Negative Mimic Antibody For Use as a Blocking Reagent In BNP Immunoassays

TECHNICAL FIELD

The present disclosure relates to isolated antibodies that do not bind to brain natriuretic peptide. The antibodies of the present disclosure can be used as reagents to reduce or remove interference in immunoassays for brain natriuretic peptide or a fragment thereof.

BACKGROUND

In the field of diagnostics, immunoassays are used for detecting analytes in biological samples. Examples of analytes that can be detected include, drugs, hormones, infectious agents, microorganisms, antibodies and the like. The identification of one or more analytes in a biological sample can be used to diagnose cancer, heart disease, etc.

Immunoassays involve a specific binding reaction between the analyte to be detected and at least one specific binding partner. The specific binding partner (which can be an antibody, antigen, etc) specifically binds to the analyte or reacts with it. The analyte and the specific binding partner form a specific binding pair complex. An example of such a specific binding pair complex is an antibody (or antibody fragment) and antigen. However, more than one analyte or more than one specific binding partner can react with each other during each reaction.

The specific binding pair complex can then be detected. Typically, at least one specific binding partner is labeled with a detectable label such as a chromogen, fluorophore, substances capable of chemi- or electrochemiluminescence, radioisotopes, haptens, enzymes labels or substances that can form another specific binding pair such as biotin and streptavidin.

As useful as immunoassays are, they are not without their problems. Such problems include nonspecific binding reactions and undesired interactions between the specific binding partners and the sample components. These interferences can cause an increase in background signal and reduced sensitivity and specificity. Depending on the kind of interference, false positive or false negative test results can occur.

When human serum/plasma is used as a biological sample in an immunoassay, heterophile or heterophilic antibodies present in a sample can interact with the specific binding partners and have an effect on the immunoassay. The existence of heterophilic antibodies and their potential for causing interference in immunoassays has been known for years. “Hetero” and “phile” are from the Greek and mean “different” and “affinity”, respectively. Taber's Medical Dictionary defines heterophilic antibodies as an “antibody response to an antigen other than a specific one” (See, Kaplan, I., et al., Clinical Chem., 45(5):616-618 (1999)). Heterophile antibodies are found in all people and are weak, multi-specific antibodies. There are a variety of possible causes for inducing heterophilic antibodies, including, exposure to animals, alternate animal contact therapy, exposure to animal products, special diets, deliberate immunization, rheumatoid factors, blood transfusions, autoimmune diseases, dialysis, maternal transfer, cardiac myopathy and gastrointestinal disease. It is apparent that heterophilic antibodies can come from natural antibodies (namely, natural polyspecific antibodies, natural idiotypic antibodies and natural rheumatoid factors (RF)) and autoantibodies (namely, autoimmune polyspecific antibodies and autoimmune RF and idiotypic antibodies). Heterophilic antibodies which bind to animal antibodies are referred to as HAAAs (human anti-animal antibodies). An example of an HAAA is a human anti-mouse antibody, also referred to as a HAMA.

A number of approaches are known in the art for reducing heterophilic antibody interference. Specifically, a number of physical and chemical techniques have been used to remove heterophile interferences. These techniques include, ultracentrifugation, removal of the interference with protein A or protein G, precipitation with trichloroacetic acid, heating at 90° C., and pretreatment with ethanol, polyethylene glycol (130 g/L), sulfhydryl agents and detergents (See, Levinson, S., et al., Clinica Chimica Acta, 325:1-15 (2002)). Alternatively, blocking agents can be used. A blocking reagent is a preparation, which, when added to an immunoassay, prevents interference by heterophilic antibodies. Two types of blocking agents are known, nonspecific blocking agents and specific blocking agents.

Historically, nonspecific blocking agents have been nonimmune globulins from the species used to produce the test antibodies. Id. The nonimmune globulin may be in the purified form, but usually, it is in the form of normal animal sera. Id. These agents have the advantage of containing a diverse mixture of proteins that can adsorb out heterophile interference from all types of sandwich and competitive assays. Id. Nonspecific binding agents are typically considered to be “passive” blocking agents because they are added in excess concentrations so that any specific anti-species antibodies present in the biological sample bind to these in preference to the specific binding partners that are present in lower concentrations.

Specific blocking agents are reagents that contain specific antibodies with activity against human immunoglobulins. Examples of commercially available specific blocking agents include immunoglobulin inhibiting reagent (IIR) and heterophile blocking reagent (HBR). IIR is a mixture of mouse monoclonal antibodies produced by using human heterophilic antibodies and HAMA as the immunogens. Id. IIR has been studied as an agent capable of blocking specific HAMA and it blocked interference in more samples than a polymerized monoclonal preparation, but not better than in a polyclonal nonimmune mouse IgG. Id.

Heterophile blocking reagents (HBR) are specific binders that are directed against the human heterophilic antibody. When HBR binds to the human heterophilic antibody, the blocking is accomplished by steric hindrance. One study suggested that HBR reduced heterophile interference from 29% to 4.8% compared with 1% normal mouse sera which reduced interference to 6.2% when an intact mouse monoclonal antibody was used for detection, while interference was reduced from 4.8% to 2.0% compared to 2.6% for the normal mouse sera when a chimeric detection antibody was used. Id. In another study, one patient's sera showed a reduction of troponin I to normal levels when HBR was added, but also to normal levels, when diluted with a preparation of normal goat serum. Id. In yet another study, HBR blocked interference in all of five sera that were previously positive prior to a modification of the reagent, but the manufacturer's modified reagent, containing bovine and goat normal sera, blocked the interference as well or better. Id. However, like nonspecific binding agents, specific binding agents do not remove interference in all cases.

Thereupon, there is a need in the art for new reagents that can be used to remove interferences from immunoassays.

SUMMARY

In one embodiment, the present disclosure relates to an isolated antibody that does not bind to BNP. The 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, a heavy chain CDR 3, a heavy framework region 1, a heavy framework region 2, a heavy framework region 3, a heavy framework region 4, the variable light domain comprising a light chain CDR 1, a light chain CDR 2, a light chain CDR 3, a light framework region 1, a light framework region 2, a light framework region 3, and a light framework region 4 wherein said antibody comprises at least one of:

(a) a heavy chain CDR 1 having an amino acid sequence of the formula of: a heavy chain CDR 1 having an amino acid sequence of the formula of:

(SEQ ID NO:1)

Gly-Tyr-Xaa₉-Phe-Thr-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄

wherein Xaa₉is threonine or alanine; Xaa₁₀is serine or alanine; Xaa₁₁is tyrosine, aspartic acid or proline; Xaa₁₂is tryptophan, cysteine, glutamine, proline, valine or asparagine; Xaa₁₃is methionine, proline, threonine, lysine or histidine; and Xaa₁₄is asparagine or glycine, provided that if (a) Xaa₉is threonine, Xaa₁₀is serine, Xaa₁₁is tyrosine, Xaa₁₂is tryptophan, Xaa₁₃is methionine then Xaa₁₄is glycine; (b) Xaa₁₀is serine, Xaa₁₁is tyrosine, Xaa₁₂is tryptophan, Xaa₁₃is methionine, Xaa₁₄is alanine, then Xaa₉is alanine; (c) Xaa₉is threonine, Xaa₁₁is tyrosine, Xaa₁₂is tryptophan, Xaa₁₃is methionine, Xaa₁₄is asparagine, then Xaa₁₀is alanine; (d) Xaa₉is threonine, Xaa₁₀is serine, Xaa₁₂is tryptophan, Xaa₁₃is methionine, Xaa₁₄is asparagine, then Xaa₁₁is aspartic acid or proline; (e) Xaa₉is threonine, Xaa₁₀is serine, Xaa₁₁is tyrosine, Xaa₁₃is methionine, Xaa₁₄is asparagine then Xaa₁₂is cysteine, glutamine, proline, valine or asparagine; or (f) Xaa₉is threonine, Xaa₁₀is serine, Xaa₁₁is tyrosine, Xaa₁₂is tryptophan, Xaa₁₄is asparagine then Xaa₁₃is proline, threonine, lysine or histidine;

(b) a heavy chain CDR 2 having an amino acid sequence of the formula of:

(SEQ ID NO:2)

Arg-Ile-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Glu-Thr-His-Tyr-

Asn-Gln-Lys-Phe-Lys-Asp

wherein Xaa₁is aspartic acid or cysteine; Xaa₂is proline or leucine; Xaa₃is tyrosine, proline or methionine; Xaa₄is aspartic acid or serine and Xaa₅is serine or leucine, provided that if: (a) Xaa₁is aspartic acid, Xaa₂is proline, Xaa₃is tyrosine, Xaa₄is aspartic acid then Xaa₅is leucine; (b) Xaa₂is proline, Xaa₃is tyrosine, Xaa₃is aspartic acid, Xaa₅is serine, then Xaa₁is cysteine; (c) Xaa₁is aspartic acid, Xaa₃is tyrosine, Xaa₄is aspartic acid, Xaa₅is serine, then Xaa₂is leucine; or (d) Xaa₁is aspartic acid, Xaa₂is proline, Xaa₄is aspartic acid, Xaa₅is serine, then Xaa₃is proline or methionine; or (e) Xaa₁is aspartic acid, Xaa₂is proline, Xaa₃is tyrosine, Xaa₅is serine, then Xaa₄is serine;

(SEQ ID NO:4)

Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈-Xaa₁₉-Xaa₂₀-Leu-Asp-Ser-

Asp-Gly-Xaa₂₁-Xaa₂₂-Xaa₂₃-Leu-Asn

wherein Xaa₁₅is lysine or leucine; Xaa₁₆is serine, valine or leucine; Xaa₁₇is serine, arginine, aspartic acid or threonine; Xaa₁₈is glutamine, glutamic acid, leucine or serine; Xaa₁₉is serine, aspartic acid, leucine; Xaa₂₀is leucine or glutamine; Xaa₂₁is lysine or glutamine; Xaa₂₂is threonine or glycine; Xaa₂₃is tyrosine or proline, provided that if: (a) Xaa₁₅is lysine; Xaa₁₆is serine; Xaa₁₇is serine, Xaa₁₈is glutamine, Xaa₁₉is serine, Xaa₂₀is leucine, Xaa₂₁is lysine, Xaa₂₂is threonine, then Xaa₂₃is proline; (b) Xaa₁₆is serine; Xaa₁₇is serine, Xaa₁₈is glutamine, Xaa₁₉is serine, Xaa₂₀is leucine, Xaa₂₁is lysine, Xaa₂₂is threonine, Xaa₂₃is tyrosine, then Xaa₁₅is leucine; (c) Xaa₁₅is lysine, Xaa₁₇is serine, Xaa₁₈is glutamine, Xaa₁₉is serine, Xaa₂₀is leucine, Xaa₂₁is lysine, Xaa₂₂is threonine, Xaa₂₃is tyrosine, then Xaa₁₆is valine or leucine; (d) Xaa₁₅is lysine, Xaa₁₆is serine; Xaa₁₈is glutamine, Xaa₁₉is serine, Xaa₂₀is leucine, Xaa₂₁is lysine, Xaa₂₂is threonine, Xaa₂₃is tyrosine, then Xaa₁₇is arginine, aspartic acid or threonine; (e) Xaa₁₅is lysine, Xaa₁₆is serine; Xaa₁₇is serine, Xaa₁₉is serine, Xaa₂₀is leucine, Xaa₂₁is lysine, Xaa₂₂is threonine, Xaa₂₃is tyrosine, then Xaa₁₈is glutamic acid; (f) Xaa₁₅is lysine, Xaa₁₆is serine; Xaa₁₇is serine, Xaa₁₈is glutamine, Xaa₂₀is leucine, Xaa₂₁is lysine, Xaa₂₂is threonine, Xaa₂₃is tyrosine, then Xaa₁₉is aspartic acid or leucine; (g) Xaa₁₅is lysine, Xaa₁₆is serine; Xaa₁₇is serine, Xaa₁₈is glutamine, Xaa₁₉is serine, Xaa₂₁is lysine, Xaa₂₂is threonine, Xaa₂₃is tyrosine, then Xaa₂₀is glutamine; (h) Xaa₁₅is lysine, Xaa₁₆is serine; Xaa₁₇is serine, Xaa₁₈is glutamine, Xaa₁₉is serine, Xaa₂₀is leucine, Xaa₂₂is threonine, Xaa₂₃is tyrosine, then Xaa₂₁is glutamine; or (i) Xaa₁₅is lysine, Xaa₁₆is serine; Xaa₁₇is serine, Xaa₁₈is glutamine, Xaa₁₉is serine, Xaa₂₀is leucine, Xaa₂₁is lysine, Xaa₂₃is tyrosine, then Xaa₂₂is glycine;

(d) a light chain CDR 2 that has an amino acid sequence having a formula of:

Val-Val-Xaa₆-Xaa₇-Xaa₈-Glu-Ser
(SEQ ID NO:5)

wherein Xaa₆is serine or glutamic acid, Xaa₇is lysine or proline and Xaa₈is leucine or glutamine, provided that if (a) Xaa₆is serine and Xaa₇is lysine, then Xaa₈is glutamine; (b) Xaa₇is lysine and Xaa₈is leucine, then Xaa₆is glutamic acid; or (c) Xaa₆is serine and Xaa₈is leucine, then Xaa₇is proline;

(e) a heavy chain framework 1 that has an amino acid sequence having a formula of:

(SEQ ID NO:114)

Gln-Val-Gln-Leu-Gln-Gln-Pro-Gly-Ala-Glu-Leu-Val-

Arg-Pro-Gly-Ala-Ser-Xaa₂₄-Lys-Leu-Ser-Cys-Lys-Ala-

Ser

wherein Xaa₂₄is alanine;

(f) a heavy chain framework 2 that has an amino acid sequence having a formula of:

(SEQ ID NO:116)

Trp-Val-Lys-Gln-Arg-Pro-Glu-Gln-Gly-Leu-Glu-Trp-

Ile-Xaa₂₅

wherein Xaa₂₅glutamic acid;

(g) a light chain framework 2 that has an amino acid sequence having a formula of:

(SEQ ID NO:121)

Trp-Xaa₂₆-Phe-Gln-Xaa₂₇-Pro-Gly-Glu-Ser-Pro-Lys-

Leu-Leu-Ile-Tyr

wherein Xaa₂₆is leucine or serine and Xaa₂₇is arginine or glycine, provided that if (a) Xaa₂₆is leucine, then Xaa₂₇is glycine; or (b) Xaa₂₇is arginine, then Xaa₂₆is serine;

(h) a light chain framework 4 that has an amino acid sequence having a formula of:

(SEQ ID NO:125)

Trp-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu-Ile-Lys-

Xaa₂₈

wherein Xaa₂₈is tryptophan; or

(i) any combinations of (a)-(h).

The above antibody 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, a DVD-Ig, a TVD-Ig, an anti-idiotypic antibody, or a functionally active epitope-binding fragment thereof.

In yet another embodiment, the present disclosure relates to an isolated antibody that does not bind to BNP. The 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, a heavy chain CDR 3, a heavy framework region 1, a heavy framework region 2, a heavy framework region 3, a heavy framework region 4, the variable light domain comprising a light chain CDR 1, a light chain CDR 2, a light chain CDR 3, a light framework region 1, a light framework region 2, a light framework region 3, and a light framework region 4 wherein said antibody comprises at least one of:

(a) the heavy chain CDR 1 has an amino acid sequence having a formula of:

(SEQ ID NO:1)

Gly-Tyr-Xaa₉-Phe-Thr-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄

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

(SEQ ID NO:2)

Arg-Ile-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Glu-Thr-His-Tyr-

Asn-Gln-Lys-Phe-Lys-Asp

wherein Xaa₁is aspartic acid or cysteine; Xaa₂is proline or leucine; Xaa₃is tyrosine, proline or methionine; Xaa₄is aspartic acid or serine and Xaa₅is serine or leucine;

Asp-Gly-Tyr;
(SEQ ID NO:3)

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

(SEQ ID NO:4)

Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈-Xaa₁₉-Xaa₂₀-Leu-Asp-Ser-

Asp-Gly-Xaa₂₁-Xaa₂₂-Xaa₂₃-Leu-Asn

(e) the light chain CDR 2 has an amino acid sequence having the formula of:

Val-Val-Xaa₆-Xaa₇-Xaa₈-Glu-Ser
(SEQ ID NO:5)

wherein Xaa₆is serine or glutamic acid, Xaa₇is lysine or proline and Xaa₈is leucine or glutamine;

(f) the light chain CDR 3 has an amino acid sequence having a formula of:

Leu-Gln-Ala-Thr-His-Phe-Pro;
(SEQ ID NO:6)

(g) the heavy chain framework 1 has an amino acid sequence having a formula of:

(SEQ ID NO:130)

Gln-Val-Gln-Leu-Gln-Gln-Pro-Gly-Ala-Glu-Leu-Val-

Arg-Pro-Gly-Ala-Ser-Xaa₂₄-Lys-Leu-Ser-Cys-Lys-Ala-

Ser

wherein Xaa₂₄is valine or alanine;

(h) the heavy chain framework 2 has an amino acid sequence having a formula of:

(SEQ ID NO:131)

Trp-Val-Lys-Gln-Arg-Pro-Glu-Gln-Gly-Leu-Glu-Trp-

Ile-Xaa₂₅

wherein Xaa₂₅is glycine or glutamic acid;

(i) the heavy chain framework 3 has an amino acid sequence having a formula of:

(SEQ ID NO:118)

Lys-Ala-Ile-Leu-Thr-Val-Asp-Lys-Ser-Ser-Ser-Thr-

Ala-Phe-Val-Gln-Leu-Thr-Ser-Leu-Thr-Ser-Glu-Asp-

Ser-Ala-Val-Tyr-Tyr-Cys-Val-Ser;

(j) the heavy chain framework 4 has an amino acid sequence having a formula of:

(SEQ ID NO:119)

Trp-Gly-Ala-Gly-Thr-Thr-Cys-Thr-Val-Ser-Ser;

(k) the light chain framework 1 has an amino acid sequence having a formula of:

(SEQ ID NO:120)

Asp-Val-Val-Met-Thr-Gln-Thr-Pro-Leu-Thr-Leu-Ser-

Val-Thr-Thr-Gly-Gln-Pro-Ala-Ser-Ile-Ser-Cys;

(l) the light chain framework 2 has an amino acid sequence having a formula of:

(SEQ ID NO:121)

Trp-Xaa₂₆-Phe-Gln-Xaa₂₇-Pro-Gly-Glu-Ser-Pro-Lys-

Leu-Leu-Ile-Tyr

wherein Xaa₂₆is leucine or serine;

wherein Xaa₂₇is arginine or glycine;

(m) the light chain framework 3 has an amino acid sequence having a formula of:

(SEQ ID NO:124)

Gly-Val-Pro-Asp-Arg-Phe-Thr-Gly-Ser-Gly-Ser-Gly-

Thr-Asp-Phe-Thr-Leu-Lys-Ile-Ser-Arg-Val-Glu-Ala-

Glu-Asp-leu-Gly-Val-Tyr-Tyr-Cys;

and

(n) the light chain framework 4 has an amino acid sequence having a formula of:

(SEQ ID NO:132)

Trp-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu-Ile-Lys-

Xaa₂₈

wherein Xaa₂₈is arginine or tryptophan;

provided that each of Xaa₁-Xaa₂₈are not simultaneously the following: Xaa₁is Asp, Xaa₂is Pro, Xaa₃is Tyr, Xaa₄is Asp, Xaa₅is Ser, Xaa₆is Ser, Xaa₇is Lys, Xaa₈is Leu, Xaa₉is Thr, Xaa₁₀is Ser, Xaa₁₁is Tyr, Xaa₁₂is Trp, Xaa₁₃is Met, Xaa₁₄is Asn, Xaa₁₅is Lys, Xaa₁₆is Ser, Xaa₁₇is Ser, Xaa₁₈is Gln, Xaa₁₉is Ser, Xaa₂₀is Leu, Xaa₂₁is Lys, Xaa₂₂is Thr, Xaa₂₃is Tyr, Xaa₂₄is Val, Xaa₂₅is Gly, Xaa₂₆is Leu, Xaa₂₇is Arg and Xaa₂₈is Arg.

In the above antibody, Xaa₁-Xaa₂₈can be as follows: (a) Xaa₁is Asp, Xaa₂is Pro, Xaa₃is Tyr, Xaa₄is Asp, Xaa₅is Ser, Xaa₆is Ser, Xaa₇is Lys, Xaa₈is Leu, Xaa₉is Thr, Xaa₁₀is Ala, Xaa₁₁is Tyr, Xaa₁₂is Cys, Xaa₁₃is Met, Xaa₁₄is Asn, Xaa₁₅is Lys, Xaa₁₆is Val, Xaa₁₇is Arg, Xaa₁₈is Glu, Xaa₁₉is Ser, Xaa₂₀is Leu, Xaa₂₁is Lys, Xaa₂₂is Thr, Xaa₂₃is Tyr, Xaa₂₄is Val, Xaa₂₅is Gly, Xaa₂₆is Leu, Xaa₂₇is Arg and Xaa₂₈is Arg; (b) Xaa₁is Cys, Xaa₂is Leu, Xaa₃is Met, Xaa₄is Asp, Xaa₅is Ser, Xaa₆is Ser, Xaa₇is Lys, Xaa₈is Leu, Xaa₉is Thr, Xaa₁₀is Ser, Xaa₁₁is Tyr, Xaa₁₂is Gln, Xaa₁₃is Pro, Xaa₁₄is Gly, Xaa₁₅is Lys, Xaa₁₆is Ser, Xaa₁₇is Asp, Xaa₁₈is Leu, Xaa₁₉is Asp, Xaa₂₀is Leu, Xaa₂₁is Lys, Xaa₂₂is Thr, Xaa₂₃is Tyr, Xaa₂₄is Val, Xaa₂₅is Gly, Xaa₂₆is Ser, Xaa₂₇is Arg and Xaa₂₈is Arg; (c) Xaa₁is Asp, Xaa₂is Pro, Xaa₃is Pro, Xaa₄is Ser, Xaa₅is Leu, Xaa₆is Glu, Xaa₇is Pro, Xaa₈is Gln, Xaa₉is Thr, Xaa₁₀is Ser, Xaa₁₁is Tyr, Xaa₁₂is Trp, Xaa₁₃is Met, Xaa₁₄is Asn, Xaa₁₅is Lys, Xaa₁₆is Ser, Xaa₁₇is Ser, Xaa₁₈is Gln, Xaa₁₉is Ser, Xaa₂₀is Leu, Xaa₂₁is Lys, Xaa₂₂is Thr, Xaa₂₃is Tyr, Xaa₂₄is Val, Xaa₂₅is Gly, Xaa₂₆is Leu, Xaa₂₇is Arg and Xaa₂₈is Arg; (d) Xaa₁is Asp, Xaa₂is Pro, Xaa₃is Tyr, Xaa₄is Asp, Xaa₅is Ser, Xaa₆is Ser, Xaa₇is Lys, Xaa₈is Leu, Xaa₉is Thr, Xaa₁₀is Ser, Xaa₁₁is Tyr, Xaa₁₂is Pro, Xaa₁₃is Thr, Xaa₁₄is Gly, Xaa₁₅is Leu, Xaa₁₆is Leu, Xaa₁₇is Thr, Xaa₁₈is Gln, Xaa₁₉is Ser, Xaa₂₀is Leu, Xaa₂₁is Lys, Xaa₂₂is Thr, Xaa₂₃is Tyr, Xaa₂₄is Val, Xaa₂₅is Glu, Xaa₂₆is Leu, Xaa₂₇is Gly and Xaa₂₈is Arg; (e) Xaa₁is Asp, Xaa₂is Pro, Xaa₃is Tyr, Xaa₄is Asp, Xaa₅is Ser, Xaa₆is Ser, Xaa₇is Lys, Xaa₈is Leu, Xaa₉is Thr, Xaa₁₀is Ser, Xaa₁₁is Asp, Xaa₁₂is Val, Xaa₁₃is Lys, Xaa₁₄is Asn, Xaa₁₅is Lys, Xaa₁₆is Ser, Xaa₁₇is Ser, Xaa₁₈is Ser, Xaa₁₉is Leu, Xaa₂₀is Gln, Xaa₂₁is Lys, Xaa₂₂is Thr, Xaa₂₃is Tyr, Xaa₂₄is Ala, Xaa₂₅is Gly, Xaa₂₆is Leu, Xaa₂₇is Arg and Xaa₂₈is Arg; (f) Xaa₁is Asp, Xaa₂is Pro, Xaa₃is Pro, Xaa₄is Ser, Xaa₅is Leu, Xaa₆is Ser, Xaa₇is Lys, Xaa₈is Leu, Xaa₉is Ala, Xaa₁₀is Ser, Xaa₁₁is Tyr, Xaa₁₂is Trp, Xaa₁₃is Met, Xaa₁₄is Asn, Xaa₁₅is Lys, Xaa₁₆is Ser, Xaa₁₇is Ser, Xaa₁₈is Gln, Xaa₁₉is Ser, Xaa₂₀is Leu, Xaa₂₁is Lys, Xaa₂₂is Thr, Xaa₂₃is Tyr, Xaa₂₄is Val, Xaa₂₅is Gly, Xaa₂₆is Leu, Xaa₂₇is Arg and Xaa₂₈is Arg; or (g) Xaa₁is Asp, Xaa₂is Pro, Xaa₃is Tyr, Xaa₄is Asp, Xaa₅is Ser, Xaa₆is Ser, Xaa₇is Lys, Xaa₈is Leu, Xaa₉is Thr, Xaa₁₀is Ser, Xaa₁₁is Pro, Xaa₁₂is Asn, Xaa₁₃is His, Xaa₁₄is Asn, Xaa₁₅is Lys, Xaa₁₆is Ser, Xaa₁₇is Ser, Xaa₁₈is Gln, Xaa₁₉is Ser, Xaa₂₀is Leu, Xaa₂₁is Gln, Xaa₂₂is Gly, Xaa₂₃is Pro, Xaa₂₄is Val, Xaa₂₅is Gly, Xaa₂₆is Leu, Xaa₂₇is Arg and Xaa₂₈is Trp.

In the above antibody, (a) the heavy chain CDR 1 has an amino acid sequence comprising SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 or SEQ ID NO:107; (b) the heavy chain CDR 2 has an amino acid sequence comprising SEQ ID NO:69, SEQ ID NO:108 or SEQ ID NO:129; (c) the light chain CDR 1 has an amino acid sequence comprising SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO:112 or SEQ ID NO:113; (d) the light chain CDR 2 has an amino acid sequence comprising SEQ ID NO:70; (e) the heavy chain framework 1 has an amino acid sequence comprising SEQ ID NO:115; (f) the heavy chain framework 2 has an amino acid sequence comprising SEQ ID NO:117; (g) the light chain framework 2 has an amino acid sequence comprising SEQ ID NO:122 or SEQ ID NO:123; or (h) the light chain framework 4 has an amino acid sequence comprising SEQ ID NO:126.

More specifically, in the above antibody, (a) the heavy chain CDR 2 has an amino acid sequence comprising SEQ ID NO:69; and (b) the light chain CDR 2 has an amino acid sequence comprising SEQ ID NO:70.

In still yet another embodiment, the present disclosure relates to an isolated polynucleotide comprising a polynucleotide sequence having a sequence of SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 and full complements thereof.

In yet still another embodiment, the present disclosure relates to a heterophilic blocking agent for use in a BNP immunoassay, the agent comprising any of the above described antibodies.

In still yet another embodiment, the present disclosure relates to a method for reducing heterophilic interference in a BNP immunoassay. The method comprises the step of adding the above described heterophilic blocking agent to an immunoassay.

In still yet another embodiment, the present disclosure relates to an immunoassay for hBNP or hBNP fragment, wherein said immunoassay comprises at least one of the above described antibodies.

In still yet another embodiment, the present disclosure relates to chinese hamster ovary (“CHO”) cell line BNP3-631-436CN4CHO that expresses the CN4 antibody, wherein said cell line comprises at least one polynucleotide sequence selected from the group consisting of: SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 and combinations thereof.

In still yet another embodiment, the present disclosure relates to an antibody made from DNA extracted from the CHO cell line BNP3-631-436CN4CHO, wherein said DNA comprises at least one polynucleotide sequence selected from the group consisting of: SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 and combinations thereof.

In still yet another embodiment, the present disclosure relates to a chimeric antibody or a fragment thereof produced by CHO cell line BNP3-631-436CN4CHO, wherein said antibody comprises DNA comprising at least one polynucleotide sequence selected from the group consisting of: SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85 and combinations thereof.

In still yet another embodiment, the present disclosure relates to a kit for use in an immunoassay for hBNP or hBNP fragment. The kit comprises:

- (a) heterophilic blocking agent comprising at least one of the above described antibodies; and
- (b) instructions for conducting the assay of the test sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of the 3-631-436 scFv protein in the orientation in which it is expressed on the surface of yeast.

FIG. 2 shows the amino acid sequence of the 3-631-436 scFv (SEQ ID NO: 66). 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. 3A-3B show the nucleotide sequence of the 3-631-436 scFv (SEQ ID NO: 67). The double-stranded sequence is depicted.

FIG. 4 is a plasmid map for vector pYD41-40-3631436-scFv containing the 3-631-436 single-chain variable fragment shown in FIG. 1.

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

FIGS. 6A-6D are the nucleotide sequence of the vector shown in FIG. 4 (SEQ ID NO: 11).

FIG. 7 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. 8 is a schematic depiction which shows how the 3-631-436 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.

FIGS. 9A-9F show the fifty (50) oligonucleotides (SEQ ID NOS:14-63) that were used to create the pYD1-41-40-3-631-436 vectors described in detail in Example 1.

FIG. 10 are graphs that show 3-631-436 WT scFv, 3-631-436 CDR light chain libraries (3631VL) and CDR heavy chain libraries (3631 VH) were incubated with or without biotinylated:cyclic BNP (1-32c) (SEQ ID NO:10) and anti-V5 followed by secondary reagents streptavidin phycoerythrin (SA:PE) and goat anti mouse-FITC (GAM:FITC). Using FACS, a sort gate (“S1” or “S2”) was set in which 3-631-436 scFv yeast cells that did not bind BNP (PE low fluorescence) but were expressed as full length proteins (FITC high fluorescence) were sorted. Selected yeast cells were grown, stained and sorted a second time as described above. Finally, round 2 sorted cells were plated on selective media and further characterized. The “pos” or positive control gate was set based on the position of the 3-631-436WT BNP binding population.

FIG. 11 shows the results of the characterization of scFv clones and wildtype (WT) clones from 3-631-436 for human brain natriuretic peptide (BNP) and anti-V5 binding pursuant to Example 1. The characterization shows that clones H1, H3, H4, L1, L2, L3 and L4 did not bind human BNP at 83 nM.

FIG. 12 shows the amino acid mutations for the 3-631-436 scFv variants.

FIG. 13 shows a 3-631-436 VH/VL combination library was incubated with or without biotinylated:cyclic BNP (1-32c) (SEQ ID NO:10) and anti-V5 followed by secondary reagents streptavidin phycoerythrin (SA:PE) and goat anti mouse-FITC (GAM:FITC). Using FACS, a sort gate (“S1” and “S2”) was set in which 3-631-436 scFv yeast cells that did not bind BNP (PE low fluorescence) but were expressed as full length proteins (FITC high fluorescence) were sorted. Selected yeast cells were grown, stained and sorted a second time from “S2” gate. Finally, round 2 sorted cells were plated on selective media and further characterized. The “WT pos” or positive control gate was set based on the position of the 3-631-436WT BNP binding population.

FIG. 14 shows the further characterization of clones CN1, CN2, CN4, CN6, CN7, CN8, CN9 and WT described in Example 1 at 2 μM human BNP.

FIG. 15 shows the amino acid mutations in the CDR heavy chain and CDR light chain for each of clones CN1, CN2, CN4, CN6, CN7, CN8 and CN9.

FIG. 16 shows KinExA® characterization of the binding of clone CN4, to human BNP. Clone CN4 did not generate a signal at both 1 nM and 10 nM monoclonal antibody (mAb) concentration demonstrating its lack of ability to bind to human BNP. Voltage values are recorded in the table to illustrate the lack of binding to BNP relative to the positive control (3-631-436 WT mAb).

FIG. 17 shows polynucleotide sequences for each of the CDR heavy chain and CDR light chain for each of clones CN1, CN2, CN4, CN6, CN7, CN8 and CN9 Specifically, FIG. 17A shows the polynucleotide sequence of the CDR heavy chain for clone CN1 (SEQ ID NOS:74 and 75). FIG. 17B shows the polynucleotide sequence of the CDR light chain for clone CN1 (SEQ ID NOS:76 and 77). FIG. 17C shows the polynucleotide sequence of the CDR heavy chain for clone CN2 (SEQ ID NOS:78 and 79). FIG. 17D shows the polynucleotide sequence of the CDR light chain for clone CN2 (SEQ ID NOS:80 and 81). FIG. 17E shows the polynucleotide sequence of the CDR heavy chain for clone CN4 (SEQ ID NOS:82 and 83). FIG. 17F shows the polynucleotide sequence of the CDR light chain for clone CN4 (SEQ ID NOS:84 and 85). FIG. 17G shows the polynucleotide sequence of the CDR heavy chain for clone CN6 (SEQ ID NOS:86 and 87). FIG. 17H shows the polynucleotide sequence of the CDR light chain for clone CN6 (SEQ ID NOS:88 and 89). FIG. 17I shows the polynucleotide sequence of the CDR heavy chain for clone CN7 (SEQ ID NOS:90 and 91). FIG. 17J shows the polynucleotide sequence of the CDR light chain for clone CN7 (SEQ ID NOS:92 and 93). FIG. 17K shows the polynucleotide sequence of the CDR heavy chain for clone CN8 (SEQ ID NOS:94 and 95). FIG. 17L shows the polynucleotide sequence of the CDR light chain for clone CN8 (SEQ ID NOS:96 and 97). FIG. 17M shows the polynucleotide sequence of the CDR heavy chain for clone CN9 (SEQ ID NOS:98 and 99). FIG. 17N shows the polynucleotide sequence of the CDR light chain for clone CN9 (SEQ ID NOS:100 and 101).

FIG. 18 shows the amino acid sequences for the variable heavy chain (SEQ ID NO:127) and variable light chain (SEQ ID NO:128) for wildtype 3-631-436. FIG. 18A shows the variable heavy chain, including framework H1 (amino acids 1-25), CDR1 (amino acids 26-35), framework H2 (amino acids 36-49), CDR2 (amino acids 50-65), framework H3 (amino acids 66-94), CDR3 (amino acids 95-97) and framework H4 (amino acids 103-113). FIG. 18B shows the variable light chain, including framework L1 (amino acids 1-23), CDR1 (amino acids 24-34), framework L2 (amino acids 35-49), CDR2 (amino acids 50-56), framework L3 (amino acids 57-88), CDR3 (amino acids 89-95) and framework L4 (amino acids 96-108).

DETAILED DESCRIPTION

The present disclosure relates to negative mimic antibodies or NEMIC antibodies. The antibodies of the present disclosure can be used as heterophilic blocking agents in BNP immunoassays. Specifically, the antibodies of the present disclosure can be used to reduce heterophilic interference in said immunoassays.

A. Definitions

As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.

a) Antibody or Antibodies

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, dual variable domain (DVD) or triple variable domain (TVD) antibodies (Dual-variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11):1290-1297 (2007) and WO2001/058956, the contents of each of which are herein incorporated by reference), 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 disclosure), 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, IgA₁and IgA₂) or subclass.

b) Binding Constants

The term “association rate constant”, “k_on” or “k_a” as used interchangeably herein, refers to the value indicating the binding rate of an antibody to its target antigen or the rate of complex formation between an antibody and antigen as shown by the equation below:

Antibody (“Ab”)+Antigen (“Ag”)→Ab-Ag.

The term “dissociation rate constant”, “k_off” or “k_d” as used interchangeably herein, refers to the value indicating the dissociation rate of an antibody from its target antigen or separation of Ab-Ag complex over time into free antibody and antigen as shown by the equation below:

Ab+Ag←Ab-Ag.

Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used.

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.

c) Epitope or Epitopes

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.

d) Human Brain Natriuretic Peptide, Human BNP, hBNP, hBNP Peptide or B-Type Natriuretic Peptide

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 naturetic peptide is shown in SEQ ID NO: 68.

e) hBNP Fragment or hBNP Peptide Fragment

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: 68). 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, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 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, 11-16, 12-32, 12-31, 12-30, 12-29, 12-28, 12-27, 12-26, 12-25, 12-24, 12-23, 12-22, 12-21, 12-20, 12-19, 12-18, 12-17, 13-32, 13-31, 13-30, 13-29, 13-28, 13-27, 13-26, 13-25, 13-24, 13-23, 13-22, 13-21, 13-20, 13-19, 13-18, 14-32, 14-31, 14-30, 14-29, 14-28, 14-27, 14-26, 14-25, 14-24, 14-23, 14-22, 14-21, 14-20, 14-19, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 16-32, 16-31, 16-30, 16-29, 16-28, 16-27, 16-26, 16-25, 16-24, 16-23, 16-22, 16-21, 17-32, 17-31, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-23, 17-22, 18-32, 18-31, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 19-32, 19-31, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 20-32, 20-31, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 22-32, 22-31, 22-30, 22-29, 22-28, 22-27, 23-32, 23-31, 23-30, 23-29, 23-28, 24-32, 24-31, 24-30, 24-29, 25-32, 25-31, 25-20, 26-32, 26-31 or 27-32 of hBNP.

f) Humanized Antibody

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.

g) Immunospecifically Binds

As used herein, the phrase “immunospecifically binds to brain natriuretic peptide”, “immunospecifically binds to a brain natriuretic fragment”, “immunospecifically binds to 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 BNP, a BNP fragment, hBNP or a hBNP fragment and do not specifically bind to other peptides. A peptide, polypeptide, protein, or antibody that immunospecifically binds to BNP, a BNP fragment, hBNP or a hBNP fragment may bind to other peptides, polypeptides, or proteins with lower binding affinity as determined by, for example, immunoassays, such as a Biacore® or KinExA® assay, or any other assays known to those of skill in the art. Antibodies or antibody fragments that immunospecifically bind to BNP, a BNP fragment, hBNP or a hBNP fragment can be identified, for example, by immunoassays, such as by using a Biacore® or KinExA® assay, or by using other techniques known to those of skill in the art. An antibody binds immunospecifically to BNP, a BNP fragment, hBNP peptide or hBNP fragment when it binds to BNP, a BNP fragment, hBNP or a 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.).

h) Isolated

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 disclosure is isolated.

i) Negative Mimic or NEMIC Antibody

As used herein, the term “negative mimic” antibody or “NEMIC” antibody refers to an antibody that does not immunospecifically bind to an antigen of interest (such as, but not limited to a protein, peptide, fragment of a protein or peptide, etc.). In other words, the NEMIC antibody of the present disclosure does not generate any detectable signal when placed in the presence of the antigen of interest under standard assay conditions. Such assay conditions are well known in the art and can include the use of a Biacore® or KinExA® assay (See, for example, the use of a KinExA® assay as described in Example 2). An example of an antigen of interest can be BNP, a BNP fragment, hBNP or a hBNP fragment. Such antibodies can be used as reagents to reduce heterophilic interference in an immunoassay.

j) Stringent Conditions

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).

k) Subject or Patient

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).

l) Test Sample

As used herein, the term “test sample” or “sample” generally refers to a biological material derived from any biological source, such as, a physiological fluid, including, but not limited to, whole blood, serum, plasma, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen and so forth. The test sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample. For example, such pretreatment may include preparing plasma from blood, diluting viscous fluids and so forth. Methods of pretreatment may also involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, etc. Moreover, it may also be beneficial to modify a solid test sample to form a liquid medium or to release the analyte.

The terminology used herein is for the purpose of describing particular embodiments only and is not otherwise intended to be limiting.

B. Negative Mimic Antibodies

In one embodiment, the present disclosure relates to negative mimic antibodies or NEMIC antibodies. Specifically, the negative mimic antibodies of the present disclosure are antibodies that do not bind, more specifically, do not immunospecifically bind to BNP or any fragment thereof. More specifically, the antibodies of the present disclosure do not immunospecifically bind to human BNP or any hBNP fragment thereof. The antibodies of the present disclosure can be used as heterophilic blocking agents to reduce heterophilic interference in an immunoassay. Preferably, the immunoassay is an immunoassay for BNP or a BNP fragment, preferably, hBNP or a hBNP fragment thereof.

The negative mimic antibodies of the present disclosure 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), 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), at least one mutation (such as deletions, additions and/or substitutions) in the heavy chain framework regions (for example, the heavy chain framework 1, heavy chain framework 2, heavy chain framework 3 and/or heavy chain framework 4) and/or at least one mutation (such as deletions, additions and/or substitutions) in the light chain framework regions (for example, the light chain framework 1, light chain framework 2, light chain framework 3 and/or light chain framework 4) when compared to the amino acid sequence of the antibody produced by hybridoma cell line 3-631-436, specifically, hybridoma cell line 3-631-436, A.T.C.C. Accession No. PTA-6476 (also referred to herein as the “wildtype”; See, FIG. 18A and FIG. 18B and SEQ ID NOS:127 and 128). Moreover, the antibodies of the present disclosure 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 or framework region of the 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 antibodies of the present disclosure do not immunospecifically bind to BNP or a fragment thereof, preferably to hBNP or a hBNP fragment, and comprises a heavy chain CDR 1 having an amino acid sequence of the formula of:

(SEQ ID NO:1)

Gly-Tyr-Xaa₉-Phe-Thr-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄

In yet a further aspect, the antibody of the present disclosure does not immunospecifically bind to hBNP or hBNP fragment and comprises a heavy chain CDR 1 having the amino acid sequence of: Gly-Tyr-Thr-Phe-Thr-Ala-Tyr-Cys-Met-Asn (SEQ ID NO:102), Gly-Tyr-Thr-Phe-Thr-Ser-Tyr-Gln-Pro-Gly (SEQ ID NO:103), Gly-Tyr-Thr-Phe-Thr-Ser-Tyr-Pro-Thr-Gly (SEQ ID NO:104), Gly-Tyr-Thr-Phe-Thr-Ser-Asp-Val-Lys-Asn (SEQ ID NO:105), Gly-Tyr-Ala-Phe-Thr-Ser-Tyr-Trp-Met-Asn (SEQ ID NO:106) or Gly-Tyr-Thr-Phe-Thr-Ser-Pro-Asn-His-Asn (SEQ ID NO:107).

In another aspect, the present disclosure relates to an antibody that does not 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:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 or SEQ ID NO:107.

In another aspect, the antibody of the present disclosure do not immunospecifically bind to BNP or a fragment thereof, preferably to hBNP or a hBNP fragment, and comprises a heavy chain CDR 2 having an amino acid sequence of the formula of:

(SEQ ID NO:2)

Arg-Ile-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Glu-Thr-His-Tyr-

Asn-Gln-Lys-Phe-Lys-Asp

wherein Xaa₁is aspartic acid or cysteine; Xaa₂is proline or leucine; Xaa₃is tyrosine, proline or methionine; Xaa₄is aspartic acid or serine and Xaa₅is serine or leucine, provided that if: (a) Xaa₁is aspartic acid, Xaa₂is proline, Xaa₃is tyrosine, Xaa₄is aspartic acid, then Xaa₅is leucine; (b) Xaa₂is proline, Xaa₃is tyrosine, Xaa₄is aspartic acid, Xaa₅is serine, then Xaa₁is cysteine; (c) Xaa₁is aspartic acid, Xaa₃is tyrosine, Xaa₄is aspartic acid, Xaa₅is serine, then Xaa₂is leucine; or (d) Xaa₁is aspartic acid, Xaa₂is proline, Xaa₄is aspartic acid, Xaa₅is serine, then Xaa₃is proline or methionine; or (e) Xaa₁is aspartic acid, Xaa₂is proline, Xaa₃is tyrosine, Xaa₅is serine, then Xaa₄is serine.

In yet a further aspect, the antibody of the present disclosure does not immunospecifically bind to hBNP or hBNP fragment and comprises a heavy chain CDR 2 having the amino acid sequence of Arg-Ile-Asp-Pro-Pro-Ser-Lys-Glu-Thr-His-Tyr-Asn-Gln-Lys-Phe-Lys (SEQ ID NO:69), Arg-Ile-Cys-Leu-Met-Tyr-Asp-Ser-Glu-Thr-His-Tyr-Asn-Gln-Lys-Phe-Lys-Asp (SEQ ID NO:108) or Arg-Ile-Asp-Pro-Pro-Ser-Leu-Glu-Thr-His-Tyr-Asn-Gln-Lys-Phe-Lys-Asp (SEQ ID NO:129). In another aspect, the present disclosure relates to an antibody that does not 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:69, SEQ ID NO:108 or SEQ ID NO:129.

In yet another aspect, the antibody of the present disclosure does not immunospecifically bind to BNP or a fragment thereof, preferably, hBNP or a hBNP fragment, and comprises a light chain CDR 1 that has an amino acid sequence having a formula of:

(SEQ ID NO:4)

Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈-Xaa₁₉-Xaa₂₀-Leu-Asp-Ser-

Asp-Gly-Xaa₂₁-Xaa₂₂-Xaa₂₃-Leu-Asn

In yet a further aspect, the antibody does not immunospecifically bind to hBNP or hBNP fragment and has a light chain CDR 1 having the amino acid sequence of:

(SEQ ID NO:109)

Lys-Val-Arg-Glu-Ser-Leu-Leu-Asp-Ser-Asp-Gly-Lys-

Thr-Tyr-Leu-Asn,

(SEQ ID NO:110)

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

Thr-Tyr-Leu-Asn,

(SEQ ID NO:111)

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

Thr-Tyr-Leu-Asn,

(SEQ ID NO:112)

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

Thr-Tyr-Leu-Asn

or

(SEQ ID NO:113)

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

Gly-Pro-Leu-Asn.

In another aspect, the present disclosure 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:109, SEQ ID NO:10, SEQ ID NO:111, SEQ ID NO:112 or SEQ ID NO:113.

In yet another aspect, the antibody of the present disclosure does not immunospecifically bind to BNP or a fragment thereof, preferably, hBNP or a hBNP fragment, and comprises a light chain CDR 2 that has an amino acid sequence having a formula of:

Val-Val-Xaa₆-Xaa₇-Xaa₈-Glu-Ser
(SEQ ID NO:5)

In yet a further aspect, the antibody does not immunospecifically bind to hBNP or hBNP fragment and has a light chain CDR 2 having the amino acid sequence of: Val-Val-Glu-Pro-Gln-Glu-Ser (SEQ ID NO:70). In another aspect, the present disclosure 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:70.

In yet a further aspect, the antibody of the present disclosure does not immunospecifically bind to BNP or a fragment thereof, preferably, hBNP or a hBNP fragment, and comprises a heavy chain framework 1 that has an amino acid sequence having a formula of:

(SEQ ID NO:114)

Gln-Val-Gln-Leu-Gln-Gln-Pro-Gly-Ala-Glu-Leu-Val-

Arg-Pro-Gly-Ala-Ser-Xaa₂₄-Lys-Leu-Ser-Cys-Lys-

Ala-Ser

wherein Xaa₂₄is alanine.

In yet a further aspect, the antibody does not immunospecifically bind to hBNP or hBNP fragment and has a heavy chain framework 1 having the amino acid sequence of: Gln-Val-Gln-Leu-Gln-Gln-Pro-Gly-Ala-Glu-Leu-Val-Arg-Pro-Gly-Ala-Ser-Ala-Lys-Leu-Ser-Cys-Lys-Ala-Ser (SEQ ID NO:115). In another aspect, the present disclosure 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:115.

In yet a further aspect, the antibody of the present disclosure does not immunospecifically bind to BNP or a fragment thereof, preferably, hBNP or a hBNP fragment, and comprises a heavy chain framework 2 that has an amino acid sequence having a formula of:

(SEQ ID NO:116)

Trp-Val-Lys-Gln-Arg-Pro-Glu-Gln-Gly-Leu-Glu-Trp-

Ile-Xaa₂₅

wherein Xaa₂₅glutamic acid.

In yet a further aspect, the antibody does not immunospecifically bind to hBNP or hBNP fragment and has a heavy chain framework 2 having the amino acid sequence of: Trp-Val-Lys-Gln-Arg-Pro-Glu-Gln-Gly-Leu-Glu-Trp-Ile-Glu (SEQ ID NO:117).

In yet a further aspect, the antibody of the present disclosure does not immunospecifically bind to BNP or a fragment thereof, preferably, hBNP or a hBNP fragment, and comprises a light chain framework 2 that has an amino acid sequence having a formula of:

(SEQ ID NO:121)

Trp-Xaa₂₆-Phe-Gln-Xaa₂₇-Pro-Gly-Glu-Ser-Pro-Lys-

Leu-Leu-Ile-Tyr

wherein Xaa₂₆is leucine or serine and Xaa₂₇is arginine or glycine, provided that if (a) Xaa₂₆is leucine, then Xaa₂₇is glycine; or (b) Xaa₂₇is arginine, then Xaa₂₆is serine.

In yet a further aspect, the antibody does not immunospecifically bind to hBNP or hBNP fragment and has a light chain framework 2 having the amino acid sequence of: Trp-Ser-Phe-Gln-Arg-Pro-Gly-Glu-Ser-Pro-Lys-Leu-Leu-Ile-Tyr (SEQ ID NO:122) or Trp-Leu-Phe-Gln-Gly-Pro-Gly-Glu-Ser-Pro-Lys-Leu-Leu-Ile-Tyr (SEQ ID NO:123).

In yet a further aspect, the antibody of the present disclosure does not immunospecifically bind to BNP or a fragment thereof, preferably, hBNP or a hBNP fragment, and comprises a light chain framework 4 that has an amino acid sequence having a formula of:

(SEQ TD NO:125)

Trp-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu-Ile-Lys-

Xaa₂₈

wherein Xaa₂₈is tryptophan.

In yet a further aspect, the antibody does not immunospecifically bind to hBNP or hBNP fragment and has a light chain framework 4 having the amino acid sequence of: Trp-Thr-Phe-Gly-Gly-Gly-Thr-Lys-Leu-Glu-Ile-Lys-Trp (SEQ ID NO:126). In another aspect, the present disclosure 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:126.

In yet a further aspect, the antibody of the present disclosure does not immunospecifically binds to BNP or a fragment thereof, preferably, hBNP or a hBNP fragment, and has a heavy chain CDR 1, heavy chain CDR 2, heavy chain CDR 3, a heavy chain framework 1, a heavy chain framework 2, a heavy chain framework 3, a heavy chain framework 4, a light chain CDR 1, a light chain CDR 2, a light variable CDR 3, a light chain framework 1, a light chain framework 2, a light chain framework 3 and a light chain framework 4 comprising the following amino acid sequences:

(a) the heavy chain CDR 1 has an amino acid sequence having a formula of:

(SEQ ID NO:1)

Gly-Tyr-Xaa₉-Phe-Thr-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄

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

(SEQ ID NO:2)

Arg-Ile-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Glu-Thr-His-Tyr-

Asn-Gln-Lys-Phe-Lys-Asp

wherein Xaa₁is aspartic acid or cysteine; Xaa₂is proline or leucine; Xaa₃is tyrosine, proline or methionine; Xaa₄is aspartic acid or serine and Xaa₅is serine or leucine;

Asp-Gly-Tyr;
(SEQ ID NO:3)

(d) the light chain CDR 1 has an amino acid sequence having a formula of: Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈-Xaa₁₉-Xaa₂₀-Leu-Asp-Ser-Asp-Gly-Xaa₂₁-Xaa₂₂-Xaa₂₃-Leu-Asn (SEQ ID NO:4)

(e) the light chain CDR 2 has an amino acid sequence having the formula of:

Val-Val-Xaa₆-Xaa₇-Xaa₈-Glu-Ser
(SEQ ID NO:5)

wherein Xaa₆is serine or glutamic acid, Xaa₇is lysine or proline and Xaa₈is leucine or glutamine;

(f) the light chain CDR 3 has an amino acid sequence having a formula of:

Leu-Gln-Ala-Thr-His-Phe-Pro;
(SEQ ID NO:6)

(g) the heavy chain framework 1 has an amino acid sequence having a formula of:

(SEQ ID NO:130)

Gln-Val-Gln-Leu-Gln-Gln-Pro-Gly-Ala-Glu-Leu-Val-

Arg-Pro-Gly-Ala-Ser-Xaa₂₄-Lys-Leu-Ser-Cys-Lys-

Ala-Ser