.3 AM1 having A.T.C.C. Accession No. PTA-6987.

In yet another aspect, the present invention relates to a chimeric antibody or a hBNP-epitope binding fragment thereof produced by CHO cell line 106.3 AM1, wherein said cell line has A.T.C.C. Accession No. PTA-6987.

In still a further aspect, the present invention relates to an isolated antibody which immunospecifically binds to hBNP, wherein said antibody has a variable heavy domain and a variable light domain, the variable heavy domain comprising a heavy chain complementary determining region (“CDR”) 1, a heavy chain CDR 2 and a heavy chain CDR 3, the variable light domain comprising a light chain CDR 1, a light chain CDR 2 and a light chain CDR 3, wherein

(a) the Heavy Chain CDR 1 has an amino acid sequence of: Gly-Tyr-Thr-Phe-Thr-His-Tyr-Gly-Ile-Asn (SEQ ID NO:6);

(b) the Heavy Chain CDR 2 has an amino acid sequence having a formula of:

(SEQ ID NO:12)

Trp-Ile-Asn-Thr-His-Thr-Gly-Glu-Xaa₁-Xaa₂-Tyr-Ala-

Asp-Asp-Phe-Lys-Gly

wherein Xaa₁is selected from the group consisting of proline and alanine;

wherein Xaa₂is selected from the group consisting of isoleucine and tyrosine;

(d) the Light Chain CDR 1 has an amino acid sequence having a formula of:

(SEQ ID NO:13)

Lys-Ala-Xaa₃-Xaa₄-Xaa₅-Val-Asp-Tyr-Asn-Gly-Asp-

Ser-Tyr-Leu-Asn

wherein Xaa₃is selected from the group consisting of: serine, alanine, asparagine, glutamine, tyrosine, threonine and arginine;

wherein Xaa₄is selected from the group consisting of: glutamine, tyrosine, tryptophan, alanine and phenylalanine;

wherein Xaa₅is selected from the group consisting of: serine, glycine, proline, alanine and aspartic acid;

(e) the Light Chain CDR 2 has an amino acid sequence having the formula of:

(SEQ ID NO:14)

Ala-Ala-Ser-Xaa₆-Xaa₇-Xaa₈-Ser

wherein Xaa₆is selected from the group consisting of: asparagine and cysteine;

wherein Xaa₇is selected from the group consisting of: leucine, glycine and alanine;

wherein Xaa₈is selected from the group consisting of glutamic acid, tryptophan and proline; and

(f) the Light Chain CDR 3 has an amino acid sequence of: Gln-Gln-Ser-Asn-Glu-Asp-Pro-Phe-Thr (SEQ ID NO:11),

wherein the heavy chain CDR 2 has an amino acid sequence other than Trp-Ile-Asn-Thr-His-Thr-Gly-Glu-Pro-Ile-Tyr-Ala-Asp-Asp-Phe-Lys-Gly (SEQ ID NO:7) when the light chain CDR 1 has the amino acid sequence of Lys-Ala-Ser-Gln-Ser-Val-Asp-Tyr-Asn-Gly-Asp-Ser-Tyr-Leu-Asn (SEQ ID NO:9) and the light chain CDR 2 has the amino acid sequence of Ala-Ala-Ser-Asn-Leu-Glu-Ser (SEQ ID NO: 10), the light chain CDR 1 has an amino acid sequence other than Lys-Ala-Ser-Gln-Ser-Val-Asp-Tyr-Asn-Gly-Asp-Ser-Tyr-Leu-Asn (SEQ ID NO:9) when the heavy chain CDR 2 has the amino acid sequence Trp-Ile-Asn-Thr-His-Thr-Gly-Glu-Pro-Ile-Tyr-Ala-Asp-Asp-Phe-Lys-Gly (SEQ ID NO:7) and the light chain CDR 2 has the amino acid sequence Ala-Ala-Ser-Asn-Leu-Glu-Ser (SEQ ID NO: 19), or the light chain CDR 2 has an amino acid sequence other than Ala-Ala-Ser-Asn-Leu-Glu-Ser (SEQ ID NO: 10) when the heavy chain CDR 2 has the amino acid sequence of Trp-Ile-Asn-Thr-His-Thr-Gly-Glu-Pro-Ile-Tyr-Ala-Asp-Asp-Phe-Lys-Gly (SEQ ID NO:7) and the light chain CDR 1 has the amino acid sequence of Lys-Ala-Ser-Gln-Ser-Val-Asp-Tyr-Asn-Gly-Asp-Ser-Tyr-Leu-Asn (SEQ ID NO:9).

More specifically, in the above-described isolated antibody:

Xaa₁can be alanine;

Xaa₂can be tyrosine;

Xaa₃can be serine;

Xaa₄can be glutamine;

Xaa₅can be serine;

Xaa₆can be asparagine;

Xaa₇can be leucine; and

Xaa₈can be glutamic acid; or

In the above-described isolated antibody:

Xaa₁can be proline;

Xaa₂can be isoleucine;

Xaa₃can be glutamine;

Xaa₄can be phenylalanine;

Xaa₅can be alanine;

Xaa₆can be asparagine;

Xaa₇can be leucine; and

Xaa₈can be glutamic acid; or

In the above-described isolated antibody:

Xaa₁can be proline;

Xaa₂can isoleucine;

Xaa₃can be tyrosine;

Xaa₄can be alanine;

Xaa₅can be serine;

Xaa₆can be asparagine;

Xaa₇can be leucine; and

Xaa₈can be glutamic acid; or

In the above-described isolated antibody

Xaa₁can be proline;

Xaa₂can be isoleucine;

Xaa₃can be glutamine;

Xaa₄can be tryptophan;

Xaa₅can be glycine;

Xaa₆can be asparagine;

Xaa₇can be leucine; and

Xaa₈can be glutamic acid; or

In the above-described isolated antibody:

Xaa₁can be proline;

Xaa₂can be isoleucine;

Xaa₃can be threonine;

Xaa₄can be tryptophan;

Xaa₅can be aspartic acid;

Xaa₆can be asparagine;

Xaa₇can be leucine; and

Xaa₈can be glutamic acid; or

In the above-described isolated antibody:

Xaa₁can be proline;

Xaa₂can be isoleucine;

Xaa₃can be arginine;

Xaa₄can be tryptophan;

Xaa₅can be proline;

Xaa₆can be asparagine;

Xaa₇can be leucine; and

Xaa₈can be glutamic acid; or

In the above-described isolated antibody:

Xaa₁can be proline;

Xaa₂can be isoleucine;

Xaa₃can be alanine;

Xaa₄can be tyrosine;

Xaa₅can be glycine;

Xaa₆can be asparagine;

Xaa₇can be leucine; and

Xaa₈can be glutamic acid; or

In the above-described isolated antibody:

Xaa₁can be proline;

Xaa₂can be isoleucine;

Xaa₃can be asparagine;

Xaa₄can be tryptophan;

Xaa₅can be proline;

Xaa₆can be asparagine;

Xaa₇can be leucine; and

Xaa₈can be glutamic acid; or

In the above-described isolated antibody:

Xaa₁can be proline;

Xaa₂can be isoleucine;

Xaa₃can be serine;

Xaa₄can be glutamine;

Xaa₅can be serine;

Xaa₆can be cysteine;

Xaa₇can be glycine; and

Xaa₈can be tryptophan; or

In the above-described isolated antibody:

Xaa₁can be proline;

Xaa₂can be isoleucine;

Xaa₃can be serine;

Xaa₄can be glutamine;

Xaa₅can be serine;

Xaa₆can be cysteine;

Xaa₇can be alanine; and

Xaa₈can be proline.

The above-described antibody can have an equilibrium dissociation constant (K_D) of between about 4.2×10⁻¹¹M and about 1.0×10⁻¹⁵M, between about 4.0×10⁻¹¹M and about 1.0×10⁻¹⁴M, between about 3.0×10⁻¹¹M and about 1.0×10⁻¹³M, between about 2.0×10⁻¹¹M and about 8.0×10⁻¹³M, or between about 1.0×10⁻¹²M and about 7.4×10⁻¹³M. Additionally, the above-described antibody can have an association rate (k_a) of between about 5.0×10⁴and about 1.0×10⁸M⁻¹s⁻¹Furthermore, the above-described antibody can have a dissociation rate (k_d) of between about 1.0×10⁻³and 1.0×10⁻⁶s⁻¹. Furthermore, the above-described antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof. Finally, the above-described antibody can immunospecifically bind to an epitope comprising amino acid residues 5 through 13 of hBNP.

In another aspect, the present invention relates to an immunoassay for hBNP or hBNP fragment, wherein said immunoassay comprises any one of the hereinbefore described antibodies of the present invention. More specifically, said immunoassay may comprise only a single antibody that immunospecifically binds to hBNP or hBNP fragment. Moreover, said immunoassay may further comprise an additional specific binding partner for hBNP or hBNP fragment.

In another aspect, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of any of the hereinbefore described antibodies of the present invention and a pharmaceutically acceptable carrier or excipient.

In yet still another aspect, the present invention relates to an isolated antibody which immunospecifically binds to an epitope comprising at least three (3) amino acids of amino acid residues 5 through 18 of human brain natriuretic peptide (“hBNP”) with at least about a two fold improvement in its equilibrium dissociation constant (K_D) when compared with at least one of (1) an antibody produced by hybridoma cell line 106.3, said cell line having A.T.C.C. Accession No. HB-12044; and (2) an antibody produced by hybridoma cell line 3-631-436 having A.T.C.C. Accession No. PTA-6476. More specifically, the antibody of the present invention exhibits at least about a two fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 106.3, at least about a two fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 3-631-436, at least about a two fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 106.3 and an antibody produced by hybridoma cell line 3-631-436, at least about a five fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 106.3, at least about a five fold improved in its K_Dwhen compared with an antibody produced by hybridoma cell line 3-631-436 or at least about a five fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 106.3 and an antibody produced by hybridoma cell line 3-631-436. Moreover, the antibody of the present invention immunospecifically binds to an epitope comprising amino acid residues 5 through 13 of hBNP. Alternatively, the antibody of the present invention immunospecifically binds to an epitope comprising amino acid residues 13 through 18 of hBNP. The isolated antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof.

In yet still a further aspect, the present invention relates to an isolated antibody which immunospecifically binds to an epitope comprising at least three (3) amino acids of amino acid residues 13 through 18 of human brain natriuretic peptide (“hBNP”) with at least about a two fold improvement in its equilibrium dissociation constant (K_D) when compared with an antibody produced by hybridoma cell line 3-631-436 having A.T.C.C. Accession No. PTA-6476. More specifically, the antibody of the present invention exhibits at least about a five fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 3-631-436, at least about a ten fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 3-631-436, at least about a fifteen fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 3-631-436, at least about a twenty fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 3-631-436 or at least about a twenty-five fold improvement in its K_Dwhen compared with an antibody produced by hybridoma cell line 3-631-436. The isolated antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof.

In yet still a further aspect, the present invention relates to an isolated antibody which immunospecifically binds to hBNP, wherein said antibody has an association rate (k_a) of between about 1.5×10⁶M⁻¹s⁻¹to about 1.0×10⁸M⁻¹s⁻¹, with the proviso that if the k_ais 1.6×10⁶M⁻¹s⁻¹then the k_dis not 5.4×10⁴s⁻¹or 6.5×10⁻⁴s⁻¹, if the k_ais 6.7×10⁶M⁻¹s⁻¹then the k_dis not 2.5×10⁻³s⁻¹, if the k_ais 8.4×10⁶M⁻¹s⁻¹then the k_dis not 2.9×10⁻³s⁻¹, if the k_ais 8.4×10⁶M⁻¹s⁻¹then the k_dis not 2.9×10⁻³s⁻¹, if the k_ais 5.8×10⁶M⁻¹s⁻¹then the k_dis not 1.6×10⁻³s⁻¹or if the k_ais 1.5×10⁶M⁻¹s⁻¹then the k_dis not 4.1×10⁻⁴s⁻¹. The isolated antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof. The isolated antibody of the present invention immunospecifically binds to an epitope comprising amino acid residues 13 through 18 of hBNP.

In yet still another aspect, the present invention relates to an isolated antibody which immunospecifically binds to hBNP, wherein said antibody has a dissociation rate (k_d) of between about 2.0×10⁻³s⁻¹to about 1.0×10⁻⁶s⁻¹. The isolated antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof. The isolated antibody of the present invention immunospecifically binds to an epitope comprising amino acid residues 13 through 18 of hBNP.

In yet another aspect, the present invention relates to an isolated antibody which immunospecifically binds to hBNP wherein said antibody has an equilibrium dissociation constant (K_D) of between about 3.5×10⁻¹⁰M to about 1.0×10⁻¹³M. The isolated antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof. The isolated antibody of the present invention immunospecifically binds to an epitope comprising amino acid residues 13 through 18 of hBNP.

In yet another aspect, the present invention relates to a chinese hamster ovary (“CHO”) cell line 3-631-436 AM5 having A.T.C.C. Accession No. PTA-______.

In yet another aspect, the present invention relates to an antibody made from DNA extracted from the CHO cell line 3-631-436 AM5 having A.T.C.C. Accession No. PTA-______.

In still yet another aspect, the present invention relates to a chimeric antibody or a hBNP-epitope binding fragment thereof produced by CHO cell line 3-631-436 AM5, wherein said cell line has A.T.C.C. Accession No. PTA-______.

In still yet another aspect, the present invention relates to a chinese hamster ovary (“CHO”) cell line 3-631-436 AM8 having A.T.C.C. Accession No. PTA-______.

In still yet another aspect, the present invention relates to an antibody made from DNA extracted from the CHO cell line 3-631-436 AM8 having A.T.C.C. Accession No. PTA-______.

In still yet another aspect, the present invention relates to a chimeric antibody or a hBNP-epitope binding fragment thereof produced by CHO cell line 3-631-436 AM8, wherein said cell line has A.T.C.C. Accession No. PTA-______.

In still yet another aspect, the present invention relates to an isolated antibody which immunospecifically binds to hBNP, wherein said antibody has a variable heavy domain and a variable light domain, the variable heavy domain comprising a heavy chain complementarity determining region (“CDR”) 1, a heavy chain CDR 2 and a heavy chain CDR 3, the variable light domain comprising a light chain CDR 1, a light chain CDR 2 and a light chain CDR 3, wherein

(a) Heavy Chain CDR 1 having an amino acid sequence of:

(SEQ ID NO:84)

Gly-Tyr-Thr-Phe-Thr-Ser-Tyr-Trp-Met-Asn;

(b) Heavy Chain CDR 2 having an amino acid sequence of:

(SEQ ID NO:85)

Arg-Ile-Asp-Pro-Tyr-Asp-Ser-Glu-Thr-His-Tyr-Asn-

Gln-Lys-Phe-Lys;

Asp-Gly-Tyr (SEQ ID NO: 86);

(d) Light Chain CDR 1 having an amino acid sequence of:

(SEQ ID NO:87)

Lys-Ser-Ser-Gln-Ser-Leu-Leu-Asp-Ser-Asp-Gly-Lys-

Thr-Tyr-Leu-Asn;

(e) Light Chain CDR 2 having an amino acid sequence having the formula of:

(SEQ ID NO:83)

Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Leu-Glu-Ser;

where Xaa₉is selected from the group consisting of valine, glutamine, histidine, tryptophan and arginine;

where Xaa₁₀is selected from the group consisting of valine, asparagine, threonine and methionine;

where Xaa₁₁is selected from the group consisting of serine, threonine, asparagine and aspartic acid;

where Xaa₁₂is selected from the group consisting of lysine and isoleucine;

provided that Xaa₉is other than valine if Xaa₁₀is Valine, Xaa₁₁is serine and Xaa₁₂is Lysine; and

(f) Light Chain CDR 3 having an amino acid sequence of:

(SEQ ID NO:89)

Leu-Gln-Ala-Thr-His-Phe-Pro.

More specifically, in the above-described isolated antibody:

Xaa₉is Glutamine;

Xaa₁₀is Asparagine;

Xaa₁₁is Threonine; and

Xaa₁₂is Lysine.

More specifically, in the above-described isolated antibody:

Xaa₉is Histidine;

Xaa₁₀is Threonine;

Xaa₁₁is Threonine; and

Xaa₁₂is Lysine.

More specifically, in the above-described isolated antibody:

Xaa₉is Tryptophan;

Xaa₁₀is Methionine;

Xaa₁₁is Threonine; and

Xaa₁₂is Lysine.

More specifically, in the above-described isolated antibody:

Xaa₉is Tryptophan;

Xaa₁₀is Methionine;

Xaa₁₁is Asparagine; and

Xaa₁₂is Lysine.

More specifically, in the above-described isolated antibody:

Xaa₉is Valine;

Xaa₁₀is Threonine;

Xaa₁₁is Aspartic Acid; and

Xaa₁₂is Lysine.

More specifically, in the above-described isolated antibody:

Xaa₉is Arginine;

Xaa₁₀is Threonine;

Xaa₁₁is Asparagine; and Xaa₁₂is Lysine.

More specifically, in the above-described isolated antibody:

Xaa₉is Tryptophan;

Xaa₁₀is Methionine;

Xaa₁₁is Aspartic Acid; and

Xaa₁₂is Lysine.

More specifically, in the above-described isolated antibody:

Xaa₉is Tryptophan;

Xaa₁₀is Threonine;

Xaa₁₁is Threonine; and

Xaa₁₂is Lysine.

More specifically, in the above-described isolated antibody:

Xaa₉is Tryptophan;

Xaa₁₀is Methionine;

Xaa₁₁is Asparagine; and

Xaa₁₂is Lysine.

More specifically, in the above-described isolated antibody:

Xaa₉is Valine;

Xaa₁₀is Threonine;

Xaa₁₁is Aspartic Acid; and

Xaa₁₂is Isoleucine.

The isolated antibody of the present invention can be a monoclonal antibody, a multispecific antibody, a human antibody, a fully humanized antibody, a partially humanized antibody, an animal antibody, a recombinant antibody, a chimeric antibody, a single-chain Fv, a single chain antibody, a single domain antibody, a Fab fragment, a F(ab′)₂fragment, a disulfide-linked Fv, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof. The isolated antibody of the present invention immunospecifically binds to an epitope comprising amino acid residues 13 through 18 of hBNP.

In yet another aspect, the present invention relates to an immunoassay for hBNP or hBNP fragment, wherein said immunoassay comprises any one of the hereinbefore described antibodies of the present invention. More specifically, said immunoassay may comprise only a single antibody that immunospecifically binds to hBNP or hBNP fragment. Moreover, said immunoassay may further comprise an additional specific binding partner for hBNP or hBNP fragment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart showing the steps used to identify and create antibodies that immunospecifically bind to human BNP with a high binding affinity.

FIG. 2 is a plasmid map for vector pYD41-40 containing the 106.3 single-chain variable fragment shown in FIG. 4.

FIGS. 3A-3E are the nucleotide sequence of the vector shown in FIG. 2.

FIG. 4 is a diagram of the 106.3 single-chain variable fragment (“scFv”).

FIG. 5 shows the amino acid sequence of the 106.3 single-chain variable fragment (“scFv”). The solid underlined sequence represents the variable heavy chain sequence (“VH”), the double underlined sequence the linker, and the stippled underline sequence the variable light chain sequence (“VL”). Italicized and bold type indicates the complementary determining regions (CDR).

FIGS. 6A-6B show the nucleotide sequence of the 106.3 scFv.

FIGS. 7A-7B show that yeast expressing full-length 106.3 single-chain variable fragment (scFv) bind to cyclic BNP (SEQ ID NO:5) More specifically, this figure shows that 106.3 scFv expressing yeast were incubated with cyclic BNP (1-32c) (SEQ ID NO:5) or anti-V5 followed by secondary reagents streptavidin phycoerythrin (SA:PE) (FIG. 7A) and goat anti mouse-phycoerythrin (GAM:PE) (FIG. 7B). The flow cytometry histograms illustrate the full-length expression of 106.3 scFv as detected by anti-V5 and the ability of 106.3 scFv to bind to cyclic BNP peptide (1-32) (SEQ ID NO:5). PE units (abscissa): 10², 10³, 10⁴, and 10⁵. Count units (ordinate): 0, 50, 100, 150 (FIG. 7A); 0, 25, 50, 75, 100, 125 (FIG. 7B).

FIG. 8 shows the 106.3 scFv off-rate measurement. More specifically, yeast expressing 106.3 scFv were incubated with a saturating concentration of biotinylated cyclic BNP (1-32c) (SEQ ID NO:5). Cells were then washed and incubated with a saturating concentration of unlabelled BNP 1-32c (SEQ ID NO:5). At each time point, cells were transferred to ice, washed and incubated with SA:PE. After 30 minutes, cells were washed again and analyzed on the flow cytometer. A first order decay equation was used to fit the individual time points where m1 was the theoretical maximum mean fluorescence units (“MFU”) at time 0, m2 was the off-rate (“koff”), m3 was the background MFU due to autofluorescence, and M0 was the time x (the x being the time that is being measured) that measurements are taken. The half-life (t_1/2) of 106.3 scFv binding to cyclic BNP (1-32c) was calculated using: t_1/2=ln 2/k_off. Three to five times the half-life was the time used to sort the 106.3 CDR mutagenic libraries.

FIG. 9 is a schematic depiction which shows how degenerate oligonucleotides were designed so that three amino acid positions of the complementarity determining region (9 nucleotides) were randomly mutated per library.

FIG. 10 is a schematic depiction which shows how the 106.3 scFv library was constructed using yeast homologous recombination. More specifically, gapped vectors were PCR generated to exclude those nucleotides that were being mutagenized in the library. The degenerate single stranded oligonucleotides were synthesized. Gapped vectors and single stranded degenerate oligonucleotides were transformed into S. cerevisiae strain EBY100. Transformed clones were selected in tryptophan deficient glucose media.

FIG. 11 is a summary showing that 106.3scFv variants isolated from CDR mutagenic libraries exhibited improvements in off-rate (namely, said variants had a slower k_off).

FIGS. 12A-C show the sequence characterization of scFv 106.3 variants. More specifically, plasmid DNA was isolated from 106.3 variants and scFv genes were sequenced.

FIG. 13 shows affinity measurements of selected 106.3 engineered, human-mouse chimeric antibodies and mouse 106.3 mAb using surface plasmon resonance using BIAcore.

FIGS. 14A-H show the fifty-four (54) oligonucleotides that were used to create the pYD41 vector discussed in Example 1.

FIG. 15 shows the results of testing to determine antibody 106.3AM1's ability to bind to human cyclic BNP 1-32 in a single antibody assay format as described in Example 3 (X signal generated with given concentration of unlabelled human cyclic BNP 1-32; A signal generated with no unlabelled human cyclic BNP 1-32; X/A=ratio of these two signals).

FIG. 16 shows an anti-hBNP antibody pair evaluation using streptavidin microparticles using antibody 106.3 AM1 and 3-631-436 as described in Example 4. In essence, the following were employed: M280 Streptavidin particles at 0.05% solids, 65 ng/mL conjugates, 100 μL sample volume, and a 2-step (18/4) sandwich format. Symbols & Abbreviations: diamonds, anti-BNP(106.3AM1)SA μP/anti-BNP(3-631-436)CPSP; squares, anti-BNP(3-631-436)SA μP/anti-BNP(106.3AM1)CPSP; RLU, Relative Light Units.

FIG. 17 shows anti-hBNP antibody pair evaluation using paramagnetic microparticles (from Polymer Labs) using antibody 106.3 AM1 and 3-631-436 as described in Example 4. Symbols & Abbreviations: diamonds, anti-BNP(106.3AM1)SA μP/anti-BNP(3-631-436)CPSP; squares, anti-BNP(3-631-436)SA μP/anti-BNP(106.3AM1)CPSP; RLU, Relative Light Units.

FIG. 18 shows the displacement of antibody 106.3 AM1 (used at about 0.01 μg/mL) with various hBNP peptides (used at about 181 nM).

FIG. 19 shows the alanine peptide mapping of antibody 106.3 AM1 using EIA.

FIG. 20 shows the alanine peptide mapping of antibody 106.3 AM1 using BIAcore. The fold increase in k_offof BNP complexes comprising various BNP peptides are displayed.

FIG. 21 is a plasmid map for vector pYD1-41-40-3631 containing the 3-631-436 single-chain variable fragment shown in FIG. 23.

FIGS. 22A-22D are the nucleotide sequence of the vector shown in FIG. 21 (SEQ ID NO:90).

FIG. 23 is a diagram of the 3-631-436 scFv.

FIG. 24 shows the amino acid sequence of the 3-631-436 scFv (SEQ ID NO:91). The solid underlined sequence represents the variable heavy chain sequence (“VH”), the double underlined sequence the linker, and the stippled underline sequence the variable light chain sequence (“VL”). Italicized and bold type indicates the complementary determining regions (CDR).

FIGS. 25A-25B show the nucleotide sequence of the 3-631-436 scFv (SEQ ID NO:92). The double-stranded sequence is depicted.

FIGS. 26A-26B show that yeast expressing full-length 3-631-436 scFv bind to cyclic BNP (SEQ ID NO:5). More specifically, this figure shows that 3-631-436 scFv expressing yeast were incubated with cyclic BNP (1-32c) (SEQ ID NO:5) or anti-V5 followed by secondary reagents streptavidin phycoerythrin (SA:PE) (FIG. 26A) and goat anti mouse-FITC (GAM:FITC) (FIG. 26B). The flow cytometry histograms illustrate the full-length expression of 3-631-436 scFv as detected by anti-V5 and the ability of 3-631-436 scFv to bind to cyclic BNP peptide (1-32) (SEQ ID NO:5). Antigen (Ag) binding or PE-A units (abscissa): 100, 101, 102, and 10³and 10⁴. Count units (ordinate): 0, 10, 20, 30, 40, 50, 60, 70, 80 (FIG. 26A); Antibody (Ab) expression or FITC-A units (abscissa) 10⁰, 10¹, 10², and 10³and 10⁴: 0, 10, 20, 30, 40, 50, 60, 70, 80 (FIG. 26B).

FIG. 27 shows the 3-631-436 scFv off-rate measurement. More specifically, yeast expressing 3-631-436 scFv were incubated with a saturating concentration of biotinylated cyclic BNP (1-32c) (SEQ ID NO:5). Cells were then washed and incubated with a saturating concentration of unlabelled BNP 1-32c (SEQ ID NO:5). At each time point, cells were transferred to ice, washed and incubated with SA:PE. After 30 minutes, cells were washed again and analyzed on the flow cytometer. A first order decay equation was used to fit the individual time points where m1 was the theoretical maximum mean fluorescence units (“MFU”) at time 0, m2 was the off-rate (“koff”), m3 was the background MFU due to autofluorescence, and M0 was the time x (the x being the time that is being measured) that measurements are taken. The half-life (t_1/2) of 3-631-436 scFv binding to cyclic BNP (1-32c) was calculated using: t_1/2=ln 2/k_off. Three to five times the half-life was the time used to sort the 3-631-436 CDR mutagenic libraries.

FIG. 28 is a summary showing that 3-631-436 scFv variants isolated from CDR mutagenic libraries exhibited improvements in off-rate (namely, said variants had a slower k_off).

FIG. 29 shows the sequence characterization of scFv 3-631-436 variants. More specifically, plasmid DNA was isolated from 3-631-436 variants and scFv genes were sequenced (SEQ ID NOS:143-151).

FIG. 30 shows affinity measurements of selected 3-631-436 engineered, mouse-mouse chimeric antibodies and wild-type mouse 3-631-436 mAb (IgG2a kappa) using surface plasmon resonance Biacore and using KinExA.

FIGS. 31A-31F show the fifty (50) oligonucleotides (SEQ ID NOS:93-142) that were used to create the pYD1-41-40-3-631-436 vector discussed in Example 5.

FIG. 32 shows anti-hBNP antibody pair evaluation using paramagnetic microparticles (from Polymer Labs) using antibody 3-631-436 AM8, antibody 106.3 AM1, antibody 106.3 AM1 Fab′2, antibody 106.3 AM2 and 3-631-436 as described in Example 7. Symbols & Abbreviations: circles anti-BNP(3-631-436 AM8) μP/anti-BNP(106.3AM1-Acrydinium); squares, anti-BNP(3-631-436 AM8) μP/anti-BNP(106.3AM1 Fab′2-Acrydinium); diamonds, anti-BNP (ms) μP/anti-BNP(106.3AM2-Acrydinium); “X's”, anti-BNP(3-631-436 AM8) μP/anti-BNP(106.3AM2-Acrydinium); RLU, Relative Light Units.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention relates to novel antibodies that immunospecifically bind to human brain natriuretic peptide with a high binding affinity. The antibodies of the present invention are highly sensitive reagents and are useful in the qualitative and/or quantitative detection of hBNP or hBNP fragments in test samples. In another embodiment, the present invention relates to immunoassays that employ the antibodies of the present invention. In yet still a further embodiment, the present invention relates to therapeutic compositions comprising the antibodies of the present invention.

DEFINITIONS

As used herein, the terms “antibody” and “antibodies” refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies (in one aspect, a bird (for example, a duck or goose), in another aspect, a shark or whale, in yet another aspect, a mammal, including a non-primate (for example, a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, mouse, etc) and a non-human primate (for example, a monkey, such as a cynomologous monkey, a chimpanzee, etc), recombinant antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, single domain antibodies, Fab fragments, F(ab′)₂fragments, disulfide-linked Fv (sdFv), and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies of the present invention), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂) or subclass.

As used herein, the term “association rate”, “k_on” or “k_a” as used interchangeably herein, refers to the value indicating the binding strength (degree) of an antibody to its target antigen or the rate of complex formation between mAb and antigen as shown by the below:

Antibody (Ab)+Antigen (Ag)→Ab−Ag

Methods for determining association constants (k_a) are well known in the art. For example, a Biacore® (Sweden) assay can be used. Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used.

As used herein, the term “dissociation rate”, “k_off” or “k_d” as used interchangeably herein, refers to the value indicating the dissociation strength (degree) of an antibody from its target antigen or separation of Ab−Ag complex over time into free mAb and antigen as shown by the below:

Antibody (Ab)+Antigen (Ag)←Ab−Ag

Methods for determining dissociation constants (k_d) are well known in the art. For example, a Biacore® (Sweden) assay can be used. Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used.

As used herein, the term “epitope” or “epitopes” refers to sites or fragments of a polypeptide or protein having antigenic or immunogenic activity in a subject. An epitope having immunogenic activity is a site or fragment of a polypeptide or protein that elicits an antibody response in an animal. An epitope having antigenic activity is a site or fragment of a polypeptide or protein to which an antibody immunospecifically binds as determined by any method well-known to those skilled in the art, for example by immunoassays.

As used herein, the term “equilibrium dissociation constant” or “K_D” as used interchangeably, herein, refers to the value obtained by dividing the dissociation rate (k_off) by the association rate (k_on). The association rate, the dissociation rate and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen.

As used herein, the term “human brain natriuretic peptide”, “human BNP”, “hBNP”, “hBNP peptide”, “B-type natriuretic peptide”, “hBNP polypeptide” hBNP 1-32 refers to a 32 amino acid molecule representing amino acids 77-108 of the 108 amino acid precursor molecule of human brain natriuretic peptide. The sequence of human brain natiuretic peptide is shown in SEQ ID NO: 155.

As used herein, the term “hBNP fragment” or “hBNP peptide fragment” as used herein refers to a polypeptide that comprises at least about five contiguous amino acids of amino acids 77-108 of the 108 amino acid BNP precursor molecule (See, SEQ ID NO: 155). In one aspect, a hBNP fragment or hBNP peptide fragment refers to a polypeptide that comprises at least about ten contiguous amino acids residues of amino acids 77-108 of the 108 amino acid BNP precursor molecule; at least about fifteen contiguous amino acids residues of amino acids 77-108 of the 108 amino acid BNP precursor molecule; at least about 20 contiguous amino acids residues of amino acids 77-108 of the 108 amino acid BNP precursor molecule; at least about 25 contiguous amino acids residues of amino acids 77-108 of the 108 amino acid BNP precursor molecule, or at least about 30 contiguous amino acid residues of amino acids 77-108 of the 108 amino acid BNP precursor molecule. Examples of hBNP fragments or hBNP peptide fragments include, but are not limited to, amino acid sequences containing amino acids residues 1-31, 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 2-32, 2-31, 2-30, 2-29, 2-28, 2-27, 2-26, 2-25, 2-24, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 3-32, 3-31, 3-30, 3-29, 3-28, 3-27, 3-26, 3-25, 3-24, 3-23, 3-32, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 4-32, 4-31, 4-30, 4-29, 4-28, 4-27, 4-26, 4-25, 4-24, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 5-32, 5-31, 5-30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 6-32, 6-31, 6-30, 6-29, 6-28, 6-27, 6-26, 6-25, 6-24, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 7-32, 7-31, 7-30, 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 9-32, 9-31, 9-30, 9-29, 9-28, 9-27, 9-26, 9-25, 9-24, 9-23, 9-22, 9-21, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 10-32, 10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 11-32, 11-31, 11-30, 11-29, 11-28, 11-27, 11-26, 11-25, 11-24, 11-23, 11-22, 11-21, 11-20, 11-19, 11-18, 11-17 or 11-16 of hBNP.

As used herein, the term “humanized” antibody refers to an immunoglobulin variant or fragment thereof, which is capable of binding to a predetermined antigen and which comprises framework regions having substantially the amino acid sequence of a human immunoglobulin and CDRs having substantially the amino acid sequence of a non-human immunoglobulin. Ordinarily, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. In general, the humanized antibody will include substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′)₂, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Generally, the antibody will contain both the light chain as well as at least the variable domain of a heavy chain. The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁, IgG₂, IgG₃and IgG₄. The humanized antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within those skilled in the art.

As used herein, the phrase “immunospecifically binds to a human brain natriuretic peptide”, “immunospecifically binds to hBNP”, “immunospecifically binds to human brain natriuretic peptide fragment” or “immunospecifically binds to hBNP fragment” and analogous terms thereof refer to peptides, polypeptides, proteins, fusion proteins and antibodies that specifically bind to hBNP or hBNP fragment and do not specifically bind to other peptides. A peptide, polypeptide, protein, or antibody that immunospecifically binds to hBNP or hBNP fragment may bind to other peptides, polypeptides, or proteins with lower binding affinity as determined by, for example, immunoassays, BIAcore, or other assays known in the art. Antibodies or antibody fragments that immunospecifically bind to hBNP or hBNP fragment can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art. An antibody binds immunospecifically to a hBNP peptide or hBNP fragment when it binds to hBNP or hBNP fragment with a higher binding affinity than to any cross-reactive antigen as determined using experimental techniques, such as, but not limited to, radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISAs) (See, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989) for a discussion regarding antibody specificity.). In one aspect of the present invention, an antibody binds immunospecifically to hBNP or hBNP fragment (such as amino acids 5-13) when it has an equilibrium dissociation constant (K_D) for the hBNP or hBNP fragment of at least about 2.0×10⁻¹¹M as determined by a BIAcore assay under standard assay conditions, and in particular the BIAcore assay described in Example 1. In another aspect of the present invention, an antibody binds immunospecifically to hBNP or hBNP fragment (such as amino acids 13-18) when it has an equilibrium dissociation constant (K_D) for the hBNP or hBNP fragment of at least about 3.5×10⁻¹⁰M as determined by a BIAcore assay under standard assay conditions, and in particular the BIAcore assay described in Example 5.

As used herein, the term “isolated” in the context of nucleic acid molecules refers to a nucleic acid molecule which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one aspect, nucleic acid molecules are isolated. In another aspect, a nucleic acid molecule encoding an antibody of the invention is isolated.

As used herein, the term “stringent conditions” refers to hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C. The term “under highly stringent conditions”, refers to hybridization to filter-bound nucleic acid in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C., or under other stringent hybridization conditions which are known to those skilled in the art (see, for example, Ausubel, F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).

As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, the terms “subject” and “subjects” refer to an animal, in one aspect, a bird (for example, a duck or goose), in another aspect, a shark or whale, or in a further aspect, a mammal including, a non-primate (for example, a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse) and a primate (for example, a monkey, such as a cynomolgous monkey, chimpanzee, and a human).

As used herein, the term “test sample” refers to a biological sample derived from serum, plasma, whole blood, lymph, CNS fluid, urine or other bodily fluids of a subject. The test sample can be prepared using routine techniques known to those skilled in the art.

As used herein, the term “therapeutically effective amount” or “pharmaceutically effective amount” means an amount of antibody or antibody portion effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. The exact dose will be ascertainable by one skilled in the art. As known in the art, adjustments based on age, body weight, sex, race, diet, time of administration, drug interaction and severity of condition may be necessary and will be ascertainable with routine experimentation by those skilled in the art. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the antibody or antibody fragment. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

II. Antibodies of the Present Invention

The present invention provides antibodies that immunospecifically bind to hBNP or hBNP fragment. In particular, the present invention provides for antibodies that have a high binding affinity for hBNP or hBNP fragment. More specifically, in one aspect, the present invention relates to isolated antibodies which immunospecifically bind to an epitope comprising at least three (3) amino acids of amino acid residues 5 through 18 of human brain natriuretic peptide (“hBNP”) (such as, but not limited, an epitope comprising amino acids 5 through 13 or amino acids 13 through 18 of hBNP) and which exhibit at least about a two fold improvement in its equilibrium dissociation constant (K_D) when compared with at least one of (1) an antibody produced by hybridoma cell line 106.3, said cell line having A.T.C.C. Accession No. HB-12044; and (2) an antibody produced by hybridoma cell line 3-631-436 having A.T.C.C. Accession No. PTA-6476. More specifically, the antibodies of the present invention immunospecifically bind to an epitope comprising at least (3) amino acid residues of amino acid residues 5 through 18 of hBNP (such as, but not limited, an epitope comprising amino acids 5 through 13 or amino acids 13 through 18 of hBNP) or hBNP fragment thereof with at least about a three fold improvement, at least about a five fold improvement, at least about a ten fold improvement, at least about a fifteen fold improvement, at least about a twenty fold improvement, at least about a twenty-five fold improvement, at least about a thirty fold improvement, at least about a thirty-five fold improvement, at least about a forty fold improvement, at least about a forty-five fold improvement, at least about a fifty fold improvement, at least about a fifty-five fold improvement, at least about a sixty fold improvement, at least about a seventy fold improvement or at least about a seventy-five fold improvement in its equilibrium dissociation constant (K_D) when compared with an antibody produced by hybridoma cell line 106.3 (the wildtype), an antibody produced by hybridoma cell line 3-631-436 (wildtype) having A.T.C.C. Accession No. PTA-6476 or both an antibody produced by hybridoma cell line 106.3 (the wildtype), an antibody produced by hybridoma cell line 3-631-436 (wildtype) having A.T.C.C. Accession No. PTA-6476.

106.3 AM1 Antibodies

Specifically, in another aspect, the present invention relates to an antibody that immunospecifically binds to an epitope comprising at least three (3) amino acids of amino acid residues 5 through 13 of hBNP or hBNP fragment with at least about a two fold improvement in its equilibrium dissociation constant (K_D) when compared with an antibody produced by hybridoma cell line 106.3, said cell line having A.T.C.C. Accession No. HB-12044 (which is also referred to herein as the “wildtype” or “106.3 wildtype”). More specifically, the antibodies of the present invention immunospecifically bind to an epitope comprising amino acid residues 5 through 13 of hBNP or a hBNP fragment thereof with at least about a three fold improvement, at least about a five fold improvement, at least about a ten fold improvement, at least about a fifteen fold improvement, at least about a twenty fold improvement, at least about a twenty-five fold improvement, at least about a thirty fold improvement, at least about a thirty-five fold improvement, at least about a forty fold improvement, at least about a forty-five fold improvement, at least about a fifty fold improvement, at least about a fifty-five fold improvement, at least about a sixty fold improvement, at least about a seventy fold improvement or at least about a seventy-five fold improvement in its equilibrium dissociation constant (K_D) when compared with an antibody produced by hybridoma cell line 106.3 (the wildtype).

In another aspect, the present invention relates to an antibody that immunospecifically binds to hBNP or hBNP fragment and has a k_on(or k_a) of at least about 2.4×10⁴M⁻¹s⁻¹, of at least about 2.5×10⁴M⁻¹s⁻¹, of at least about 3.3×10⁴M 1s⁻¹, of at least about 5.0×10⁴M⁻¹s⁻¹, of at least about 1.25×10⁷M⁻¹s⁻¹of at least about 1.35×10⁷M⁻¹-1, of at least about 1.0×10⁸M⁻¹s⁻¹, of at least about 1.0×10⁹M⁻¹s⁻¹, or has a k_on(or k_a) ranging from about 5.0×10⁴M⁻¹s⁻¹to about 1.0×10⁸M⁻¹s⁻¹, from about 3.3×10⁴M⁻¹s⁻¹to about 1.0×10⁹M⁻¹s⁻¹, from about 2.5×10⁴M⁻¹s⁻¹to about 1.25×10⁷M⁻¹s⁻¹, from about 2.4×10⁴M⁻¹s⁻¹to about 1.35×10⁷M⁻¹s⁻¹.

In another aspect, an antibody of the present invention immunospecifically binds to the amino acid residues 5 through 13 of human BNP or hBNP fragment at a k_on(or k_a) of at least about 2.4×10⁴M⁻¹s⁻¹, of at least about 2.5×10⁴M⁻¹s⁻¹, of at least about 3.3×10⁴M⁻¹s⁻¹, of at least about 5.0×10⁴M⁻¹s⁻¹, of at least about 1.25×10⁷M⁻¹s⁻¹of at least about 1.35×10⁷M⁻¹s⁻¹, of at least about 1.0×10⁸M⁻¹s⁻¹, of at least about 1.0×10⁹M⁻¹s⁻¹, or has a k_on(or k_a) ranging from about 5.0×10⁴M⁻¹s⁻¹to about 1.0×10⁸M⁻¹s⁻¹, from about 3.3×10⁴M⁻¹s⁻¹to about 1.0×10⁹M⁻¹s⁻¹, from about 2.5×10⁴M⁻¹s⁻¹to about 1.25×10⁷M⁻¹s⁻¹, from about 2.4×10⁴M⁻¹s⁻¹to about 1.35×10⁷M⁻¹s⁻¹.

In another aspect, the present invention provides antibodies produced by Chinese hamster ovary cell line 106.3 AM1 (also known as 106.3 L1 B24/H2288). Antibodies produced by this cell line bind to amino acid residues 5 thorough 13 of hBNP or hBNP fragment at a k_on(or k_a) of at least about 2.4×10⁴M⁻¹s⁻¹, of at least about 2.5×10⁴M⁻¹s⁻¹, of at least about 3.3×10⁴M⁻¹s⁻¹, of at least about 5.0×10⁴M⁻¹s⁻¹, of at least about 1.25×10⁷M⁻¹s⁻¹of at least about 1.35×10⁷M⁻¹s⁻¹, of at least about 1.0×10⁸M⁻¹s⁻¹, of at least about 1.0×10⁹M⁻¹s⁻¹, or has a k_on(or k_a) ranging from about 5.0×10⁴M⁻¹s⁻¹to about 1.0×10⁸M⁻¹s⁻¹, from about 3.3×10⁴M⁻¹s⁻¹to about 1.0×10⁹M⁻¹s⁻¹, from about 2.5×10⁴M⁻¹s⁻¹to about 1.25×10⁷M⁻¹s⁻¹, from about 2.4×10⁴M⁻¹s⁻¹to about 1.35×10⁷M⁻¹s⁻¹.

The present invention provides antibodies that immunospecifically bind to hBNP or hBNP fragment. In particular, the present invention provides for antibodies that have a high binding affinity for hBNP or hBNP fragment. More specifically, in one aspect, an antibody that immunospecifically binds to hBNP or hBNP fragment and has a k_off(or k_d) of at least about 1.0×10⁻³s⁻¹, of at least about 1.0×10⁻⁴s⁻¹, of at least about 1.0×10⁻⁵s⁻¹, of at least about 1.0×10⁻⁶s⁻¹or has a k_off(or k_d) ranging from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁶s⁻¹, from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁵s⁻¹or from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁴s⁻¹.

In another aspect, an antibody of the present invention immunospecifically binds to the amino acid residues 5 through 13 of human BNP or hBNP fragment at a k_off(or k_off) of at least about 1.0×10⁻³s⁻¹, of at least about 1.0×10⁴s⁻¹, of at least about 1.0×10⁻⁵s⁻¹, of at least about 1.0×10⁻⁶s⁻¹or has a k_off(or k_d) ranging from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁶s⁻¹, from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁵s⁻¹or from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁴s⁻¹.

In another aspect, the present invention provides antibodies produced by Chinese hamster ovary cell line 106.3 AM1. Antibodies produced by this cell line bind to amino acid residues 5 thorough 13 of hBNP or hBNP fragment at a k_off(or k_d) of at least about 1.0×10⁻³s⁻¹, of at least about 1.0×10⁻⁴s⁻¹, of at least about 1.0×10⁻⁵s⁻¹, of at least about 1.0×10⁻⁶s⁻¹or has a k_off(or k_d) ranging from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁶s⁻¹, from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁵s⁻¹or from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁴s⁻¹

In another aspect, an antibody of the present invention immunospecifically binds to the amino acid residues 5 through 13 of human BNP at a K_Dof at least about 4.2×10⁻¹¹M, of at least about 4.0×10⁻¹¹M, of at least about 3.0×10⁻¹¹M, of at least about 2.0×10⁻¹¹M, of at least about 1.0×10⁻¹²M of at least about 8.0×10⁻¹³M, of at least about 7.4×10⁻¹³M, of at least about 1.0×10⁻¹³M, of at least about 1.0×10⁻¹⁴M, of at least about 1.0×10⁻¹⁵M, or has a K_Dranging from 4.2×10⁻¹¹M to 1.0×10⁻¹⁵M, from 4.0×10⁻¹¹M to 1.0×10⁻¹⁴M, from 3×10⁻¹¹. M to 1.0×10⁻¹³M, from 2×10⁻¹¹M to 8.0×10⁻¹³M, or from 1.0×10⁻¹²M to 7.4×10⁻¹³M.

In another aspect, the present invention provides antibodies produced by Chinese hamster ovary (CHO) cell line 106.3 AM1. Antibodies produced by this cell line bind to amino acid residues 5 thorough 13 of hBNP or hBNP fragment at a K_Dof from 4.2×10⁻¹¹M to 7.4×10⁻¹³M.

In another aspect, the antibodies of the present invention are derivatives or variants of the antibodies produced by hybridoma cell line 106.3 (A.T.C.C. Accession No. HB-12044). More specifically, the inventors of the present invention have discovered that antibodies that are derivatives or variants of the antibodies produced by hybridoma cell line 106.3 can be produced which exhibit a high binding affinity to hBNP or hBNP fragment. More specifically, the antibodies of the present invention exhibit a k_on(or k_a) of at least about 2.4×10⁴M⁻¹s⁻¹, of at least about 2.5×10⁴M⁻¹s⁻¹, of at least about 3.3×10⁴M⁻¹s⁻¹, of at least about 5.0×10⁴M⁻¹s⁻¹, of at least about 1.25×10⁷M⁻¹s⁻¹of at least about 1.35×10⁷M⁻¹s⁻¹, of at least about 1.0×10⁸M⁻¹s⁻¹, of at least about 1.0×10⁹M⁻¹s⁻¹, or have a k_on(or k_a) ranging from about 5.0×10⁴M⁻¹s⁻¹to about 1.0×10⁸M⁻¹s⁻¹, from about 3.3×10⁴M⁻¹s⁻¹to about 1.0×10⁹M⁻¹s⁻¹, from about 2.5×10⁴M⁻¹s⁻¹to about 1.25×10⁷M⁻¹s⁻¹, from about 2.4×10⁴M⁻¹s⁻¹to about 1.35×10⁷M⁻¹s⁻¹, a k_off(or k_d) of at least about 1.0×10⁻³s⁻¹, of at least about 1.0×10⁴s⁻¹, of at least about 1.0×10⁻⁵s⁻¹, of at least about 1.0×10⁻⁶s⁻¹or have a k_off(or k_d) ranging from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁶s⁻¹, from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁵s⁻¹or from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁴s⁻¹and a K_Dof at least about 4.2×10⁻¹¹M, of at least about 4.0×10⁻¹¹M, of at least about 3.0×10⁻¹¹M, of at least about 2.0×10⁻¹¹M, of at least about 1.0×10⁻¹²M of at least about 8.0×10⁻¹³M, of at least about 7.4×10⁻¹³M, of at least about 1.0×10⁻¹³M, of at least about 1.0×10⁻¹⁴M, of at least about 1.0×10⁻¹⁵M, or has a K_Dranging from 4.2×10⁻¹¹M to 1.0×10⁻¹⁵M, from 4.0×10⁻¹¹M to 1.0×10¹⁴M, from 3×10⁻¹M to 1.0×10⁻¹³M, from 2×10⁻¹M to 8.0×10¹³M, or from 1.0×10⁻¹²M to 7.4×10⁻¹³M. The derived or variant antibodies of the present invention comprise at least one mutation (such as deletions, additions and/or substitutions) in at least one of the heavy chain complementary determining (“CDR”) regions (for example, the heavy chain CDR 1, heavy chain CDR 2 and/or heavy chain CDR 3), and/or at least one mutation (such as deletions, additions and/or substitutions) in the light chain CDR regions (for example, the light chain CDR 1, light chain CDR 2, and/or light chain CDR 3) when compared to the amino acid sequence of the antibody produced by hybridoma cell line 106.3 (also referred to herein as the “wildtype”). Moreover, the antibodies of the present invention may also contain one or more other mutations (such as deletions, additions and/or substitutions) in a part or portion of the antibody other than the CDR, such as, but not limited to, the framework region of an antibody. Methods for creating such derivatives are well known in the art and include the use of site-directed mutagenesis and PCR-mediated mutagenesis, which will be discussed in more detail infra.

More specifically, in another aspect, the antibody of the present invention immunospecifically binds to hBNP or hBNP fragment and comprises a heavy chain CDR 2 having an amino acid sequence of the formula of:

(SEQ ID NO:12)

Trp-Ile-Asn-Thr-His-Thr-Gly-Glu-Xaa₁-Xaa₂-Tyr-Ala-

Asp-Asp-Phe-Lys-Gly

In yet a further aspect, the antibody of the present invention immunospecifically binds to hBNP or hBNP fragment and comprises a heavy chain CDR 2 having the amino acid sequence shown in SEQ ID NO: 15. In another aspect, the present invention relates to an antibody that immunospecifically binds to hBNP or hBNP fragment that comprises an amino acid sequence that is at least 35%, preferably at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence of SEQ ID NO: 15.

In yet another aspect, the antibody of the present invention immunospecifically binds to hBNP or hBNP fragment and comprises a light chain CDR 1 that has an amino acid sequence having a formula of:

(SEQ ID NO:13)

Lys-Ala-Xaa₃-Xaa₄-Xaa₅-Val-Asp-Tyr-Asn-Gly-Asp-

Ser-Tyr-Leu-Asn

In yet a further aspect, the antibody immunospecifically binds to hBNP or hBNP fragment and has a light chain CDR 1 having the amino acid sequence of SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO: 21 or SEQ ID NO:22. In another aspect, the present invention relates to an antibody that immunospecifically binds to hBNP or hBNP fragment that comprises an amino acid sequence that is at least 35%, preferably at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence of SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO: 21 or SEQ ID NO:22.

In yet another aspect, the antibody of the present invention immunospecifically binds to hBNP or hBNP fragment and comprises a light chain CDR 2 that has an amino acid sequence having a formula of:

(SEQ ID NO:14)

Ala-Ala-Ser-Xaa₆-Xaa₇-Xaa₈-Ser

In yet a further aspect, the antibody immunospecifically binds to hBNP or hBNP fragment and has a light chain CDR 2 having the amino acid sequence of SEQ ID NO:23 or SEQ ID NO: 24. In another aspect, the present invention relates to an antibody that immunospecifically binds to hBNP or hBNP fragment that comprises an amino acid sequence that is at least 35%, preferably at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence of SEQ ID NO:23 or SEQ ID NO:24.

In yet a further aspect, the antibody of the present invention immunospecifically binds to hBNP or hBNP fragment and has a heavy chain CDR 1, heavy chain CDR 2, heavy chain CDR 3, a light chain CDR 1, a light chain CDR 2 and a light variable CDR 3 comprising the following amino acid sequences:

(a) Heavy Chain CDR 1 having an amino acid sequence of: Gly-Tyr-Thr-Phe-Thr-His-Tyr-Gly-Ile-Asn (SEQ ID NO:6);

(b) Heavy Chain CDR 2 having an amino acid sequence having a formula of:

(SEQ ID NO:12)

Trp-Ile-Asn-Thr-His-Thr-Gly-Glu-Xaa₁-Xaa₂-Tyr-Ala-

Asp-Asp-Phe-Lys-Gly

where Xaa₁is selected from the group consisting of proline and alanine;

where Xaa₂is selected from the group consisting of isoleucine and tyrosine;

(d) Light Chain CDR 1 having an amino acid sequence having a formula of:

(SEQ ID NO:13)

Lys-Ala-Xaa₃-Xaa₄-Xaa₅-Val-Asp-Tyr-Asn-Gly-Asp-

Ser-Tyr-Leu-Asn

where Xaa₃is selected from the group consisting of: serine, alanine, asparagine, glutamine, tyrosine, threonine and arginine;

where Xaa₄is selected from the group consisting of: glutamine, tyrosine, tryptophan, alanine and phenylalanine;

where Xaa₅is selected from the group consisting of: serine, glycine, proline, alanine and aspartic acid;

(e) Light Chain CDR 2 has an amino acid sequence having the formula of:

(SEQ ID NO:14)

Ala-Ala-Ser-Xaa₆-Xaa₇-Xaa₈-Ser

where Xaa₆is selected from the group consisting of: asparagine and cysteine;

where Xaa₇is selected from the group consisting of: leucine, glycine and alanine;

where Xaa₈is selected from the group consisting of glutamic acid, tryptophan and proline; and

(f) Light Chain CDR 3 has an amino acid sequence of: Gln-Gln-Ser-Asn-Glu-Asp-Pro-Phe-Thr (SEQ ID NO:11),

where the heavy chain CDR 2 has an amino acid sequence other than Trp-Ile-Asn-Thr-His-Thr-Gly-Glu-Pro-Ile-Tyr-Ala-Asp-Asp-Phe-Lys-Gly (SEQ ID NO:7) when the light chain CDR 1 has the amino acid sequence of Lys-Ala-Ser-Gln-Ser-Val-Asp-Tyr-Asn-Gly-Asp-Ser-Tyr-Leu-Asn (SEQ ID NO:9) and the light chain CDR 2 has the amino acid sequence of Ala-Ala-Ser-Asn-Leu-Glu-Ser (SEQ ID NO: 10), the light chain CDR 1 has an amino acid sequence other than Lys-Ala-Ser-Gln-Ser-Val-Asp-Tyr-Asn-Gly-Asp-Ser-Tyr-Leu-Asn (SEQ ID NO:9) when the heavy chain CDR 2 has the amino acid sequence Trp-Ile-Asn-Thr-His-Thr-Gly-Glu-Pro-Ile-Tyr-Ala-Asp-Asp-Phe-Lys-Gly (SEQ ID NO:7) and the light chain CDR 2 has the amino acid sequence Ala-Ala-Ser-Asn-Leu-Glu-Ser (SEQ ID NO: 19), or the light chain CDR 2 has an amino acid sequence other than Ala-Ala-Ser-Asn-Leu-Glu-Ser (SEQ ID NO:10) when the heavy chain CDR 2 has the amino acid sequence of Trp-Ile-Asn-Thr-His-Thr-Gly-Glu-Pro-Ile-Tyr-Ala-Asp-Asp-Phe-Lys-Gly (SEQ ID NO:7) and the light chain CDR 1 has the amino acid sequence of Lys-Ala-Ser-Gln-Ser-Val-Asp-Tyr-Asn-Gly-Asp-Ser-Tyr-Leu-Asn (SEQ ID NO:9).

Preferably, the antibodies having the above-described formulas comprise a heavy chain CDR 1, heavy chain CDR 2, heavy chain CDR 3, light chain CDR 1, light chain CDR 2 and light chain CDR 3 where Xaa₁-Xaa₈in the above described formulas have the amino acid residues shown below in Table 1.

TABLE 1

Xaa₁
Xaa₂
Xaa₃
Xaa₄
Xaa₅
Xaa₆
Xaa₇
Xaa₈

Alanine
Tyrosine
Serine
Glutamine
Serine
Asparagines
Leucine
Glutamic

Acid

Proline
Isoleucine
Glutamine
Phenylalanine
Alanine
Asparagines
Leucine
Glutamic

Acid

Proline
Isoleucine
Tyrosine
Alanine
Serine
Asparagines
Leucine
Glutamic

Acid

Proline
Isoleucine
Glutamine
Tryptophan
Glycine
Asparagines
Leucine
Glutamic

Acid

Proline
Isoleucine
Threonine
Tryptophan
Aspartic
Asparagines
Leucine
Glutamic

Acid

Acid

Proline
Isoleucine
Arginine
Tryptophan
Proline
Asparagines
Leucine
Glutamic

Acid

Proline
Isoleucine
Alanine
Tyrosine
Glycine
Asparagines
Leucine
Glutamic

Acid

Proline
Isoleucine
Asparagine
Tryptophan
Proline
Asparagines
Leucine
Glutamic

Acid

Proline
Isoleucine
Serine
Glutamine
Serine
Cysteine
Glycine
Tryptophan

Proline
Isoleucine
Serine
Glutamine
Serine
Cysteine
Alanine
Proline

3-631-436 AM5 and 3-631-436 AM8 Antibodies

In another aspect, the present invention relates to an antibody that immunospecifically binds to an epitope comprising at least three (3) amino acids of amino acid residues 13 through 18 of hBNP or a hBNP fragment with at least about a two fold improvement in its equilibrium dissociation constant (K_D) when compared with an antibody produced by 3-631-436 which was deposited with the American Type Culture Collection (A.T.C.C.) on Dec. 21, 2004 and assigned A.T.C.C. Accession No. PTA-6476 and is described in U.S. Patent Publication 2006/0183154 published on Aug. 17, 2006 (which is also referred to herein as the “wildtype” and “3-631-436”). More specifically, the antibodies of the present invention immunospecifically bind to an epitope comprising amino acid residues 13 through 18 of hBNP or a hBNP fragment thereof with at least about a two fold improvement, at least about a three fold improvement, at least about a four fold improvement, at least about a five fold improvement, at least about a ten fold improvement, at least about a fifteen fold improvement, at least about a twenty fold improvement, at least about a twenty-five fold improvement, at least about a thirty fold improvement, at least about a thirty-five fold improvement, at least about a forty fold improvement, at least about a forty-five fold improvement, at least about a fifty fold improvement, at least about a fifty-five fold improvement, at least about a sixty fold improvement, at least about a seventy fold improvement or at least about a seventy-five fold improvement in its equilibrium dissociation constant (K_D) when compared with an antibody produced by hybridoma cell line 3-631-436.

In another aspect, the present invention relates to an antibody that immunospecifically binds to hBNP or hBNP fragment and has a k_on(or k_a) of at least about 1.5×10⁶M⁻¹s⁻¹, of at least about 3.5×10⁶M⁻¹s⁻¹, of at least about 7.8×10⁶M⁻¹s⁻¹, of at least about 8.0×10⁶M⁻¹s⁻¹of at least about 1.0×10⁷M⁻¹s⁻¹, of at least about 2.0×10⁷M⁻¹s⁻¹, of at least about 5.0×10⁷M⁻¹s⁻¹, of at least about 7.5×10⁷M⁻¹s⁻¹, of at least about 1.0×10⁸M⁻¹s⁻¹, or has a k_on(or k_a) ranging from about 1.5×10⁶M⁻¹s⁻¹to about 1.0×10⁸M⁻¹s⁻¹, from about 1.95×10⁶M⁻¹s⁻¹to about 1.0×10⁷M⁻¹s⁻¹, from about 2.70×10⁶M⁻¹s⁻¹to about 9.0×10⁶M⁻¹s⁻¹, or from about 7.0×10⁶M⁻¹s⁻¹to about 9.0×10⁶M⁻¹s⁻¹with the proviso that if the k_onis 1.6×10⁶M⁻¹s⁻¹then the k_offis not 5.4×10⁻⁴s⁻¹or 6.5×10⁻⁴s⁻¹, if the k_onis 6.7×10⁶M⁻¹s⁻¹then the k_offis not 2.5×10⁻³s⁻¹, if the k_onis 8.4×10⁶M⁻¹s⁻¹then the k_offis not 2.9×10⁻³s⁻¹, if the k_onis 8.4×10⁶M⁻¹s⁻¹then the k_offis not 2.9×10⁻³s⁻¹, if the k_onis 5.8×10⁶M⁻¹s⁻¹then the k_offis not 1.6×10⁻³s⁻¹or if the k_onis 1.5×10⁶M⁻¹s⁻¹then the k_offis not 4.1×10⁻⁴s⁻¹.

In another aspect, an antibody of the present invention immunospecifically binds to at least three (3) amino acid residues of amino acid residues 13 through 18 of human BNP or hBNP fragment at a k_on(or k_a) of at least about 1.5×10⁶M⁻¹s⁻¹, of at least about 3.5×10⁶M⁻¹s⁻¹, of at least about 7.8×10⁶M⁻¹s⁻¹, of at least about 8.0×10⁶M⁻¹s⁻¹of at least about 1.0×10⁷M⁻¹s⁻¹, of at least about 2.0×10⁷M⁻¹s⁻¹, of at least about 5.0×10⁷M⁻¹s⁻¹, of at least about 7.5×10⁷M⁻¹s⁻¹, of at least about 1.0×10⁸M⁻¹s⁻¹, or has a k_on(or k_a) ranging from about 1.5×10⁶M⁻¹s⁻¹to about 1.0×10⁸M⁻¹s⁻¹, from about 1.95×10⁶M⁻¹s⁻¹to about 1.0×10⁷M⁻¹s⁻¹, from about 2.70×10⁶M⁻¹s⁻¹to about 9.0×10⁶M⁻¹s⁻¹, or from about 7.0×10⁶M⁻¹s⁻¹to about 9.0×10⁶M⁻¹s⁻¹with the proviso that if the k_onis 1.6×10⁶M⁻¹s⁻¹then the k_offis not 5.4×10⁻⁴s⁻¹or 6.5×10⁻⁴s⁻¹, if the k_onis 6.7×10⁶M⁻¹s⁻¹then the k_offis not 2.5×10⁻³s⁻¹, if the k_onis 8.4×10⁶M⁻¹s⁻¹then the k_offis not 2.9×10⁻³s⁻¹, if the k_onis 8.4×10⁶M⁻¹s⁻¹then the k_offis not 2.9×10⁻³s⁻¹, if the k_onis 5.8×10⁶M⁻¹s⁻¹then the k_offis not 1.6×10⁻³s⁻¹or if the k_onis 1.5×10⁶M⁻¹s⁻¹then the k_offis not 4.1×10⁻⁴s⁻¹.

In another aspect, the present invention provides antibodies produced by Chinese hamster ovary cell line 3-631-436 AM5 (also known as BNP3-631-436AM5CHO893-214), Chinese hamster ovary cell line 3-631-436 AM8 (also known as BNP3-631-436AM8CHO974-211) or a combination of antibodies produced by Chinese hamster ovary cell line 3-631-436 AM5 and Chinese hamster ovary cell line 3-631-436 AM8. Antibodies produced by the CHO cell line 3-631-436 AM5 bind to amino acid residues 13 thorough 18 of hBNP or hBNP fragment having a k_on(or k_a) of at least about 7.6×10⁶M⁻¹s⁻¹, of at least about 7.7×10⁶M⁻¹s⁻¹, of at least about 7.8×10⁶M⁻¹s⁻¹, of at least about 7.9×10⁶M⁻¹s⁻¹or have a k_on(or k_a) ranging from about 7.6×10⁶M⁻¹s⁻¹to about 7.9×10⁶M⁻¹s⁻¹. Antibodies produced by the CHO cell line3-631-436 AM8 bind to amino acid residues 13 thorough 18 of hBNP or hBNP fragment having a k_on(or k_a), of at least about 8.0×10⁶M⁻¹s⁻¹, of at least about 8.1×10⁶M⁻¹s⁻¹, of at least about 8.2×10⁶M⁻¹s⁻¹, of at least about 8.3×10⁶M⁻¹s⁻¹, of at least about 8.4×10⁶M⁻¹s⁻¹, or has a k_on(or k_a) ranging from about 8.0×10⁶M⁻¹s⁻¹to about 8.4×10⁶M⁻¹s⁻¹.

The present invention provides antibodies that immunospecifically bind to hBNP or hBNP fragment. In particular, the present invention provides for antibodies that have a high binding affinity for hBNP or hBNP fragment. More specifically, in one aspect, an antibody that immunospecifically binds to hBNP or hBNP fragment and has a k_off(or k_d) of at least about 1.0×10⁻³s⁻¹, of at least about 6.5×10⁻⁴s⁻¹, of at least about 5.0×10⁻⁴s⁻¹, of at least about 4.0×10⁻⁴s⁻¹, of at least about 1.0×10⁻⁴s⁻¹, of at least about 7.5×10⁻⁵s⁻¹, 5.0×10⁻⁵s⁻¹, of at least about 2.0×10⁻⁵s⁻¹, of at least about 1×10⁻⁵s⁻¹, of at least about 1.0×10⁻⁶s⁻¹or has a k_off(or k_d) ranging from 2.0×10⁻³s⁻¹to 1.0×10⁻⁶s⁻¹, from 1.0×10⁻³s⁻¹to 1.0×10⁻⁵s⁻¹or from 1.0×10⁻³s⁻¹to 8.5×10⁴s⁻¹.

In another aspect, an antibody of the present invention immunospecifically binds to at least three amino acid residues of amino acid residues 13 through 18 of human BNP or hBNP fragment at a k_off(or k_off) k_off(or k_d) of at least about 1.0×10⁻³s⁻¹, of at least about 6.5×10⁻⁴s⁻¹, of at least about 5.0×10⁻⁴s⁻¹, of at least about 4.0×10⁻⁴s⁻¹, of at least about 1.0×10⁻⁴s⁻¹, of at least about 7.5×10⁻⁵s⁻¹, 5.0×10⁻⁵s⁻¹, of at least about 2.0×10⁻⁵s⁻¹, of at least about 1×10⁻⁵s⁻¹, of at least about 1.0×10⁻⁶s⁻¹or has a k_off(or k_d) ranging from 2.0×10⁻³s⁻¹to 1.0×10⁻⁶s⁻¹, from 1.0×10⁻³s⁻¹to 1.0×10⁻⁵s⁻¹or from 1.0×10⁻³s⁻¹to 8.5×10⁻⁴s⁻¹.

In another aspect, the present invention provides antibodies produced by Chinese hamster ovary cell line 3-631-436 AM5. Antibodies produced by this cell line bind to amino acid residues 13 thorough 18 of hBNP or hBNP fragment at a k_off(or k_d) of at least about 1.5×10⁻³s⁻¹, of at least about 1.2×10⁻³s⁻¹, of at least about 1.1×10⁻³s⁻¹, or of at least about 1.0×10⁻³s⁻¹or has a k_off(or k_d) ranging from about 1.0×10⁻³s⁻¹to about 1.5×10⁻³s⁻¹or from about 1.1×10⁻³s⁻¹to about 1.3×10⁻³s⁻¹.

In another aspect, the present invention provides antibodies produced by Chinese hamster ovary cell line 3-631-436 AM8. Antibodies produced by this cell line bind to amino acid residues 13 thorough 18 of hBNP or hBNP fragment at a k_off(or k_d) of at least about 8.5×10⁻⁴s⁻¹, of at least about 8.2×10⁴s⁻¹, of at least about 8.1×10⁻⁴s⁻¹, or of at least about 8.0×10⁻⁴s⁻¹or has a k_off(or k_d) ranging from about 8.0×10⁻⁴s⁻¹to about 8.5×10⁻⁴s⁻¹or from about 8.2×10⁻⁴s⁻¹to about 8.4×10⁻⁴s⁻¹.

In another aspect, an antibody of the present invention immunospecifically binds to at least three (3) amino acid residues of the amino acid residues 13 through 18 of human BNP at a K_Dranging from about 3.5×10⁻¹⁰M to about 1.0×10⁻¹³M, from about 2.8×10⁻¹⁰M to about 1.0×10⁻¹²M, from about 2.15×10⁻¹⁰M to about 1.0×10⁻¹¹M or from about 1.6×10⁻¹⁰M to about 1.0×10⁻¹⁰M.

In another aspect, the present invention provides antibodies produced by Chinese hamster ovary (CHO) cell line 3-631-436 AM5. Antibodies produced by this cell line bind to at least three (3) amino acid residues of amino acid residues 13 thorough 18 of hBNP or hBNP fragment at a K_Dof from 1.6×10⁻¹⁰M to 1.2×10⁻¹⁰M.

In another aspect, the present invention provides antibodies produced by Chinese hamster ovary (CHO) cell line 3-631-436 AM8. Antibodies produced by this cell line bind to at least three (3) amino acid residues of amino acid residues 13 thorough 18 of hBNP or hBNP fragment at a K_Dof from 1.2×10⁻¹⁰M to 1.0×10⁻¹⁰M.

In another aspect, the antibodies of the present invention are derivatives or variants of the antibodies produced by hybridoma cell line 3-631-436 (A.T.C.C. Accession No. PTA-6476). More specifically, the inventors of the present invention have discovered that antibodies that are derivatives or variants of the antibodies produced by hybridoma cell line 3-631-436 can be produced which exhibit a high binding affinity to hBNP or hBNP fragment. More specifically, the antibodies of the present invention exhibit a k_on(or k_a) of at least about 1.5×10⁶M⁻¹s⁻¹, of at least about 3.5×10⁶M⁻¹s⁻¹, of at least about 7.8×10⁶M⁻¹s⁻¹, of at least about 8.0×10⁶M⁻¹s⁻¹of at least about 1.0×10⁷M⁻¹s⁻¹, of at least about 2.0×10⁷M⁻¹s⁻¹, of at least about 5.0×10⁷M⁻¹s⁻¹, of at least about 7.5×10⁷M⁻¹s⁻¹, of at least about 1.0×10⁸M⁻¹s⁻¹, or has a k_on(or k_a) ranging from about 1.5×10⁶M⁻¹s⁻¹to about 1.0×10⁸M⁻¹s⁻¹, from about 1.95×10⁶M⁻¹s⁻¹to about 1.0×10⁷M⁻¹s⁻¹, from about 2.70×10⁶M⁻¹s⁻¹to about 9.0×10⁶M⁻¹s⁻¹, or from about 7.0×10⁶M⁻¹s⁻¹to about 9.0×10⁶M⁻¹s⁻¹with the proviso that if the k_onis 1.6×10⁶M⁻¹s⁻¹then the k_offis not 5.4×10⁻⁴s⁻¹or 6.5×10⁻⁴s⁻¹, if the k_onis 6.7×10⁶M⁻¹s⁻¹then the k_offis not 2.5×10⁻³s⁻¹, if the k_onis 8.4×10⁶M⁻¹s⁻¹then the k_offis not 2.9×10⁻³s⁻¹, if the k_onis 8.4×10⁶M⁻¹s⁻¹then the k_offis not 2.9×10⁻³s⁻¹, if the k_onis 5.8×10⁶M⁻¹s⁻¹then the k_offis not 1.6×10⁻³-1 or if the k_onis 1.5×10⁶M⁻¹s⁻¹then the k_offis not 4.1×10⁻⁴s⁻¹, or has a k_off(or k_d) of at least about 1.0×10⁻³s⁻¹, of at least about 6.5×10⁻⁴s⁻¹, of at least about 5.0×10⁻⁴s⁻¹of at least about 4.0×10⁻⁴s⁻¹, of at least about 1.0×10⁻⁴s⁻¹, of at least about 7.5×10⁻⁵s⁻¹, of least about 5.0×10⁻⁵s⁻¹, of at least about 2.0×10⁻⁵s⁻¹, of at least about 1.0×10⁻⁵s⁻¹, of at least about 1.0×10⁻⁶s⁻¹or has a k_off(or k_d) ranging from 2.0×10⁻³s⁻¹to 1.0×10⁻⁶s⁻¹, from 1.0×10⁻³s⁻¹to 1.0×10⁻⁵s⁻¹or from 1.0×10⁻³s⁻¹to 8.5×10⁻⁴s⁻¹or has a K_Dranging from about 3.5×10⁻¹⁰M to about 1.0×10⁻¹³M, from about 2.8×10⁻¹⁰M to about 1.0×10⁻¹²M, from about 2.15×10⁻¹⁰M to about 1.0×10⁻¹¹M or from about 1.6×10⁻¹⁰M to about 1.0×10⁻¹⁰M.

The derived or variant antibodies of the present invention comprise at least one mutation (such as deletions, additions and/or substitutions) in at least one of the heavy chain complementary determining (“CDR”) regions (for example, the heavy chain CDR 1, heavy chain CDR 2 and/or heavy chain CDR 3), and/or at least one mutation (such as deletions, additions and/or substitutions) in the light chain CDR regions (for example, the light chain CDR 1, light chain CDR 2, and/or light chain CDR 3) when compared to the amino acid sequence of the antibody produced by hybridoma cell line 3-631-436 (also referred to herein as the “wildtype” and “3-631-436”). Moreover, the antibodies of the present invention may also contain one or more other mutations (such as deletions, additions and/or substitutions) in a part or portion of the antibody other than the CDR, such as, but not limited to, the framework region of an antibody. Methods for creating such derivatives are well known in the art and include the use of site-directed mutagenesis and PCR-mediated mutagenesis, which will be discussed in more detail infra.

More specifically, in another aspect, the antibody of the present invention immunospecifically binds to hBNP or hBNP fragment and comprises a light chain CDR 2 having an amino acid sequence of the formula of:

Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Leu-Glu-Ser (SEQ ID NO:83)

where Xaa₉is selected from the group consisting of valine, glutamine, histidine, tryptophan and arginine;

where Xaa₁₀is selected from the group consisting of valine, asparagine, threonine and methionine;

where Xaa₁₁is selected from the group consisting of serine, threonine, asparagine and aspartic acid;

where Xaa₁₂is selected from the group consisting of lysine and isoleucine;

provided that Xaa₉is other than valine if Xaa₁₀is Valine, Xaa₁is serine and Xaa₁₂is lysine.

In yet a further aspect, the antibody of the present invention immunospecifically binds to hBNP or hBNP fragment and comprises a light chain CDR 2 having the amino acid sequence shown in SEQ ID NO:83. In another aspect, the present invention relates to an antibody that immunospecifically binds to hBNP or hBNP fragment that comprises an amino acid sequence that is at least about 35%, preferably at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to an amino acid sequence of SEQ ID NO:83.

In yet a further aspect, the antibody immunospecifically binds to hBNP or hBNP fragment and has a light chain CDR 2 having the amino acid sequence of SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 149, SEQ ID NO:150 or SEQ ID NO: 151. In another aspect, the present invention relates to an antibody that immunospecifically binds to hBNP or hBNP fragment that comprises an amino acid sequence that is at least about 35%, preferably at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to an amino acid sequence of SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO:147, SEQ ID NO: 148, SEQ ID NO:149, SEQ ID NO:149, SEQ ID NO:150 or SEQ ID NO: 151.

(a) Heavy Chain CDR 1 having an amino acid sequence of:

(SEQ ID NO:84)

Gly-Tyr-Thr-Phe-Thr-Ser-Tyr-Trp-Met-Asn;

(b) Heavy Chain CDR 2 having an amino acid sequence having an amino acid sequence of:

(SEQ ID NO:85)

Arg-Ile-Asp-Pro-Tyr-Asp-Ser-Glu-Thr-His-Tyr-Asn-

Gln-Lys-Phe-Lys;

(SEQ ID NO:86)

Asp-Gly-Tyr

(d) Light Chain CDR 1 having an amino acid sequence having an amino acid sequence of:

(SEQ ID NO:87)

Lys-Ser-Ser-Gln-Ser-Leu-Leu-Asp-Ser-Asp-Gly-Lys-

Thr-Tyr-Leu-Asn

(e) Light Chain CDR 2 has an amino acid sequence having the formula of:

(SEQ ID NO:83)

Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Leu-Glu-Ser

where Xaa₉is selected from the group consisting of valine, glutamine, histidine, tryptophan and arginine;

where Xaa₁₀is selected from the group consisting of valine, asparagine, threonine and methionine;

where Xaa₁₁is selected from the group consisting of serine, threonine, asparagine and aspartic acid;

where Xaa₁₂is selected from the group consisting of lysine and isoleucine;

provided that Xaa₉is other than valine if Xaa₁₀is Valine, Xaa₁₁is serine and Xaa₁₂is Lysine,

(f) Light Chain CDR 3 has an amino acid sequence of:

(SEQ ID NO:89)

Leu-Gln-Ala-Thr-His-Phe-Pro.

Preferably, the antibodies having the above-described formulas comprise a heavy chain CDR 1, heavy chain CDR 2, heavy chain CDR 3, light chain CDR 1, light chain CDR 2 and light chain CDR 3 where Xaa₉-Xaa₁₂in the above described formulas have the amino acid residues shown below in Table 2.

TABLE 2

Xaa₉
Xaa₁₀
Xaa₁₁
Xaa₁₂

Glutamine
Asparagine
Threonine
Lysine

Histidine
Threonine
Threonine
Lysine

Tryptophan
Methionine
Threonine
Lysine

Tryptophan
Methionine
Asparagine
Lysine

Valine
Threonine
Aspartic
Lysine

Acid

Arginine
Threonine
Asparagine
Lysine

Tryptophan
Methionine
Aspartic
Lysine

Acid

Tryptophan
Threonine
Threonine
Lysine

Tryptophan
Methionine
Asparagine
Lysine

Valine
Threonine
Aspartic
Isoleucine

Acid

III. Nucleic Acid Molecules

The present invention provides for a nucleic acid molecule, generally isolated, encoding an antibody of the present invention that immunospecifically binds to hBNP or hBNP fragment.

106.3 AM1 Nucleic Acid Molecules

In one aspect, the invention provides an isolated nucleic acid molecule encoding an antibody that binds to an epitope comprising amino acid residues 5 through 13 of hBNP or a hBNP fragment thereof with at least about a two fold improvement, at least about a three fold improvement, at least about a five fold improvement, at least about a ten fold improvement, at least about a fifteen fold improvement, at least about a twenty fold improvement, at least about a twenty-five fold improvement, at least about a thirty fold improvement, at least about a thirty-five fold improvement, at least about a forty fold improvement, at least about a forty-five fold improvement, at least about a fifty fold improvement, at least about a fifty-five fold improvement, at least about a sixty fold improvement, at least about a seventy fold improvement or at least about a seventy-five fold improvement in its equilibrium dissociation constant (K_D) when compared with an antibody produced by hybridoma cell line 106.3, said cell line having A.T.C.C. Accession No. HB-12044. The present invention also provides an isolated nucleic acid molecule that comprises a nucleotide sequence that hybridizes, under stringent conditions, to the nucleic acid molecule described herein that encodes an antibody that binds to an epitope comprising amino acid residues 5 through 13 of hBNP or hBNP fragment with at least about a two fold improvement, at least about a three fold improvement, at least about a five fold improvement, at least about a ten fold improvement, at least about a fifteen fold improvement, at least about a twenty fold improvement, at least about a twenty-five fold improvement, at least about a thirty fold improvement, at least about a thirty-five fold improvement, at least about a forty fold improvement, at least about a forty-five fold improvement, at least about a fifty fold improvement, at least about a fifty-five fold improvement, at least about a sixty fold improvement, at least about a seventy fold improvement or at least about a seventy-five fold improvement in its equilibrium dissociation constant (K_D) when compared with an antibody produced by hybridoma cell line 106.3, said cell line having A.T.C.C. Accession No. HB-12044.

In another aspect, the invention provides an isolated nucleic acid molecule encoding an antibody that immunospecifically binds to hBNP or hBNP fragment and that has a K_Dof at least about 4.2×10⁻¹¹M, of at least about 4.0×10⁻¹¹M, of at least about 3.0×10⁻¹¹M, of at least about 2.0×10⁻¹¹M, of at least about 1.0×10⁻¹²M of at least about 8.0×10⁻¹³M, of at least about 7.4×10⁻¹³M, of at least about 1.0×10⁻¹³M, of at least about 1.0×10⁻¹⁴M, of at least about 1.0×10⁻¹⁵M, or has a K_Dranging from 4.2×10⁻¹¹M to 1.0×10⁻¹⁵M, from 4.0×10⁻¹¹M to 1.0×10⁻¹⁴M, from 3×10⁻¹¹M to 1.0×10¹³M, from 2×10⁻¹¹M to 8.0×10¹³M, or from 1.0×10⁻¹²M to 7.4×10⁻¹³M. The present invention also provides an isolated nucleic acid molecule that comprises a nucleotide sequence that hybridizes, under stringent conditions, to the nucleic acid molecule described herein that encodes an antibody that immunospecifically binds to hBNP or hBNP fragment and that has a K_Dof at least about 4.2×10⁻¹¹M, of at least about 4.0×10⁻¹¹M, of at least about 3.0×10⁻¹¹M, of at least about 2.0×10⁻¹¹M, of at least about 1.0×10⁻¹²M of at least about 8.0×10⁻¹³M, of at least about 7.4×10⁻¹³M, of at least about 1.0×10⁻¹³M, of at least about 1.0×10⁻¹⁴M, of at least about 1.0×10⁻¹⁵M, or has a K_Dranging from 4.2×10⁻¹¹M to 1.0×10⁻¹⁵M, from 4.0×10⁻¹¹M to 1.0×10⁻¹⁴M, from 3×10⁻¹¹M to 1.0×10⁻¹³M, from 2×10⁻¹¹M to 8.0×10⁻¹³M, or from 1.0×10⁻¹²M to 7.4×10⁻¹³M.

In another aspect, an isolated nucleic acid molecule encodes an antibody that immunospecifically binds to amino acid residues 5 through 13 of human BNP or hBNP fragment at a K_Dof at least about 4.2×10⁻¹¹M, of at least about 4.0×10⁻¹¹M, of at least about 3.0×10⁻¹¹M, of at least about 2.0×10⁻¹¹M, of at least about 1.0×10⁻¹²M of at least about 8.0×10⁻¹³M, of at least about 7.4×10⁻¹³M, of at least about 1.0×10⁻¹³M, of at least about 1.0×10⁻¹⁴M, of at least about 1.0×10⁻¹⁵M, or has a K_Dranging from 4.2×10⁻¹¹M to 1.0×10⁻¹⁵M, from 4.0×10⁻¹¹M to 1.0×10⁻¹⁴M, from 3×10⁻¹¹M to 1.0×10⁻¹³M, from 2×10⁻¹¹M to 8.0×10⁻¹³M, or from 1.0×10⁻¹²M to 7.4×10⁻¹³M. The present invention also provides an isolated nucleic acid molecule that comprises a nucleotide sequence that hybridizes, under stringent conditions, to the nucleic acid molecule described herein that encodes an antibody that immunospecifically binds to amino acid residues 5 through 13 of hBNP or hBNP fragment at a K_Dof at least about 4.2×10⁻¹¹M, of at least about 4.0×10⁻¹¹M, of at least about 3.0×10⁻¹¹M, of at least about 2.0×10⁻¹¹M, of at least about 1.0×10⁻¹²M of at least about 8.0×10⁻¹³M, of at least about 7.4×10⁻¹³M, of at least about 1.0×10⁻¹³M, of at least about 1.0×10⁻¹⁴M, of at least about 1.0×10⁻¹⁵M, or has a K_Dranging from 4.2×10⁻¹¹M to 1.0×10⁻¹⁵M, from 4.0×10⁻¹¹M to 1.0×10⁻¹⁴M, from 3×10⁻¹¹M to 1.0×10⁻¹³M, from 2×10⁻¹¹M to 8.0×10⁻¹³M, or from 1.0×10⁻¹²M to 7.4×10⁻¹³M.

In yet another aspect, the invention provides an isolated nucleic acid molecule encoding an antibody that immunospecifically binds to amino acid residues 5 through 13 of hBNP or hBNP fragment at a K_Dof from 4.2×10⁻¹¹M to 7.4×10⁻¹³M, wherein said nucleic acid molecule comprises the nucleotide sequence of antibody produced by CHO cell line 106.3 AM1. The present invention also provides an isolated nucleic acid molecule that comprises a nucleotide sequence that hybridizes, under stringent conditions, to the nucleic acid molecule described herein that encodes an antibody that immunospecifically binds to amino acid residues 5 through 13 of hBNP or hBNP fragment at a K_Dof from 4.2×10⁻¹¹M to 7.4×10⁻¹³M, wherein said nucleic acid molecule comprises the nucleotide sequence of antibody produced by CHO cell line 106.3 AM1.

In another aspect, the present invention provides an isolated nucleic acid molecule that encodes antibodies that immunospecifically bind to hBNP or hBNP fragment, wherein said antibodies comprise derivatives or variants of antibodies produced by hybridoma cell line 106.3 (A.T.C.C. Accession No. HB-12044). As discussed previously herein, the inventors of the present invention have discovered that antibodies that are derivatives or variants of the antibodies produced by hybridoma cell line 106.3 can be produced which exhibit a high binding affinity, specifically a k_on(or k_a) of at least about 2.4×10⁴M⁻¹s⁻¹, of at least about 2.5×10⁴M⁻¹s⁻¹, of at least about 3.3×10⁴M⁻¹s⁻¹, of at least about 5.0×10⁴M⁻¹s⁻¹, of at least about 1.25×10⁷M⁻¹s⁻¹of at least about 1.35×10⁷M⁻¹s⁻¹, of at least about 1.0×10⁸M¹s¹, of at least about 1.0×10⁹M⁻¹s¹, or have a k_on(or k_a) ranging from about 5.0×10⁴M⁻¹s⁻¹to about 1.0×10⁸M⁻¹s¹, from about 3.3×10⁴M⁻¹s⁻¹to about 1.0×10⁹M⁻¹s⁻¹, from about 2.5×10⁴M⁻¹s⁻¹to about 1.25×10⁷M⁻¹s⁻¹, from about 2.4×10⁴M⁻¹s⁻¹to about 1.35×10⁷M⁻¹s⁻¹, a k_off(or k_d) of at least about 1.0×10⁻³s⁻¹, of at least about 1.0×10⁻⁴s⁻¹, of at least about 1.0×10⁻⁵s⁻¹, of at least about 1.0×10⁻⁶s⁻¹or have a k_off(or k_d) ranging from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁶s⁻¹, from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁵s⁻¹or from about 1.0×10⁻³s⁻¹to about 1.0×10⁻⁴s⁻¹and a K_Dof at least about 4.2×10⁻¹¹M, of at least about 4.0×10⁻¹¹M, of at least about 3.0×10⁻¹¹M, of at least about 2.0×10⁻¹¹M, of at least about 1.0×10⁻¹²M of at least about 8.0×10⁻¹³M, of at least about 7.4×10⁻¹³M, of at least about 1.0×10⁻¹³M, of at least about 1.0×10⁻¹⁴M, of at least about 1.0×10⁻¹⁵M, or has a K_Dranging from 4.2×10⁻¹¹M to 1.0×10⁻¹⁵M, from 4.0×10⁻¹¹M to 1.0×10⁻¹⁴M, from 3×10⁻¹¹M to 1.0×10⁻¹³M, from 2×10⁻¹¹M to 8.0×10⁻¹³M, or from 1.0×10⁻¹²M to 7.4×10⁻¹³M. The derived or variant antibodies of the present invention comprises at least one mutation (such as deletions, additions and/or substitutions) in at least one of the heavy chain complementary determining (“CDR”) regions (for example, the heavy chain CDR 1, heavy chain CDR 2, or heavy chain CDR 3), at least one mutation (such as deletions, additions and/or substitutions) in the light chain CDR regions (for example, the light chain CDR 1, light chain CDR 2, or light chain CDR 3) when compared to the amino acid sequence of the antibody produced by hybridoma cell line 106.3. Standard techniques known to those of skill in the art can be used to introduce mutations (such as deletions, additions, and/or substitutions) in the nucleic acid molecule encoding an antibody of the present invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. In one aspect, the derivatives include less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original antibody produced by hybridoma cell line 106.3. In one aspect, the derivatives have conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues (i.e., amino acid residues which are not critical for the antibody to immunospecifically bind to hBNP or hBNP fragment). A “conservative amino acid substitution” is one in which the amino acid residue is replaced with the amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that exhibit enhanced binding affinity to hBNP or hBNP fragment. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined.

In another aspect, the present invention provides an isolated nucleic acid molecule encoding an antibody that immunospecifically binds to hBNP or hBNP fragment, said antibody having a heavy chain CDR 2 having an amino acid sequence of the formula of:

(SEQ ID NO:12)

Trp-Ile-Asn-Thr-His-Thr-Gly-Glu-Xaa₁-Xaa₂-Tyr-Ala-

Asp-Asp-Phe-Lys-Gly

where Xaa₁is selected from the group consisting of proline and alanine and Xaa₂is selected from the group consisting of isoleucine and tyrosine, provided that when Xaa₁is proline, Xaa₂is not isoleucine. The present invention also provides an isolated nucleic acid molecule that comprises a nucleotide sequence that hybridizes, under stringent conditions, to the nucleic acid molecule described herein that encodes an antibody having a heavy chain CDR 2 having an amino acid sequence of the above-described formula.

In another aspect, the present invention provides an isolated nucleic acid molecule encoding an antibody that immunospecifically binds to hBNP or hBNP fragment, said antibody having a light chain CDR 1 that has an amino acid sequence having a formula of:

(SEQ ID NO:13)

Lys-Ala-Xaa₃-Xaa₄-Xaa₅-Val-Asp-Tyr-Asn-Gly-Asp-

Ser-Tyr-Leu-Asn

where Xaa₃is selected from the group consisting of: serine, alanine, asparagine, glutamine, tyrosine, threonine and arginine; where Xaa₄is selected from the group consisting of: glutamine, tyrosine, tryptophan, alanine and phenylalanine and where Xaa₅is selected from the group consisting of: serine, glycine, proline, alanine and aspartic acid, provided that Xaa₃is not serine when Xaa₄is glutamine and Xaa₅is serine. The present invention also provides an isolated nucleic acid molecule that comprises a nucleotide sequence that hybridizes, under stringent conditions, to the nucleic acid molecule described herein that encodes an antibody having a light chain CDR 1 having an amino acid sequence of the above-described formula.

In another aspect, the invention provides an isolated nucleic acid molecule encoding an antibody that immunospecifically binds to hBNP or hBNP fragment, said antibody comprising (alternatively, consisting of) a light chain CDR 1 having an amino acid sequence of SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22. The present invention also provides an isolated nucleic acid molecule that comprises a nucleotide sequence that hybridizes, under stringent conditions, to the nucleic acid molecule described herein that encodes an antibody comprising a light chain CDR 1 having the amino acid sequence of SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.

In another aspect, the present invention provides an isolated nucleic acid molecule encoding an antibody that immunospecifically binds to hBNP or hBNP fragment, said antibody having a light chain CDR 2 that has an amino acid sequence having a formula of:

(SEQ ID NO:14)

Ala-Ala-Ser-Xaa₆-Xaa₇-Xaa₈-Ser

where Xaa₆is selected from the group consisting of: asparagine and cysteine, where Xaa₇is selected from the group consisting of: leucine, glycine and alanine and where Xaa₈is selected from the group consisting of glutamic acid, tryptophan and proline, provided that Xaa₆is not asparagine when Xaa₇is leucine and Xaa₈is glutamic acid. The present invention also provides an isolated nucleic acid molecule that comprises a nucleotide sequence that hybridizes, under stringent conditions, to the nucleic acid molecule described herein that encodes an antibody having a light chain CDR 2 having an amino acid sequence of the above-described formula.

In another aspect, the invention provides an isolated nucleic acid molecule that encodes an antibody that immunospecifically binds to hBNP or hBNP fragment, said antibody comprising (alternatively, consisting) a heavy chain CDR 2 having an amino acid sequence of SEQ ID NO: 15, a light chain CDR 1 having an amino acid sequence of SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22, a light chain CDR 2 having an amino acid sequence of SEQ ID NO:23 or SEQ ID NO:24 or any combinations these amino acid sequences. The present invention also provides an isolated nucleic acid molecule that comprises a nucleotide sequence that hybridizes, under stringent conditions, to the nucleic acid molecule described herein that encodes an antibody comprising a heavy chain CDR 2 having an amino acid sequence of SEQ ID NO: 15, a light chain CDR 1 having an amino acid sequence of SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22, a light chain CDR 2 having an amino acid sequence of SEQ ID NO:23 or SEQ ID NO:24 or any combinations these amino acid sequences.