WO2009086126A2

WO2009086126A2 - Natriuretic polypeptides

Info

Publication number: WO2009086126A2
Application number: PCT/US2008/087714
Authority: WO
Inventors: John C. Burnett, Jr.; Candace Y.W. Lee
Original assignee: Mayo Foundation For Medical Education And Research
Priority date: 2007-12-21
Filing date: 2008-12-19
Publication date: 2009-07-09
Also published as: WO2009086126A3

Abstract

This document provides natriuretic polypeptides. For example, this document provides polypeptides having a natriuretic activity. In some cases, a polypeptide provided herein can have natriuretic activities without lowering blood pressure. This document also provides methods and materials for inducing natriuretic activities within a mammal.

Description

NATRIURETIC POLYPEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority from U.S. Provisional Application Serial No. 61/015,903, filed on December 21, 2007.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant HL036634 awarded by the National Institutes of Heart, Lung, and Blood Institute. The government has certain rights in the invention.

BACKGROUND

1. Technical Field

This document relates to natriuretic polypeptides. For example, this document provides methods and materials related to natriuretic polypeptides and the use of natriuretic polypeptides to treat cardiovascular and renal conditions.

2. Background Information

Natriuretic polypeptides are polypeptides that can cause natriuresis (increased sodium excretion in the urine). Such polypeptides can be produced by brain, heart, kidney, and/or vascular tissue.

SUMMARY

This document relates to natriuretic polypeptides. For example, this document provides methods and materials related to natriuretic polypeptides and the use of natriuretic polypeptides to treat cardiovascular conditions, renal conditions, or both cardiovascular conditions and renal conditions. In some cases, a polypeptide provided herein can have diuretic activity, natriuretic activity, the ability to activate cyclic GMP (cGMP), the ability to increase glomerular filtration rate, the ability to reduce renin production, the ability to reduce angiotensin production, the ability to reduce aldosterone production, the ability to reduce abnormally elevated cardiac filling pressures, the ability to optimize renal blood flow, or a combination thereof. In some cases, a polypeptide provided herein can increase endogenous atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) levels. In some cases, a polypeptide provided herein can lack the ability to lower blood pressure and can lack the ability to cause systemic hypotension (i.e., can preserve blood pressure). In some cases, a polypeptide provided herein can be an agonist for natriuretic peptide receptor- A (NPR-A), natriuretic peptide receptor-B (NPR-B), or both NPR-A and NPR-B.

In general, one aspect of this document features a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus:

(a) the sequence set forth in SEQ ID NO: 1 or the sequence set forth in SEQ ID NO: 1 with no more than three additions, subtractions, or substitutions,

(b) the sequence set forth in SEQ ID NO: 2 or the sequence set forth in SEQ ID NO:2 with no more than five additions, subtractions, or substitutions, and

(c) the sequence set forth in SEQ ID NO: 3 or the sequence set forth in SEQ ID NO: 3 with no more than three additions, subtractions, or substitutions. The polypeptide can comprise natriuretic activity. The polypeptide can comprise a cGMP- activating property. The polypeptide can preserve glomerular filtration rate and renal perfusion pressure. The polypeptide can have minimal effects on systemic blood pressure. The polypeptide can comprise renin-angiotensin system (RAS) suppressing activity. The polypeptide can comprise the sequence set forth in SEQ ID NO: 1. The polypeptide can comprise the sequence set forth in SEQ ID NO:2. The polypeptide can comprise the sequence set forth in SEQ ID NO:3. The polypeptide can comprise the sequence set forth in SEQ ID NO: 1, the sequence set forth in SEQ ID NO:2, and the sequence set forth in SEQ ID NO:3. The polypeptide can comprise the sequence set forth in SEQ ID NO: 1 with no more than three conservative amino acid substitutions. The polypeptide can comprise the sequence set forth in SEQ ID NO:2 with no more than five conservative amino acid substitutions. The polypeptide can comprise the sequence set forth in SEQ ID NO: 3 with no more than three conservative amino acid substitutions. The polypeptide can be a substantially pure polypeptide. The polypeptide can comprise a plasma angiotensin II-suppressing activity. The polypeptide can comprise plasma BNP and CNP immunoreactivity- augmenting activity.

In another aspect, this document features a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus:

(a) the sequence set forth in SEQ ID NO: 6 or the sequence set forth in SEQ ID NO:6 with no more than three additions, subtractions, or substitutions,

(c) the sequence set forth in SEQ ID NO: 7 or the sequence set forth in SEQ ID NO: 7 with no more than three additions, subtractions, or substitutions. The polypeptide can comprise natriuretic activity. The polypeptide can comprise a cGMP- activating property. The polypeptide can comprise a natriuretic and diuretic activity, and can preserve glomerular filtration rate and renal perfusion pressure. The polypeptide can comprise a cardiac-unloading activity. The polypeptide can comprise an RAS-suppressing activity. The polypeptide can have minimal effects on systemic blood pressure. The polypeptide can comprise the sequence set forth in SEQ ID NO:6. The polypeptide can comprise the sequence set forth in SEQ ID NO:2. The polypeptide can comprise the sequence set forth in SEQ ID NO:7. The polypeptide can comprise the sequence set forth in SEQ ID NO:6, the sequence set forth in SEQ ID NO:2, and the sequence set forth in SEQ ID NO:7. The polypeptide can comprise the sequence set forth in SEQ ID NO: 6 with no more than three conservative amino acid substitutions. The polypeptide can comprise the sequence set forth in SEQ ID NO:2 with no more than five conservative amino acid substitutions. The polypeptide can comprise the sequence set forth in SEQ ID NO: 7 with no more than three conservative amino acid substitutions. The polypeptide can be a substantially pure polypeptide.

In another aspect, this document features an isolated nucleic acid encoding (I) a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus:

(a) the sequence set forth in SEQ ID NO: 1 or the sequence set forth in SEQ ID NO:1 with no more than three additions, subtractions, or substitutions,

(c) the sequence set forth in SEQ ID NO: 3 or the sequence set forth in SEQ ID NO:3 with no more than three additions, subtractions, or substitutions; or (II) a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus:

(c) the sequence set forth in SEQ ID NO: 7 or the sequence set forth in SEQ ID NO: 7 with no more than three additions, subtractions, or substitutions.

In another aspect, this document features a vector comprising a nucleic acid encoding (I) a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus:

(b) the sequence set forth in SEQ ID NO: 2 or the sequence set forth in SEQ ID NO:2 with no more than five additions, subtractions, or substitutions, and (c) the sequence set forth in SEQ ID NO:3 or the sequence set forth in SEQ ID

NO:3 with no more than three additions, subtractions, or substitutions; or (II) a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus:

(c) the sequence set forth in SEQ ID NO: 7 or the sequence set forth in SEQ ID NO: 7 with no more than three additions, subtractions, or substitutions. In another aspect, this document features a host cell comprising a nucleic acid encoding (I) a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus:

(a) the sequence set forth in SEQ ID NO: 1 or the sequence set forth in SEQ ID NO: 1 with no more than three additions, subtractions, or substitutions, (b) the sequence set forth in SEQ ID NO: 2 or the sequence set forth in SEQ ID

NO:2 with no more than five additions, subtractions, or substitutions, and

(a) the sequence set forth in SEQ ID NO: 6 or the sequence set forth in SEQ ID NO:6 with no more than three additions, subtractions, or substitutions, (b) the sequence set forth in SEQ ID NO: 2 or the sequence set forth in SEQ ID

NO:2 with no more than five additions, subtractions, or substitutions, and

(c) the sequence set forth in SEQ ID NO: 7 or the sequence set forth in SEQ ID NO: 7 with no more than three additions, subtractions, or substitutions. The host cell can be a eukaryotic host cell. In another aspect, this document features a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (I) a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus:

In another aspect, this document features a method for increasing natriuretic activity within a mammal using chimeric peptides, which consist entirely of human amino acid sequences from endogenous natriuretic peptides (ANP, BNP, CNP) that exist in the human cardiovascular system, with minimal effects on blood pressure. The method can comprise administering, to the mammal, (I) a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus:

(c) the sequence set forth in SEQ ID NO: 3 or the sequence set forth in SEQ ID NO:3 with no more than three additions, subtractions, or substitutions; or (II) a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:6 or the sequence set forth in SEQ ID

NO:6 with no more than three additions, subtractions, or substitutions,

In another aspect, this document features a method for treating a mammal having a cardiovascular condition or renal condition. The method can comprise administering, to the mammal, a polypeptide under conditions wherein the severity of a manifestation of the cardiovascular condition or renal condition is reduced. The polypeptide can be (I) a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises, in an order from amino terminus to carboxy terminus:

NO:2 with no more than five additions, subtractions, or substitutions, and

(c) the sequence set forth in SEQ ID NO: 7 or the sequence set forth in SEQ ID NO: 7 with no more than three additions, subtractions, or substitutions. Administration of the polypeptide to the mammal can have minimal effects on systemic blood pressure in the mammal.

In another aspect, this document features a polypeptide less than 44 amino acid residues in length, wherein the polypeptide comprises amino acid sequences from endogenous natriuretic peptides native to the human cardiovascular system. The polypeptide can lack immunogenicity when administered to a human. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of a CAA-NP polypeptide that is 28 amino acid residues in length (SEQ ID NO:4). The first six amino acid residues of SEQ ID NO:4 correspond to amino acid residues 1 to 6 of human ANP and are designated as SEQ ID NO: 1. Amino acid residues 7 to 23 of SEQ ID NO:4 correspond to amino acid residues 6 to 22 of human mature CNP and are designated as SEQ ID NO:2. Amino acid residues 24 to 28 of SEQ ID NO:4 correspond to amino acid residues 24 to 28 of human ANP and are designated as SEQ ID NO:3.

Figure 2 is a schematic diagram of a CBB-NP polypeptide that is 32 amino acid residues in length (SEQ ID NO:5). The first nine amino acid residues of SEQ ID NO: 5 correspond to amino acid residues 1 to 9 of human BNP and are designated as SEQ ID NO:6. Amino acid residues 10 to 26 of SEQ ID NO:5 correspond to amino acid residues 6 to 22 of human mature CNP and are designated as SEQ ID NO:2. Amino acid residues 27 to 32 of SEQ ID NO:4 correspond to amino acid residues 27 to 32 of human BNP and are designated as SEQ ID NO:7.

Figure 3 is a diagram of experimental protocols for CAA-NP infusion for 45 minutes (upper panel) and for CBB-NP infusion 75 minutes (lower panel).

Figure 4 is a graph plotting plasma cyclic GMP in response to CAA-NP infusion. Figure 5 is a graph plotting a time course of cyclic GMP activation during and after CAA-NP infusion.

Figure 6 is a graph plotting urinary cyclic GMP excretion in response to CAA- NP infusion.

Figure 7 is a graph plotting net renal generation of cGMP in response to CAA- NP infusion.

Figures 8A and 8B are graphs plotting urinary sodium excretion for animals treated with CAA-NP. As one of the dogs had an exceedingly high baseline urinary sodium level (approximately 4-fold greater than the baselines values of the other dogs), an additional analysis was performed using data from the remaining four dogs (Figure 8B). A significant natriuretic response was observed (from 36 ± 8 at pre- infusion to 219 ± 55 μEq/mL at 30 minutes of infusion, P<0.05).

Figure 9 is a graph plotting urine flow for animals treated with CAA-NP.

Figure 10 is a graph plotting glomerular filtration rate for animals treated with CAA-NP. Figure 11 is a graph plotting pulmonary capillary wedge pressure for animals treated with CAA-NP.

Figure 12 is a graph plotting right atrial pressure for animals treated with CAA-NP.

Figures 13A and 13B are graphs plotting mean arterial pressure for animals treated with CAA-NP. Exploratory testing of CAA-NP at a higher dosage level (28.28 pmol/kg/minute i.v.) was performed in two additional dogs (Figure 13B).

Figure 14 is a graph plotting pulmonary arterial pressure for animals treated with CAA-NP. Figure 15 is a graph plotting renal blood flow for animals treated with CAA- NP.

Figure 16 is a graph plotting renal perfusion pressure for animals treated with CAA-NP. Figure 17 is a graph plotting renal vascular resistance for animals treated with

CAA-NP.

Figure 18 is a graph plotting proximal fractional reabsorption of sodium for animals treated with CAA-NP.

Figure 19 is a graph plotting distal fractional reabsorption of sodium for animals treated with CAA-NP.

Figure 20 is a graph plotting plasma renin activity for animals treated with CAA-NP.

Figure 21 is a graph plotting plasma angiotensin II levels for animals treated with CAA-NP. Figure 22 is a graph plotting plasma aldosterone levels for animals treated with CAA-NP.

Figure 23 is a graph plotting plasma ANP immunoreactivity for animals treated with CAA-NP.

Figure 24 is a graph plotting plasma BNP immunoreactivity for animals treated with CAA-NP.

Figure 25 is a graph plotting plasma CNP immunoreactivity for animals treated with CAA-NP.

Figure 26 is a graph plotting hematocrit for animals treated with CAA-NP.

Figure 27 is a graph plotting cardiac output in response to CAA-NP infusion. Figure 28 is a graph plotting cyclic GMP response to ANP, BNP, CNP, or

CAA-NP in human aortic endothelial cells.

Figure 29 is a graph plotting cyclic GMP response to ANP, BNP, CNP, or CAA-NP in the absence or presence of an NRP-A antagonist or an antibody against the ligand binding domain of NPR-B. Figure 30 is a graph plotting plasma GMP levels for animals treated with

CBB-NP.

Figure 31 is a graph plotting a time course of cyclic GMP activation during and after CBB-NP infusion. Figure 32 is a graph plotting urinary cyclic GMP excretion for animals treated with CBB-NP.

Figure 33 is a graph plotting net renal cyclic GMP generation for animals treated with CBB-NP. Figure 34 is a graph plotting urine flow for animals treated with CBB-NP.

Figure 35 is a graph plotting urinary sodium excretion for animals treated with CBB-NP.

Figure 36 is a graph plotting glomerular filtration rate for animals treated with CBB-NP. Figure 37 is a graph plotting pulmonary capillary wedge pressure for animals treated with CBB-NP.

Figure 38 is a graph plotting right atrial pressure for animals treated with CBB-NP.

Figure 39 is a graph plotting mean arterial pressure for animals treated with CBB-NP.

Figure 40 is a graph plotting renal perfusion pressure for animals treated with CBB-NP. Results are not statistically significant.

Figure 41 is a graph plotting renal blood flow for animals treated with CBB- NP. Figure 42 is a graph plotting proximal fractional reabsorption of sodium for animals treated with CBB-NP.

Figure 43 is a graph plotting distal fractional reabsorption of sodium for animals treated with CBB-NP.

Figure 44 is a graph plotting renal vascular resistance for animals treated with CBB-NP.

Figure 45 is a graph plotting cardiac output for animals treated with CBB-NP. Results are not statistically significant.

Figure 46 is a graph plotting pulmonary arterial pressure for animals treated with CBB-NP. Figure 47 is a graph plotting hematocrit levels for animals treated with CBB-

NP.

Figure 48 is a graph plotting systemic vascular resistance for animals treated with CBB-NP. Results are not statistically significant. Figure 49 is a graph plotting plasma renin activity for animals treated with CBB-NP.

Figure 50 is a graph plotting plasma angiotensin II for animals treated with CBB-NP. Figure 51 is a graph plotting plasma aldosterone for animals treated with

CBB-NP. Results are not statistically significant.

Figure 52 is a graph plotting plasma ANP immunoreactivity for animals treated with CBB-NP.

Figure 53 is a graph plotting plasma BNP immunoreactivity for animals treated with CBB-NP. Results are not statistically significant.

Figure 54 is a graph plotting plasma CNP immunoreactivity for animals treated with CBB-NP.

Figure 55 is a graph plotting urinary ANP excretion for animals treated with CBB-NP. Figure 56 is a graph plotting urinary BNP excretion for animals treated with

CBB-NP.

Figure 57 is a graph plotting urinary CNP excretion for animals treated with CBB-NP.

Figure 58 is a graph plotting cyclic GMP response to ANP, BNP, CNP, and CBB-NP in human aortic endothelial cells.

Figure 59 is a graph plotting cyclic GMP response to CBB-NP in the absence or presence of an NPR-A antagonist or an antibody to the ligand-binding domain of NPR-B.

DETAILED DESCRIPTION

This document relates to natriuretic polypeptides. For example, this document provides methods and materials related to natriuretic polypeptides and the use of natriuretic polypeptides to treat cardiovascular conditions (e.g., acute decompensated heart failure, acute coronary syndromes, and ventricular remodeling post-myocardial infarction) and renal conditions (e.g., perioperative renal dysfunction, renal dysfunction secondary to heart failure, and diabetic nephropathy). In some cases, a polypeptide provided herein can have diuretic activity, natriuretic activity, the ability to activate cGMP, the ability to increase glomerular filtration rate, the ability to reduce renin production, the ability to reduce angiotensin production, the ability to reduce aldosterone production, the ability to reduce cardiac filling pressures, the ability to optimize renal blood flow, or a combination thereof. In some cases, a polypeptide provided herein can increase endogenous ANP, BNP, and CNP levels. In some cases, a polypeptide provided herein can lack the ability to lower blood pressure or cause systemic hypotension. In some cases, a polypeptide provided herein can be an agonist for NPR-A, NPR-B, or both NPR-A and NPR-B. A polypeptide provided herein can have any sequence and can have any length. For example, a polypeptide provided herein can include the sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some cases, a polypeptide provided herein can contain an amino acid sequence that aligns to (a) the sequence set forth in SEQ ID NO:1 with three or less (e.g., two or less, one, or zero) amino acid additions, deletions, substitutions, or combinations thereof, (b) the sequence set forth in SEQ ID NO:2 with five or less (e.g., four or less, three or less, two or less, one, or zero) amino acid additions, deletions, substitutions, or combinations thereof, and (c) the sequence set forth in SEQ ID NO: 3 with three or less (e.g., two or less, one, or zero) amino acid additions, deletions, substitutions, or combinations thereof. For example, a polypeptide provided herein can contain the sequence set forth in SEQ ID NO: 1 with the exception that the first threonine residue or the last serine residue of SEQ ID NO: 1 is deleted or replaced with a different amino acid residue.

In some cases, a polypeptide provided herein can include the sequences set forth in SEQ ID NO:6, SEQ ID NO:2, and SEQ ID NO:7. In some cases, a polypeptide provided herein can contain an amino acid sequence that aligns to (a) the sequence set forth in SEQ ID NO:6 with three or less (e.g., two or less, one, or zero) amino acid additions, deletions, substitutions, or combinations thereof, (b) the sequence set forth in SEQ ID NO:2 with five or less (e.g., four or less, three or less, two or less, one, or zero) amino acid additions, deletions, substitutions, or combinations thereof and (c) the sequence set forth in SEQ ID NO: 7 with three or less (e.g., two or less, one, or zero) amino acid additions, deletions, substitutions, or combinations thereof. For example, a polypeptide provided herein can contain the sequence set forth in SEQ ID NO: 6 with the exception that the first serine residue or the last serine residue of SEQ ID NO: 6 is deleted or replaced with a different amino acid residue.

Amino acid substitutions can be conservative amino acid substitutions. Conservative amino acid substitutions can be, for example, aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids. Conservative amino acid substitutions also include groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. After making an amino acid substitution, the activities of the polypeptide containing the amino acid substitution can be assessed using the assays described herein.

In some cases, a polypeptide provided herein can contain (a) a first amino acid sequence that either is set forth in SEQ ID NO: 1 or aligns to the sequence set forth in SEQ ID NO: 1 with three or less (e.g., two or less, one, or zero) amino acid deletions, substitutions, or combinations thereof, (b) a second amino acid sequence that either is set forth in SEQ ID NO:2 or aligns to the sequence set forth in SEQ ID NO:2 with five or less (e.g., four or less, three or less, two or less, one, or zero) amino acid additions, substitutions, or combinations thereof, and (a) a third amino acid sequence that either is set forth in SEQ ID NO: 3 or aligns to the sequence set forth in SEQ ID NO:3 with three or less (e.g., two or less, one, or zero) amino acid deletions, substitutions, or combinations thereof. For example, a polypeptide provided herein can comprise or consist of the sequence set forth in SEQ ID NO:4. In some cases, a polypeptide provided herein can contain (a) a first amino acid sequence that either is set forth in SEQ ID NO: 6 or aligns to the sequence set forth in SEQ ID NO:6 with three or less (e.g., two or less, one, or zero) amino acid deletions, substitutions, or combinations thereof, (b) a second amino acid sequence that either is set forth in SEQ ID NO:2 or aligns to the sequence set forth in SEQ ID NO:2 with five or less (e.g., four or less, three or less, two or less, one, or zero) amino acid additions, substitutions, or combinations thereof, and (a) a third amino acid sequence that either is set forth in SEQ ID NO: 7 or aligns to the sequence set forth in SEQ ID NO:7 with three or less (e.g., two or less, one, or zero) amino acid deletions, substitutions, or combinations thereof. For example, a polypeptide provided herein can comprise or consist of the sequence set forth in SEQ ID NO:5.

A polypeptide provided herein can have any length. For example, a polypeptide provided herein can be between 23 and 45 (e.g., between 25 and 45, between 26 and 44, between 27 and 43, between 28 and 42, between 29 and 41, between 30 and 40, between 31 and 39, between 23 and 35, between 25 and 30, or between 30 and 35) amino acid residues in length. It will be appreciated that a polypeptide with a length of 25 or 45 amino acid residues is a polypeptide with a length between 25 and 45 amino acid residues. In some cases, a polypeptide provided herein can be a substantially pure polypeptide. As used herein, the term "substantially pure" with reference to a polypeptide means that the polypeptide is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. Thus, a substantially pure polypeptide is any polypeptide that is removed from its natural environment and is at least 60 percent pure or is any chemically synthesized polypeptide. A substantially pure polypeptide can be at least about 60, 65, 70, 75, 80, 85, 90, 95, or 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.

A polypeptide provide herein can be obtained by expression of a recombinant nucleic acid encoding the polypeptide or by chemical synthesis (e.g., using solid phase polypeptide synthesis methods or an peptide synthesizer such as an ABI 43 IA Peptide Synthesizer; Applied Biosystems; Foster City, CA). For example, standard recombinant technology using expression vectors encoding a polypeptide provided herein can be used. The resulting polypeptides then can be purified using, for example, affinity chromatographic techniques and HPLC. The extent of purification can be measured by any appropriate method, including but not limited to: column chromatography, polyacrylamide gel electrophoresis, or high-performance liquid chromatography. A polypeptide provide herein can be designed or engineered to contain a tag sequence that allows the polypeptide to be purified (e.g., captured onto an affinity matrix). For example, a tag such as c-myc, hemagglutinin, polyhistidine, or FLAG™ tag (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino termini. Other fusions that can be used include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase.

A polypeptide provided herein can be produced to contain three regions, a first region that includes an N-terminus (e.g., an N-terminus sequence from a human ANP or BNP polypeptide), a second region that includes a ring structure of a mature natriuretic polypeptide such as a human CNP polypeptide, and third region that includes a C-terminus (e.g., a C-terminus sequence from a human ANP or BNP polypeptide). For example, a polypeptide provided herein can be produced to contain a first region that includes an N-terminus sequence from a human ANP polypeptide, a second region that includes a ring structure of a mature natriuretic polypeptide such as a human CNP polypeptide, and third region that includes a C-terminus sequence from a human ANP polypeptide. In some cases, a polypeptide provided herein can be produced to contain a first region that includes an N-terminus sequence from a human BNP polypeptide, a second region that includes a ring structure of a mature natriuretic polypeptide such as a human CNP polypeptide, and third region that includes a C- terminus sequence from a human BNP polypeptide.

A polypeptide provided herein can be used to treat cardiovascular diseases, congestive heart failure, myocardial infarction, coronary artery diseases, renal diseases, hepatic diseases, cancer, metabolic diseases, or combinations thereof. For example, a CAA-NP polypeptide having the amino acid sequence set forth in SEQ ID NO:4 or a CBB-NP polypeptide having the amino acid sequence set forth in SEQ ID NO: 5 can be administered to a human having coronary artery disease under conditions wherein the severity of the human's coronary artery disease symptoms is reduced.

A polypeptide provided herein can be formulated as a pharmaceutical composition by admixture with pharmaceutically acceptable non-toxic excipients or carriers. Such compositions can be administered to a subject in need thereof in an amount effective to treat, for example, heart, liver, kidney, or other sodium retaining conditions. Pharmaceutical compositions can be prepared for topical administration; for parenteral administration, particularly in the form of liquid solutions or suspensions in aqueous physiological buffer solutions; for oral administration, particularly in the form of tablets or capsules; or for intranasal administration, particularly in the form of powders, nasal drops, or aerosols. Compositions for other routes of administration can be prepared as desired using appropriate methods. Formulations for topical administration include, for example, sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents and other suitable additives. Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be useful. In some embodiments, transdermal delivery of natriuretic peptides as provided herein can be particularly useful. Methods and compositions for transdermal delivery include those described in the art (e.g., in Wermeling et ah, Proc. Natl. Acad. Sci. USA 105:2058-2063 (2008); Goebel and Neubert, Skin Pharmacol. Physiol. 21 :3-9 (2008); Banga, Pharm. Res. 24:1357-1359 (2007); Malik et al, Curr. Drug Deliv. 4:141-151 (2007); and Prausnitz, Nat. Biotechnol. 24:416-417 (2006)).

Formulations for parenteral administration can include as common excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and combinations thereof. In some cases, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, polyoxethylene-polyoxypropylene copolymers, or combinations thereof can be used as excipients for controlling the release of the polypeptide in vivo. Other suitable parenteral delivery systems that can be used include, without limitation, ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, liposomes, and combinations thereof. Formulations for inhalation administration can include excipients such as lactose. Inhalation formulations can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, deoxycholate, or combinations thereof, or they can be oily solutions for administration in the form of nasal drops. If desired, a composition containing a polypeptide provided herein can be formulated as gel to be applied intranasally. Formulations for parenteral administration can include glycocholate for buccal administration. For oral administration, tablets or capsules can be prepared using appropriate methods with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated using appropriate methods. Preparations for oral administration can be formulated to give controlled release of the polypeptide. Nasal preparations can be presented in a liquid form or as a dry product. Nebulised aqueous suspensions or solutions can include carriers or excipients to adjust pH and/or tonicity.

In some embodiments, a natriuretic peptide can be modified by linkage to a polymer such as polyethylene glycol (PEG), or by fusion to another polypeptide such as albumin, for example. For example, one or more PEG moieties can be conjugated to a NP via lysine residues. Linkage to PEG or another suitable polymer, or fusion to albumin or another suitable polypeptide can result in a modified NP having an increased half life as compared to an unmodified NP. Without being bound by a particular mechanism, an increased serum half life can result from reduced proteolytic degradation, immune recognition, or cell scavanging of the modified NP. Methods for modifying a polypeptide by linkage to PEG (also referred to as "PEGylation") or other polymers are known in the art, and include those set forth in U.S. Patent No. 6,884,780; Cataliotti et al., Trends Cardiovasc. Med. 17:10-14 (2007); Veronese and Mero, BioDrugs 22:315-329 (2008); Miller et al., Bioconjugate Chem. 17:267-274 (2006); and Veronese and Pasut, Drug Discov. Today 10:1451-1458 (2005), all of which are incorporated herein by reference in their entirety. Methods for modifying a polypeptide by fusion to albumin also are known in the art, and include those set forth in U.S. Patent Publication No. 20040086976, and Wang et al., Pharm. Res. 21 :2105- 2111 (2004), both of which are incorporated herein by reference in their entirety.

Nucleic Acids Encoding Polypeptides

This document also provides isolated nucleic acids that encode one or more of the polypeptides provided herein. The term "isolated" as used herein with reference to nucleic acid refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

The term "isolated" as used herein with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid (e.g., a nucleic acid encoding a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5) can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid. As used herein, the term "nucleic acid" refers to both RNA and DNA, including mRNA, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, and nucleic acid analogs. The nucleic acid can be double-stranded or single- stranded, and where single-stranded, can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of a nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'- deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller Antisense Nucleic Acid Drug Dev., 7:187-195 (1997); and Hyrup et al. Bioorgan. Med. Chem., 4:5-23 (1996). In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

A nucleic acid provided herein can comprise or consist of a sequence that encodes the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. For example, such a nucleic acid can contain the human nucleic acid sequence for CNP and ANP engineered to encode the amino acid sequence set forth in SEQ ID NO:4. In some cases, such a nucleic acid can contain the human nucleic acid sequence for CNP and BNP engineered to encode the amino acid sequence set forth in SEQ ID NO:5.

Typically, an isolated nucleic acid provided herein is at least 10 nucleotides in length (e.g., 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 200, 300, 350, 400, or more nucleotides in length). Nucleic acid molecules that are less than full-length can be useful, for example, as primers or probes for diagnostic purposes. Isolated nucleic acid molecules can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used. PCR refers to a procedure or technique in which target nucleic acids are enzymatically amplified. Sequence information from the ends of the region of interest or beyond typically is employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 15 to 50 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. For example, a primer can be 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or 45 nucleotides in length. A primer can be purified from a restriction digest by conventional methods, or can be chemically synthesized. Primers typically are single- stranded for maximum efficiency in amplification, but a primer can be double- stranded. Double-stranded primers are first denatured (e.g., treated with heat) to separate the strands before use in amplification. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self- sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids as described elsewhere (Lewis, Genetic Engineering News, 12(9): 1 (1992); Guatelli et al, Proc. Natl. Acad. ScL USA, 87:1874-1878 (1990); and Weiss, Science, 254:1292 (1991)).

Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.

Isolated nucleic acids also can be obtained by mutagenesis. For example, a nucleic acid sequence encoding a polypeptide having the sequence set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, or 7 can be mutated using standard techniques such as, for example, oligonucleotide-directed mutagenesis and/or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology, Chapter 8, Green

Publishing Associates and John Wiley & Sons, Edited by Ausubel et al., 1992. Such mutations include additions, deletions, substitutions, and combinations thereof. Vectors and Host Cells

This document also provides vectors containing a nucleic acid provided herein. As used herein, a "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted so as to bring about the replication of the inserted segment. A vector can be an expression vector. An "expression vector" is a vector that includes one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.

In an expression vector provided herein, the nucleic acid can be operably linked to one or more expression control sequences. As used herein, "operably linked" means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it can be necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is "operably linked" and "under the control" of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the polypeptide encoded by the coding sequence.

Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, poxviruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech Laboratories (Mountain View, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA). An expression vector can include a tag sequence designed to facilitate subsequent manipulation of the expressed nucleic acid sequence (e.g., purification or localization). Tag sequences, such as green fluorescent protein (GFP), glutathione S- transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.

This document also provides host cells containing a nucleic acid molecule and/or nucleic acid vector provided herein. The term "host cell" refers to prokaryotic cells and eukaryotic cells into which a nucleic acid molecule or vector can be introduced. Any method can be used to introduce nucleic acid into a cell. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer can be used introduce nucleic acid into cells. In addition, naked DNA can be delivered directly to cells in vivo as described elsewhere (U.S. Patent Nos. 5,580,859 and 5,589,466).

Detecting Polypeptides

This document provides methods and materials for detecting a polypeptide provided herein. Such methods and materials can be used to monitor polypeptide levels within a mammal receiving the polypeptide as a therapeutic. A polypeptide provided herein (e.g., a CAA-NP polypeptide having the amino acid sequence set forth in SEQ ID NO:4 or a CBB-NP polypeptide having the amino acid sequence set forth in SEQ ID NO:5) can be detected, for example, immunologically using one or more antibodies. As used herein, the term "antibody" includes intact molecules as well as fragments thereof that are capable of binding to an epitopic determinant of a polypeptide provided herein. The term "epitope" refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes generally have at least five contiguous amino acids (a continuous epitope), or alternatively can be a set of noncontiguous amino acids that define a particular structure (e.g., a conformational epitope). The term "antibody" includes polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab)₂ fragments. Polyclonal antibodies are heterogeneous populations of antibody molecules that are contained in the sera of the immunized animals. Monoclonal antibodies are homogeneous populations of antibodies to a particular epitope of an antigen.

Antibody fragments that have specific binding affinity for a polypeptide provided herein (e.g., a CAA-NP polypeptide having the amino acid sequence set forth in SEQ ID NO:4 or a CBB-NP polypeptide having the amino acid sequence set forth in SEQ ID NO:5) can be generated by known techniques. For example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule; Fab fragments can be generated by reducing the disulfide bridges of F(ab')2 fragments. In some cases, Fab expression libraries can be constructed. See, for example, Huse et al., Science, 246:1275 (1989). Once produced, antibodies or fragments thereof can be tested for recognition of a polypeptide provided herein by standard immunoassay methods including ELISA techniques, radioimmunoassays, and Western blotting.

See, Short Protocols in Molecular Biology, Chapter 11, Green Publishing Associates and John Wiley & Sons, Edited by Ausubel, F.M et al., 1992.

In immunological assays, an antibody having specific binding affinity for a polypeptide provided herein or a secondary antibody that binds to such an antibody can be labeled, either directly or indirectly. Suitable labels include, without limitation, radionuclides (e.g., ¹²⁵I, ¹³¹1, ³⁵S, ³H, ³²P, ³³P, or ¹⁴C), fluorescent moieties (e.g., fluorescein, FITC, PerCP, rhodamine, or PE), luminescent moieties (e.g., QDOT™ nanoparticles supplied by Invitrogen (Carlsbad, CA)), compounds that absorb light of a defined wavelength, or enzymes (e.g., alkaline phosphatase or horseradish peroxidase). Antibodies can be indirectly labeled by conjugation with biotin then detected with avidin or streptavidin labeled with a molecule described above. Methods of detecting or quantifying a label depend on the nature of the label and are known in the art. Examples of detectors include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers. Combinations of these approaches

(including "multi-layer" assays) familiar to those in the art can be used to enhance the sensitivity of assays. Immunological assays for detecting a polypeptide provided herein can be performed in a variety of known formats, including sandwich assays, competition assays (competitive RIA), or bridge immunoassays. See, for example, U.S. Patent Nos. 5,296,347; 4,233,402; 4,098,876; and 4,034,074. Methods of detecting a polypeptide provided herein generally include contacting a biological sample with an antibody that binds to a polypeptide provided herein and detecting binding of the polypeptide to the antibody. For example, an antibody having specific binding affinity for a polypeptide provided herein can be immobilized on a solid substrate by any of a variety of methods known in the art and then exposed to the biological sample. Binding of the polypeptide to the antibody on the solid substrate can be detected by exploiting the phenomenon of surface plasmon resonance, which results in a change in the intensity of surface plasmon resonance upon binding that can be detected qualitatively or quantitatively by an appropriate instrument, e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden). In some cases, the antibody can be labeled and detected as described above. A standard curve using known quantities of a polypeptide provided herein can be generated to aid in the quantitation of the levels of the polypeptide.

In some embodiments, a "sandwich" assay in which a capture antibody is immobilized on a solid substrate can be used to detect the presence, absence, or level of a polypeptide provided herein. The solid substrate can be contacted with the biological sample such that any polypeptide of interest in the sample can bind to the immobilized antibody. The presence, absence, or level of the polypeptide bound to the antibody can be determined using a "detection" antibody having specific binding affinity for the polypeptide. In some embodiments, a capture antibody can be used that has binding affinity for ANP, BNP, or CNP as well as a polypeptide provided herein. In this embodiment, a detection antibody can be used that has specific binding affinity for a particular polypeptide provided herein (e.g., a CAA-NP polypeptide having the amino acid sequence set forth in SEQ ID NO:4 or a CBB-NP polypeptide having the amino acid sequence set forth in SEQ ID NO:5). It is understood that in sandwich assays, the capture antibody should not bind to the same epitope (or range of epitopes in the case of a polyclonal antibody) as the detection antibody. Thus, if a monoclonal antibody is used as a capture antibody, the detection antibody can be another monoclonal antibody that binds to an epitope that is either physically separated from or only partially overlaps with the epitope to which the capture monoclonal antibody binds, or a polyclonal antibody that binds to epitopes other than or in addition to that to which the capture monoclonal antibody binds. If a polyclonal antibody is used as a capture antibody, the detection antibody can be either a monoclonal antibody that binds to an epitope that is either physically separated from or partially overlaps with any of the epitopes to which the capture polyclonal antibody binds, or a polyclonal antibody that binds to epitopes other than or in addition to that to which the capture polyclonal antibody binds. Sandwich assays can be performed as sandwich ELISA assays, sandwich Western blotting assays, or sandwich immunomagnetic detection assays.

Suitable solid substrates to which an antibody (e.g., a capture antibody) can be bound include, without limitation, microtiter plates, tubes, membranes such as nylon or nitrocellulose membranes, and beads or particles (e.g., agarose, cellulose, glass, polystyrene, polyacrylamide, magnetic, or magnetizable beads or particles). Magnetic or magnetizable particles can be particularly useful when an automated immunoassay system is used.

Antibodies having specific binding affinity for a polypeptide provided herein can be produced through standard methods. For example, a polypeptide can be recombinantly produced as described above, can be purified from a biological sample (e.g., a heterologous expression system), or can be chemically synthesized, and used to immunize host animals, including rabbits, chickens, mice, guinea pigs, or rats. For example, a polypeptide having the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5, or fragments thereof that are at least six amino acids in length, can be used to immunize an animal. Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Monoclonal antibodies can be prepared using a polypeptide provided herein and standard hybridoma technology. In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described by Kohler et al., Nature, 256:495 (1975), the human B-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72 (1983); Cote et al., Proc. Natl. Acad. Sci. USA, 80:2026 (1983)), and the EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc., pp. 77-96 (1983)). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridoma producing the monoclonal antibodies can be cultivated in vitro and in vivo.

Other techniques for detecting a polypeptide provided herein include mass- spectrophotometric techniques such as electrospray ionization (ESI), and matrix- assisted laser desorption-ionization (MALDI). See, for example, Gevaert et al., Electrophoresis, 22(9): 1645-51 (2001); Chaurand et al, J. Am. Soc. Mass Spectrom., 10(2):91-103 (1999). Mass spectrometers useful for such applications are available from Applied Biosystems (Foster City, CA); Bruker Daltronics (Billerica, MA); and Amersham Pharmacia (Sunnyvale, CA).

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1 - Generating Chimeric Natriuretic Polypeptides A polypeptide with the sequence set forth in Figure 1 was designed and synthesized using an ABI 43 IA Peptide Synthesizer. This polypeptide is referred to as a CAA-NP polypeptide (Figure 1). The synthesized CAA-NP polypeptide was confirmed by high-performance liquid chromatography/mass spectrometry. Its molecular weight was 31 11.62, and its amino acid sequence is Ser-Leu-Arg-Arg-Ser- Ser-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys-Asn- Ser-Phe-Arg-Tyr (SEQ ID NO:4), with a disulfide bridge joining the Cys residues. A polypeptide with the sequence set forth in Figure 2 was designed and synthesized using an ABI 43 IA Peptide Synthesizer. This polypeptide is referred to as a CBB-NP polypeptide (Figure 2). The synthesized CBB-NP polypeptide was confirmed by high-performance liquid chromatography/mass spectrometry. Its molecular weight was 3419.13, and its amino acid sequence is Ser-Pro-Lys-Met-Val- Gln-Gly-Ser-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu- Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO:5), with a disulfide bridge joining the Cys residues. Example 2 - In Vivo Assays

CAA-NP and CBB-NP were infused intravenously into normal anesthetized dogs as continuous intravenous infusions (Figure 3). Cardiorenal and neurohumoral responses to continuous intravenous infusion of CAA-NP and CBB-NP were assessed. CAA-NP (14.14 pmol/kg/minute i.v.) as a 45-minute continuous infusion was studied in five normal anesthetized dogs. Clearances were performed at pre- infusion (pre-I) and during the last 30 minutes of infusion. Blood and urine samples were collected during the clearances. Moreover, in two of these dogs, the time course of cGMP response to CAA-NP infusion and post-infusion was quantified by sampling blood at baseline, at 10, 15, 30, 45 minutes of CAA-NP infusion and at 1, 2, 4, 6, 10, 20, 30, 45, 60 minutes following cessation of CAA-NP infusion (i.e., at 46, 47, 49, 51, 55, 65, 75, 90, 105 minutes from the start of the infusion). In two additional dogs, exploratory testing of a higher dose of CAA-NP (28.28 pmol/kg/minute) on systemic blood pressure was performed as a 45-minute continuous infusion. CBB-NP (14.14 pmol/kg/minute) was studied as a continuous infusion for 75 minutes in seven normal anesthetized dogs. Clearances were performed at pre-I and at 30 and 60 minutes of infusion. In two preliminary experiments, CBB-NP (14.14 pmol/kg/minute) was tested as a continuous infusion for 45 minutes. The time course of cGMP response to CBB-NP infusion and post-infusion was quantified by sampling blood at baseline, at 10, 15, 30, 45 minutes of CBB-NP infusion and at 1, 2, 4, 6, 10, 20, 30, 45, 60 minutes following cessation of CBB-NP infusion (i.e., at 46, 47, 49, 51, 55, 65, 75, 90, 105 minutes from the start of the infusion).

Insulin clearance was used for assessment of glomerular filtration rate (GFR) in response to CAA-NP or CBB-NP infusion. A lithium clearance technique was used for measurement of proximal and distal fractional reabsorption of Na⁺ (PFR_Na and DFR_Na, respectively). Cyclic GMP, neurohormones, and natriuretic peptide levels were quantified by radioimmunoassays. Renal perfusion pressure was estimated by the difference between mean arterial pressure and right atrial pressure. See Weinfeld et al, Am. Heart J. 138:285-90 (1999). For statistical comparisons, 2- tailed paired t-test was used for comparing data on CAA-NP at 30 minutes of infusion versus pre -infusion data. Repeated measures one-way ANOVA followed by Dunnett's multiple comparison test was used for comparing data on CBB-NP at 30 minutes and 60 minutes of infusion versus pre -infusion data. Statistical significance was defined as P<0.05. Results of in vivo experiments on CAA-NP and CBB-NP are presented in Figures 4-27 and 30-57, respectively.

Both CAA-NP and CBB-NP activated cyclic GMP (the second messenger for natriuretic peptides) in plasma, increased urinary cyclic GMP excretion, and increased net renal generation of cGMP. Both CAA-NP and CBB-NP were natriuretic and preserved glomerular filtration rate and renal perfusion pressure. Further, CBB-NP increased urine flow reduced cardiac filling pressures. Both CAA-NP and CBB-NP suppressed the renin-angiotensin system, with minimal effects on systemic blood pressure. Thus, these results demonstrate that a polypeptide containing CNP and ANP or BNP amino acid sequences can activate cyclic GMP, enhance natriuresis and/or diuresis, and preserve the glomerular filtration rate, without inducing excessive hypotension. Moreover, CBB-NP also significantly decreased cardiac filling pressures and inhibited the renin-angiotensin system. The tubular effects of the synthesized CAA-NP and CBB-NP polypeptides are consistent with actions at the of the proximal tubule and the inner medullary collecting duct cells, given the significant reductions in proximal and distal fractional reabsorption of sodium, respectively, by both peptides.

Example 3 - In Vitro Findings in Human Aortic Endothelial Cells Human aortic endothelial cells were incubated with ANP, BNP, CNP, CAA-

NP, or CBB-NP for 10 minutes (10^~6 M) in the absence or presence of a NPR-A antagonist, A71915 (10^~6 M) or a specific antibody against the ligand-binding domain ofNPR-B (1 : 100). See Delporte et al, Eur. J. Pharmacol. 207:81-88 (1991). The NPR-B antibody to the ligand-binding domain was produced in rabbits and characterized by both ELISA and Western blotting methods. Cyclic GMP response was quantified by radioimmunoassay. For data analyses, one-way ANOVA followed by Bonferroni's multiple comparison tests were used. Statistical significance was defined as P<0.05.

Results of in vitro experiments on CAA-NP and CBB-NP are presented in Figures 28-29 and 58-59, respectively. In human aortic endothelial cells, the cyclic GMP responses (pmol/mL) elicited by ANP, BNP, CNP and CAA-NP were 0.027 ± 0.002, 0.054 ± 0.003, 0.174 ± 0.036* and 0.251 ± 0.029^ϊ§*, respectively (^JP<0.001 vs. control 0.001 ± 0.001; ^§P<0.001 vs. ANP and BNP; *P<0.05 vs. CNP) (Figure 28). With the NPR-A antagonist, the cyclic GMP responses were 0.025 ± 0.001, 0.036 ± 0.004, 0.137 ± 0.024 and 0.172 ± 0.027*, respectively (*P<0.05 vs. no antagonist) (Figure 29). With the NPR-B antibody, the responses were 0.021 ± 0.002, 0.044 ± 0.004, 0.082 ± 0.007^r and 0.155 ± 0.016^r, respectively (^rP<0.01 vs. no antibody) (Figure 29).

In human aortic endothelial cells, CBB-NP and CNP increased cyclic GMP (pmol/mL) vs. control (0.108 ± 0.020 and 0.174 ± 0.036 vs. 0.001 ± 0.001, P<0.01 and <0.001, respectively). CBB-NP elicited a greater cyclic GMP response vs. ANP (0.027 ± 0.002, P<0.05), and comparable responses vs. BNP and CNP (Figure 58). In the presence of A71915 or the NPR-B antibody, cGMP response was attenuated to 0.09 ± 0.01 and 0.06 ± 0.002* pmol/mL, respectively (*P<0.05 vs. no antibody) (Figure 59).

In summary, in human aortic endothelial cells, CAA-NP stimulates cyclic GMP to a significantly greater extent than the native human natriuretic peptides (ANP, BNP, and CNP), and its cyclic GMP-stimulating action may involve both

NPR-A and NPR-B. CBB-NP stimulates cGMP in human aortic endothelial cells to a comparable extent vs. the native BNP and CNP, but to a significantly greater extent vs. ANP. These data suggest that the cyclic GMP stimulating actions of CBB-NP involves NPR-B, and possibly NPR-A. The surprising finding of significantly greater cyclic GMP activation by CAA-NP as compared to native natriuretic peptides ANP, BNP, and CNP raise the possibility that CAA-NP may have particular potential as a unique therapeutic in heart failure, in that it not only exerts favorable cardiorenal and neurohumoral actions (including cGMP activation, natriuresis, preservation of glomerular filtration rate and renal perfusion pressure, suppression of plasma angiotensin II, augmentation of endogenous BNP and CNP levels, and without inducing systemic hypotension), but also may potentially ameliorate endothelial dysfunction in heart failure. It is noteworthy that CAA-NP and CBB-NP are composed of amino acid sequences from endogenous natriuretic peptides that are native to the human cardiovascular system, which would minimize the risk of immunogenicity that may be observed with protein therapeutics. See, e.g., Haller et al. (2008) CHn. Pharmacol. Ther. 84:624-627. OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A polypeptide less than 44 amino acid residues in length, wherein said polypeptide comprises, in an order from amino terminus to carboxy terminus:

(c) the sequence set forth in SEQ ID NO: 3 or the sequence set forth in SEQ ID NO: 3 with no more than three additions, subtractions, or substitutions.

2. The polypeptide of claim 1, wherein said polypeptide comprises natriuretic activity.

3. The polypeptide of claim 1, wherein said polypeptide comprises a cGMP- activating property.

4. The polypeptide of claim 1 , wherein said polypeptide preserves glomerular filtration rate and renal perfusion pressure.

5. The polypeptide of claim 1, wherein said polypeptide has minimal effects on systemic blood pressure.

6. The polypeptide of claim 1, wherein said polypeptide comprises the sequence set forth in SEQ ID NO: 1.

7. The polypeptide of claim 1, wherein said polypeptide comprises the sequence set forth in SEQ ID NO:2.

8. The polypeptide of claim 1, wherein said polypeptide comprises the sequence set forth in SEQ ID NO:3.

9. The polypeptide of claim 1, wherein said polypeptide comprises a plasma angiotensin II-suppressing activity.

10. The polypeptide of claim 1, wherein said polypeptide comprises a plasma BNP and CNP immunoreactivity-augmenting activity.

11. The polypeptide of claim 1 , wherein said polypeptide comprises the sequence set forth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:2, and the sequence set forth in SEQ ID NO:3.

12. The polypeptide of claim 1, wherein said polypeptide comprises the sequence set forth in SEQ ID NO: 1 with no more than three conservative amino acid substitutions.

13. The polypeptide of claim 1, wherein said polypeptide comprises the sequence set forth in SEQ ID NO:2 with no more than five conservative amino acid substitutions.

14. The polypeptide of claim 1, wherein said polypeptide comprises the sequence set forth in SEQ ID NO: 3 with no more than three conservative amino acid substitutions.

15. The polypeptide of claim 1 , wherein said polypeptide is a substantially pure polypeptide.

16. A polypeptide less than 44 amino acid residues in length, wherein said polypeptide comprises, in an order from amino terminus to carboxy terminus:

17. The polypeptide of claim 16, wherein said polypeptide comprises natriuretic activity.

18. The polypeptide of claim 16, wherein said polypeptide comprises a cGMP- activating property.

19. The polypeptide of claim 16, wherein said polypeptide comprises a natriuretic and diuretic activity, and preserves glomerular filtration rate and renal perfusion pressure.

20. The polypeptide of claim 16, wherein said polypeptide comprises a cardiac- unloading activity.

21. The polypeptide of claim 16, wherein said polypeptide comprises a renin- angiotensin system suppressing activity.

22. The polypeptide of claim 16, wherein said polypeptide has minimal effects on systemic blood pressure.

23. The polypeptide of claim 16, wherein said polypeptide comprises the sequence set forth in SEQ ID NO:6.

24. The polypeptide of claim 16, wherein said polypeptide comprises the sequence set forth in SEQ ID NO:2.

25. The polypeptide of claim 16, wherein said polypeptide comprises the sequence set forth in SEQ ID NO:7.

26. The polypeptide of claim 16, wherein said polypeptide comprises the sequence set forth in SEQ ID NO:6, the sequence set forth in SEQ ID NO:2, and the sequence set forth in SEQ ID NO:7.

27. The polypeptide of claim 16, wherein said polypeptide comprises the sequence set forth in SEQ ID NO: 6 with no more than three conservative amino acid substitutions.

28. The polypeptide of claim 16, wherein said polypeptide comprises the sequence set forth in SEQ ID NO:2 with no more than five conservative amino acid substitutions.

29. The polypeptide of claim 16, wherein said polypeptide comprises the sequence set forth in SEQ ID NO: 7 with no more than three conservative amino acid substitutions.

30. The polypeptide of claim 16, wherein said polypeptide is a substantially pure polypeptide.

31. A polypeptide less than 44 amino acid residues in length, wherein said polypeptide comprises an amino terminal amino acid sequence from a first endogenous natriuretic peptide native to the human cardiovascular system, an intermediate amino acid sequence from a second endogenous natriuretic peptide native to the human cardiovascular system, and a carboxy terminal amino acid sequence from said first endogenous natriuretic peptide or from a third endogenous natriuretic peptide endogenous to the human cardiovascular system.

32. The polypeptide of claim 31 , wherein said polypeptide lacks immunogenicity when administered to a human.

33. The polypeptide of claim 31 , wherein said amino terminal amino acid sequence and said carboxy terminal amino acid sequence are from human atrial natriuretic peptide (ANP), and said intermediate amino acid sequence is from human C -type natriuretic peptide (CNP).

34. The polypeptide of claim 33, wherein said polypeptide has the amino acid sequence set forth in SEQ ID NO:4.

35. The polypeptide of claim 31 , wherein said amino terminal amino acid sequence and said carboxy terminal amino acid sequence are from human B-type natriuretic peptide (BNP), and said intermediate amino acid sequence is from human CNP.

36. The polypeptide of claim 35, wherein said polypeptide has the amino acid sequence set forth in SEQ ID NO:5.

37. An isolated nucleic acid encoding the polypeptide of claim 1, claim 16, or claim 31.

38. A vector comprising a nucleic acid encoding the polypeptide of claim 1 , claim 16, or claim 31.

39. A host cell comprising a nucleic acid encoding the polypeptide of claim 1, claim 16, or claim 31.

40. The host cell of claim 39, wherein said host cell is a eukaryotic host cell.

41. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the polypeptide of claim 1, claim 16, or claim 31.

42. A method for increasing natriuretic activity within a mammal without significantly lowering blood pressure, wherein said method comprises administering a polypeptide of claim 1, claim 16, or claim 31 to said mammal.

43. A method for treating a mammal having a cardiovascular condition or renal condition, wherein said method comprises administering, to said mammal, a polypeptide of claim 1, claim 16, or claim 31 under conditions wherein the severity of a manifestation of said cardiovascular condition or renal condition is reduced.

44. The method of claim 43, wherein administration of said polypeptide to said mammal has minimal effects on systemic blood pressure of said mammal.