Immunoglobulin protein that binds to NPR1 agonists

Abstract

The present disclosure provides novel immunoglobulin proteins that bind to human natriuretic peptide receptor 1 (NPR1) agonists, preferably anti-NPR1 antibodies. In certain embodiments, the proteins of the present disclosure comprise at least one immunoglobulin variable domain that binds to an anti-NPR1 antibody. In certain embodiments, the proteins of the present disclosure are useful for blocking and / or reversing the effects of an administered anti-NPR1 antibody. In certain embodiments, the antigen binding proteins are useful for effective management of blood pressure and hemodynamics in humans.

Description

[Technical field]

[0001] The present disclosure relates to immunoglobulin proteins that specifically bind to natriuretic peptide receptor 1 (NPR1) agonists and therapeutic methods using those proteins.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application has been filed as a PCT International Patent Application on December 17, 2021. This application claims priority to U.S. Provisional Patent Application No. 63 / 127,959, filed December 18, 2020, the entire contents of which are incorporated herein by reference.

[0003] Sequence Statement This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy, created on December 13, 2021, is named 40848-0104WOU1-SeqListing.txt and is 71 kilobytes in size. [Background technology]

[0004] Natriuretic peptide receptor 1 (NPR1; also known as NPR-A) belongs to the cell surface family of guanylyl cyclase receptors, an enzyme that catalyzes the conversion of GTP to cyclic GMP. NPR1 is highly expressed in the kidney, lung, adrenal gland, vasculature, brain, liver, endothelial tissue and adipose tissue, and at lower levels in the heart. It is activated by binding to atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP). NPR1 activation and signaling stimulate many physiological responses involving many tissues. The ANP-NPR1 system has been well studied for its role in vascular relaxation, natriuresis, diuresis, endothelial permeability, and noncardiovascular functions such as lipolysis and immune cell function (Non-Patent Document 1). Activation of NPR1 causes natriuresis (excretion of salt by the kidney) and reduces blood pressure.

[0005] Currently approved therapeutic agents aimed at NPR1 agonism face multiple clinical challenges.

[0006] Monoclonal antibodies against NPR1 were first described by Kitano et al. (Non-Patent Document 2). Activating or agonistic anti-NPR1 antibodies are disclosed, for example, in US Pat. No. 5,399,411, US Pat. No. 5,499,421, and US Pat. No. 5,523,636. A fully human agonistic antibody that specifically binds to and activates the NPR1 protein with high affinity is described in US Pat. No. 5,523,636. R5381 is an agonist of NPR1 that has shown long-term efficacy in lowering systemic blood pressure compared to the current standard of care.

[0007] In vivo studies have shown that R5381 induces a significant and sustained reduction in systemic blood pressure without evidence of adverse hypotension (i.e., syncope, altered exercise capacity, death). Because the primary mechanism of action of certain anti-NPR1 antibodies has been found to be hemodynamic, there is a need for reversal agents that block their hemodynamic effects. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 9,090,695 [Patent Document 2] U.S. Patent Publication No. 20160168251 [Patent Document 3] WO2010065293 [Patent Document 4] U.S. Patent Publication No. 20200123263 [Non-patent literature]

[0009] [Non-Patent Document 1] Potter 2011, Pharmacol.Ther.130:71-82 [Non-Patent Document 2] 1995 Immunol Lett 47:215~22 Summary of the Invention [Problem to be solved by the invention]

[0010] In an effort to address possible concerns regarding the use of NPR1 agonists (e.g., activating or agonistic anti-NPR1 antibodies), reversal agents that specifically bind to such NPR1 agonists have been developed, as disclosed herein.

[0011] Anti-NPR1 antibodies have been described for the treatment and / or prevention of diseases, disorders or conditions associated with NPR1, and / or ameliorating at least one symptom associated with such diseases, disorders or conditions (see, for example, WO2020 / 086406). The primary mechanism of action of anti-NPR1 antibodies is hemodynamic. Potential adverse events associated with reduced blood pressure include persistent symptomatic hypotension, reflex tachycardia due to compensatory sympathetic nervous system responses (which may increase the risk of myocardial infarction, stroke, arrhythmias, and heart failure), and reduced cardiac output and end-organ perfusion in subjects with normal (low) venous pressure. Thus, there is a need for reversal agents (or rescue agents) that can target and stabilize, or reduce or reverse the hemodynamic effects of anti-NPR1 antibodies. [Means for solving the problem]

[0012] Thus, the present disclosure provides agents that reverse the hemodynamic effects of natriuretic peptide receptor 1 (NPR1) agonists, also referred to herein as reversal or rescue agents.

[0013] In another aspect, the disclosure provides an agent that reverses the drop in blood pressure associated with administration of an NPR1 agonist in a subject.

[0014] In one embodiment, the agent is selected from the group consisting of immunoglobulin proteins, vasopressors, alpha-adrenergic receptor agonists, steroids, antidiuretic hormones, angiogenesis inhibitors, and small molecule agents that increase blood pressure.

[0015] In one embodiment, the agent is an immunoglobulin protein. In one embodiment, the agent specifically binds to an NPR1 agonist. In one embodiment, the NPR1 agonist is an antibody or antigen-binding fragment thereof that specifically binds to NPR1. In one embodiment, the anti-NPR1 antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising SEQ ID NO: 48; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising SEQ ID NO: 52. In one embodiment, the anti-NPR1 antibody comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) comprising SEQ ID NO: 49, 50 and 51, respectively; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) comprising SEQ ID NO: 53, 54 and 55, respectively. In one embodiment, the anti-NPR1 antibody or antigen-binding fragment thereof comprises the HCVR of SEQ ID NO: 48 and the LCVR of SEQ ID NO: 52. In one embodiment, the anti-NPR1 antibody is a monoclonal antibody. In one embodiment, the anti-NPR1 antibody is an IgG1 or IgG4 antibody. In one embodiment, the anti-NPR1 antibody or antigen-binding fragment thereof comprises a heavy chain comprising SEQ ID NO: 56 and a light chain comprising SEQ ID NO: 57. In one embodiment, the anti-NPR1 antibody is R5381.

[0016] In one embodiment, the rescue agent is an immunoglobulin protein. In one embodiment, the immunoglobulin protein comprises a monoclonal antibody or an antigen-binding fragment thereof. In another embodiment, the immunoglobulin protein comprises a bivalent antibody. In another embodiment, the immunoglobulin protein comprises a monovalent or "one-arm" antibody. In another embodiment, the immunoglobulin protein comprises a recombinant monoclonal antibody. In another embodiment, the immunoglobulin protein comprises a fully human monoclonal antibody that is bivalent or monovalent. In another embodiment, the immunoglobulin protein is a fully human monoclonal antibody that is of the IgG1 or IgG4 isotype. In one embodiment, the immunoglobulin protein comprises a Fab fragment. In an embodiment, the immunoglobulin protein comprises a monoclonal antibody or an antigen-binding fragment thereof, a bivalent monoclonal antibody, a monovalent monoclonal antibody, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a Fd fragment, a scFv, or a dAb. In one embodiment, the immunoglobulin protein comprises at least one immunoglobulin variable domain comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR). In one embodiment, the immunoglobulin protein comprises one immunoglobulin variable domain comprising three heavy chain CDRs contained within a HCVR and three light chain CDRs contained within a LCVR. In one embodiment, the immunoglobulin protein further comprises a multimerization component comprising at least one Fc fragment. In one embodiment, the multimerization component comprises a first Fc fragment and a second Fc fragment, wherein the first Fc fragment or the second Fc fragment, but not both, comprises a modification in the CH3 domain that reduces binding of the immunoglobulin protein to Protein A compared to an immunoglobulin protein lacking the modification. In one embodiment, the modifications include a H315R and a Y316F substitution (EU numbering).

[0017] In one embodiment, the immunoglobulin protein comprises a monovalent antibody, the monovalent antibody comprising a heavy chain comprising a heavy chain constant region and an HCVR, and a light chain comprising a light chain constant region and an LCVR, the heavy chain being of a human IgG1 or IgG4 isotype. In one embodiment, the heavy chain constant region comprises a modification in the CH3 domain that reduces binding of the immunoglobulin protein to Protein A compared to an immunoglobulin protein lacking the modification. In one embodiment, the modification comprises a H315R substitution and a Y316F substitution (EU numbering). In one embodiment, the immunoglobulin protein further comprises a multimerization component comprising an Fc fragment. In one embodiment, the Fc fragment is of a human IgG1 or IgG4 isotype.

[0018] In one embodiment, the immunoglobulin protein comprises a Fab fragment comprising one immunoglobulin variable domain comprising three heavy chain CDRs contained within HCVRs and three light chain CDRs contained within LCVRs. In one embodiment, the immunoglobulin protein further comprises a multimerization component. In one embodiment, the multimerization component comprises at least one Fc fragment. In one embodiment, the Fc fragment is of isotype IgG1, IgG4 or variants thereof. In one embodiment, the multimerization component comprises a first Fc fragment and a second Fc fragment, where the first Fc fragment or the second Fc fragment, but not both, comprises a modification in the CH3 domain that reduces binding of the immunoglobulin protein to Protein A compared to an immunoglobulin protein lacking the modification. In one embodiment, the modification comprises a H315R substitution and a Y316F substitution (EU numbering) in an Fc fragment of an IgG1 or IgG4 isotype.

[0019] Table 1 lists the amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining region (HCDR) (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining region (LCDR) (LCDR1, LCDR2, and LCDR3) of exemplary immunoglobulin proteins. Table 2 lists the nucleic acid sequence identifiers for the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary immunoglobulin proteins.

[0020] Typical conventions used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0021] In certain embodiments, the immunoglobulin protein of the disclosure is an antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence listed in Table 1 with no more than 12 amino acid substitutions, and / or the LCVR comprises an amino acid sequence listed in Table 1 with no more than 10 amino acid substitutions. For example, the disclosure provides an antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence set forth in Table 1, wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions. In other examples, the disclosure provides an antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the LCVR comprises an amino acid sequence set forth in Table 1, wherein the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In one embodiment, the disclosure provides an immunoglobulin protein or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein said HCVR comprises an amino acid sequence listed in Table 1, said amino acid sequence having at least one amino acid substitution, and / or said LCVR comprises an amino acid sequence listed in Table 1, said amino acid sequence having at least one amino acid substitution.

[0022] In certain embodiments, the immunoglobulin protein of the disclosure is an antibody or antigen-binding fragment thereof that comprises an HCVR that comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0023] In certain embodiments, the immunoglobulin protein of the disclosure is an antibody or antigen-binding fragment thereof that comprises an LCVR that comprises an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0024] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0025] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0026] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0027] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0028] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0029] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0030] In certain embodiments, the immunoglobulin protein of the disclosure is an antibody or antigen-binding fragment thereof that comprises an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) that includes any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that comprises an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary immunoglobulin proteins listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 8 / 16 and 28 / 36.

[0031] In certain embodiments, the immunoglobulin protein of the disclosure is an antibody or antigen-binding fragment thereof encoded by a nucleic acid molecule disclosed herein. For example, the disclosure provides a nucleic acid molecule encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0032] The disclosure also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0033] The present disclosure also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0034] The present disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0035] The present disclosure also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0036] The present disclosure also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0037] The present disclosure also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0038] The present disclosure also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0039] In certain embodiments, the immunoglobulin protein of the present disclosure is an antibody or antigen-binding fragment thereof with an altered glycosylation pattern. In certain embodiments, modifications to remove undesired glycosylation sites may be useful, or antibodies lacking fucose moieties are displayed on the oligosaccharide chains, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications are made to modify complement-dependent cytotoxicity (CDC).

[0040] In one aspect, the disclosure provides: (i) an immunoglobulin protein comprising one immunoglobulin variable domain comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within an HCVR, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within an LCVR. In one embodiment, the HCVR of the immunoglobulin protein comprises an amino acid sequence selected from any of the HCVR sequences in Table 1. In one embodiment, the LCVR of the immunoglobulin protein comprises an amino acid sequence selected from any of the LCVR sequences in Table 1. In one embodiment, the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22; and the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30. In one embodiment, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 24; HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 26; HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 28; LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 32; LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 34; and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 36. In one embodiment, HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 comprises an amino acid sequence selected from (i) SEQ ID NOs: 4, 6, 8, 12, 14 and 16; or (ii) SEQ ID NOs: 24, 26, 28, 32, 34 and 36. In one embodiment, the immunoglobulin protein further comprises a multimerization component, the multimerization component comprising at least one Fc fragment, in one embodiment, the Fc fragment is of the IgG1 or IgG4 isotype.In one embodiment, the multimerization component comprises a first Fc fragment and a second Fc fragment, where the first Fc fragment or the second Fc fragment, but not both, comprises a modification in the CH3 domain that reduces binding of the immunoglobulin protein to Protein A compared to an immunoglobulin protein lacking the modification. In one embodiment, the modification comprises a H315R substitution and a Y316F substitution (EU numbering) in an Fc fragment of an IgG1 or IgG4 isotype. In one embodiment, the multimerization component comprises an Fc fragment comprising the amino acid sequence of SEQ ID NO:46 and an Fc fragment comprising the amino acid sequence of SEQ ID NO:58. SEQ ID NO:58: ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSPGK

[0041] In one embodiment, the immunoglobulin protein comprises a bivalent antibody or antigen-binding fragment thereof. In one embodiment, the immunoglobulin protein comprises a monovalent ("one-arm") antibody or antigen-binding fragment thereof. In one embodiment, the immunoglobulin protein comprises a heavy chain comprising an HCVR and a light chain comprising an LCVR, wherein the heavy chain is of a human IgG1 or IgG4 isotype. In one embodiment, the heavy chain comprises a modification in the CH3 domain that reduces binding of the immunoglobulin protein to Protein A compared to an immunoglobulin protein lacking the modification. In one embodiment, the modification comprises a H315R substitution and a Y316F substitution (EU numbering) in the heavy chain of an IgG1 or IgG4 isotype. In one embodiment, the heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 38; and the light chain has an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 40. In one embodiment, the immunoglobulin protein further comprises an Fc fragment. In one embodiment, the Fc fragment is of an IgG1 or IgG4 isotype. In one embodiment, the Fc fragment comprises an amino acid sequence comprising SEQ ID NO: 46.

[0042] In one embodiment, the immunoglobulin protein specifically binds to an anti-NPR1 antibody. In one embodiment, the anti-NPR1 antibody is R5381.

[0043] In one embodiment, the immunoglobulin protein is REGN9035. In one embodiment, the immunoglobulin protein is REGN9037.

[0044] In one aspect, the disclosure provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding a heavy chain variable region (HCVR) of an immunoglobulin protein disclosed herein. In another aspect, the disclosure provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding a light chain variable region (LCVR) of an immunoglobulin protein disclosed herein. In one aspect, the disclosure provides a vector comprising a polynucleotide molecule disclosed herein. In certain embodiments, the vector is a recombinant expression vector capable of expressing a polynucleotide comprising a heavy chain variable region and / or a light chain variable region of an immunoglobulin protein. For example, the disclosure includes a recombinant expression vector comprising any of the nucleic acid molecules disclosed herein, i.e., any of the nucleic acid molecules encoding any of the HCVR, LCVR and / or CDR sequences listed in Table 2. In another aspect, the disclosure provides a host cell expressing a vector disclosed herein. For example, the disclosure provides a host cell comprising a first recombinant expression vector capable of expressing a polypeptide comprising a heavy chain variable region of an immunoglobulin protein as disclosed herein; a second expression vector capable of expressing a polypeptide comprising a light chain variable region of an immunoglobulin protein as disclosed herein. In one embodiment, the present disclosure provides a host cell comprising a first isolated polynucleotide molecule comprising a polynucleotide sequence encoding a heavy chain variable region (HCVR) of an immunoglobulin protein disclosed herein, and a second isolated polynucleotide molecule comprising a polynucleotide sequence encoding a light chain variable region (LCVR) of an immunoglobulin protein disclosed herein. In certain embodiments, the host cell comprises a mammalian cell or a prokaryotic cell. In certain embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell or an Escherichia coli (E. coli) cell.

[0045] In one aspect, the present disclosure provides a method for producing an immunoglobulin protein or fragment thereof that specifically binds to an anti-NPR1 antibody or antigen-binding fragment thereof, comprising growing a host cell disclosed herein under conditions that allow for the production of the antibody or fragment, and recovering the immunoglobulin protein or fragment thus produced. In certain embodiments, the present disclosure provides a method for producing an immunoglobulin protein or fragment thereof of the present disclosure, comprising introducing into a host cell an expression vector comprising a nucleic acid sequence encoding the HCVR and / or LCVR of an immunoglobulin protein or fragment thereof of the present disclosure operably linked to a promoter; culturing the host cell under conditions favorable for the expression of the nucleic acid sequence; and isolating the immunoglobulin protein or fragment thereof from the culture medium and / or the host cell. The isolated immunoglobulin protein or fragment thereof can be purified using any of the methods known in the art. In one embodiment, the immunoglobulin protein of the present disclosure can be purified using reagents and methods that utilize different binding to Protein A, as disclosed elsewhere herein.

[0046] In one embodiment, the rescue agent is a vasopressor. In another embodiment, the vasopressor is midodrine.

[0047] In one aspect, the present disclosure provides a pharmaceutical composition comprising the rescue agent disclosed herein and a pharma- ceutical acceptable carrier or diluent.In one embodiment, the composition comprises a combination of rescue agent and a second therapeutic agent.In one embodiment, the second therapeutic agent is any agent that is advantageously combined with rescue agent.Additional combination therapy and co-formulations that comprise the rescue agent disclosed herein are disclosed elsewhere herein.

[0048] In one aspect, the disclosure provides a method for reversing the hemodynamic effects of an agonist antibody or antigen-binding fragment that specifically binds to natriuretic peptide receptor 1 (NPR1) protein, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a relief agent disclosed herein.

[0049] In another aspect, the present disclosure provides a method for reversing the drop in blood pressure associated with administration of an agonist antibody or antigen-binding fragment that specifically binds to natriuretic peptide receptor 1 (NPR1) protein, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a rescue agent disclosed herein.

[0050] In certain embodiments, the pharmaceutical composition is administered to the subject subcutaneously, intravenously, intradermally, intraperitoneally, intramuscularly, or orally.

[0051] In another aspect, the disclosure provides for the use of a rescue agent disclosed herein in the manufacture of a medicament for reversing the hemodynamic effects associated with administration of an anti-NPR1 antibody in a subject in need thereof.

[0052] In one embodiment, the subject has an NPR1-related disease or disorder. In one embodiment, the disease or disorder is hypertension, heart failure and / or chronic kidney disease.

[0053] In one aspect, the present disclosure provides a composition comprising: (i) an immunoglobulin protein disclosed herein; and (ii) an NPR1 agonist. In one embodiment, the NPR1 agonist is an anti-NPR1 antibody (e.g., R5381). In one embodiment, the composition is used in a method for effectively regulating blood pressure in a subject in need thereof. In one embodiment, the subject has an NPR1-related disease or disorder. In one embodiment, the disease or disorder is hypertension, heart failure and / or chronic renal failure.

[0054] In one aspect, the disclosure provides antibodies or antigen-binding fragments thereof that compete for binding with the immunoglobulin proteins disclosed herein.

[0055] In another aspect, the disclosure provides antibodies or antigen-binding fragments thereof that bind to the same epitope as the immunoglobulin proteins disclosed herein.

[0056] Other embodiments will become apparent from consideration of the following detailed description. [Brief description of the drawings]

[0057] [Figure 1] FIG. 1 provides a comparison of the dissociation half-life (t1 / 2) of anti-R5381 antibodies, REGN9035 and REGN9037, in pH 7.4, pH 6.5, pH 6.0, and pH 5.0 buffers in bar graph format. [Diagram 2] FIG. 1 provides the pharmacokinetic profiles of anti-R5381 antibodies REGN9035, REGN9037, REGN6580, and REGN6581 in NPR1 hu / hu mice in line graph format. [Diagram 3] FIG. 13 shows in line graph format the effect of bivalent anti-R5381 mAb on reversal of R5381-induced systolic blood pressure reduction in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on systolic blood pressure and body weight. Animals were given a single 5 mg / kg subcutaneous injection of R5381 or PBS as described in Table 18. Animals were then given a single 50 mg / kg intravenous injection of anti-R5381 bivalent mAb or PBS as described in Table 18. All values ​​are mean pressures ± SEM over 24 hours from days -2 to 20, n=4-5 per group. Statistics-2-way ANOVA with Dunnett; *p<0.05 vs. isotype control mAb. [Figure 4]FIG. 13 shows in line graph format the effect of bivalent anti-R5381 mAb on reversal of R5381-induced diastolic blood pressure fall in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on systolic blood pressure and body weight. Animals were given a single 5 mg / kg subcutaneous injection of R5381 or PBS as described in Table 18. Animals were then given a single 50 mg / kg intravenous injection of anti-R5381 bivalent mAb or PBS as described in Table 18. All values ​​are mean pressures ± SEM over 24 hours from days -2 to 20, n=4-5 per group. Statistics-2-way ANOVA with Dunnett; *p<0.05 vs. isotype control. [Diagram 5] FIG. 13 shows in line graph format the effect of bivalent anti-R5381 mAb on reversal of R5381 mAb-induced heart rate effects in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on systolic blood pressure and body weight. Animals were given a single 5 mg / kg subcutaneous injection of R5381 or PBS as described in Table 18. Animals were then given a single 50 mg / kg intravenous injection of anti-R5381 bivalent mAb or PBS as described in Table 18. All values ​​are mean heart rate ± SEM over 24 hours from days -2 to 20, n=4-5 per group. Statistics-2-way ANOVA with Dunnett. [Figure 6] FIG. 13 shows in line graph format the effect of bivalent anti-R5381 mAb on reversal of R5381 mAb-induced mean arterial blood pressure reduction in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on systolic blood pressure and body weight. Animals were given a single 5 mg / kg subcutaneous injection of R5381 or PBS as described in Table 18. Animals were then given a single 50 mg / kg intravenous injection of anti-R5381 bivalent mAb or PBS as described in Table 18. All values ​​are mean pressures ± SEM over 24 hours from days -2 to 20, n=4-5 per group. Statistics-2-way ANOVA with Dunnett; *p<0.05 vs. isotype control mAb. [Figure 7] FIG. 2 shows in line graph format the effect of bivalent anti-R5381 mAb on reversal of R5381-induced systolic blood pressure reduction in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on systolic blood pressure and body weight. Animals were given a single 5 mg / kg subcutaneous injection of R5381 or PBS as described in Table 23. Animals were then given a single 50 mg / kg subcutaneous injection of anti-R5381 bivalent mAb or isotype control mAb as described in Table 23. All values ​​are mean pressure ± SEM over 24 hours from days -3 to 21, n=4-5 per group. Statistics- Two-way ANOVA with Dunnett; *p<0.05 PBS vs. isotype control mAb; **p<0.01 PBS vs. isotype control mAb; !p<.05 REGN6580 vs. isotype control mAb; #p<0.05 REGN6581 vs. isotype control. [Figure 8] FIG. 2 shows in line graph format the effect of monovalent anti-R5381 mAbs on reversal of R5381-induced systolic blood pressure reduction in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on body weight. Animals were given a single 5 mg / kg subcutaneous injection of NPR1 agonist mAb or isotype control mAb as described in Table 28. Animals were then given a single 50 mg / kg intravenous injection of anti-R5381 bivalent (REGN6580) or monovalent (REGN9035 or REGN9037) mAb or PBS as described in Table 28. All values ​​are mean ± SEM, n=4-5 per group. Statistics- Two-way ANOVA with Dunnett; *p<0.05 REGN6580 sc vs isotype control mAb; #p<0.05 REGN9035 sc vs isotype control mAb; !p<0.05 REGN9037 sc vs isotype control mAb; @p<0.05 REGN6580 iv vs isotype control mAb; &p<0.05 REGN9035 iv vs isotype control mAb; >p<0.05 REGN9037 iv vs isotype control mAb. [Figure 9]FIG. 2 shows in line graph format the acute effect of monovalent and bivalent anti-R5381 mAbs on reversal of R5381-induced blood pressure reduction in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on body weight. Animals were given a single 5 mg / kg subcutaneous injection of R5381 or PBS control as described in Table 28. Animals were then given a single 50 mg / kg intravenous injection of anti-R5381 bivalent (REGN6580) or monovalent (REGN9035 or REGN9037) mAbs or isotype control mAbs as described in Table 28. All values ​​are mean ± SEM, n=4-5 per group. [Figure 10] FIG. 2 shows in line graph format the effect of monovalent anti-R5381 mAbs on reversal of R5381-induced cGMP production in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on body weight. Animals were given a single 5 mg / kg subcutaneous injection of NPR1 agonist mAb or isotype control mAb as described in Table 28. Animals were then given a single 50 mg / kg intravenous injection of anti-R5381 bivalent (REGN6580) or monovalent (REGN9035 or REGN9037) mAb or PBS as described in Table 28. Urine was collected overnight from day 21 to day 22 of the study. All values ​​are mean ± SEM, n=5-6 per group. Statistics- ANOVA with Dunnett; ****p<0.0001 vs. PBS+PBS; ####p<0.0001 vs. R5381+isotype control mAb. [Figure 11]Figure 1 shows that the blood pressure lowering effect of R5381 is reversed by three doses of 2.5 mg / kg midodrine administered 3 days after a single dose of 25 mg / kg R5381. Male cynomolgus monkeys weighing 3-5 kg ​​were surgically implanted with radiotelemetry transmitters. On day 0, each animal received a single IV bolus of saline (PBS; n=10) or 25 mg / kg R5381 (n=13). On day 3, each animal received three doses of 2.5 mg / kg / dose midodrine (n=6 in saline group; n=7 in R5381 group) or water / vehicle (n=4 in saline group; n=6 in R5381 group) administered by oral gavage, with each dose spaced 3-4 hours apart, as indicated by the dotted lines on the x-axis. Blood pressure measurements were collected for each animal pre-dose (for baseline measurements) and during a 4-day monitoring period after dosing. The mean change from baseline systolic blood pressure for each treatment group between 35 and 72 hours after R5381 dosing is shown. Data are presented as group mean ± standard error of the mean. [Figure 12] Figure 1. R5381-induced heart rate effects are reversed by three doses of 2.5 mg / kg midodrine administered 3 days after a single dose of 25 mg / kg. Male cynomolgus monkeys, age 3-5 kg, were surgically implanted with radiotelemetry transmitters. On day 0, each animal received a single IV bolus of saline (PBS; n=10) or 25 mg / kg R5381 (n=13). On day 3, each animal received three doses of 2.5 mg / kg / dose midodrine (n=6 in saline group; n=7 in R5381 group) or water / vehicle (n=4 in saline group; n=6 in R5381 group) administered by oral gavage, with each dose spaced 3-4 hours apart, as indicated by the dotted lines on the x-axis. Heart rate measurements were collected for each animal pre-dose (for baseline measurements) and during a 4-day monitoring period after dosing. The mean change from baseline heart rate for each treatment group between 35 and 72 hours after R5381 administration is shown. Data are presented as group mean ± standard error of the mean. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Before describing the method, it is to be understood that the disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the disclosure will be limited only by the appended claims.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods and materials are described here.All publications mentioned herein are incorporated herein by reference in their entirety.

[0060] definition The term "NPR1," also referred to as "NPRA," refers to natriuretic peptide receptor 1 (also known as natriuretic peptide receptor A). NPR1 is a homodimeric transmembrane guanylate cyclase, an enzyme that catalyzes cGMP synthesis. The protein has four distinct regions, including an extracellular ligand-binding domain, a single transmembrane region, an intracellular protein kinase-like homology domain, and a guanylyl cyclase catalytic domain. The amino acid sequence of the full-length NPR1 protein is exemplified by the amino acid sequence provided in UniProtKB / Swiss-Prot under accession number P16066.1 (SEQ ID NO:59): 1 mpgprrpags rlrllllllll pplllllrgs hagnltvavv lplantsypw swarvgpave 61 lalaqvkarp dllpgwtvrt vlgssenalg vcsdtaapla avdlkwehnp avflgpgcvy 121 aaapvgrfta hwrvplltag apalgfgvkd eyalttragp syaklgdfva alhrrlgwer 181 qalmlyayrp gdeehcfflv eglfmrvrdr lnitvdhlef aeddlshytr llrtmprkgr 241 viyicsspda frtlmllale aglcgedyvf fhldifgqsl qggqgpaprr pwergdgqdv 301 sarqafqaak iitykdpdnp eyleflkqlk hlayeqfnft medglvntip asfhdgllly 361 iqavtetlah ggtvtdgeni tqrmwnrsfq gvtgylkids sgdretdfsl wdmdpengaf 421 rvvlnyngts qelvavsgrk lnwplgyppp dipkcgfdne dpacnqdhls tlevlalvgs 481 lsllgilivs ffiyrkmqle kelaselwrv rwedvepssl erhlrsagsr ltlsgrgsny 541 gsllttegqf qvcepatyyk gnlvavkrvn rkrieltrkv lfelkhmrdv qnehltrfvg 601 actdppnici lteycprgsl qdilenesit ldwmfryslt ndivkgmlfl hngaicshgn 661 lkssncvvdg rfvlkitdyg lesfrdldpe qghtvyakkl wtapellrma sppvrgsqag 721 dvysfgiilq eialrsgvfh vegldlspke iiervtrgeq ppfrpslalq shleelgllm 781 qrcwaedpqe rppfqqirlt lrkfnrenss nildnllsrm eqyannleel veertqayle 841 ekrkaeally qilphsvaeq lkrgetvqae afdsvtiyfs divgftalsa estpmqvvtl 901 lndlytcfda vidnfdvykv etigdaymvv sglpvrngrl hacevarmal alldavrsfr 961 irhrpqeqlr lrigihtgpv cagvvglkmp ryclfgdtvn tasrmesnge alkihlsset 1021 kavleefggf elelrgdvem kgkgkvrtyw llgergsstr g

[0061] The term "NPR1" includes recombinant NPR1 protein or fragments thereof. The term also encompasses NPR1 protein or fragments thereof linked to, for example, a histidine tag, mouse or human Fc, or a signal sequence.

[0062] As used herein, the term "NPR1 agonist" refers to a molecule that activates, increases or enhances NPR1 activity or stabilizes the activated conformation of NPR1. In a preferred embodiment, the term "NPR1 agonist" refers to an antibody or antigen-binding fragment thereof that specifically binds to NPR1 and activates or increases at least one biological activity of NPR1. Such agonist anti-NPR1 antibodies can bind to NPR1 either in the presence or absence of a ligand (e.g., ANP or BNP). In certain embodiments, the biological activity includes, but is not limited to, a decrease or reduction in blood pressure in a subject upon administration of the agonist anti-NPR1 antibody. In certain embodiments, the biological activity includes hemodynamic changes (e.g., a reduction in blood pressure) in a subject with a disease or disorder such as hypertension, heart failure or chronic kidney disease. This term includes, for example, agonist anti-NPR1 antibodies disclosed in US Publication No. 20200123263. In a specific embodiment, the term refers to an anti-NPR1 antibody comprising the HCVR of SEQ ID NO: 48 and the LCVR of SEQ ID NO: 52. In another specific embodiment, the term refers to R5381 (also known as REGN5381). R5381 is a fully human anti-NPR1 monoclonal antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 56 and a light chain comprising the amino acid sequence of SEQ ID NO: 57.

[0063] As used herein, a "reversing agent" or "rescue agent" is an agent that reverses the hemodynamic effects of an NPR1 agonist. The terms "reversing agent" and "rescue agent" are used interchangeably herein. In certain embodiments, a reversing agent referred to herein reverses the hemodynamic changes associated with administration (to a subject) of an agonist antibody or antigen-binding fragment that specifically binds to NPR1. The term "reversing" includes increasing blood pressure in a subject whose blood pressure has decreased as a result of administration of an NPR1 agonist. The increase in blood pressure can be measured using any standard blood pressure assessment means known in the art (e.g., a blood pressure monitor). This increase can be to the treatment level of the preagonist antibody or to a level that provides sufficient hemodynamic stability. Hemodynamic effects can include indirect effects, which are effects related to blood pressure reduction. These effects can also be reversed using the agents disclosed herein. Hemodynamic effects referred to herein include physiological parameters such as blood pressure and heart rate, or clinical signs such as dizziness, lightheadedness, blurred vision, nausea, fatigue, etc. In certain embodiments, the reversing agent specifically binds to an NPR1 agonist and reverses the hemodynamic effects caused by the NPR1 agonist. In certain embodiments, the reversing agent comprises an immunoglobulin protein disclosed herein.

[0064] As used herein, the term "immunoglobulin protein" refers to an antigen-binding molecule that comprises at least one immunoglobulin variable domain. The at least one variable domain is an antigen-binding domain and comprises a heavy chain variable region and a light chain variable region. In certain embodiments, the variable domain is comprised in a Fab, Fv, single chain Fv, or any other antigen-binding fragment of an antibody, as disclosed elsewhere herein. In certain embodiments, the variable domain is comprised in a monovalent or bivalent antibody. The term includes, but is not limited to, antibodies and antigen-binding fragments thereof, monovalent antibodies, bivalent antibodies and antigen-binding fragments thereof. The immunoglobulin protein may also comprise a multimerization component linked to the variable domain. As disclosed elsewhere herein, the multimerization component may comprise an Fc fragment of an antibody or a truncated heavy chain of an antibody. For example, the immunoglobulin protein of the present disclosure may comprise a single variable domain within a Fab, which is linked to at least one Fc fragment. In certain embodiments, the immunoglobulin protein comprises a polypeptide comprising (i) a heavy chain comprising a heavy chain constant region and a heavy chain variable region, (ii) a light chain comprising a light chain constant region and a light chain variable region, and (iii) an Fc fragment or truncated heavy chain. In certain embodiments, the Fc domain polypeptide is a "dummy Fc", which refers to an Fc domain polypeptide that is not linked to an antigen-binding domain. An immunoglobulin protein comprising a single variable domain may be referred to as a "one-arm" or "single-arm" or "monovalent" antibody. In the context of the present disclosure, the term refers to an antibody or antigen-binding fragment thereof that binds to the variable region of another antibody ("anti-idiotypic antibody"). In certain embodiments, the term refers to an antibody (or antigen-binding fragment thereof) comprising a single variable domain that specifically binds to an anti-NPR1 antibody or antigen-binding fragment thereof. In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to an anti-NPR1 antibody is a competitive binder. The one-arm antibody of the present disclosure may comprise any of the HCVR / LCVR or CDR amino acid sequences set forth in Table 1 herein.Exemplary one-arm anti-R5381 antibodies disclosed herein include REGN9035 and REGN9037.

[0065] As used herein, the term "antibody", unless specifically indicated otherwise, is intended to refer to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds (i.e., a "complete antibody molecule"), as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. In the context of the present disclosure, the term "bivalent antibody" refers to a "complete antibody molecule", i.e., one comprising two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region ("HCVR" or "V H "), and the heavy chain constant region (domain C H 1. C H 2, and C H Each light chain comprises a light chain variable region ("LCVR" or "V L "), and the light chain constant region (C L ) is included. V H and V L The regions are further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H And V L comprises three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the disclosure, the FRs of the antibody (or antigen-binding fragment thereof) are identical to human germline sequences or are naturally or artificially modified. The amino acid contiguous sequence is defined based on a side-by-side analysis of two or more CDRs.

[0066] In certain embodiments, the term "antibody" or "antigen-binding molecule" includes a monovalent antigen-binding molecule. A monovalent antigen-binding molecule includes a single antigen-binding domain formed by a single heavy chain and a single light chain. A monovalent antigen-binding molecule further includes a polypeptide including at least one Fc domain of a heavy chain. In certain embodiments, the Fc domain polypeptide is a "dummy Fc", which refers to an Fc domain polypeptide that is not linked to an antigen-binding domain. In certain embodiments, a monovalent antibody has a complete heavy chain, a complete light chain, and a truncated heavy chain.

[0067] As used herein, "Fc fragment" or "Fc region" refers to the crystallizable region of a fragment of an immunoglobulin, which is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In IgG, IgA, and IgD antibody isotypes, the Fc region or Fc domain is derived from the second and third constant domains (CH2 and CH3) of the antibody heavy chain; IgM and IgE Fc regions have three heavy chain constant domains (CH1 and CH2) in the polypeptide chain. H In the context of the present disclosure, the term refers to an Fc region derived from a human Fc domain, unless specifically indicated otherwise. In certain embodiments, the Fc fragment is derived from a human IgG1, IgG2, IgG3, or IgG4 isotype.

[0068] As used herein, a "multimerization component" refers to any macromolecule that has the ability to associate with a second macromolecule of the same or similar structure or composition. For example, a multimerization component may be an immunoglobulin C H A non-limiting example of a multimerization component is a polypeptide comprising the Fc portion of an antibody (C H 2-C H3 domains), e.g., an Fc domain of an IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4, and any allotype within each isotype group. In certain embodiments, the immunoglobulin protein of the present disclosure comprises a multimerization component comprising at least one Fc fragment. In certain embodiments, the immunoglobulin protein comprises two Fc fragments. The first and second Fc fragments may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second Fc fragments may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4. In certain embodiments, the two Fc fragments have identical sequences. In certain embodiments, the two Fc fragments differ from each other by one or more amino acids. In certain embodiments, the first Fc fragment or the second Fc fragment, but not both, comprises a modification in the CH3 domain that reduces binding of the immunoglobulin protein to Protein A compared to the immunoglobulin protein lacking the modification. In certain embodiments, the multimerization component is an Fc fragment, or an amino acid sequence of 1 to about 200 amino acids in length that contains at least one cysteine ​​residue. In other embodiments, the multimerization component is a cysteine ​​residue, or a short cysteine-containing peptide. Other multimerization components include peptides or polypeptides that include or consist of a leucine zipper, a helix loop motif, or a coiled-coil motif. For example, a multimerization component that includes an Fc domain can include one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring form of the Fc domain, as disclosed elsewhere herein.

[0069] Substitution of one or more CDR residues or omission of one or more CDRs are also possible. Antibodies that bind even when one or two CDRs are omitted have been described in the scientific literature. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact areas between an antibody and its antigen based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs have no amino acids in contact with the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0070] CDR residues that are not in contact with the antigen are identified by molecular modeling and / or experimentally from regions of the Kabat CDRs that are outside the Chothia CDRs, based on previous studies (e.g., residues H60-H65 in CDRH2 are often not necessary). If a CDR or its residues are excluded, it is usually replaced with an amino acid that occupies the corresponding position in another human antibody sequence or a common such sequence. The positions for substitution within the CDRs and the amino acids to replace are also selected experimentally. The experimental substitutions are conservative or non-conservative substitutions.

[0071] The fully human immunoglobulin proteins disclosed herein (specifically bind to anti-NPR1 antibodies) may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be easily ascertained by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residues in the germline sequence from which the antibody is derived, or to the corresponding residues in another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations").

[0072] Starting from the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments that contain one or more individual germline mutations or combinations thereof. H and / or V LAll of the framework and / or CDR residues in the domain are mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues, for example, mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues found in CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residues in a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody is originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations are readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist biological properties, reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present disclosure.

[0073] The present disclosure also includes fully human immunoglobulin proteins (that specifically bind to anti-NPR1 antibodies) that include variants of any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present disclosure includes fully human immunoglobulin proteins (that specifically bind to anti-NPR1 antibodies) that have HCVR, LCVR and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein.

[0074] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an anti-NPR1 antibody. Antibody fragments include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments of antibodies can be obtained from complete antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or synthesized. The DNA is sequenced and manipulated chemically or by using molecular biology techniques, e.g., to place one or more variable and / or constant domains in the appropriate position, or to introduce codons, create cysteine ​​residues, or modify, add, or delete amino acids.

[0075] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the expression "antigen-binding fragment" as used herein.

[0076] Antigen-binding fragments of antibodies typically contain at least one variable domain. A variable domain may be of any size or amino acid composition and generally contains at least one CDR and is flanked or in-frame by one or more framework sequences. L V connected to the domain H In an antigen-binding fragment having a domain, H and V L The domains may be positioned relative to one another in any suitable arrangement. For example, the variable region may be a dimer, with the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of the antibody contains a monomeric V H or V L Contains a domain.

[0077] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains found in the antigen-binding fragment of an antibody of the present disclosure include: (i) V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any arrangement of variable and constant domains, including any of the exemplary arrangements listed above, the variable and constant domains are either directly linked to each other or linked by a complete or partial hinge or linker region. A hinge region consists of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids and provides a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure can be linked (e.g., by disulfide bonds) to each other and / or to one or more monomeric V domains. H Or V L The invention also includes homo- or hetero-dimers (or other multimers) of any of the above listed variable and constant domain arrangements non-covalently associated with the domain.

[0078] As used herein, the term "human antibody" or "fully human antibody" or "fully human immunoglobulin protein" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the present disclosure include, for example, in the CDRs, particularly CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, the term "human antibody" or "fully human antibody" as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or in cells of non-human mammals. The term is not intended to include antibodies isolated from or raised in a human subject.

[0079] The term "recombinant" as used herein refers to an antibody or antigen-binding fragment thereof of the present disclosure that is made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term refers to antibodies expressed in non-human mammalian (including transgenic non-human mammalian, e.g., transgenic mice) or cellular (e.g., CHO cell) expression systems, or isolated from a recombinant combinatorial human antibody library.

[0080] The terms "specifically bind" or "specifically bind to" and the like mean that an antibody or antigen-binding fragment thereof forms a complex with an anti-NPR1 antibody that is relatively stable, for example, under physiological conditions. Specific binding is at least about 1×10 -8 Characterized by an equilibrium dissociation constant M or less (e.g., K D (A smaller value indicates a stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, an antibody is identified by surface plasmon resonance, e.g., BIACORE™, and specifically binds to an anti-NPR1 antibody (e.g., R5381). Furthermore, a multispecific antibody that binds to one domain in an anti-NPR1 antibody and one or more additional antigens, or a bispecific antibody that binds to two different regions of an anti-NPR1 antibody, is nevertheless considered to be an antibody that "specifically binds" as used herein.

[0081] The term "high affinity" antibody refers to an antibody that has an affinity of at least 10 as measured by surface plasmon resonance, e.g., BIACORE™, or solution affinity ELISA. -8 M; preferably 10 -9 M; more preferably, 10 -10 M, and even more preferably 10 -11 K of M D The binding affinity of the mAb to the anti-NPR1 antibody is expressed as

[0082] The term "slow off rate", "Koff", or "kd" refers to a slow off rate of 1×10 as determined by surface plasmon resonance, e.g., BIACORE™. -3 s -1 or less, preferably 1×10 -4 s -1 It means an antibody that dissociates from the anti-NPR1 antibody with a rate constant equal to or lower than that.

[0083] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The "antigen" of the immunoglobulin protein described herein is an anti-NPR1 antibody (e.g., R5381) or an antigen-binding fragment thereof (i.e., a fragment of the antibody that binds to NPR1). As used herein, the terms "antigen-binding fragment" of an antibody, or "antibody fragment" refer to one or more fragments of an antibody that retain the ability to bind to an anti-NPR1 antibody.

[0084] In certain embodiments, the antibodies or antibody fragments of the present disclosure are conjugated to a moiety, such as a ligand or therapeutic moiety (an "immunoconjugate"), a second rescue agent, or any other therapeutic moiety useful for reversing the hemodynamic effects of an anti-NPR1 antibody.

[0085] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds to an anti-NPR1 antibody is substantially free of Abs that specifically bind to antigens other than the anti-NPR1 antibody).

[0086] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biomolecular interactions by detection of changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0087] As used herein, the term "K D " is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction. In the context of this disclosure, an "antigen" of an immunoglobulin protein is an anti-NPR1 antibody (e.g., R5381) or an antigen-binding fragment thereof (i.e., a fragment of the antibody that binds to NPR1).

[0088] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, also known as a paratope. The "antigen" of an immunoglobulin protein is an anti-NPR1 antibody (e.g., R5381) or an antigen-binding fragment thereof (i.e., a fragment of an antibody that binds to NPR1). A single antigen may have more than one epitope. Thus, different immunoglobulin proteins (antibodies) may bind to different regions on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B and / or T cells respond. It also refers to the region of an antigen that is bound by an antibody. Epitopes are defined as structural or functional. Functional epitopes are generally a subset of structural epitopes, with their residues directly involved in the affinity of the interaction. Epitopes are also conformational, i.e., they contain non-linear amino acids. In certain embodiments, epitopes include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and in certain embodiments, have specific three-dimensional structural characteristics, and / or specific alteration characteristics.

[0089] As used herein, the term "cross-compete" means that an antibody or an antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or an antigen-binding fragment thereof. The "antigen" of an immunoglobulin protein is an anti-NPR1 antibody (e.g., R5381) or an antigen-binding fragment thereof (i.e., a fragment of an antibody that binds to NPR1). The term also includes competition between two antibodies in both directions, i.e., a first antibody that binds to a second antibody and blocks the binding of the second antibody, and vice versa. In certain embodiments, the first and second antibodies bind to the same epitope. Alternatively, the first and second antibodies bind to different but overlapping epitopes such that the binding of one inhibits or blocks the binding of the second antibody, for example, through steric hindrance. Cross-competition between antibodies is measured by methods known in the art, for example, by real-time, label-free biolayer interferometry assays. Cross-competition between two antibodies is expressed as the binding of the second antibody that is less than the background signal due to self-to-self binding (where the first and second antibodies are the same antibody). Cross-competition between two antibodies is expressed as the % binding of the second antibody that is less than, for example, baseline self-to-self background binding (where the first and second antibodies are the same antibody).

[0090] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is a nucleotide sequence identity of at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule, in certain instances, encodes a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0091] The term "substantial similarity" or "substantially similar" as applied to polypeptides means that two peptide sequences, when optimally aligned, for example by the programs GAP or BESTFIT with default gap weights, share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity. Preferably, residue positions are not identical but differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity is adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0092] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using a measure of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which are used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as between homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG Version 6.1. Polypeptide sequences are also compared using FASTA, a program in GCG Version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) results in alignment and percent sequence identity of the region of optimal overlap between the query and search sequences (Pearson, 2000, supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410, and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0093] By the phrase "therapeutically effective amount" is meant an amount that produces the desired effect in those to whom it is administered. The exact amount depends on the purpose of the treatment and can be ascertained by those skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). For example, a therapeutically effective amount of a rescue agent according to the present invention is, in one embodiment, an amount that produces some degree of reversal of the hemodynamic effect of a natriuretic peptide receptor 1 (NPR1) agonist.

[0094] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human. In certain embodiments of the present disclosure, the subject has experienced, is experiencing, or may experience a drop in blood pressure or other changes in hemodynamic parameters associated with the administration of an agonist antibody or antigen-binding fragment thereof that specifically binds to NPR1.

[0095] As used herein, the terms "treat", "treating" or "treatment" refer to the reduction or amelioration of the severity of at least one symptom or sign of a disease or disorder by administering a therapeutic agent, such as an immunoglobulin protein described herein, to a subject in need thereof. The term includes the inhibition of progression of the disease or the inhibition of the worsening of symptoms / signs. The term may also include a positive prognosis of the disease, i.e., the subject is free of the disease or has a remission of the disease upon administration of a therapeutic agent, such as an antibody of the present disclosure. The therapeutic agent is administered to the subject in a therapeutic dose.

[0096] The terms "prevent," "preventing," or "prevention" refer to the inhibition of the onset of a disease or disorder or any symptom or sign of such a disease or disorder upon administration of a therapeutic agent.

[0097] Preparation of human immunoglobulin proteins Immunoglobulin variable (antigen-binding) domains specific for a particular antigen are prepared by any antibody generation technique known in the art. Once obtained, they are appropriately arranged to produce the immunoglobulin protein molecules of the present disclosure using conventional methods. (Descriptions of exemplary immunoglobulin protein components and formats that can be used to construct the immunoglobulin protein molecules of the present disclosure are provided elsewhere herein.) In certain embodiments, one or more of the individual components of the proteins of the present disclosure (e.g., heavy and light chain variable regions) are derived from chimeric, humanized, or fully human antibodies. Methods for making such antibodies are well known in the art. For example, human antibodies are generated in transgenic mice.

[0098] Any such known method can be used in the context of the present disclosure to generate human antibodies that specifically bind to an anti-NPR1 antibody. In one embodiment, the anti-NPR1 antibody is R5381.

[0099] An immunogen comprising any one of the following is used to generate antibodies against anti-NPR1 antibodies. In certain embodiments, the immunoglobulin proteins (antibodies) of the present disclosure are obtained from mice immunized with full-length anti-NPR1 antibodies (e.g., R5381) or DNA encoding the protein or a fragment thereof. Alternatively, the protein or a fragment thereof can be produced and modified using standard biochemical techniques and used as an immunogen.

[0100] In certain embodiments, the immunogen may be a recombinant anti-NPR1 antibody or antigen-binding fragment thereof expressed in E. coli or any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells.

[0101] One or more of the heavy and / or light chains of the immunoglobulin protein of the present disclosure are prepared using VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®), or any other known method for generating monoclonal antibodies. Using VELOCIMMUNE® technology, a high affinity chimeric antibody against an anti-NPR1 antibody (e.g., R5381) having a human variable region and a mouse constant region is first isolated. VELOCIMMUNE® technology involves the creation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region sites such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[0102] As in the experimental section below, antibodies are characterized and selected for desired characteristics including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to generate a fully human antibody of the disclosure, e.g., wild-type or modified IgG1 or IgG4. While the constant region selected will vary according to the particular use, the high affinity antigen binding and target specificity characteristics reside in the variable regions.

[0103] biological equivalent The immunoglobulin proteins of the present disclosure include proteins that have a different amino acid sequence from the described antibodies, but maintain the ability to bind to an anti-NPR1 antibody, e.g., R5381. Such variant proteins contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity that is substantially equivalent to that of the described protein (e.g., an antibody). Similarly, the DNA sequences of immunoglobulin proteins (e.g., encoding antibodies) include sequences that contain one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encode antibodies or antibody fragments that are substantially biologically equivalent to the immunoglobulin proteins (e.g., antibodies or antibody fragments) of the present disclosure.

[0104] Two antigen-binding proteins, or antibodies, are considered bioequivalents if, for example, their rate and extent of absorption are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences when administered under similar experimental conditions at the same molar, single or multiple doses. An antibody is considered to be equivalent in its extent of absorption but not in its rate of absorption, and would still be considered to be bioequivalent because such differences in rate of absorption are intentional, reflected in the label, and are not essential, for example, to achieving effective body drug concentrations during chronic use, and are not considered to be pharmaceutical significant for the particular drug product being studied.

[0105] In one embodiment, two antigen binding proteins are biologically equivalent if there are no clinically meaningful differences in the safety, purity, or efficacy of the two immunoglobulin proteins.

[0106] In one embodiment, two immunoglobulin proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without expecting a clinically significant change in immunogenicity or an increased risk of side effects, including impaired efficacy, compared to continuing therapy without switching.

[0107] In one embodiment, two immunoglobulin proteins are biologically equivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, so long as such mechanisms are known.

[0108] Bioequivalence is demonstrated by in vivo and / or in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo studies in humans or other mammals where the concentration of the immunoglobulin protein or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro studies that correlate with and sufficiently predict human in vivo bioavailability data; (c) in vivo studies in humans or other mammals where the relevant acute pharmacological effects of the immunoglobulin protein (e.g., an antibody (or its target)) are measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the immunoglobulin protein.

[0109] Biologically equivalent variants of the immunoglobulin proteins (e.g., antibodies) of the present disclosure are constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. The "biological activity" of the immunoglobulin proteins (e.g., antibodies or antibody fragments that specifically bind to R5381) of the present disclosure includes, but is not limited to, specifically binding to an antigen (e.g., anti-NPR1 antibody R5381), reversing the hemodynamic effects of the anti-NPR1 antibody, and increasing blood pressure that has fallen as a result of administration of the anti-NPR1 antibody. Blood flow, cardiac load, and / or heart rate are also positively affected in certain embodiments. For example, cysteine ​​residues that are not essential for biological activity are deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during refolding. In other contexts, biologically equivalent immunoglobulin proteins include antibody variants that include amino acid changes to modify the glycosylation characteristics of the antibody, for example, mutations that remove or eliminate glycosylation.

[0110] Immunoglobulin Proteins Containing Fc Variants According to certain embodiments of the present disclosure, there are provided immunoglobulin proteins comprising an Fc domain that comprises one or more mutations that enhance or decrease binding of the antibody to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides immunoglobulin proteins that bind to anti-NPR1 antibodies and have a C-terminal region of the Fc domain that enhances or decreases binding of the antibody to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HThe immunoglobulin proteins include those containing mutations in the 3 region that increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., within an endosome where the pH ranges from about 5.5 to about 6.0). Such mutations may result in an increased serum half-life of the immunoglobulin protein when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0111] For example, the disclosure includes immunoglobulin proteins that include an Fc domain that includes one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations, as well as other mutations in the antibody variable domains disclosed herein, are contemplated within the scope of this disclosure.

[0112] The present disclosure also relates to a first C H 3 domain and second Ig C H It contains three domain immunoglobulin proteins, the first and second Ig C H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the immunoglobulin protein to Protein A compared to a protein lacking the amino acid difference. H The 3 domain binds to protein A and the second Ig C H The C3 domain contains a mutation that reduces or abolishes Protein A binding, such as H315R (EU numbering). H In one embodiment, the first Ig C 3 can further comprise Y316F according to EU numbering. H The 3 domain binds to protein A and the second Ig C H The C3 domain contains a mutation that reduces or abolishes Protein A binding, such as the H95R modification (according to the IMGT exon numbering; H435R by EU numbering). H3 can further include a Y96F modification (by IMGT; Y436F by EU). See, e.g., U.S. Patent No. 8,586,713. H Additional modifications found within 3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU); in the case of IgG2 antibodies, N44S, K52N, and V82I (by IMGT; N384S, K392N, and V422I by EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

[0113] The present disclosure also provides a chimeric heavy chain constant (C H ) region, wherein the chimeric C H A region may contain more than one C H For example, the immunoglobulin proteins of the present disclosure include segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H C from a human IgG1, human IgG2 or human IgG4 molecule in combination with some or all of the three domains H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibodies of the present disclosure may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2 or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues at positions 228 to 236 according to EU numbering) derived from a human IgG1, human IgG2 or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric C described herein may be H Antibodies comprising the region can, in certain embodiments, exhibit modified Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Patent Application Publication No. 2014 / 0243504, the disclosure of which is incorporated herein by reference in its entirety).

[0114] Biological Characteristics of Immunoglobulin Proteins In general, the immunoglobulin proteins of the present disclosure function by binding to and reversing the hemodynamic effects of an NPR1 agonist (such as an anti-NPR1 antibody). For example, the present disclosure provides immunoglobulin proteins with a K of less than about 3 nM at 25° C. and less than about 7 nm at 37° C. as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 3 herein. D In certain embodiments, the antibody or antigen-binding fragment thereof has a K of less than about 25 nM, less than about 10 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.75 nM, or less than about 0.5 nM, as measured by surface plasmon resonance, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay. D It binds to the parent hybridoma of the anti-NPR1 antibody.

[0115] The present disclosure also includes immunoglobulin proteins and fragments thereof that bind to NPR1 agonists in a pH-sensitive manner. For example, the k d The k and dissociation half-life (t1 / 2) change in buffers at pH 7.4, pH 6.5, pH 6.0, and pH 5.0, as shown in Example 5 herein. For REGN9035, the dissociation half-life (t1 / 2) decreases significantly with decreasing pH, from about 12 minutes to about 0.6 minutes; for REGN9037, the dissociation half-life (t1 / 2) changes slightly with decreasing pH, from about 13 minutes to about 17 minutes. d also varies similarly, between about 9.96E-04 and 1.97E-02 for REGN9035 and between about 8.67E-04 and 6.64E-04 for REGN9037.

[0116] The present disclosure also includes immunoglobulin proteins and fragments thereof that inhibit the activation of hNPR1 induced by anti-NPR1 antibodies. For example, bivalent and monovalent anti-R5381 antibodies and antigen-binding fragments thereof block R5381-induced hNPR1 activation in the presence and absence of endogenous ligands (e.g., ANP, BNP). Maximum inhibition (about 97% to about 106%) of R5381-induced NPR1 activation is measured by cGMP accumulation, for example, using an assay format as defined in Example 6 herein, or a substantially similar assay. In certain embodiments, an antibody or antigen-binding fragment of the disclosure inhibits anti-NPR1 antibody-induced hNPR1 activation by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, or at least about 105%, at least about 106%.

[0117] The present disclosure also includes immunoglobulin proteins and fragments thereof that block the binding of anti-NPR1 antibodies to hNPR1. For example, bivalent and monovalent anti-R5381 antibodies and antigen-binding fragments thereof block the binding of biotin-R5381 to human NPR1, as assessed using a blocking ELISA, or a substantially similar assay, as described in Example 7. Blocking of about 99% and about 100% was assessed for monovalent / one arm and bivalent anti-R5381 antibodies, respectively. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure block the binding of anti-NPR1 antibodies to hNPR1 by about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0118] The present disclosure also includes immunoglobulin proteins and fragments thereof that form minimal or no circulating immune complexes (CICs) with agonist anti-NPR1 antibodies.For example, bivalent and monovalent anti-R5381 antibodies and antigen-binding fragments thereof do not form detectable CICs with R5381 when assessed using Clq-CIC assay, or a substantially similar assay, as described in Example 8.In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure do not form CICs with anti-NPR1 antibodies.The presence of CICs in circulation is statistically correlated with disease activity.

[0119] The present disclosure also includes immunoglobulin proteins and fragments thereof that reverse the hemodynamic effects of anti-NPR1 antibodies. For example, bivalent anti-R5381 antibodies and antigen-binding fragments thereof can clear R5381 from mice more quickly and more effectively than isotype controls, as assessed by immunoassays, or substantially similar assays, as described in Example 10. Serum concentrations of R5381 dropped significantly by day 7 and were undetectable by day 22. As another example, bivalent and monovalent anti-R5381 antibodies and antigen-binding fragments thereof rapidly and persistently reversed the blood pressure lowering effects of R5381, as assessed by collecting systolic, diastolic, pulse, and mean arterial pressures, and heart rate, or substantially similar assays, as described in Examples 11 and 12. Bivalent and monovalent anti-R5381 antibodies and antigen-binding fragments thereof also inhibited NPR1-induced cGMP production, as assessed in urine using ELISA, or substantially similar assays, as described in Examples 11 and 12. In certain embodiments, the antibodies or antigen-binding fragments of the disclosure increase blood pressure back to baseline levels (ie, back to the levels prior to the drop in blood pressure due to administration of the anti-NPR1 antibody).

[0120] In one embodiment, the disclosure provides an isolated recombinant immunoglobulin protein or antigen-binding fragment thereof that specifically binds to an anti-NPR1 antibody (such as R5381), where the immunoglobulin protein exhibits one or more of the following characteristics: (a) comprises a fully human monoclonal antibody; (b) comprises a fully human monovalent or one-armed antibody; (c) comprises a multimerization component comprising a single immunoglobulin domain and at least one Fc fragment; (d) has a dissociation constant (K) of less than 7 nM at 25° C. and 37° C. as measured by surface plasmon resonance assay. D) binds to anti-NPR1 antibodies; (e) exhibits pH-dependent dissociation; (f) inhibits anti-NPR1 antibody-induced activation of NPR1 by about 97% to about 106%; (g) blocks at least some biotin anti-NPR1 agonist antibodies from binding to human NPR1; (h) does not form a detectable CIC with anti-NPR1 antibodies; (i) clears anti-NPR1 antibodies from serum more rapidly than an isotype control; (j) reverses the blood pressure lowering effect of anti-NPR1 antibodies; and (k) comprises a HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1, and a LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

[0121] The immunoglobulin proteins of the present disclosure may have one or more of the biological characteristics described above, or any combination thereof. Other biological properties of the immunoglobulin proteins of the present disclosure will be apparent to those of skill in the art from a consideration of this disclosure, including the Examples herein.

[0122] Epitope mapping and related techniques The present disclosure includes immunoglobulin proteins that interact with one or more amino acids found in one or more regions of an anti-NPR1 antibody molecule. The epitope that the immunoglobulin protein binds to consists of a single continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located in any of the aforementioned domains of the anti-NPR1 antibody molecule (e.g., a linear epitope in a domain). Alternatively, the epitope consists of a number of non-contiguous amino acids (or amino acid sequences) located in any or both of the aforementioned domains of the antibody molecule (e.g., a conformational epitope).

[0123] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" in a polypeptide or protein. Exemplary techniques include conventional cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide truncation analysis crystallography studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens are utilized (Tomer (2000) Prot. Sci. 9:487-496). Another method used to identify the amino acids in a polypeptide that an antibody interacts with is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium labeling of a protein of interest, followed by binding of an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo back exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface retain deuterium and therefore exhibit a relatively large mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to proteolytic enzyme cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0124] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. In the context of this disclosure, the antigen is an anti-NPR1 antibody. B cell epitopes are formed from both contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically include at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation.

[0125] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) directed against the same antigen by the similarity of the binding profile of each antibody to a chemically or enzymatically modified antigen surface (US2004 / 0101920, specifically incorporated herein by reference in its entirety). Each category reflects a unique epitope that is either distinct or partially overlapping with the epitope represented by another category. This technique allows for rapid sorting of genetically matched antibodies so that characterization can be focused on genetically defined antibodies. When applied to hybridoma screening, MAP facilitates the identification of rare hybridoma clones that produce mAbs with desired characteristics. Using MAP, the antibodies of the present disclosure are classified into groups of antibodies that bind to different epitopes.

[0126] In certain embodiments, the disclosure includes immunoglobulin proteins and fragments thereof that interact with one or more epitopes found within the extracellular domain of an anti-NPR1 antibody. An epitope may consist of one or more contiguous sequences of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids found within the extracellular domain of an anti-NPR1 antibody. Alternatively, an epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within an anti-NPR1 antibody.

[0127] The present disclosure includes antibodies that bind to the same epitope, or a portion of an epitope, as any of the specific representative immunoglobulin proteins listed in Table 1. Similarly, the present disclosure also includes antibodies that compete with any of the specific representative immunoglobulin proteins listed in Table 1 for binding to an anti-NPR1 antibody or fragment thereof. For example, the present disclosure includes antibodies that cross-compete with one or more of the antibodies listed in Table 1 for binding to an anti-NPR1 antibody or fragment thereof.

[0128] By using routine methods known in the art, it can be easily determined whether an antibody binds to the same epitope as a reference immunoglobulin protein or competes with a reference immunoglobulin protein for binding. For example, to determine whether a test antibody binds to the same epitope as a reference immunoglobulin protein of the present disclosure, the reference antibody is allowed to bind to an anti-NPR1 antibody protein or peptide under saturating conditions. The ability of the test antibody to bind to the anti-NPR1 antibody protein molecule is then evaluated. If the test antibody can bind to the anti-NPR1 antibody after saturation binding with the reference immunoglobulin protein, it is concluded that the test antibody binds to a different epitope than the reference anti-immunoglobulin protein. On the other hand, if the test antibody cannot bind to the anti-NPR1 antibody protein after saturation binding with the reference immunoglobulin protein, then the test antibody binds to the same epitope as the epitope bound by the reference immunoglobulin protein of the present disclosure.

[0129] To determine whether an antibody competes with a reference immunoglobulin protein for binding, the binding methodology described above is carried out in two ways. In the first way, the reference antibody is allowed to bind to the anti-NPR1 antibody protein under saturating conditions, and then the binding of the test antibody to the anti-NPR1 antibody molecule is evaluated. In the second way, the test antibody is allowed to bind to the anti-NPR1 antibody molecule under saturating conditions, and then the binding of the reference antibody to the anti-NPR1 antibody molecule is evaluated. In both ways, if only the first (saturating) antibody is capable of binding to the anti-NPR1 antibody molecule, then it is concluded that the test antibody and the reference antibody compete for binding to the anti-NPR1 antibody. As will be understood by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but sterically blocks the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0130] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. The antigen is an anti-NPR1 antibody. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0131] Further routine experiments (e.g., peptide mutations and binding analysis) are then performed to determine whether the observed lack of binding of the test antibody is due in fact to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This screening experiment is performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0132] Immunoconjugates The present disclosure further encompasses human immunoglobulin proteins conjugated to therapeutic moieties ("immunoconjugates") for treating NPR1-related diseases or disorders (e.g., hypertension) and / or improving hemodynamic effects associated with therapeutic use of anti-NPR1 antibodies. As used herein, the term "immunoconjugate" refers to an immunoglobulin protein that is chemically or biologically bound to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The protein is bound to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide, or therapeutic agent at any position along the molecule as long as it can bind to its target, the anti-NPR1 antibody. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent is a second, different antibody against the NPR1 protein. The type of therapeutic moiety bound to the rescue agent takes into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art, see, for example, WO05 / 103081.

[0133] Therapeutic Administration and Formulation The present disclosure provides therapeutic compositions comprising the immunoglobulin proteins of the present disclosure. The therapeutic compositions are administered with suitable carriers, excipients, and other agents that are incorporated into the formulation to effect transport, delivery, improved tolerance, and the like, in accordance with the present disclosure. A large number of suitable formulations can be found in a formulary known to any medicinal chemist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0134] The dose of the immunoglobulin protein may vary depending on the age and size of the subject to be administered, the condition, the route of administration, etc. When the protein of the present disclosure is used to reverse the hemodynamic effects of anti-NPR1 antibodies in adult patients or to prevent such hemodynamic effects, it is advantageous to administer the immunoglobulin protein of the present disclosure at a single dose, usually about 0.1 to about 100 mg per kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment are adjusted. In certain embodiments, the immunoglobulin protein of the present disclosure is administered as a starting dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 500 mg, or about 10 to about 400 mg. In certain embodiments, the initial dose is followed by administration of a second or multiple subsequent doses of an immunoglobulin protein or antigen-binding fragment thereof in an amount that is about the same as or less than that of the initial dose, where the subsequent doses are separated by at least 1 to 3 days; at least 1 week; at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0135] A variety of delivery systems are known and are used to administer the pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions are administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and are administered together with other biologically active agents. Administration is systemic or local. Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (see, eg, Langer (1990) Science 249:1527-1533).

[0136] The use of nanoparticles to deliver the immunoglobulin proteins of the present disclosure is also contemplated herein. Antibody-conjugated nanoparticles are used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles, as well as preparation and use methods, are described in detail in Arruebo, M. et al., 2009 ("Antibody-conjugated nanoparticles for biomedical applications," J. Nanomat, Vol. 2009, Article No. 439389, p. 24, doi:10.1155 / 2009 / 439389), which is incorporated herein by reference. Nanoparticles are developed and conjugated to antibodies contained in pharmaceutical compositions for targeting cells. Nanoparticles for drug delivery are also described, for example, in US8257740, or US8246995, each of which is incorporated herein in its entirety.

[0137] In some situations, pharmaceutical compositions are delivered in controlled release systems.In one embodiment, pumps are used.In another embodiment, polymeric materials are used.In yet another embodiment, the controlled release system is placed close to the target of the composition, and therefore only a small systemic dose is required.

[0138] The injectable preparations include intravenous, subcutaneous, intracranial, intraperitoneal and intramuscular injection forms, drip infusion forms, etc. These injectable preparations are prepared by publicly known methods.

[0139] The pharmaceutical composition of the present disclosure is delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is easily applied in the delivery of the pharmaceutical composition of the present disclosure. Such a pen delivery device may be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered, the cartridge is emptied, and the empty cartridge is easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device is then reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition that is held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0140] Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are prepared in dosage forms in unit doses suitable for the dosage of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of immunoglobulin protein contained is generally about 5 to about 500 mg per dosage form in unit doses; in particular, about 5 to about 300 mg for other dosage forms, and about 10 to about 300 mg of immunoglobulin protein is preferably contained per dosage form.

[0141] Therapeutic Uses of Immunoglobulin Proteins The present disclosure includes a method comprising administering a therapeutic composition comprising an immunoglobulin protein to a subject in need thereof.The therapeutic composition can comprise any of the immunoglobulin proteins disclosed herein and a pharma- ceutically acceptable carrier or diluent.As used herein, the term "subject in need thereof" refers to a human or non-human animal that will benefit from the increase in blood pressure or the reversal of the effect of administering an NPR1 agonist.

[0142] The immunoglobulin protein of the present disclosure (and therapeutic compositions comprising same) is useful, inter alia, for the treatment of any disease or disorder in which an increase in blood pressure is beneficial. In particular, the immunoglobulin protein of the present disclosure is used for the treatment, prevention, and / or amelioration of any disease or disorder related to or mediated by the expression or activity of NPR1. The mechanism of action by which the therapeutic method of the present disclosure is achieved involves binding to agonist anti-NPR1 antibody and removal / clearance of the agonist antibody. Removal of agonist anti-NPR1 antibody results in an increase in blood pressure.

[0143] The immunoglobulin protein of the present disclosure can be used to treat any NPR1-related disease or disorder, for example, in a subject administered an NPR1 agonist, and reversal of the hemodynamic effects of the NPR1 agonist is desired. Examples of NPR1-related diseases or disorders include, but are not limited to, hypertension, heart failure, obesity, renal failure, chronic kidney disease, macular edema, glaucoma, stroke, lung injury, pulmonary fibrosis, inflammation, asthma, skeletal growth disorder, fracture, diabetes, and cancer. Administration of a therapeutic composition comprising the immunoglobulin protein of the present disclosure can lead to the prevention of one or more adverse events associated with lowering blood pressure. Potential adverse events associated with lowering blood pressure include persistent symptomatic hypotension, reflex tachycardia due to compensatory sympathetic nervous system response (which may increase the risk of myocardial infarction, stroke, arrhythmia, heart failure), and reduced cardiac output and end-organ perfusion in subjects with normal (low) venous pressure.

[0144] In one embodiment, the immunoglobulin proteins of the present disclosure are used for the manufacture of a pharmaceutical composition or medicament for treating a patient with hypotension as a result of administration of an anti-NPR1 antibody. In another embodiment, the immunoglobulin proteins are used as adjunctive therapy with any other agent or treatment known to one of skill in the art that is useful for increasing blood pressure and / or addressing symptoms associated with lowering blood pressure.

[0145] The present disclosure also provides a composition comprising: (i) a therapeutic amount of an immunoglobulin protein disclosed herein; and (ii) an agonist anti-NPR1 antibody for use in a method for effectively regulating blood pressure and / or hemodynamic changes in a subject suffering from an NPR1-related disease or disorder.

[0146] Non-immunoglobulin protein reversal agent In certain embodiments of the present disclosure, the agent for use in reversing the hemodynamic effects of an anti-NPR1 antibody or antigen-binding fragment thereof (i.e., a reversal agent) is selected from the group consisting of hypertensive agents, alpha-adrenergic receptor agonists, steroids, antidiuretic hormones, vascular endothelial growth factor (VEGF) antagonists / angiogenesis inhibitors, and small molecule agents that increase blood pressure.

[0147] A vasopressor is a drug that constricts blood vessels and increases blood pressure. Alpha-adrenergic receptor agonists (α-agonists) bind to α-receptors on vascular smooth muscle, inducing smooth contraction and vasoconstriction, and increasing blood pressure. Antidiuretic hormone is a hormone released from the posterior pituitary gland that acts on the kidney to increase water reabsorption and cause vasoconstriction in the cardiovascular system. Vasopressors and antidiuretic hormone are known in the art.

[0148] In another embodiment, the reversal agent is a drug for treating anti-NPR1 antibody-induced hemodynamic effects selected from the group consisting of, but not limited to, Midodrine, Levophed, Norepinephrine, Phenylephrine, Fludrocortisone, Orvaten, Northera, Ephedrine, Vazculep, Droxidopa, Akovaz, Biorphen, Corphedra, and Emerphed.

[0149] Thus, the present disclosure includes reversal agents other than immunoglobulin proteins (including antibodies) that can reverse the hemodynamic effects of anti-NPR1 antibody therapy. For example, midodrine, an α1-adrenergic receptor agonist, can reverse the blood pressure and heart rate effects of R5381, as described in Example 14 herein.

[0150] Combination therapy In certain embodiments, it is contemplated that the immunoglobulin protein of the present disclosure and other reversal agents are used in combination with one or more other known antihypertensive therapies to control the blood pressure of a subject.In certain embodiments, the immunoglobulin protein of the present disclosure is used in combination with an NPR1 agonist, preferably an anti-NPR1 antibody.In one particular embodiment, the immunoglobulin protein of the present disclosure is used in combination with R5381 for effective control of blood pressure in a subject in need thereof.

[0151] Combination therapy can include the immunoglobulin proteins of the present disclosure and any additional therapeutic agent that can be advantageously combined with the immunoglobulin proteins of the present disclosure, or biologically active fragments of the immunoglobulin proteins of the present disclosure. The immunoglobulin proteins of the present disclosure can be synergistically combined with one or more drugs or therapies used to increase blood pressure or address effects related to lowering blood pressure.

[0152] As used herein, the term "in combination with" means that an additional therapeutically active ingredient can be administered prior to, simultaneously with, or after administration of the immunoglobulin protein or other reversing agent of the present disclosure. The term "in combination with" also includes sequential or simultaneous administration of the immunoglobulin protein or other reversing agent and a second therapeutic agent.

[0153] In certain embodiments, the immunoglobulin protein of the present disclosure is administered to a subject in combination with an NPR1 agonist, such as R5381. In further embodiments, the immunoglobulin protein of the present disclosure and the NPR1 agonist, such as R5381, are administered to a subject simultaneously (at the same time point), either together in one composition or separately in more than one composition. In yet further embodiments, the immunoglobulin protein of the present disclosure and the NPR1 agonist, such as R5381, are administered to a subject sequentially, with the immunoglobulin protein of the present disclosure administered after the NPR1 agonist, such as R5381.

[0154] The additional therapeutic active component is administered to the subject prior to administration of the immunoglobulin protein or other reversal agent of the present disclosure. For example, if the first component is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less than 30 minutes before administration of the second component, the first component is considered to be administered "before" the second component. In other embodiments, the additional therapeutic active component is administered to the subject after administration of the immunoglobulin protein or other reversal agent of the present disclosure. For example, if the first component is administered 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or more after administration of the second component, the first component is considered to be administered "after" the second component. In yet other embodiments, an additional therapeutically active ingredient is administered to the subject simultaneously with administration of the immunoglobulin protein or other reversal agent of the present disclosure.

[0155] For purposes of this disclosure, "concurrent" administration includes administration of an immunoglobulin protein or other reversal agent and an additional therapeutically active ingredient to a subject in a single dosage form, for example, administered to the subject within about 30 minutes of each other, or in separate dosage forms. When administered in separate dosage forms, each dosage form is administered via the same route (e.g., both the immunoglobulin protein or other reversal agent and the additional therapeutically active ingredient are administered intravenously); or, each dosage form is administered via a different route (e.g., the immunoglobulin protein or other reversal agent is administered intravenously and the additional therapeutically active ingredient is administered orally). In any case, administration of the ingredients in a single dosage form, or separate dosage forms, or separate dosage forms by the same route, are all considered "concurrent administration" for purposes of this disclosure. For purposes of this disclosure, administration of an immunoglobulin protein or other reversal agent "before," "concurrently," or "after" (as these terms are defined herein above) administration of an additional therapeutically active ingredient is considered administration of the immunoglobulin protein or other reversal agent "in combination with" the additional therapeutically active ingredient.

[0156] The present disclosure includes pharmaceutical compositions in which the immunoglobulin proteins or other reversal agents of the present disclosure are co-formulated with one or more additional therapeutically active ingredients as described elsewhere herein.

[0157] Diagnostic Uses of Immunoglobulin Proteins The rescue agent of the present disclosure is used to detect and / or measure anti-NPR1 antibodies in a sample, for example, for diagnostic purposes. An exemplary diagnostic assay for anti-NPR1 antibodies can include, for example, contacting a sample obtained from a patient with a rescue agent of the present disclosure, where the rescue agent is labeled with a detectable label or reporter molecule or is used as a capture ligand to selectively isolate anti-NPR1 antibodies from a patient sample. Alternatively, an unlabeled rescue agent can be used for diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C.32 P, 35 S, or 125 The antibody may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure anti-NPR1 antibodies in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0158] The sample that can be used in the anti-NPR1 antibody diagnostic assay according to the present disclosure includes any tissue or fluid sample that can be obtained from a patient, which contains detectable amounts of anti-NPR1 antibody protein or any of its fragments after administration to a subject.Generally, the level of anti-NPR1 antibody protein in a particular sample obtained from a healthy patient (e.g., a patient who has not received anti-NPR1 antibody) is measured to first establish a baseline or standard level of anti-NPR1 antibody.This baseline level of anti-NPR1 antibody can then be compared with the level of anti-NPR1 antibody measured in a sample obtained from an individual suspected of receiving anti-NPR1 antibody.

[0159] The immunoglobulin proteins specific for anti-NPR1 antibody protein may not contain additional labels or moieties, or they may contain N-terminal or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if present) can determine the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, the peptide containing N-terminal biotin is oriented so that the C-terminal part of the peptide is distal to the surface. EXAMPLES

[0160] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the disclosed methods and compositions, and are not intended to limit the scope of what the inventors regard as the invention. Attempts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric. EXAMPLES

[0161] Example 1: Generation of human antibodies against agonist antibodies that specifically bind to natriuretic peptide receptor 1 (NPR1) Preparation of anti-R5381 antibody Human antibodies against an anti-NPR1 antibody protein, which contains DNA encoding a human immunoglobulin heavy chain variable region and a kappa light chain variable region, were generated in VELOCIMMUNE® mice, which were immunized with the anti-NPR1 antibody R5381 (described elsewhere herein).

[0162] Antibody immune response was monitored by anti-R5381 specific immunoassay. When the desired immune response was achieved, splenocytes were collected and fused with mouse myeloma cells to maintain viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produce anti-R5381 specific antibodies. The cell lines were used to obtain several anti-R5381 chimeric antibodies (e.g., antibodies with human variable domains and mouse constant domains).

[0163] Anti-R5381 antibodies were also isolated directly from antigen-positive mouse B cells without fusion with myeloma cells, as described in U.S. Patent No. 7,582,298, the entirety of which is specifically incorporated herein by reference. Using this method, several fully human anti-R5381 antibodies (i.e., antibodies with human variable and constant domains) were obtained.

[0164] Exemplary antibodies generated as disclosed above were designated mAb36312 and mAb36313.

[0165] Generation of "one-arm" anti-R5381 antibody The selected antibodies generated above were used to generate monovalent or "one-arm" antibodies or antigen-binding molecules. Such monovalent antigen-binding molecules contain a single R5381 binding moiety that includes an HCVR and an LCVR. Monovalent antibodies were constructed using standard methodologies (see WO2010151792) that include a full-length heavy chain, a full-length light chain, and an additional Fc domain polypeptide, with the heavy chain constant region differing from the Fc domain polypeptide by at least two amino acids. Such modifications are useful for purifying monovalent antibodies (see WO2010151792).

[0166] Exemplary one-arm antibodies with an IgG4 Fc domain were generated and designated REGN9035 and REGN9037.

[0167] The biological properties of exemplary antibodies generated according to the methods of this Example are described in detail in the Examples below. EXAMPLES

[0168] Example 2: Heavy and light chain variable region amino acid and nucleotide sequences Table 1 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-R5381 antibodies of the disclosure.

[0169] [Table 1]

[0170] The corresponding nucleic acid sequence identifiers are shown in Table 2.

[0171] [Table 2]

[0172] Antibodies referred to herein typically have fully human variable regions, but may have human or mouse constant regions. As one of skill in the art will appreciate, an antibody with a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted to an antibody with a human IgG4, etc.), but in each case the variable domains (including CDRs) indicated by the numerical identifiers shown in Table 2 remain the same, and the antigen binding characteristics are expected to be consistent or substantially similar, regardless of the nature of the Fc domain. In certain embodiments, selected antibodies with mouse IgG1 Fc were converted to antibodies with human IgG4 Fc. In one embodiment, the IgG4 Fc domain contains two or more amino acid changes as disclosed in US20100331527. In one embodiment, the human IgG4 Fc contains a serine to proline mutation in the hinge region (S108P) to promote dimer stabilization. Unless otherwise indicated, all antibodies used in the following examples contain the human IgG4 isotype.

[0173] An exemplary bivalent antibody comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO:2 / 10 is REGN6580. REGN6580 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:18 and a light chain comprising the amino acid sequence of SEQ ID NO:20.

[0174] An exemplary bivalent antibody comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO:22 / 30 is REGN6581. REGN6581 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38 and a light chain comprising the amino acid sequence of SEQ ID NO:40.

[0175] The selected anti-R5381 antibody was used to construct a one-arm antibody comprising an anti-R5381 binding arm and an additional Fc polypeptide (or truncated heavy chain). In certain embodiments, the anti-R5381 binding arm comprises a heavy chain constant region of an IgG1, IgG2, IgG3, IgG4 isotype or a variant thereof. In one embodiment, the additional Fc polypeptide is of an IgG1 isotype or a variant thereof. In one embodiment, the additional Fc polypeptide is of an IgG4 isotype or a variant thereof.

[0176] Tables 3A, 3B, and 3C list the HCVRs, LCVRs, CDRs, and heavy and light chain sequence identifiers for selected one-arm antibodies.

[0177] [Table 3]

[0178] [Table 4]

[0179] [Table 5]

[0180] Unless otherwise indicated, all antibodies used in the following examples are of the human IgG4 isotype. EXAMPLES

[0181] Example 3: Biacore binding kinetics of selected antibodies The equilibrium dissociation constants (K D) was determined using a real-time surface plasmon resonance (SPR)-based Biacore T200 biosensor. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-ET) running buffer at 25 °C and 37 °C. The Biacore CM5 sensor chip surface was first derivatized by amine coupling with an anti-mouse Fc-specific antibody (GE Healthcare, #BR100838) to capture H2aM22033N. Different concentrations of mAbs (100 nM to 3.7 nM, 3-fold serial dilutions) prepared in HBS-ET running buffer were injected for 3 min at a flow rate of 50 μL / min. Dissociation of different mAbs bound to H2aM22033N was monitored for 10 min in HBS-ET running buffer. At the end of each cycle, the H2aM22033N capture surface was regenerated using a 60 s injection of 10 mM glycine-HCl, pH 1.5. The association rate (k a ) and dissociation rate (k d ) was determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve fitting software. D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rates as follows:

number

[0182] The binding kinetic parameters for the binding of different mAbs to H2aM22033N of the present disclosure at 25° C. and 37° C., respectively, are shown in Tables 4 and 5 below.

[0183] [Table 6]

[0184] [Table 7] EXAMPLES

[0185] Example 4: Cross-competition between different anti-R5381 antibodies Binding competition between anti-R5381 antibodies (mAbs) was determined using a real-time, label-free biolayer interferometry (BLI) assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was carried out at 25°C with plate shaking at a speed of 1000 rpm in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, 1 mg / mL BSA, pH 7.4 (HBS-EBT) buffer. To assess whether the two mAbs could compete with each other for binding to their respective epitopes on R5381, first about 0.47 nm of R5381 was captured on an Octet biosensor chip (Fortebio Inc, #18-5064) coated with anti-human antibody (AHC) by immersing the biosensor chip in a well containing 1.7 μg / mL of R5381 solution for 1 min. The R5381-captured biosensor chip was then saturated with the first anti-R5381 mAb (hereafter referred to as mAb-1) by immersing it in a well containing 50 μg / mL of a solution of mAb-1 for 4 min. The biosensor chip was then subsequently immersed in a well containing 50 μg / mL of a solution of the second anti-R5381 mAb (hereafter referred to as mAb-2) for 3 min. The biosensor chip was washed with HBS-ETB buffer between each step of the experiment. The real-time binding response was monitored during the entire course of the experiment, and the binding response was recorded at the end of each step. The response of mAb-2 binding to R5381 pre-complexed with mAb-1 was compared to determine the competitive / non-competitive behavior of different anti-R5381 mAbs. There was cross-competition between the anti-R5381 mAbs. EXAMPLES

[0186] Example 5: pH sensitivity of anti-R5381 antibodies binding to R5381 Dissociation rate constants (k d ) was determined using a real-time surface plasmon resonance (SPR)-based Biacore 4000 biosensor. All binding studies were performed at 37 °C using four running buffers: (i) PBS, 0.05% v / v of surfactant Tween-20, pH 7.4 (PBS-T-pH7.4), (ii) PBS, 0.05% v / v of surfactant Tween-20, pH 6.5 (PBS-T-pH6.5), (iii) PBS, 0.05% v / v of surfactant Tween-20, pH 6.0 (PBS-T-pH6.0), and (iv) PBS, 0.05% v / v of surfactant Tween-20, pH 5.0 (PBS-T-pH5.0). The Biacore CM5 sensor chip surface was first derivatized by amine coupling with anti-mouse Fc-specific antibody (GE Healthcare, #BR100838) to capture H2aM22033N (parent hybridoma of R5381). Different concentrations of anti-R5381 mAb (100 nM to 11.11 nM, 3-fold serial dilutions) prepared in PBS-T-pH7.4 buffer were injected for 3 min at a flow rate of 30 μL / min, followed by dissociation of bound anti-R5381 mAb for 10 min in PBS-T-pH7.4, PBS-T-pH6.5, PBS-T-pH6.0 or PBS-T-pH5.0 running buffer.

[0187] Dissociation rate constants (k d ) was determined by fitting the real-time binding sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. The dissociation half-life (t1 / 2) was determined by K d The values ​​were calculated as follows:

number

[0188] k for selected anti-R5381 mAbs binding to H2aM22033N in PBS-T, pH 7.4d The t values ​​and subsequent dissociation in PBS-T-pH 7.4, PBS-T-pH 6.5, PBS-T-pH 6.0 or PBS-T-pH 5.0 of the present disclosure at 37° C. are shown in Tables 6, 7, 8, and 9, respectively.

[0189] [Table 8]

[0190] [Table 9]

[0191] [Table 10]

[0192] [Table 11]

[0193] A comparison of the dissociation half-life (t1 / 2) of the R5381 anti-idiotypic monoclonal antibodies REGN9035 and REGN9037 in buffers of pH 7.4, pH 6.5, pH 6.0, and pH 5.0 is shown in Figure 1. EXAMPLES

[0194] Example 6: Inhibition of R5381-induced NPR1 activation To assess regulation of human NPR1 (hNPR1), we developed a stable HEK293 cell line stably expressing hNPR1 with a C-terminal myc and FLAG tag. The cell lines were selected for high expression of hNPR1, abbreviated as HEK293 / hNPR1.MycDDK HS or HEK29 / hNPR1, and maintained in DMEM containing 10% FBS, NEAA, pen / strep / glut, and G418 sulfate 500 μg / mL. Binding of ligand to NPR1 activates the guanylate cyclase domain of the receptor, which catalyzes the production of cGMP from GTP (Zois et al., 2014 Nature 11(7):403-412). NPR1 activity was assessed using a homogeneous time-resolved fluorescence (HTRF) assay that measures cGMP levels.

[0195] For the cGMP assay, HEK293 / hNPR1 cells were seeded at 20,000 cells / well in 96-well half-area plates containing 50 μl of complete growth medium and cultured overnight. The next day, the medium was replaced with 10 μl of dilution buffer (OptiMEM with 0.1% FBS), followed by the addition of 10 μl of 2× agonist at various concentrations, including samples without agonist (see Table 10 below), made in dilution buffer, to induce activation of hNPR1. To evaluate the reversal of R5381-induced hNPR1 activation, cells were treated with 10 μl of 2× anti-R5381 at various concentrations (see Table 10) made in dilution buffer, followed by the addition of 10 μl of 70 nM R5381 made in dilution buffer alone or in dilution buffer containing 40 pM ANP or 80 pM BNP. Treated cells were incubated at 37° C. for 30 minutes. HTRF assay was performed using the cGMP HTRF kit from Cisbio according to the manufacturer's protocol (#62GM2PEH). Briefly, 20 μl of 1× cGMP serial dilutions were added to blank wells for cGMP standard curve. To measure cGMP concentrations in samples containing test articles, 10 μl of cGMP-d2 and 10 μl of anti-cGMP antibody conjugated with cryptate diluted in lysis buffer were added to each well in sequence for 60 min at room temperature in the dark. Fluorescence intensity was detected using an EnVision multilabel plate reader (excitation=320 nm, emission=620 nm / 665 nm, PerkinElmer), and the fluorescence resonance energy transfer (FRET) ratio was calculated using the formula described below:

number

[0196] FRET ratios were converted to cGMP concentrations according to a cGMP standard curve and analyzed using a four-parameter logistic equation across an 11-point dose-response curve to calculate the half effective concentration (EC ) of the tested agonists using GraphPad Prism 8. 50 ) values ​​and the half inhibitory concentration (IC 50) values ​​were obtained. Maximum inhibition was calculated by the formula given below:

number

[0197] In this formula, [cGMP, nM] ベースライン , [cGMP, nM] 試験抗体 , and [cGMP, nM] 70nM R5381 are the cGMP concentration values ​​from cells treated with dilution buffer, the highest concentration of anti-R5381 antibody, and 70 nM R5381 with or without 40 pM ANP or 80 pM BNP, respectively.

[0198] [Table 12]

[0199] result R5381 activated hNPR1 expressed in HEK293 / hNPR1 cells to stimulate cGMP accumulation with EC50 values ​​of 1.78-31.2 nM in the presence or absence of ANP or BNP, and basal levels of cGMP without antibody were increased by a constant amount of ANP or BNP (Table 11 below). The isotype control, REGN1945, showed no measurable activation in dilution buffer. EC50 values ​​for ANP and BNP were not calculated due to the limit of quantitation at high concentrations in this assay compared to the cGMP standard curve.

[0200] All mono-armed and bivalent anti-R5381 antibodies blocked 70 nM R5381-induced hNPR1 activation in the presence or absence of 40 pM ANP or 80 pM BNP, with IC50 values ​​ranging from 15.5 to 57.5 nM and maximum inhibition rates ranging from 97% to 106% (Table 11). The isotype control, REGN1945, did not show significant inhibition of 70 nM R5381-induced hNPR1 activation in the presence or absence of ANP or BNP (Table 11).

[0201] Taken together, all anti-R5381 antibodies showed significant inhibition of R5381-induced hNPR1 activation in the presence or absence of endogenous ligand, as measured by cGMP accumulation.

[0202] [Table 13]

[0203] Anti-R5381 antibodies were found to inhibit R5381-induced hNPR1 activation in the absence of endogenous ligands or in the presence of 40 pM ANP or 80 pM BNP. Cells were incubated with increasing concentrations of ANP, BNP, R5381 or isotype control (REGN1945) alone, or 70 nM R5381, in the presence or absence of a constant concentration of ANP or BNP. Fluorescence intensity was detected using EnVision (excitation = 320 nm, emission = 620 nm / 665 nm), and FRET ratios and cGMP concentrations were calculated as described in the Experimental Procedures. EXAMPLES

[0204] Example 7: Blocking ELISA Assay 1.0 μg / mL of hNPR1.ecto.mmh (of which amino acids 1-441: human NPR1 amino acids G32-E473 from the translation of NM_000906.3, and amino acids 442-469: Myc-Myc-hexahistidine tag; SEQ ID NO: 47) in PBS was coated onto a 96-well microtiter plate and incubated overnight at 4 °C. Subsequently, nonspecific binding sites were blocked using a 0.5% (w / v) solution of BSA in PBS (assay buffer). Anti-R5381 mAb and isotype control mAb were serially diluted 3-fold from 500 nM to 8.46 pM in assay buffer. In a 96-well dilution plate, 285 pM of biotin-R5381 was mixed with the 3-fold serially diluted antibodies and allowed to pre-bind for 1 h at room temperature (RT). The final concentrations of anti-R5381 and isotype control mAbs ranged from 333.33 nM to 5.64 pM, and the final concentration of biotin-R5381 was 95 pM. After incubation at room temperature for 1 h, the pre-binding reaction mixture was transferred to a microtiter plate coated with hNPR1.ecto.mmh. The microtiter plate was incubated at room temperature for 1 h and then washed with plate washing solution. Binding of biotin-R5381 was detected using poly-HRP-streptavidin protein. The plate was incubated with the detection protein for 1 h at room temperature and then washed with plate washing solution. The assay plate was developed using TMB colorimetric substrate according to the manufacturer's recommended procedure.

[0205] The absorbance at 450 nm was recorded for each well and plotted as a function of antibody concentration. Data were analyzed with GraphPad Prism software using a four-parameter logistic equation over an 11-point dose-response curve to calculate IC 50 The calculated IC value, defined as the concentration of antibody required to reduce the binding of biotin-R5381 to immobilized hNPR1.ecto.mmh by 50%, was 50The values ​​were used as an index of blocking potency. The percent blocking at the highest anti-R5381 concentration was calculated as an index of the antibody's ability to block biotin-R5381 binding to NPR1 compared to the baseline of the assay. The baseline signal of the assay, defined as 0% binding of biotin-R5381, was determined from the OD450nm reading from poly-HRP-streptavidin binding in wells containing only assay buffer. The binding signal of 95 pM biotin-R5381 in the absence of anti-R5381 was defined as 100% binding or 0% blocking.

[0206] result The ability of anti-R5381 antibodies to block the binding of biotin-R5381 to human NPR1 was assessed using a blocking ELISA assay. The blocking results are summarized in Table 12 below. Percent blocking calculated at the highest antibody concentration tested (333.33 nM) is reported for all antibodies. REGN9035 had an IC of 2.04 nM. 50 [M] blocked the binding of 95 pM biotin-R5381, demonstrating 99.29% blocking at the highest concentration tested. REGN9037 had an IC of 2.40 nM. 50 [M] blocked binding of 95 pM biotin-R5381, demonstrating 99.05% blockade at the highest concentration tested.

[0207] The anti-R5381 bivalent antibodies REGN6580 and REGN6581 had IC of 381 pM and 543 pM, respectively. 50 Both mAbs blocked biotin-R5381 binding at [M] values ​​of 95 pM, with both mAbs demonstrating 100% blockade at the highest concentration. An isotype control mAb (REGN1945) showed no blockade of biotin-R5381 under identical assay conditions.

[0208] [Table 14]

[0209] An ELISA-based method was used to assess blocking of biotin-R5381 binding to hNPR1.ecto.mmh (SEQ ID NO: 47) coated on ELISA plates in the presence of various concentrations of one-arm anti-R5381 mAbs, REGN9035 and REGN9037. As controls, the respective bivalent mAbs REGN6580 and REGN6581, as well as an isotype control mAb, were also tested. Percentage of blocking at the highest mAb concentration (333.33 nM) and blocking IC of the mAbs 50 [M] values ​​are summarized above in Table 12. Molar concentration [M] indicates the antibody concentration of the mAb. EXAMPLES

[0210] Example 8: Circulating immune complex formation between R5381 and anti-R5381 The possibility of forming circulating immune complexes (CIC) between R5381 and anti-R5381 antibodies was tested using the Microvue C1q-CIC kit developed by Quidel. The assay was performed according to the manufacturer's instructions. Antigen and antibody samples were combined in a 1:1 or 1:10 ratio and incubated at 37°C for 30 minutes to initiate complex formation. The antibody-antigen samples, as well as positive and negative heat-aggregated gamma globin (HAGG) controls, were then diluted 1:50 in the C1q-coated test plate. The kit standards were added directly to the C1q-coated test plate. The test plate was then incubated at room temperature for 1 hour. Unbound antibody, antigen or complex was washed from the plate using 1x wash buffer. HRP-conjugated detection antibody was added to the test plate and incubated at room temperature for 30 minutes, after which unbound HRP-conjugated detection antibody was washed from the plate using 1x wash buffer. HRP substrate was added to the test plate and incubated at room temperature for 30 minutes. An acidic stop solution was then applied to inactivate the HRP enzyme.The plates were then read at 405 nM on a Perkin Elmer Victor X5 plate reader.

[0211] The raw data was background subtracted and a linear standard curve was plotted using MicroVue C1q-CIC kit standards, which was analyzed by linear regression. Sample and HAGG standard values ​​(μgEq / mL) were then calculated using the linear regression equation.

[0212] result HAGG high and low controls were included on each plate. According to the manufacturer's instructions, all samples with values ​​below 4.0 μgEq / mL were considered negative. The possibility of R5381 forming CIC with anti-R5381 antibodies was examined using the MicroVue C1q-CIC kit. The results are summarized in Tables 13A and 13B below. The final values ​​and presence of CIC are shown. No CIC was detected in any of the experimentally tested conditions.

[0213] [Table 15]

[0214] [Table 16] EXAMPLES

[0215] Example 9: NPR1 hu / hu Pharmacokinetic evaluation of anti-R5381 antibody in mice The pharmacokinetics of two one-armed anti-R5381 antibodies, REGN9035 and REGN9037, and their respective bivalent parental counterparts, REGN6580 and REGN6581, were evaluated in humanized NPR1 mice (mice homozygous for the humanized NPR1 allele, NPR1 hu / hu) in vitro. Cohorts included 5 mice per antibody tested. Mice administered REGN6580 and REGN6581 were given a single subcutaneous (SC) dose of 1 mg / kg. Mice administered REGN9035 and REGN9037 were given a single normalized SC dose based on the molar equivalent to the parental counterpart (0.67 mg / kg). Blood samples were collected 6 hours after dosing and on days 1, 2, 3, 7, 10, 14, 21, and 30. Blood was processed to serum and frozen at -80°C until analysis. All hIgG and functional hIgG serum concentrations of REGN9035, REGN9037, REGN6580, and REGN6581 were measured using the GyroLab xPlore platform (Gyros).

[0216] The Gyros technology uses an affinity flow-through format for automated immunoassays with laser-induced fluorescence detection. Samples were loaded onto a compact disc (CD) containing multiple nanoliter-scale affinity capture columns arranged radially. Liquid flow was controlled by centrifugal and capillary forces.

[0217] To measure total and functional concentrations of REGN9035, REGN9037, REGN6580 and REGN6581 in serum, test article-specific biotinylated capture reagents (Table 14 below) were added to a Gyrolab Bioaffy 200 CD containing affinity columns pre-packed with streptavidin-coated beads (Dynospheres). Standards used for calibration (Table 14) were run at concentrations ranging from 0.488 to 2000 ng / mL. Serial dilutions of serum samples were prepared in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). Serial dilutions of standards were prepared in PBS + 0.5% BSA containing 2% normal mouse serum (NMS). Singlets of serum samples diluted 1:50 and duplicates of standards were added to the affinity columns coated with capture reagent at room temperature. Captured human IgG was detected using Alexa-647-conjugated mouse anti-human IgG1 / hIgG4 monoclonal antibody (at 0.5 μg / mL) diluted in Rexxip F buffer (Gyros); the resulting fluorescent signal was recorded in response units (RU) by a GyroLab xPlore instrument. The lower limit of quantification (LLOQ) of 0.05 μg / mL for each assay was defined as the lowest concentration on the standard curve at which a quality control (QC) sample was determined to consistently deviate less than 25% from the expected concentration (Table 14). Sample concentrations were determined by interpolation from a standard curve constructed using a four-parameter logistic curve fit in Gyrolab Evaluator Software. The mean concentration from two replicates was used to calculate the final concentration.

[0218] [Table 17]

[0219] PK parameters were determined by noncompartmental analysis (NCA) using Phoenix® WinNonlin® software version 6.3 (Certara, LP, Princeton, NJ) and an extravascular administration model. Maximum observed serum concentrations (C) were calculated using the respective mean concentration values ​​for each antibody (all hIgG). max ), the estimated observed half-life (t 1 / 2 ), area under the concentration curve versus time to last measurable concentration (AUC last All PK parameters, including the antibody clearance rate (Cl), and antibody clearance rate (Cl), were determined using the linear trapezoidal rule with linear interpolation and uniform weighting.

[0220] result NPR1 hu / hu Following SC administration of 1 mg / kg (or dose equivalent) of anti-R5381 Ab in mice, REGN9035, REGN9037, REGN6580, and REGN6581 increased the maximum concentration of all hIgG in serum normalized to similar doses (C max / D = 10.3, 9.23, 9.3, and 11.4 mg / mL). In addition, REGN9035, REGN9037, REGN6580, and REGN6581 also showed similar half-life values ​​(T 1 / 2 = 18.1, 17.1, 16.1, and 15.4 days), and dose-normalized drug exposure values ​​(AUC last / D = 204, 174, 165, and 205(d * The NPR1 activity (mg / mL / (mg / kg)), and clearance rates (Cl = 5.0, 6.1, 4.9, and 3.7 mL / day / kg, respectively) were comparable across all time points. hu / hu No measurable differences were observed in the PK profiles of the one-arm antibodies REGN9035 and REGN9037 compared to their bivalent counterparts REGN6580 and REGN6581 in mice.

[0221] A summary of the data for total antibody concentrations and functional antibody concentrations is summarized below in Table 15, mean PK parameters are set forth below in Table 16, and mean total antibody concentrations versus time are shown in FIG. 2.

[0222] [Table 18]

[0223] [Table 19] EXAMPLES

[0224] Example 10: Serum concentration analysis of all R5381 (anti-NPR1 mAbs) after rescue with anti-R5381 antibodies Serum sample concentration analysis from an in vivo study evaluating the efficacy of bivalent anti-R5381 mAbs, REGN6580 and REGN6581, compared to the isotype control antibody, REGN1945, in reversing the blood pressure lowering effect of R5381. The study was performed in mice homozygous for the humanized NPR1 allele (NPR1), which was telemetered normotensive. hu / hu ) mice. Briefly, cohorts included 5 mice per test antibody. All mice received a single 5 mg / kg dose of R5381 subcutaneously (SC). Three days later, mice received a single 50 mg / kg dose of REGN6580, REGN6581 or the isotype control REGN1945 intravenously (IV). Blood samples were collected 7 and 22 days after the first R5381 dose. Blood was processed to serum and frozen at -80°C until analysis. All R5381 serum concentrations were measured using a GyroLab xPlore platform (Gyros).

[0225] The Gyros technology uses an affinity flow-through format for automated immunoassays with laser-induced fluorescence detection. Samples were loaded onto a compact disc (CD) containing multiple nanoliter-scale affinity capture columns arranged radially. Liquid flow was controlled by centrifugal and capillary forces.

[0226] Immunoassays were performed to measure total R5381 in serum. Mice administered R5381 followed by anti-R5381 mAb are presumed to form antibody:anti-idiotype complexes in serum (R5381:REGN6580 or R5381:REGN6581). A dissociation step was performed at the beginning of the assay to accurately measure total R5381 antibody concentrations in these samples. Briefly, to measure total R5381, REGN6712, a biotinylated capture reagent specific for the test article, at a concentration of 75 μg / mL was pre-incubated with serum samples diluted 1:250 or standards diluted to concentrations ranging from 0.244 to 1000 ng / mL for 4 hours at 37°C. Dilutions of serum samples were prepared in phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) (+ capture reagent) and serial dilutions of the standard (R5381) were prepared in PBS + 0.5% BSA containing 0.4% normal mouse serum (NMS) (+ capture reagent). After pre-incubation of the capture reagent with the samples or standards for 4 hours at 37°C, diluted singlets of serum samples (+ capture reagent) and diluted duplicates of the standard (+ capture reagent) were added to a Gyrolab Bioaffy 200 CD containing an affinity column pre-packed with streptavidin-coated beads (Dynospheres). Captured human IgG was detected using 0.5 μg / mL of Alexa-647-conjugated mouse anti-human IgG1 / hIgG4 monoclonal antibody diluted in Rexxip F buffer (Gyros); the resulting fluorescent signal was recorded in response units (RU) by a GyroLab xPlore instrument. The lower limit of quantification (LLOQ) of 0.1 μg / mL for each assay was defined as the lowest concentration on the standard curve at which pre-complexed quality control (QC) (R5381:REGN6580, R5381:REGN6581) samples were determined to consistently deviate by less than 25% from the expected concentration.

[0227] For the measurement of total R5381 concentrations in serum from mice administered R5381 followed by unbound control antibody (REGN1945), no dissociation step was required. Total R5381 concentrations were measured as follows. Briefly, mAb36313, a biotinylated capture reagent specific for the test article, was added at 50 μg / mL to a Gyrolab Bioaffy 200 CD containing an affinity column pre-packed with streptavidin-coated beads (Dynospheres) at room temperature. The standard (R5381) used for calibration in this assay was diluted at concentrations ranging from 0.488 to 2000 ng / mL. Serial dilutions of the standard were prepared in PBS + 0.5% BSA containing 1% normal mouse serum (NMS). Singlets of serum samples diluted 1:100 and duplicates of the standard were added to the affinity column coated with the capture reagent at room temperature. Captured human IgG was detected using 0.5 μg / mL of Alexa-647-conjugated mouse anti-human IgG1 / hIgG4 monoclonal antibody diluted in Rexxip F buffer (Gyros); the resulting fluorescent signal was recorded in RU by a GyroLab xPlore instrument. The LLOQ of 0.05 μg / mL for each assay was defined as the lowest concentration on the standard curve at which QC samples were determined to consistently deviate by less than 25% from the expected concentration.

[0228] Sample concentrations were determined by interpolation from a standard curve constructed using a four-parameter logistic curve fit in Gyrolab Evaluator Software. The average concentration from two replicates was used to calculate the final concentration.

[0229] Calculation of average concentration Individual concentrations and mean concentrations below the lower limit of quantitation (<LLOQ) were reported as below the limit of blank (BLQ). When more than 50% of the individual values were at the BLQ, the mean value at that time was reported as the BLQ. Using zero as the BLQ value, when less than 50% of the individual values within the treatment group were at the BLQ and the mean value was arithmetically at the BLQ, the mean value was reported as the BLQ. Using zero as the BLQ value, when less than 50% of the individual values within the treatment group were at the BLQ and the mean value was arithmetically above the LLOQ, this arithmetic value was reported.

[0230] Results NPR1 hu / hu Seven days after administration of R5381 at 5 mg / kg to mice, followed by administration of the rescue reagents REGN6580, REGN6581, or isotype control mAb (REGN1945) at 50 mg / kg three days later, lower mean serum concentrations of all R5381 occurred in mice that received anti-idiotype antibodies compared to mice that received the isotype control. This indicates that the anti-idiotype antibodies REGN6580 and REGN6581 are involved in the more rapid clearance of R5381 seen in mice when these anti-idiotype mAbs are given, compared to mice that received the isotype control.

[0231] Mice administered R5381 and then a dose of REGN6580 or REGN6581 three days later (seven days after the first R5381 administration) had total R5381 concentrations of 3.6 or 7.4 μg / mL, respectively. In comparison, mice administered the isotype control three days later had total R5381 concentrations of 30.5 μg / mL. Analyzing serum collected 22 days after administration of R5381 followed by the anti-idiotype antibody resulted in undetectable concentrations of all R5381, while the serum concentrations of all R5381 in the group administered the isotype control remained at approximately 6 μg / mL.

[0232] A summary of all data for R5381 serum concentrations in mice rescued by IV administration of REGN6580, REGN6581, or the isotype control, REGN1945, is found in Table 17 below.

[0233] [Table 20] EXAMPLES

[0234] Example 11: Evaluation of reversal of R5381-induced blood pressure reduction using a single 50 mg / kg intravenous dose of bivalent anti-R5381 mAb in normotensive NPR1 hu / hu mice Telemetric normotensive NPR1 hu / hu To evaluate the efficacy of bivalent anti-R5381 antibodies in reversing R5381-induced blood pressure reduction in mice, we used 18- to 20-week-old male NPR1 mice. hu / hu Mice (n=20) were implanted with PA-C10 telemetry devices (DSI, St. Paul, MN) and allowed to recover for at least 7 days. Animals were stratified into groups (groups 1-4) based on pre-study systolic blood pressure and body weight. Animals were housed individually under standard conditions (temperature 64°F-84°F (18°C-29°C); relative humidity 30%-70%) and maintained on a 12-h light / 12-h dark cycle. Food (research diet standard pelleted chow) and water were provided ad libitum.

[0235] Test proteins were administered to the appropriate animals by a single subcutaneous injection on day 0. Rescue agents were administered to the appropriate animals by a single intravenous injection on day 3. Dose volumes for each animal were based on the most recent body weight measurement.

[0236] [Table 21]

[0237] Systolic, diastolic, pulse, mean arterial pressure and heart rate were collected for 10 seconds every 10 minutes for the duration of the study. Data were binned and assessed accordingly for acute (hourly binning) and chronic (24 hour binning) reversal of R5381-induced blood pressure reduction. Urinary cyclic guanosine monophosphate (cGMP) concentrations on days 21 / 22 were assessed by ELISA. Total absolute and relative heart weights were collected at necropsy. All data are presented as mean ± SEM.

[0238] result In vivo screening of the bivalent anti-R5381 antibodies, REGN6580 and REGN6581, demonstrated rapid and sustained reversal of the blood pressure lowering effects of R5381 (Figure 3). Both REGN6580 and REGN6581 were able to increase pressure back to baseline levels when administered intravenously 3 days after the first dose of R5381. The initial pressure drop of 6-8 mmHg compared to time-matched controls (Table 19 below) was reversed within 3 days after administration of either REGN6580 or REGN6581 (Figure 3).

[0239] [Table 22]

[0240] Durability of reversal was maintained over the 22 day study period, with statistically significant differences in all hemodynamic parameters for animals treated with REGN6580 or REGN6581 when compared to animals treated with R5381 and isotype control mAb (Table 20 below).

[0241] [Table 23]

[0242] No statistically significant differences were observed following administration of either REGN6580 or REGN6581 when compared to PBS control animals (Table 20, Figures 3-6), indicating a complete and sustained reversal of the R5381-induced blood pressure lowering effect.

[0243] Both bivalent anti-R5381 antibodies, REGN6580 and REGN6581, attenuated the decrease in relative heart weight, as shown by the lack of statistically significant differences in heart weight relative to tibia weight in groups treated with either reversal agent compared to PBS-treated animals (Table 21 below).

[0244] [Table 24]

[0245] No effect was observed on urinary volume with either the test article or the rescue article delivered (Table 22 below).

[0246] [Table 25]

[0247] When assessed in urine on day 22, cGMP production was unaffected by administration of the reversal agent (Table 22).

[0248] Both bivalent anti-R5381 antibodies, REGN6580 and REGN6581, inhibited normotensive NPR1 cells treated with a single dose of R5381. hu / hu Following a single intravenous injection into mice on study day 3, there was a rapid and sustained reversal of the blood pressure lowering effects of R5381 through study day 21. EXAMPLES

[0249] Example 12: Normotensive NPR1 hu / hu Assessment of reversal of R5381-induced hypotension using a single 50mg / kg subcutaneous dose of bivalent anti-R5381 mAb in mice Telemetric normotensive NPR1 hu / huTo evaluate the efficacy of bivalent anti-R5381 antibodies in reversing R5381-induced blood pressure reduction in mice, male NPR1 mice approximately 10–12 weeks of age were treated with hu / hu (n=20) Mice were implanted with PA-C10 telemetry devices (DSI, St. Paul, MN) and allowed to recover for at least 7 days. Animals were stratified into groups (Groups 1-4) based on pre-study systolic blood pressure and body weight. Animals were housed individually under standard conditions (temperature 64°F-84°F (18°C-29°C); relative humidity 30%-70%) and maintained on a 12-h light / 12-h dark cycle. Food (research diet standard pelleted chow) and water were provided ad libitum.

[0250] Test proteins were administered to the appropriate animals by a single subcutaneous injection on day 0. Rescue agents were administered to the appropriate animals by a single subcutaneous injection on day 3. Dose volumes for each animal were based on the most recent body weight measurement.

[0251] [Table 26]

[0252] Systolic, diastolic, pulse, mean arterial pressure and heart rate were collected for 10 seconds every 10 minutes for the duration of the study. Data were binned and assessed accordingly for acute (hourly binning) and chronic (24 hour binning) reversal of R5381-induced blood pressure reduction. Urinary cyclic guanosine monophosphate (cGMP) concentrations on days 21 / 22 were assessed by ELISA. All data are presented as mean ± SEM.

[0253] result In vivo screening of bivalent anti-R5381 antibodies demonstrated sustained reversal of the blood pressure lowering effect of R5381 (Figure 7 and Table 24 below).

[0254] [Table 27]

[0255] Both REGN6580 and REGN6581 were able to increase pressure back to baseline levels when administered subcutaneously 3 days after the first dose of R5381 (Figure 7 and Table 25 below).

[0256] [Table 28]

[0257] The durability of reversal was maintained over the 22 day study period, with statistically significant differences in all hemodynamic parameters for animals administered REGN6580 or REGN6581 when compared to animals administered R5381 and isotype control mAb (Figure 7 and Table 25 below). No statistically significant differences were observed following administration of either REGN6580 or REGN6581 when compared to PBS control animals (Table 25, Figure 7), indicating a complete and sustained reversal of the R5381-induced blood pressure lowering effect.

[0258] Both REGN6580 and REGN6581 demonstrated attenuation of NPR1 signaling as shown by a statistically significant decrease in urinary cGMP levels after 22 days of subcutaneous administration of either REGN6580 or REGN6581 (Table 26 below).

[0259] [Table 29]

[0260] A trend towards non-statistically significant reductions in absolute and relative heart weights was observed following administration of R5381 and isotype control (Table 27 below).

[0261] [Table 30]

[0262] This change is likely due to the hemodynamic effects of R5381. Although not significant, animals given R5381 followed by either REGN6580 or REGN6581 had absolute and relative heart weights closer to those of control animals, consistent with attenuation of R5381-induced hemodynamic effects.

[0263] After a single subcutaneous injection on study day 3, both bivalent anti-R5381 antibodies, REGN6580 and REGN6581, significantly reduced remotely measured normotensive NPR1 hu / hu When assessed in mice, it rapidly and persistently reversed the blood pressure lowering effects of R5381. Both agents also functioned to inhibit NPR1-induced cGMP production through day 22 of the study. EXAMPLES

[0264] Example 13: Normotensive NPR1 hu / hu Assessment of reversal of R5381-induced hypotension using a single 50mg / kg dose of monovalent anti-R5381 mAb in mice Telemetric normotensive NPR1 hu / hu In an attempt to evaluate the efficacy of monovalent anti-R5381 antibodies in reversing R5381-induced blood pressure reduction in mice, we administered monovalent anti-R5381 antibodies to approximately 13-14 week-old male NPR1 mice. hu / hu (n=48) Mice were implanted with PA-C10 telemeters (DSI, St. Paul, MN) and allowed to recover for at least 7 days. Animals were stratified into groups (Table 28 below) (Groups 1-8) based on pre-study systolic blood pressure and body weight (Table 29 below).

[0265] [Table 31]

[0266] [Table 32]

[0267] Animals were housed individually under standard conditions (temperature 64°F-84°F (18°C-29°C); relative humidity 30%-70%) and maintained on a 12-h light / 12-h dark cycle. Food (laboratory diet standard pelleted chow) and water were provided ad libitum.

[0268] Test proteins were administered to appropriate animals by a single subcutaneous injection on day 0. Rescue agents were administered to appropriate animals by a single subcutaneous or intravenous injection on day 3. Dose volumes for each animal were based on the most recent body weight measurement. Overnight urine collections were performed on study days 20 and 21.

[0269] Systolic, diastolic, pulse, mean arterial pressure and heart rate were collected for 10 seconds every 10 minutes for the duration of the study. Data were binned and assessed accordingly for acute (hourly binning) and chronic (24 hour binning) reversal of R5381-induced blood pressure reduction. Urinary cyclic guanosine monophosphate (cGMP) concentrations on days 21 / 22 were assessed by ELISA. All data are presented as mean ± SEM.

[0270] result In vivo screening of monovalent anti-R5381 antibodies demonstrated rapid and sustained reversal of the blood pressure lowering effect of R5381 (Table 30 below; Figures 8 and 9).

[0271] [Table 33]

[0272] Both REGN9035 and REGN9037 were able to increase pressure back to baseline levels when administered intravenously or subcutaneously 3 days after the first dose of R5381 (Table 30; Figure 8 and Figure 9). The initial drop in absolute pressure of 3-6 mmHg (Table 29) and relative pressure of approximately 10 mmHg (Figure 8) compared to time-matched controls was reversed within hours after intravenous administration of REGN9035, REGN9037 or REGN6580 (Figure 9). Subcutaneous delivery of REGN9035, REGN9037 or REGN6580 achieved complete reversal within 24 hours after administration of the reversal agent (Figure 9). The persistence of reversal was maintained over the 20-day study period, with statistically significant differences in all hemodynamic parameters for animals administered REGN9035 or REGN9037 when compared to animals administered R5381 and isotype control mAb (Table 30, Figure 8). No statistically significant differences were observed following administration of either REGN9035, REGN9037 or REGN6580 when compared to PBS control animals (Table 30, FIG. 8), indicating a complete and sustained reversal of the R5381-induced blood pressure lowering effect. The reduction in pressure induced by R5381 resulted in lower heart weights (Table 31 below), likely a result of the reduction in left ventricular afterload induced by NPR1 agonism.

[0273] [Table 34]

[0274] The changes in myocardial structure were ameliorated following administration of either the reversing agents, REGN9035, REGN9037 or REGN6580 (Table 31). Finally, all anti-R5381 mAbs demonstrated attenuation of NPR1 signaling as shown by a statistically significant decrease in cGMP following subcutaneous or intravenous administration after 22 days (Figure 10).

[0275] Both bivalent and monovalent anti-R5381 antibodies, REGN6580, as well as REGN9035 and REGN9037, respectively, rapidly and persistently reversed the blood pressure lowering effect of R5381 and significantly improved the blood pressure in normotensive NPR1 patients receiving a single dose of R5381. hu / hu Following a single subcutaneous or intravenous injection into mice on study day 3, NPR1-induced cGMP production was inhibited through study day 21. EXAMPLES

[0276] Example 14: Midodrine rescue following a single intravenous dose of R5381 in telemetered cynomolgus monkeys An in vivo study was conducted to evaluate midodrine, an alpha-adrenergic receptor agonist, as an effective agent to temporarily reverse the blood pressure lowering effects of R5381 in cynomolgus monkeys. Animals were given a single IV bolus of 25 mg / kg R5381, followed 3 days later by three doses of 2.5 mg / kg midodrine administered by oral gavage with 3-4 hours between each dose. Animals were monitored for 4 days after R5381 administration, including 1 day after midodrine administration, to evaluate hemodynamic changes; pre-dose measurements served as the baseline for each animal. Midodrine temporarily reversed the R5381-induced decrease in mean systolic blood pressure to time-matched control levels. Midodrine also temporarily reversed the R5381-induced increase in mean heart rate, resulting in a mean decrease from baseline heart rate in R5381-treated animals.

[0277] Specifically, evaluation of the utility of vasopressor administration as an agent to temporarily reverse the blood pressure effects of R5381 in normotensive telemetered male cynomolgus monkeys is particularly relevant in clinical settings where patients receiving R5381 may need to increase their blood pressure (e.g., shock-induced hypotension). Prior to administering a dose, each animal was surgically implanted with a radiotelemetry transmitter. On day 0, each animal received a single IV bolus of saline (PBS; n = 10) or 25 mg / kg R5381 (n = 13). On day 3, each animal received three doses of the alpha-adrenergic receptor agonist midodrine at a dose of 2.5 mg / kg (n = 6 in the saline group; n = 7 in the R5381 group), or water / vehicle (n = 4 in the saline group; n = 6 in the R5381 group), with each dose administered by oral gavage 3-4 hours apart. Animals were monitored for 48 hours to assess cardiovascular hemodynamic changes. Blood pressure and heart rate measurements were collected for each animal from day -3 before R5381 administration through day 4 after R5381 administration. Pre-administration measurements served as the baseline for each animal.

[0278] result Midodrine reversed the R5381-induced drop in blood pressure and heart rate in cynomolgus monkeys. Three doses of midodrine temporarily reversed the R5381-induced drop in mean systolic blood pressure, and R5381-treated animals that received midodrine showed a similar mean change from baseline systolic blood pressure compared to that observed in saline-treated animals that did not receive midodrine (Figure 11).

[0279] Furthermore, three doses of midodrine reversed the R5381-induced increase in mean heart rate. Midodrine administration caused a mean decrease in baseline heart rate in R5381-treated animals; a similar effect was observed in saline-treated animals treated with midodrine (Figure 12).

[0280] The present disclosure should not be limited in scope by the specific embodiments described herein. Indeed, various modifications in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

Claims

1. Agents that reverse the hemodynamic effects of natriuretic peptide receptor 1 (NPR1) agonists.

2. An agent that reverses the fall in blood pressure associated with administration of natriuretic peptide receptor 1 (NPR1) agonists.

3. 3. The agent of claim 1 or 2, which is selected from the group consisting of immunoglobulin proteins, vasopressors, alpha-adrenergic receptor agonists, steroids, antidiuretic hormones, angiogenesis inhibitors, and small molecule drugs that increase blood pressure.

4. The agent according to any one of claims 1 to 3, which is an immunoglobulin protein.

5. The agent according to any one of claims 1 to 4, which specifically binds to an NPR1 agonist.

6. The agent according to any one of claims 1 to 5, wherein the NPR1 agonist is an antibody or an antigen-binding fragment thereof that specifically binds to NPR1.

7. The agent of claim 6, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising SEQ ID NO:48; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising SEQ ID NO:

52.

8. The agent of claim 6 or 7, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) comprising SEQ ID NOs: 49, 50 and 51, respectively; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) comprising SEQ ID NOs: 53, 54 and 55, respectively.

9. The agent of claim 8, wherein the antibody or antigen-binding fragment thereof comprises the HCVR of SEQ ID NO:48 and the LCVR of SEQ ID NO:

52.

10. The agent according to any one of claims 6 to 9, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

11. The method of claim 10, wherein the antibody is an IgG1 antibody.

12. The method of claim 10, wherein the antibody is an IgG4 antibody.

13. The agent of claim 10, wherein the antibody comprises a heavy chain comprising SEQ ID NO:56 and a light chain comprising SEQ ID NO:

57.

14. The method according to any one of claims 1 to 13, wherein the NPR1 agonist is R5381.

15. The agent of any one of claims 4 to 14, wherein the immunoglobulin protein comprises a monoclonal antibody or an antigen-binding fragment thereof, a Fab fragment, an F(ab)2 fragment, an Fv fragment, an Fd fragment, an scFv, a dAb, a bivalent monoclonal antibody or a monovalent monoclonal antibody.

16. The agent of claim 15, wherein the immunoglobulin protein comprises at least one immunoglobulin variable domain comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR).

17. The agent of claim 15 or 16, wherein the immunoglobulin protein comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) comprising amino acid sequences selected from SEQ ID NOs: 4, 6, 8, 12, 14 and 16; or SEQ ID NOs: 24, 26, 28, 32, 34 and 36.

18. 18. The method of claim 17, wherein the HCVR comprises an amino acid sequence having at least 90%, optionally 95%, 98% or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2 and 22.

19. 19. The agent of claim 17 or 18, wherein the LCVR comprises an amino acid sequence having at least 90%, optionally 95%, 98% or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 10 and 30.

20. The method of any one of claims 17 to 19, wherein HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22; and LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30.

21. The agent according to any one of claims 17 to 20, wherein HCVR comprises the amino acid sequence of SEQ ID NO:2 and LCVR comprises the amino acid sequence of SEQ ID NO:

10.

22. The agent according to any one of claims 17 to 20, wherein HCVR comprises the amino acid sequence of SEQ ID NO: 22 and LCVR comprises the amino acid sequence of SEQ ID NO:

30.

23. The agent according to any one of claims 17 to 22, wherein the immunoglobulin protein comprises a human monoclonal antibody comprising at least one immunoglobulin variable domain.

24. The agent of claim 23, wherein the human monoclonal antibody is of the IgG1 or IgG4 isotype.

25. The agent of claim 23 or 24, wherein the human monoclonal antibody comprises a heavy chain and a light chain, and the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 38.

26. The agent according to any one of claims 23 to 25, wherein the human monoclonal antibody comprises a heavy chain and a light chain, and the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 40.

27. The agent according to any one of claims 4 to 15, wherein the immunoglobulin protein comprises one immunoglobulin variable domain.

28. 28. The agent of claim 27, wherein one immunoglobulin variable domain comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR), wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 24; HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 26; HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 28; LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 32; LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 34; and LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 36.

29. 29. The agent of claim 28, wherein HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 comprises an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 4, 6, 8, 12, 14, and 16; and (ii) SEQ ID NOs: 24, 26, 28, 32, 34, and 36.

30. 30. The agent of claim 28 or 29, wherein the HCVR comprises an amino acid sequence having at least 90%, optionally 95%, 98% or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2 and 22.

31. The agent of any one of claims 28 to 30, wherein the LCVR comprises an amino acid sequence having at least 90%, optionally 95%, 98% or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 10 and 30.

32. The method of any one of claims 28 to 31, wherein HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22; and LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30.

33. The agent according to any one of claims 28 to 32, wherein HCVR comprises the amino acid sequence of SEQ ID NO:2 and LCVR comprises the amino acid sequence of SEQ ID NO:

10.

34. The agent according to any one of claims 28 to 32, wherein HCVR comprises the amino acid sequence of SEQ ID NO: 22 and LCVR comprises the amino acid sequence of SEQ ID NO:

30.

35. The agent according to any one of claims 26 to 34, wherein one immunoglobulin variable domain is comprised within a Fab fragment.

36. The agent of any one of claims 26 to 35, further comprising a multimerization component.

37. The agent of claim 36, wherein the multimerization component comprises at least one Fc fragment.

38. 38. The agent of claim 37, wherein the Fc fragment is of isotype IgG1, IgG4 or a variant thereof.

39. 39. The agent of claim 38, wherein the Fc fragment is of the IgG4 isotype.

40. 39. The agent of claim 38, wherein the Fc fragment is of the IgG1 isotype.

41. 41. The agent of any one of claims 37 to 40, comprising a first Fc fragment and a second Fc fragment, wherein the first Fc fragment or the second Fc fragment, but not both Fc fragments, comprises a modification in the CH3 domain that reduces binding of the immunoglobulin protein to Protein A compared to an immunoglobulin protein lacking the modification.

42. 42. The agent of claim 41, wherein the modification comprises a H315R substitution and a Y316F substitution (EU numbering) in the Fc fragment.

43. The agent according to any one of claims 37 to 42, wherein the multimerisation component comprises an Fc fragment comprising the amino acid sequence of SEQ ID NO:46 and an Fc fragment comprising the amino acid sequence of SEQ ID NO:

58.

44. The agent according to any one of claims 16 to 22 or 26 to 34, wherein the immunoglobulin variable domain is comprised in a monovalent monoclonal antibody.

45. 45. The agent of claim 44, wherein the monovalent monoclonal antibody comprises a heavy chain comprising a heavy chain constant region and a HCVR, and a light chain comprising a LCVR.

46. 46. ​​The agent of claim 45, wherein the heavy chain constant region comprises a modification in the CH3 domain that reduces binding of the immunoglobulin protein to Protein A compared to an immunoglobulin protein lacking the modification.

47. 47. The agent of claim 46, wherein the modification comprises a H315R substitution and a Y316F substitution (EU numbering) in the heavy chain constant region of the IgG1 or IgG4 isotype.

48. The agent according to any one of claims 45 to 47, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 42 and the light chain comprises the amino acid sequence of SEQ ID NO:

20.

49. The agent according to any one of claims 45 to 47, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 44 and the light chain comprises the amino acid sequence of SEQ ID NO:

40.

50. The agent according to any one of claims 44 to 49, wherein the immunoglobulin protein further comprises an Fc fragment.

51. 51. The agent of claim 50, wherein the Fc fragment is of the IgG1 or IgG4 isotype.

52. 52. The agent of claim 50 or 51, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:

46.

53. The agent of any one of claims 1 to 52, which is REGN9035 or REGN9037.

54. 54. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the heavy chain variable region (HCVR) of an immunoglobulin protein according to any one of claims 15 to 53.

55. 54. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the light chain variable region (LCVR) of an immunoglobulin protein according to any one of claims 15 to 53.

56. 56. A vector comprising the polynucleotide molecule of claim 54 and / or the polynucleotide molecule of claim 55.

57. A host cell expressing the vector of claim 56.

58. 58. The host cell of claim 57, which is a CHO cell.

59. 60. A method for producing an immunoglobulin protein, comprising growing a host cell according to claim 57 under conditions allowing production of the protein, and recovering the protein so produced.

60. 60. The method of claim 59, wherein the host cell is a CHO cell.

61. The drug according to any one of claims 1 to 3, which is a vasopressor.

62. The method of claim 61, wherein the hypertensive agent is midodrine.

63. A pharmaceutical composition comprising an agent according to any one of claims 1 to 53 and a pharma- ceutically acceptable carrier or diluent.

64. 64. The pharmaceutical composition of claim 63 for use in a method of reversing the hemodynamic effects of an NPR1 agonist in a subject in need thereof.

65. 64. The pharmaceutical composition of claim 63 for use in a method of reversing hemodynamic changes associated with administration of an NPR1 agonist in a subject in need thereof.

66. 66. The pharmaceutical composition of claim 64 or 65, wherein the composition is administered to the subject subcutaneously, intravenously, intradermally, intraperitoneally, intramuscularly, or orally.

67. The pharmaceutical composition of any one of claims 64 to 66, wherein the NPR1 agonist is an antibody or an antigen-binding fragment thereof that specifically binds to NPR1.

68. The pharmaceutical composition of claim 67, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising SEQ ID NO: 48; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising SEQ ID NO:

52.

69. The pharmaceutical composition according to any one of claims 64 to 68, wherein the NPR1 agonist is R5381.

70. The pharmaceutical composition of any one of claims 64 to 69, wherein the subject has a disease or disorder selected from the group consisting of hypertension, heart failure and chronic kidney disease.

71. An antibody or antigen-binding fragment thereof that competes for binding with an agent according to any one of claims 15 to 53.

72. An antibody or antigen-binding fragment thereof that binds to the same epitope as the agent of any one of claims 15 to 53.

73. (a) a single immunoglobulin variable domain comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22, and a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30. An immunoglobulin protein comprising:

74. (a) an immunoglobulin protein comprising a single immunoglobulin variable domain comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR), where HCDR1-HCDR2-HCDR3 are selected from the group consisting of SEQ ID NOs: 4, 6 and 8, and 24, 26 and 28, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR), where LCDR1-LCDR2-LCDR3 are selected from the group consisting of SEQ ID NOs: 12, 14 and 16, and 32, 34 and 36.

75. (i) HCDR1 comprises the amino acid sequence of SEQ ID NO:4, HCDR2 comprises the amino acid sequence of SEQ ID NO:6, HCDR3 comprises the amino acid sequence of SEQ ID NO:8, LCDR1 comprises the amino acid sequence of SEQ ID NO:12, LCDR2 comprises the amino acid sequence of SEQ ID NO:14, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16; or (ii) the immunoglobulin protein of claim 74, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:24, HCDR2 comprises the amino acid sequence of SEQ ID NO:26, HCDR3 comprises the amino acid sequence of SEQ ID NO:28, LCDR1 comprises the amino acid sequence of SEQ ID NO:32, LCDR2 comprises the amino acid sequence of SEQ ID NO:34, and LCDR3 comprises the amino acid sequence of SEQ ID NO:

36.

76. 76. The immunoglobulin protein of claim 74 or 75, wherein the HCVR comprises an amino acid sequence having at least 90%, optionally 95%, 98% or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 2 and 22.

77. 77. The immunoglobulin protein of any one of claims 74 to 76, wherein the LCVR comprises an amino acid sequence having at least 90%, optionally 95%, 98% or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 10 and 30.

78. The HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22, The immunoglobulin protein of any one of claims 74 to 77, wherein the CVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30.

79. (1) the HCVR comprises the amino acid sequence of SEQ ID NO:2 and the LCVR comprises the amino acid sequence of SEQ ID NO:10; or (2) The immunoglobulin protein according to any one of claims 73 to 78, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 22 and the LCVR comprises the amino acid sequence of SEQ ID NO:

30.

80. 80. The immunoglobulin protein of any one of claims 73 to 79, further comprising a multimerization component.

81. The immunoglobulin protein of claim 80, wherein the multimerization component comprises at least one Fc fragment.

82. The immunoglobulin protein of claim 81, wherein the Fc fragment is of the human IgG1 or IgG4 isotype.

83. 83. The immunoglobulin protein of claim 81 or 82, comprising a first Fc fragment and a second Fc fragment, wherein the first Fc fragment or the second Fc fragment, but not both Fc fragments, comprises a modification in the CH3 domain that reduces binding of the immunoglobulin protein to Protein A compared to an immunoglobulin protein lacking the modification.

84. The immunoglobulin protein of claim 83, wherein the modification comprises a H315R substitution and a Y316F substitution (EU numbering) in the Fc fragment.

85. 85. The immunoglobulin protein of any one of claims 80 to 84, comprising a first Fc fragment comprising the amino acid sequence of SEQ ID NO:46 and a second Fc fragment comprising the amino acid sequence of SEQ ID NO:

58.

86. 86. The immunoglobulin protein of any one of claims 73 to 85, comprising an HCVR of SEQ ID NO:2, an LCVR of SEQ ID NO:10 and a multimerization component, the multimerization component comprising a first Fc fragment comprising the amino acid sequence of SEQ ID NO:46 and a second Fc fragment comprising the amino acid sequence of SEQ ID NO:

58.

87. 86. The immunoglobulin protein of any one of claims 73 to 85, comprising an HCVR of SEQ ID NO: 22, an LCVR of SEQ ID NO: 30 and a multimerization component, the multimerization component comprising a first Fc fragment comprising the amino acid sequence of SEQ ID NO: 46 and a second Fc fragment comprising the amino acid sequence of SEQ ID NO:

58.

88. 80. The immunoglobulin protein of any one of claims 73 to 79, wherein the immunoglobulin protein comprises a heavy chain comprising an HCVR and a heavy chain constant region, and a light chain comprising an LCVR and a light chain constant region, wherein the heavy chain is of the human IgG1 or IgG4 isotype.

89. The immunoglobulin protein of claim 88, wherein the heavy chain constant region comprises a modification in the CH3 domain that reduces binding of the immunoglobulin protein to Protein A compared to an immunoglobulin protein lacking the modification.

90. The immunoglobulin protein of claim 89, wherein the modification comprises a H315R substitution and a Y316F substitution (EU numbering) in the heavy chain constant region of an IgG1 or IgG4 isotype.

91. The immunoglobulin protein of any one of claims 88 to 90, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42 and 44.

92. The immunoglobulin protein of any one of claims 88 to 91, wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 and 40.

93. (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 42 and the light chain comprises the amino acid sequence of SEQ ID NO: 20; or (ii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 44 and the light chain comprises the amino acid sequence of SEQ ID NO:

40.

94. 94. The immunoglobulin protein of any one of claims 88 to 93, further comprising a multimerization component.

95. The immunoglobulin protein of claim 94, wherein the multimerization component comprises an Fc fragment.

96. The immunoglobulin protein of claim 96, wherein the Fc fragment is of the human IgG1 or IgG4 isotype.

97. 97. The immunoglobulin protein of claim 95 or 96, wherein the Fc fragment comprises the amino acid sequence of SEQ ID NO:

46.

98. 98. The immunoglobulin protein of any one of claims 88 to 97, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 42, a light chain comprising the amino acid sequence of SEQ ID NO: 20, and an Fc fragment comprising the amino acid sequence of SEQ ID NO:

46.

99. 98. The immunoglobulin protein of any one of claims 88 to 97, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:44, a light chain comprising the amino acid sequence of SEQ ID NO:40 and an Fc fragment comprising the amino acid sequence of SEQ ID NO:

46.

100. 100. The immunoglobulin protein of any one of claims 73 to 99, which specifically binds to an NPR1 agonist.

101. The immunoglobulin protein of claim 100, wherein the NPR1 agonist is an antibody or an antigen-binding fragment thereof that specifically binds to NPR1.

102. The immunoglobulin protein of claim 101, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO:48 and an LCVR comprising the amino acid sequence of SEQ ID NO:

52.

103. The immunoglobulin protein according to any one of claims 100 to 102, wherein the NPR1 agonist is R5381.

104. The immunoglobulin protein of any one of claims 73 to 103, which is REGN9035 or REGN9037.

105. 105. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding an HCVR of an immunoglobulin protein according to any one of claims 73 to 104.

106. 105. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the LCVR of an immunoglobulin protein according to any one of claims 73 to 104.

107. A vector comprising the polynucleotide molecule of claim 105 and / or the polynucleotide molecule of claim 106.

108. A host cell expressing the vector of claim 107.

109. The host cell of claim 108, which is a CHO cell.

110. 109. A method for producing an immunoglobulin protein, comprising growing a host cell according to claim 108 under conditions allowing production of the protein, and recovering the protein so produced.

111. 111. The method of claim 110, wherein the host cell is a CHO cell.

112. A pharmaceutical composition comprising an immunoglobulin protein according to any one of claims 73 to 104 and a pharma- ceutically acceptable carrier or diluent.

113. 113. The pharmaceutical composition of claim 112 for use in a method for reversing the hemodynamic effects of an NPR1 agonist in a subject in need thereof.

114. 113. The pharmaceutical composition of claim 112 for use in a method for reversing hemodynamic changes associated with administration of an NPR1 agonist in a subject in need thereof.

115. 115. The pharmaceutical composition of claim 113 or 114, wherein the composition is administered to the subject subcutaneously, intravenously, intradermally, intraperitoneally, intramuscularly, or orally.

116. The pharmaceutical composition of any one of claims 113 to 115, wherein the NPR1 agonist is an antibody or an antigen-binding fragment thereof that specifically binds to NPR1.

117. The pharmaceutical composition of claim 116, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO:48 and an LCVR comprising the amino acid sequence of SEQ ID NO:

52.

118. The pharmaceutical composition according to any one of claims 113 to 117, wherein the NPR1 agonist is R5381.

119. The pharmaceutical composition of any one of claims 113 to 118, wherein the subject has a disease or disorder selected from the group consisting of hypertension, heart failure and chronic kidney disease.

120. An antibody or antigen-binding fragment thereof that competes for binding to the immunoglobulin protein of any one of claims 73 to 104.

121. An antibody or antigen-binding fragment thereof that binds to the same epitope as the immunoglobulin protein of any one of claims 73 to 104.

122. A composition comprising: (i) an immunoglobulin protein according to any one of claims 73 to 104; and (ii) an NPR1 agonist.

123. The composition of claim 122, wherein the NPR1 agonist is an antibody or an antigen-binding fragment thereof that specifically binds to NPR1.

124. The composition of claim 123, wherein the antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO:48 and an LCVR comprising the amino acid sequence of SEQ ID NO:

52.

125. The composition according to any one of claims 122 to 124, wherein the NPR1 agonist is R5381.

126. 126. The composition of any one of claims 122 to 125 for use in a method for the effective regulation of blood pressure in a subject in need thereof.

127. The composition of claim 126, wherein the subject has an NPR1-associated disease or disorder.

128. 128. The composition of claim 127, wherein the disease or disorder is selected from the group consisting of hypertension, heart failure and chronic kidney disease.