Antagonist anti-NPR1 antibodies and methods of use thereof

Abstract

The present disclosure provides monoclonal antibodies that bind to natriuretic peptide receptor 1 (NPR1) protein, and methods of using the same. In various embodiments of the present disclosure, the antibodies are fully human antagonistic antibodies that bind to NPR1. In certain embodiments, the antibodies of the present disclosure are useful for blocking NPR1 signaling and / or activity, thus providing a means of treating or preventing diseases, disorders, or conditions associated with NPR1, including hypotension, in humans.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed as a PCT International patent application on December 6, 2022, and claims priority to U.S. Provisional Patent Application No. 63 / 286,476, filed December 6, 2021, and U.S. Provisional Patent Application No. 63 / 310,078, filed February 14, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] Reference to sequence table XML This application contains a Sequence Listing that has been submitted electronically in XML format. The Sequence Listing XML is incorporated herein by reference. The XML file, created on November 24, 2022, is named 40848_0112WOU1_SL.xml and is 82,654 bytes in size.

[0003] The present disclosure relates to antagonist antibodies and antigen-binding fragments of antibodies that specifically bind to natriuretic peptide receptor 1 (NPR1), and therapeutic and diagnostic methods using those antibodies. [Background technology]

[0004] Natriuretic peptide receptor 1 (NPR1; also known as NPR-A) is a membrane-bound guanylate cyclase that mediates the intracellular conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP) (Non-Patent Document 1). NPR1 is widely expressed in the kidney, lung, adrenal gland, vasculature, brain, liver, endothelial tissue, and adipose tissue, and at low 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 non-cardiovascular functions such as lipolysis and immune cell function (Non-Patent Document 2). Agonism of NPR1 leads to changes in systemic blood pressure (BP) through cGMP-mediated effects on intravascular volume, vascular relaxation, natriuresis, and diuresis.

[0005] Monoclonal antibodies against NPR1 were first described by Kitano et al. (Non-Patent Document 3) in 1995. Activating or agonistic anti-NPR1 antibodies are disclosed, for example, in US Pat. Nos. 5,993,333, 5,993,542, 5,993,613, 5,993,721, and 6,043,636, as well as in US Pat. No. 5,113,663.

[0006] Hypotension, or low blood pressure, can be a relatively benign, asymptomatic condition, but can become problematic if pump pressure is not sufficient to perfuse major organs with oxygenated blood (Non-Patent Document 4, available at https: / / www.ncbi.nlm.nih.gov / books / NBK499961 / ). Complications of untreated hypotension with inadequate cardiac output can be severe and even fatal. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 9,090,695 [Patent Document 2] U.S. Patent Publication No. 20160168251 [Patent Document 3] U.S. Patent Publication No. 20200123263 [Patent Document 4] WO2010065293 [Non-patent literature]

[0008] [Non-Patent Document 1] Martinez-Rumayor et al. 2008 Am J Cardiol 101(3a): pp. 3-8 [Non-Patent Document 2] Potter, 2011 Pharmacol.Ther.130:71-82 [Non-Patent Document 3] Immunol.Lett.47:215-22 [Non-Patent Document 4] Sharma et al., Hypotension, updated 2021 Summary of the Invention [Problem to be solved by the invention]

[0009] Disorders characterized by hypotension represent a high unmet medical need for safe, long-acting therapies. Relatively few drugs exist that increase blood pressure and intravascular volume. Most of the existing drugs have limitations, for example, oral agents have a short duration of action and require multiple daily administrations, and intravenous vasopressors require frequent infusions in the ICU with monitoring due to a narrow therapeutic index. [Means for solving the problem]

[0010] In one aspect, the disclosure provides an antagonist antibody and antigen-binding fragment thereof that specifically binds to natriuretic peptide receptor 1 (NPR1) protein. In one embodiment, an isolated antibody or antigen-binding fragment thereof that specifically binds to natriuretic peptide receptor 1 (NPR1) protein is provided, where the antibody or antigen-binding fragment thereof binds to and blocks NPR1. In a further embodiment, blocking NPR1 includes inhibiting and / or blocking NPR1 signaling and / or activity. In certain embodiments, the anti-NPR1 antibody is a fully human antibody that binds to NPR1 with high affinity and blocks NPR1. The antibodies of the disclosure are useful, inter alia, for blocking or reducing the hypotensive effect of NPR1 signaling and / or NPR1 protein. In certain embodiments, the antibodies are useful for preventing, treating, or ameliorating at least one symptom or sign of an NPR1-related disease or disorder in a subject, including hypotension. In certain embodiments, the antibodies are administered prophylactically or therapeutically to a subject having or at risk of having an NPR1-related disease or disorder. In certain embodiments, the antibodies are used to increase systemic blood pressure in a subject suffering from hypotension. Such antibodies, when administered to a subject in need thereof, can be used as a therapy for disorders or conditions associated with hypotension.

[0011] The antagonistic antibodies disclosed herein bind to NPR1 with high affinity and have improved pharmacokinetic properties (compared to standard therapeutics). A single dose of the antibodies disclosed herein results in a sustained increase in blood pressure. In fact, the antibodies disclosed herein are effective in increasing and maintaining increased blood pressure for as long as 28 days when administered as a single dose. Such antibodies can be used to provide superior efficacy with less frequent administration in subjects with NPR1-related diseases or disorders (e.g., hypotension).

[0012] The antibodies of the disclosure may be full length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2 or scFv fragments) and may be modified to affect functionality, e.g., to increase persistence in the host or to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, the antibodies may be bispecific.

[0013] In a first aspect, the disclosure provides an isolated, recombinant, monoclonal antagonist antibody or antigen-binding fragment thereof that specifically binds to NPR1.

[0014] In certain embodiments, the antibodies are fully human monoclonal antibodies.

[0015] Exemplary anti-NPR1 antibodies of the disclosure are listed in Tables 1 and 2 herein. Table 1 lists the amino acid sequence identifiers of 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 antibodies. Table 2 lists the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary antibodies.

[0016] The present disclosure provides antibodies or antigen-binding fragments thereof comprising an HCVR comprising 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.

[0017] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising an LCVR comprising 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.

[0018] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, 38, and 55.

[0019] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, 46, and 63.

[0020] The disclosure also provides an antibody or antigen-binding fragment thereof comprising three complementarity determining regions (CDRs) contained within a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 2, 22, 38, and 55; and three CDRs contained within a light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 10, 30, 46, and 63.

[0021] The disclosure also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR / LCVR amino acid sequence pair contained within any of the exemplary anti-NPR1 antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 2 / 10 (e.g., mAb38067), 22 / 30 (e.g., mAb38072), 38 / 46 (e.g., mAb38090), and 55 / 63 (e.g., mAb22034).

[0022] The present disclosure also provides antibodies or antigen-binding fragments 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 present disclosure provides antibodies or antigen-binding fragments 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 present disclosure provides antibodies or antigen-binding fragments 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 anti-NPR1 antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence listed in Table 1, the amino acid sequence having at least one amino acid substitution, and / or the LCVR comprises an amino acid sequence listed in Table 1, the amino acid sequence having at least one amino acid substitution.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The present disclosure also provides (a) a heavy chain determining region (HCDR) 1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 24, 40, and 57; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 26, 42, and 59; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 28, 44, and 61; (d) a light chain determining region (LCDR) 1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 48, and 65; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of AAS and GAS; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, and 69. The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0030] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising 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 present disclosure provides an antibody or antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-NPR1 antibodies 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 (e.g., mAb38067), 28 / 32 (e.g., mAb38072), 44 / 16 (e.g., mAb38090), and 61 / 69 (e.g., mAb22034).

[0031] The disclosure also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR, wherein the HCVR comprises an HCDR1 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, an HCDR2 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, and an HCDR3 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1. In certain embodiments, the disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR, wherein the LCVR comprises an LCDR1 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, an LCDR2 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1, and an LCDR3 comprising an amino acid sequence that differs by one amino acid from the amino acid sequence listed in Table 1. For example, the disclosure provides an antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an HCDR1 comprising an amino acid sequence of SEQ ID NO: 24 or an amino acid sequence that differs from SEQ ID NO: 24 by one amino acid, an HCDR2 comprising an amino acid sequence of SEQ ID NO: 26 or an amino acid sequence that differs from SEQ ID NO: 26 by one amino acid, and an HCDR3 comprising an amino acid sequence of SEQ ID NO: 28 or an amino acid sequence that differs from SEQ ID NO: 28 by one amino acid. In another exemplary embodiment, the disclosure provides an antibody or antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the LCVR comprises an LCDR1 comprising an amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that differs from SEQ ID NO: 12 by one amino acid, an LCDR2 comprising an amino acid sequence of AAS or an amino acid sequence that differs from AAS by one amino acid, and an LCDR3 comprising an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence that differs from SEQ ID NO: 32 by one amino acid.

[0032] The present disclosure also provides an antibody or antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NO:4-SEQ ID NO:6-SEQ ID NO:8-SEQ ID NO:12-AAS-SEQ ID NO:16 (e.g., mAb38067), SEQ ID NO:24-SEQ ID NO:26-SEQ ID NO:28-SEQ ID NO:12-AAS-SEQ ID NO:32 (e.g., mAb38072), SEQ ID NO:40-SEQ ID NO:42-SEQ ID NO:44-SEQ ID NO:48-AAS-SEQ ID NO:16 (e.g., mAb38090), and SEQ ID NO:57-SEQ ID NO:59-SEQ ID NO:61-SEQ ID NO:65-GAS-SEQ ID NO:69 (e.g., mAb22034).

[0033] In related embodiments, the disclosure provides an antibody or antigen-binding fragment thereof that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR / LCVR amino acid sequence pair defined by any of the exemplary antibodies listed in Table 1. For example, the disclosure includes an antibody or antigen-binding fragment thereof that comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within the HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 8 / 16 (e.g., mAb38067), 28 / 32 (e.g., mAb38072), 44 / 16 (e.g., mAb38090), and 61 / 69 (e.g., mAb22034). In a related embodiment, the disclosure provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) / light chain variable region (LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 38 / 46, and 55 / 63.

[0034] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed herein. 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.

[0035] In certain embodiments, the disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to NPR1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the light chain is selected from the group consisting of SEQ ID NOs: 20, 36, 53, and 73.

[0036] In a further embodiment, the disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to NPR1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain is selected from the group consisting of SEQ ID NOs: 18, 34, 51, and 71; and the light chain is selected from the group consisting of SEQ ID NOs: 20, 36, 53, and 73.

[0037] In further embodiments, the disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to NPR1, wherein the antibody or antigen-binding fragment thereof binds to a residue in the lower lobe of the extracellular domain of NPR1. In still further embodiments, the disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to NPR1, wherein the antibody or antigen-binding fragment thereof interacts with at least one of NPR1 residues selected from the group consisting of Arg143, Leu144, Glu384, Leu401, Val402, Ala103, Ser405, Gly406, Arg407, Lys408, Trp411, Leu413, Gly414, Tyr415, and Pro416.

[0038] In a further embodiment, the disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to NPR1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 18; and the light chain comprises the amino acid sequence of SEQ ID NO: 20.

[0039] In a further embodiment, the disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to NPR1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:34; and the light chain comprises the amino acid sequence of SEQ ID NO:36.

[0040] In a further embodiment, the disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to NPR1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:51; and the light chain comprises the amino acid sequence of SEQ ID NO:53.

[0041] In a further embodiment, the disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to NPR1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:71; and the light chain comprises the amino acid sequence of SEQ ID NO:73.

[0042] In certain embodiments, the disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to NPR1, the antibody or antigen-binding fragment thereof 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), the HCVR comprising: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, 38, and 55; (ii) an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, 38, and 55; (iii) an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, 38, and 55. or (iv) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, 38, and 55, wherein said amino acid sequence has no more than 12 amino acid substitutions; LCVR comprises: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, 46, and 63; (b) an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, 46, and 63; (c) an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, 46, and 63; or (d) an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, 46, and 63, wherein said amino acid sequence has no more than 10 amino acid substitutions.

[0043] In certain preferred embodiments, the disclosure includes antibodies that specifically bind to NPR1 in an antagonistic manner, ie, block or reduce NPR1 binding and / or activity.

[0044] The present disclosure includes anti-NPR1 antibodies with modified glycosylation patterns. In some embodiments, modification to remove undesired glycosylation sites may be useful, or antibodies lacking fucose moieties present on oligosaccharide chains may be useful, for example, to increase antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation may be performed to modify complement-dependent cytotoxicity (CDC).

[0045] In certain embodiments, the present disclosure provides antibodies and antigen-binding fragments thereof that exhibit pH-dependent binding to NPR1. For example, the present disclosure includes antibodies and antigen-binding fragments thereof that bind to NPR1 with higher affinity at neutral pH than at basic pH (i.e., reduced binding at basic pH).

[0046] The present disclosure also provides antibodies and antigen-binding fragments thereof that compete for specific binding to NPR1 with an antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein each of the HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0047] The present disclosure also provides antibodies and antigen-binding fragments thereof that cross-compete for binding to NPR1 with a reference antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, each of the HCVR and LCVR having an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0048] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as a reference antibody or antigen-binding fragment thereof that comprises three CDRs of an HCVR and three CDRs of an LCVR, each of the HCVR and LCVR having an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0049] In certain embodiments, an antibody or antigen-binding fragment of the present disclosure is bispecific, comprising a first binding specificity for a first epitope of NPR1 and a second binding specificity for a second epitope of NPR1, wherein the first and second epitopes are distinct and non-overlapping.

[0050] In certain embodiments, the disclosure provides an isolated antagonist anti-NPR1 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof having one or more of the following characteristics: (a) being a fully human monoclonal antibody; (b) having a dissociation constant (K) of less than 1.7 nM at 25° C. and 37° C. as measured by surface plasmon resonance assay. D ) binds to human NPR1; (c) has a K of less than 1.99 nM at 25° C. and 37° C. as measured by surface plasmon resonance assays; D (d) binds to monkey NPR1 with a K of less than 1.52 nM in the presence of ANP at 25° C. and 37° C. as measured by surface plasmon resonance assays. D (e) inhibits ligand-induced NPR1 activation (e.g., induced by ANP or BNP) as measured by a cGMP accumulation assay; (f) has an EC of less than 2.9 nM in the presence or absence of ANP or BNP as measured by an electrochemiluminescence-based immunoassay. 50 (g) binds to human NPR1 with an EC of less than 4.2 nM in the presence or absence of ANP or BNP as measured by electrochemiluminescence-based immunoassay. 50(h) increases systemic blood pressure (including systolic pressure, diastolic pressure, mean arterial pressure, and pulse pressure) when administered to normotensive and hypotensive mice, where the increase in systemic blood pressure persists for up to about 28 days following administration of a single dose; (i) increases systemic blood pressure when administered to ANP overexpression-induced hypotensive mice, where the increase in systemic blood pressure persists for up to about 28 days following administration of a single dose; (j) increases systemic blood pressure in LPS-induced hypotensive mice; 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.

[0051] In a second aspect, the present disclosure provides a nucleic acid molecule encoding an anti-NPR1 antibody or a portion thereof. In certain embodiments, an isolated polynucleotide molecule is provided that includes a polynucleotide sequence encoding a heavy chain variable region (HCVR) of an isolated antibody or antigen-binding fragment thereof that specifically binds to a natriuretic peptide receptor 1 (NPR1) protein, the antibody or antigen-binding fragment thereof binds to and blocks NPR1. In a further embodiment, an isolated polynucleotide molecule is provided that includes a polynucleotide sequence encoding a light chain variable region (LCVR) of an isolated antibody or antigen-binding fragment thereof that specifically binds to a natriuretic peptide receptor 1 (NPR1) protein, the antibody or antigen-binding fragment thereof binds to and blocks NPR1. For example, the present 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 includes 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] The present disclosure also provides a nucleic acid molecule encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the set of HCDR1-HCDR2-HCDR3 amino acid sequences is as defined by any of the exemplary antibodies listed in Table 1.

[0060] The present disclosure also provides a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the set of LCDR1-LCDR2-LCDR3 amino acid sequences is as defined by any of the exemplary antibodies listed in Table 1.

[0061] The disclosure also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises the amino acid sequence of 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 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, and a polynucleotide sequence selected from any of the LCVR nucleic 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. In certain embodiments according to this aspect of the disclosure, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same anti-NPR1 antibody listed in Table 1.

[0062] In a related embodiment, the present disclosure provides a vector comprising a polynucleotide molecule encoding the HCVR of an isolated antibody or antigen-binding fragment thereof that specifically binds to the natriuretic peptide receptor 1 (NPR1) protein, wherein the antibody or antigen-binding fragment thereof binds to and blocks NPR1. In another related embodiment, the present disclosure provides a vector comprising a polynucleotide molecule encoding the LCVR of an isolated antibody or antigen-binding fragment thereof that specifically binds to the natriuretic peptide receptor 1 (NPR1) protein, wherein the antibody or antigen-binding fragment thereof binds to and blocks NPR1. In yet another related embodiment, the present disclosure provides a recombinant expression vector capable of expressing a polypeptide comprising the heavy and / or light chain variable region of the antibody. For example, the present disclosure includes a recombinant expression vector comprising any of the above-mentioned nucleic acid molecules, i.e., any of the HCVR, LCVR, and / or CDR sequences listed in Table 2. In certain embodiments, the disclosure provides an expression vector comprising: (a) a nucleic acid molecule comprising a nucleic acid sequence encoding an HCVR of an antibody that binds to NPR1, the HCVR comprising an amino acid sequence selected from the group consisting of sequences listed in Table 1; and / or (b) a nucleic acid molecule comprising a nucleic acid sequence encoding an LCVR of an antibody that binds to NPR1, the LCVR comprising an amino acid sequence selected from the group consisting of sequences listed in Table 1. Also included within the scope of the disclosure are host cells comprising vectors according to the disclosure, as well as methods of producing antibodies or portions thereof by culturing host cells under conditions that allow for the production of antibodies or antibody fragments, and methods of recovering the antibodies and antibody fragments so produced. In certain embodiments, the method of production according to the disclosure further comprises formulating the antibody or antigen-binding fragment thereof as a pharmaceutical composition comprising an acceptable carrier. 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.In certain embodiments, the present disclosure provides a method of producing an antibody or antigen-binding 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 the antibody or antigen-binding fragment thereof operably linked to a promoter; culturing the host cell under conditions favorable for expression of the nucleic acid sequence; and isolating the antibody or antigen-binding fragment thereof from the culture medium and / or the host cell. The isolated antibody or antigen-binding fragment thereof can be purified using any of the methods known in the art.

[0063] In a third aspect, the disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that specifically binds to natriuretic peptide receptor 1 (NPR1) protein, the antibody or antigen-binding fragment thereof binding to and blocking NPR1, and a pharma- ceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises a therapeutically effective amount of at least one recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to NPR1, and a pharma- ceutically acceptable carrier. In a related aspect, the disclosure features a composition that is a combination of an anti-NPR1 antibody and a second therapeutic agent or therapy. In one embodiment, the second therapeutic agent or therapy is any agent or therapy that is advantageously combined with an anti-NPR1 antibody. Exemplary agents or therapies that may be advantageously combined with antagonist anti-NPR1 antibodies include, but are not limited to, other agents that bind and / or block NPR1 signaling and / or activity (including other antibodies or antigen-binding fragments thereof, etc.), and / or agents that do not directly bind to NPR1 but that nevertheless treat or ameliorate at least one symptom or sign of a disease or disorder associated with NPR1 (as disclosed elsewhere herein). Additional combination therapies and co-formulations that include the anti-NPR1 antibodies of the present disclosure are disclosed elsewhere herein.

[0064] In a fourth aspect, the present disclosure provides a method of treatment for a disease or disorder associated with NPR1 in a subject using an anti-NPR1 antibody or an antigen-binding portion of the antibody of the present disclosure, the method of treatment comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an antibody or an antigen-binding fragment of the antibody according to the present disclosure. In certain embodiments, the disorder to be treated is any disease or condition (e.g., hypotension) that is improved, ameliorated, inhibited or prevented by blocking NPR1 activity. In certain embodiments, the NPR1-related disease or disorder is selected from the group consisting of hypotension, circulatory shock, septic shock, neurogenic orthostatic hypotension, postural orthostatic tachycardia syndrome (POTS), heart failure, cardiogenic shock, obesity, renal failure, chronic kidney disease, macular edema, glaucoma, stroke, lung disorder, pulmonary fibrosis, inflammation, asthma, skeletal growth disorder, bone fracture, diabetes, hypoglycemia, and cancer.

[0065] In certain embodiments, the present disclosure provides a method for preventing or treating an NPR1-related disease or disorder, comprising administering a therapeutically effective amount of an anti-NPR1 antibody or antigen-binding fragment thereof of the present disclosure to a subject in need thereof. In certain embodiments, the antibody or antigen-binding fragment thereof is administered prophylactically or therapeutically to a subject (e.g., a subject having or at risk of having an NPR1-related disease or disorder). In certain embodiments, the antibody or antigen-binding fragment thereof, or a composition comprising an antibody or antigen-binding fragment thereof according to the present disclosure, is administered in combination with a second therapeutic agent or therapy. The second therapeutic agent or therapy, in certain embodiments, is selected from the group consisting of angiogenesis inhibitors, vasoconstrictors / vasopressors, immunosuppressants, ascorbic acid, calcineurin inhibitors, corticosteroids, VEGF inhibitors, decongestants, antidepressants, hormonal contraceptives, stimulants (including cardiac stimulants), caffeine, extracorporeal membrane oxygenation, ventricular assist devices, intra-aortic balloon pumps, lifestyle modifications, dietary supplements, antibacterial agents, insulin, and anti-inflammatory agents. In certain embodiments, the second therapeutic agent or therapy can be an agent or therapy that helps to attenuate or reduce any possible side effects associated with the antibody or antigen-binding fragment thereof of the present disclosure, if such side effects occur. The antibody or fragment thereof, or a composition comprising the antibody or antigen-binding fragment thereof according to the present disclosure, is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, or intramuscularly. The antibody or fragment thereof is administered at a dose of about 0.1 mg / kg of subject's body weight to about 100 mg / kg of subject's body weight. In certain embodiments, the antibody of the present disclosure is administered at one or more doses, including between 10 mg and 600 mg.

[0066] The present disclosure also includes the use of an anti-NPR1 antibody, or antigen-binding fragment thereof, of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder that benefits from blocking NPR1 binding and / or activity.

[0067] Other embodiments will become apparent from consideration of the following detailed description. [Brief description of the drawings]

[0068] [Figure 1] 1A and 1B show that anti-NPR1 antibodies inhibited hNPR1 activation induced by (FIG. 1A) ANP and (FIG. 1B) BNP. Cells were pretreated with increasing concentrations of anti-NPR1 antibodies, control mAb, or dilution buffer alone for 15 min at 37° C., followed by increasing concentrations of ANP, BNP, 0.2 nM ANP, or 0.7 nM BNP for 30 min at 37° C. Experiments were performed in duplicate. Open symbols indicate conditions in which no test article was added or only a fixed concentration of ANP or BNP was added, and filled symbols indicate conditions in which test articles were added at various concentrations; dilution buffer: OptiMEM with 0.1% FBS. [Figure 2-1] Figures 2A-2E show noncompetitive inhibition of ANP-mediated NPR1 activation by (Figure 2A) H4H22034N, (Figure 2B) REGN7541, (Figure 2C) REGN7544, and (Figure 2D) REGN7548 using HEK293 / hNPR1 cells. Data from panels 2A to 2D were analyzed by Schild plot analysis (Figure 2E) to evaluate the Schild slope for each of the anti-NPR1 antibodies. Fluorescence intensity and detection of cGMP concentration were calculated as described in Experimental Procedures. Experiments were performed in duplicate. No ligand stimulation for each indicated antibody concentration was plotted at 0.1 pM and included in the Schild plot analysis. Open symbols indicate conditions with ANP alone without test article; filled symbols indicate conditions when test article was added at the indicated concentrations of 50 nM, 150 nM, or 450 nM with ANP in the concentration range of 1 pM to 1 μM; Dilution buffer: OptiMEM with 0.1% FBS; CR: concentration ratio. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 3-1]3A-3C show that anti-NPR1 antibodies induced NPR1 internalization as measured by cytotoxicity assays with secondary ADCs in the absence of ligand (Fig. 3A) or in the presence of 100 nM ANP (Fig. 3B) or 100 nM BNP (Fig. 3C). HEK293 / hNPR1 cells were pretreated with increasing concentrations of anti-NPR1 antibodies, control mAb, or dilution buffer alone in the presence or absence of 100 nM ANP or 100 nM BNP for 5 min at 37 °C, followed by secondary ADC treatment for 3 days at 37 °C. Experiments were performed in duplicate. Open symbols indicate conditions when no test article was added or only a fixed concentration of ANP or BNP was added, and filled symbols indicate conditions when test article was added at various concentrations; dilution buffer: OptiMEM with 0.1% FBS. [Figure 3-2] Continued from Figure 3-1. [Figure 4] Figure 2 shows the acute effect of NPR1 antagonist mAb on pulse pressure in normotensive NPR1 hu / hu mice - a single 25 mg / kg dose. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on body weight. Animals were given a single 25 mg / kg intravenous injection of NPR1 antagonist mAb or PBS as described in Table 25. All values ​​are mean ± SEM, n=6 per group. [Diagram 5] Figure 2 shows the effect of NPR1 antagonist mAb on systolic blood pressure in normotensive NPR1 hu / hu mice - a single 25 mg / kg dose. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on body weight. Animals were given a single 25 mg / kg intravenous injection of NPR1 antagonist mAb or PBS as described in Table 25. All values ​​are mean ± SEM, n=6 per group. [Figure 6]FIG. 2 shows the effect of NPR1 antagonist mAbs on systolic blood pressure in normotensive NPR1 hu / hu mice - a single 1 mg / kg dose. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on body weight. Animals received a single 1 mg / kg subcutaneous injection of NPR1 antagonist mAb or a single 25 mg / kg injection of IgG4P isotype control mAb as described in Table 28. All values ​​are mean ± SEM, n=4-5 per group. [Figure 7] Figure 2. Normotensive NPR1 hu / hu mice - Effect of NPR1 antagonist mAb on systolic blood pressure in a single 25 mg / kg dose. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on body weight. Animals were given a single 25 mg / kg subcutaneous injection of NPR1 antagonist mAb or IgG4P isotype control mAb as described in Table 28. All values ​​are mean ± SEM, n=4-5 per group. [Figure 8] Figure 3. Effect of NPR1 antagonist mAb on systolic blood pressure in ANP overexpression-induced hypotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on body weight. Animals received a single 50ug HDD dose of control or ANP plasmid as described in Table 31, followed by a single 25mg / kg intravenous injection of NPR1 antagonist mAb or isotype control. All values ​​are mean ± SEM, n=4-7 per group. [Figure 9] Figures 9A and 9B show the effect of NPR1 antagonist mAbs on absolute (Figure 9A) and relative heart weight (Figure 9B) of hypotensive NPR1 hu / hu mice after induction of overexpression. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on body weight. Animals were given a single 25 mg / kg subcutaneous injection of NPR1 antagonist mAb or IgG4P isotype control mAb as described in Table 31. All values ​​are mean ± SEM, n = 5-8 per group. [Figure 10]Figure 3. Effect of NPR1 antagonist mAb on pulse pressure in LPS-induced hypotensive NPR1 hu / hu mice - a single 25 mg / kg prophylactic or therapeutic intravenous dose. Telemetered NPR1 hu / hu mice were randomized into groups based on body weight. Animals received a single 5 mg / kg intraperitoneal injection of LPS or saline and a single intravenous injection of NPR1 antagonist mAb or PBS approximately 24 hours before or 8 hours after LPS administration, as described in Table 35. All values ​​are mean ± SEM, n = 7-10 per group. [Figure 11] 1 shows the mean change in pulse pressure normalized to baseline for each treatment group from 3 days prior to administration of REGN7544 or vehicle control to the end of the experiment. Data are presented as group mean ± standard error of the mean. [Figure 12] Figure 1 shows the mean change in systolic blood pressure normalized to baseline for each treatment group from 3 days prior to administration of REGN7544 or vehicle control to the end of the experiment. Data are presented as group mean ± standard error of the mean. Vertical dotted lines indicate administration of drug as indicated by route of administration (i.e., SC or PO). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] 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.

[0070] 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.

[0071] 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. NPR1 is a receptor for both atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and undergoes a conformational change in the extracellular domain upon ligand binding (Ogawa et al., 2004 J. Biol. Chem. 279:28625-31). 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. The term "NPR1" includes recombinant NPR1 protein or fragments thereof. The term also encompasses, for example, NPR1 protein or a fragment thereof linked to a histidine tag, mouse or human Fc, or a signal sequence such as ROR1 (eg, SEQ ID NOs: 74-78).

[0072] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule (i.e., an "intact antibody molecule") composed of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "VH "), 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.

[0073] 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).

[0074] 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.

[0075] The fully human anti-NPR1 monoclonal antibodies disclosed herein 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 are readily ascertained by comparing the amino acid sequences disclosed herein to 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"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art will readily create a large number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V 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.

[0076] The present disclosure also includes fully human anti-NPR1 monoclonal 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 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.

[0077] As used herein, the term "human antibody" or "fully human antibody" 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) are 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.

[0078] 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.

[0079] 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 antigen that is relatively stable 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™, to specifically bind to NPR1. Furthermore, a multispecific antibody that binds to one domain in NPR1 and one or more additional antigens, or a bispecific antibody that binds to two different regions of NPR1, is nevertheless considered an antibody that "specifically binds" as used herein.

[0080] 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 for NPR1 is expressed as .

[0081] 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 NPR1 with a rate constant equal to or lower than that.

[0082] 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. 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 the NPR1 protein.

[0083] In certain embodiments, an antibody or antibody fragment of the disclosure is conjugated to a moiety, such as a ligand or therapeutic moiety (an "immunoconjugate"), a second anti-NPR1 antibody, or any other therapeutic moiety useful for treating an NPR1-associated disease or disorder.

[0084] 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 NPR1 is substantially free of Abs that specifically bind to antigens other than NPR1).

[0085] As used herein, an "antagonist antibody" (or an "antibody that blocks or reduces NPR1 activity") is intended to refer to an antibody that blocks or reduces NPR1 signaling and / or at least one biological activity of NPR1 upon binding to NPR1. For example, an antagonist anti-NPR1 antibody can increase systemic blood pressure upon administration to a subject in need thereof. The anti-NPR1 antibodies disclosed herein are antagonist antibodies.

[0086] As used herein, an "activating antibody" or "agonist antibody" (or an "antibody that increases or enhances NPR1 activity" or an "antibody that stabilizes the activated conformation") is intended to refer to an antibody that, upon binding to NPR1, results in activation of at least one biological activity of NPR1. For example, an activating or agonist anti-NPR1 antibody can reduce systemic blood pressure upon administration to a subject in need thereof.

[0087] 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).

[0088] As used herein, the term "K D " is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0089] 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. A single antigen may have more than one epitope. Thus, different 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, and have their residues that are directly involved in the affinity of the interaction. Epitopes are also conformational, i.e., they include 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 features, and / or specific alteration features.

[0090] As used herein, the term "cross-compete" means that an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in both directions, i.e., a first antibody 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, via 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 being less than the background signal due to self-self binding (where the first and second antibodies are the same antibody). Cross-competition between two antibodies is expressed, for example, as the percent binding of a second antibody that is less than baseline self-to-self background binding (where the first and second antibodies are the same antibody).

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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 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). As used herein, this phrase refers to an amount that blocks NPR1 (e.g., NPR1 signaling, NPR1 activity) and / or increases systemic blood pressure.

[0095] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, in need of amelioration, prevention and / or treatment of an NPR1-related disease or disorder, such as hypotension. This term includes human subjects having or at risk of having such a disease or disorder.

[0096] As used herein, the term "treat", "treating" or "treatment" refers to the reduction or amelioration of the severity of at least one symptom or sign of an NPR1-related disease or disorder by administering a therapeutic agent, such as an antagonist antibody of the present disclosure, 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. Disorders or diseases include hypotension, and / or disorders or diseases associated with hypotension, and / or diseases or disorders, such as hypotension associated with septic shock and neurodegenerative diseases. Disorders or diseases also include postural orthostatic tachycardia syndrome (POTS).

[0097] The terms "prevent," "preventing," or "prevention" refer to the inhibition of the onset of an NPR1-related disease or disorder, such as hypotension, or any symptoms or signs of such a disease or disorder, upon administration of an antibody of the disclosure.

[0098] As used herein, the phrase "blood pressure" may refer to any one of systolic blood pressure, diastolic blood pressure, mean arterial pressure (area under the arterial pressure / time curve divided by cardiac cycle duration), and pulse pressure (difference between systolic and diastolic pressure). Methods for measuring blood pressure are known in the art. Blood pressure is measured in millimeters of mercury (mmHg) and is usually expressed in terms of systolic (blood) pressure relative to diastolic (blood) pressure. Measurement methods include auscultation, oscillometric, ultrasound, and finger cuff methods. It can generally be measured using, for example, a digital blood pressure monitor or a sphygmomanometer.

[0099] Antigen-binding fragment of an antibody As used herein, the term "antibody" shall be understood to include antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "complete antibody molecules"), as well as antigen-binding fragments thereof, unless otherwise specifically indicated. 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 NPR1 protein. 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 whole 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, for example, to place one or more variable and / or constant domains in the appropriate position, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids.

[0100] 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.

[0101] 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.

[0102] 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 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.

[0103] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each of which can specifically bind to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art.

[0104] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method may be used in the context of the present disclosure to generate human antibodies that specifically bind to NPR1.

[0105] An immunogen comprising any one of the following is used to generate antibodies against the NPR1 protein. In certain embodiments, the antibodies of the present disclosure are obtained from mice immunized with the full-length native NPR1 protein (see, e.g., UniProtKB / Swiss-Prot Accession No. P16066.1), or DNA encoding the protein or a fragment thereof. Alternatively, the protein or a fragment thereof may be produced and modified using standard biochemical techniques and used as an immunogen.

[0106] In one embodiment, the immunogen may be a recombinant NPR1 protein or a fragment thereof (e.g., SEQ ID NOs: 74-78) expressed in Escherichia coli or any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells.

[0107] Using VELOCIMMUNE® technology (see, e.g., US6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®), or any other known method for generating monoclonal antibodies, a high affinity chimeric antibody against NPR1 is first isolated, having a human variable region and a mouse constant region. VELOCIMMUNE® technology involves the creation of a transgenic mouse with a genome that includes human heavy and light chain variable regions operably linked to endogenous mouse constant region sites, such that the mouse produces antibodies that include human variable regions and mouse constant regions in response to antigenic stimulation. DNA encoding the heavy and light chain variable regions 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.

[0108] Generally, VELOCIMMUNE® mice are challenged with an antigen of interest, and lymphoid cells (such as B cells) are harvested from the mice that express antibodies. The lymphoid cells are fused with myeloma cell lines to prepare immortalized hybridoma cell lines, which are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy and light chains is isolated and linked to constant regions of the desired isotypes of heavy and light chains. Such antibody proteins are produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains is directly isolated from antigen-specific lymphocytes.

[0109] First, a high affinity chimeric antibody with human variable region and mouse constant region is isolated. As in the experimental section below, the antibody is 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 present disclosure, e.g., wild type or modified IgG1 or IgG4. While the constant region selected varies according to the particular use, the high affinity antigen binding and target specificity characteristics reside in the variable region.

[0110] biological equivalent The antagonist anti-NPR1 antibodies and antibody fragments of the present disclosure include proteins that have a different amino acid sequence from the described antibodies but maintain the ability to bind to NPR1 protein. Such variant antibodies and antibody fragments 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 antibodies. Similarly, the DNA sequences encoding the antibodies of the present disclosure 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 antibodies or antibody fragments of the present disclosure.

[0111] 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.

[0112] 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 antigen binding proteins.

[0113] In one embodiment, two antigen binding 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.

[0114] In one embodiment, two antigen binding 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.

[0115] 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 antibody 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 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 antibody.

[0116] Biologically equivalent variants of the 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. 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 renaturation. In other contexts, biologically equivalent antibodies include antibody variants that contain amino acid changes to modify the glycosylation characteristics of the antibody, for example, mutations that remove or eliminate glycosylation.

[0117] Anti-NPR1 Antibodies Containing Fc Variants According to certain embodiments of the present disclosure, there are provided anti-NPR1 antibodies comprising an Fc domain that includes 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 an anti-NPR1 antibody comprising an Fc domain that includes 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. H 2 or C HThe present invention includes anti-NPR1 antibodies that contain mutations in region 3 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 antibody 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.

[0118] For example, the disclosure includes anti-NPR1 antibodies 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.

[0119] The present disclosure also provides a chimeric heavy chain constant (C H ) region, and a chimeric C H A region may contain more than one C H For example, the antibodies 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).

[0120] Antibody Biology Generally, the antagonist antibodies of the present disclosure function by binding to the NPR1 protein and blocking its signaling and / or activity. For example, the present disclosure provides antibodies that have a dissociation constant (K) of less than 1.7 nM at 25° C. and 37° C. as measured by surface plasmon resonance assay, 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 1.27 nM, less than about 0.34 nM, less than about 0.08 nM, or less than about 0.06 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 human NPR1.

[0121] The present disclosure also relates to a compound having a dissociation constant (K) of less than 1.99 nM at 25° C. and 37° C., as measured by a surface plasmon resonance assay, 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 1.23 nM, less than about 0.32 nM, less than about 0.1 nM, or less than about 0.07 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 monkey NPR1.

[0122] The present disclosure also provides a method for the preparation of a medicament for the treatment of cancer, comprising administering to the patient a medicament for which the medicament has a K of less than 1.52 nM in the presence of ANP at 25° C. and 37° C. as measured by a surface plasmon resonance assay, e.g., using an assay format as defined in Example 3 herein. D In certain embodiments, the antibodies or antigen-binding fragments thereof have a K of less than about 1.1 nM, less than about 0.8 nM, less than about 0.6 nM, and less than about 0.5 nM in the presence of ANP at 25° C. and 37° C., as measured by a surface plasmon resonance assay, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay. D It binds to human NPR1.

[0123] The present disclosure also includes antibodies and antigen-binding fragments of antibodies that inhibit ligand-induced NPR1 activation (e.g., induced by ANP or BNP) as measured by a cGMP accumulation assay, for example, using an assay format as defined in Example 6 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof inhibit ligand-induced NPR1 activation by at least about 80%, at least about 90%, at least about 92%, at least about 99%, and at least about 100%, as measured by a cGMP accumulation assay, for example, using an assay format as defined in Example 6 herein, or a substantially similar assay.

[0124] The present disclosure also provides a method for treating rheumatoid arthritis, comprising administering to a patient a therapeutically effective amount of rheumatoid arthritis (RA) or rheumatoid arthritis (BNP) having an EC of less than 2.9 nM in the presence or absence of ANP or BNP, as measured by an electrochemiluminescence-based immunoassay, e.g., using an assay format as defined in Example 7 herein. 50 In certain embodiments, the antibodies or antigen-binding fragments thereof have an EC of less than about 2.1 nM, less than about 1.2 nM, and less than about 0.6 nM in the presence or absence of ANP or BNP, as measured by electrochemiluminescence-based immunoassay, e.g., using an assay format as defined in Example 7 herein, or a substantially similar assay. 50 It binds to human NPR1.

[0125] The present disclosure also provides a method for treating a subject with an EC value of less than 4.2 nM in the presence or absence of ANP or BNP, as measured by an electrochemiluminescence-based immunoassay, e.g., using an assay format as defined in Example 7 herein. 50 In certain embodiments, the antibodies or antigen-binding fragments thereof have an EC of less than about 2.9 nM, less than about 2.1 nM, and less than about 0.7 nM in the presence or absence of ANP or BNP, as measured by electrochemiluminescence-based immunoassay, e.g., using an assay format as defined in Example 7 herein, or a substantially similar assay. 50 It binds to monkey NPR1.

[0126] The present disclosure also includes antibodies and antigen-binding fragments of antibodies that, when administered to normotensive and hypotensive mice, increase systemic blood pressure (including systolic pressure, diastolic pressure, mean arterial pressure, and pulse pressure), with the increase in systemic blood pressure persisting for up to about 28 days upon administration of a single dose, e.g., as described herein in Example 9.

[0127] The present disclosure also includes antibodies and antigen-binding fragments of antibodies that increase systemic blood pressure when administered to ANP overexpression-induced hypotensive mice, where the increase in systemic blood pressure persists for up to about 28 days upon administration of a single dose, e.g., as described in Example 10 herein.

[0128] The present disclosure also includes antibodies and antigen-binding fragments thereof that increase systemic blood pressure in LPS-induced hypotensive mice, as described in Example 11 herein.

[0129] In one embodiment, the disclosure provides an isolated, recombinant antibody or antigen-binding fragment thereof that specifically binds to NPR1 protein in the presence or absence of ANP or BNP and reduces or blocks NPR1 signaling and / or activity, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) has a dissociation constant (K) of less than 1.7 nM at 25° C. and 37° C. as measured by surface plasmon resonance assay. D ) binds to human NPR1; (c) has a K of less than 1.99 nM at 25° C. and 37° C. as measured by surface plasmon resonance assays; D (d) binds to monkey NPR1 with a K of less than 1.52 nM in the presence of ANP at 25° C. and 37° C. as measured by surface plasmon resonance assays. D (e) inhibits ligand-induced NPR1 activation as measured by a cGMP accumulation assay; (f) has an EC of less than 2.9 nM in the presence or absence of ANP or BNP as measured by an electrochemiluminescence-based immunoassay. 50 (g) binds to human NPR1 with an EC of less than 4.2 nM in the presence or absence of ANP or BNP as measured by electrochemiluminescence-based immunoassay. 50(h) when administered to normotensive and hypotensive mice, increases systemic blood pressure, the increase in systemic blood pressure persisting for up to about 28 days following administration of a single dose; (i) when administered to ANP overexpression-induced hypotensive mice, increases systemic blood pressure, the increase in systemic blood pressure persisting for up to about 28 days following administration of a single dose; (j) increases systemic blood pressure in LPS-induced hypotensive mice; 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.

[0130] The antibodies of the present disclosure may have one or more of the above biological characteristics, or any combination thereof. Other biological properties of the antibodies of the present disclosure will be apparent to those of skill in the art from a consideration of this disclosure, including the Examples herein.

[0131] Epitope mapping and related techniques The present disclosure includes antagonist anti-NPR1 antibodies that interact with one or more amino acids found in one or more regions of the NPR1 protein molecule. The epitope that the antibody 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 NPR1 protein molecule (e.g., linear epitope in a domain). Alternatively, the epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) located in any or both of the aforementioned domains of the protein molecule (e.g., conformational epitope).

[0132] 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.

[0133] The term "epitope" refers to a site on an antigen to which B and / or T cells respond. 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. An epitope typically comprises at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation.

[0134] 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.

[0135] In certain embodiments, the disclosure includes antagonist anti-NPR1 antibodies and antigen-binding fragments thereof that interact with one or more epitopes found within the extracellular domain of NPR1. 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 located within the extracellular domain of NPR1. Alternatively, an epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within NPR1.

[0136] The present disclosure includes antagonist anti-NPR1 antibodies that bind to the same epitope, or a portion of an epitope, as any of the specific representative antibodies listed in Table 1. Similarly, the present disclosure also includes antagonist anti-NPR1 antibodies that compete with any of the specific representative antibodies listed in Table 1 for binding to the NPR1 protein or a fragment thereof. For example, the present disclosure includes antagonist anti-NPR1 antibodies that cross-compete with one or more antibodies listed in Table 1 for binding to the NPR1 protein.

[0137] By using routine methods known in the art, it can be easily determined whether an antibody binds to the same epitope as a reference anti-NPR1 antibody or competes with the reference anti-NPR1 antibody for binding. For example, to determine whether a test antibody binds to the same epitope as a reference anti-NPR1 antibody of the present disclosure, the reference antibody is allowed to bind to NPR1 protein or peptide under saturating conditions. Then, the ability of the test antibody to bind to NPR1 protein molecule is evaluated. If the test antibody can bind to NPR1 after saturation binding with the reference anti-NPR1 antibody, it is concluded that the test antibody binds to a different epitope than the reference anti-NPR1 antibody. On the other hand, if the test antibody cannot bind to NPR1 protein after saturation binding with the reference anti-NPR1 antibody, then the test antibody binds to the same epitope as the epitope bound by the reference anti-NPR1 antibody of the present disclosure.

[0138] To determine whether an antibody competes with a reference anti-NPR1 antibody 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 NPR1 protein under saturating conditions, and then the binding of the test antibody to the NPR1 molecule is evaluated. In the second way, the test antibody is allowed to bind to the NPR1 molecule under saturating conditions, and then the binding of the reference antibody to the NPR1 molecule is evaluated. In both ways, if only the first (saturating) antibody is capable of binding to the NPR1 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to NPR1. 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.

[0139] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. 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, e.g., 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.

[0140] 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.

[0141] In certain embodiments, the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to natriuretic peptide receptor 1 (NPR1) protein and blocks NPR1.

[0142] Immunoconjugates The present disclosure encompasses human antagonist anti-NPR1 monoclonal antibodies conjugated to a therapeutic moiety (immunoconjugates) for treating NPR1-related diseases or disorders (e.g., hypotension). As used herein, the term "immunoconjugate" refers to an antibody that is chemically or biologically bound to a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide or protein, or a therapeutic agent. The antibody is bound to a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide, or a therapeutic agent at any position along the molecule, so long as it can bind to its target. 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 anti-NPR1 antibody takes into consideration the condition to be treated and the desired therapeutic effect to be achieved. Examples of agents suitable for forming immunoconjugates are known in the art, see, for example, WO05 / 103081.

[0143] multispecific antibody The antagonist antibody of the present disclosure can be monospecific, bispecific, or multispecific.Multispecific antibodies can be specific to different epitopes of one target polypeptide, or can contain antigen binding domains specific to more than one target polypeptide.See, for example, Tutt et al., 1991, J.Immunol.147:60-69; Kufer et al., 2004, Trends Biotechnol.22:238-244.

[0144] Any of the multispecific antigen-binding molecules or variants thereof of the present disclosure can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques), as is well known to those skilled in the art.

[0145] In one embodiment, NPR1-specific antibodies are produced in a bispecific format ("bispecific") in which variable regions that bind different domains of the NPR1 protein are linked together to confer dual domain specificity within a single binding molecule. Variable regions with specificity for individual domains (e.g., segments of the N-terminal domain), or variable regions that can bind different regions within one domain, are paired on a structural scaffold that allows each region to bind to a separate epitope or to different regions within one domain simultaneously. In one example of a bispecific, a heavy chain variable region (V) from a binder with specificity for one domain is linked together with a heavy chain variable region (V H ) but that V H without destroying the original singularity for the original V H Non-cognate V paired with L To identify partners, we selected light chain variable regions (V) from a series of binders with specificity for the second domain. L ) can be recombined with a single V L Segment (e.g. V L 1) consists of two bond "arms" (V H 1-V L 1 and V H 2-V L 1) to generate a bispecific consisting of two different V H Domain (e.g., VH 1 and V H 2) is combined with a single V L The use of segments reduces the complexity of the system, thereby simplifying and improving the efficiency of the cloning, expression, and purification processes used to generate bispecifics (see, e.g., US2011 / 0195454 and US2010 / 0331527).

[0146] Alternatively, antibodies that bind to more than one domain and a second target, such as, but not limited to, a second different anti-NPR1 antibody, can be prepared in a bispecific format using the techniques described herein or other techniques known to those skilled in the art. Antibody variable regions that bind to different regions are linked together, for example, with variable regions that bind to related sites on the extracellular domain of NPR1, to provide dual antigen specificity within a single binding molecule. Properly designed bispecifics of this nature perform dual functions. A variable region with specificity for the extracellular domain is combined with a variable region with specificity for the outside of the extracellular domain, and paired on a structural scaffold that allows each variable region to bind to a separate antigen.

[0147] Other exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knob-into-hole, common light chain (such as a common light chain with knob-into-hole), CrossMab, CrossFab, (SEED)body, Leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2Bispecific formats are included (for a review of the aforementioned formats, see, e.g., Klein et al., 2012, mAbs 4:6, pp. 1-11, and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0148] Therapeutic Administration and Formulation The present disclosure provides therapeutic compositions comprising the antagonist anti-NPR1 antibodies or antigen-binding fragments thereof 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, tolerance improvement, and the like, according to the present disclosure. Many suitable formulations can be found in formularies known to all medicinal chemists: 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, emulsion 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.

[0149] The dose of the antibody may vary depending on the age and size of the subject to be administered, the target disease, condition, route of administration, etc. When the antibody of the present disclosure is used to treat a disease or disorder in an adult patient or to prevent such a disease, it is advantageous to administer the antibody of the present disclosure at a single dose of typically about 0.1 to about 100 mg per kg of body weight. In one embodiment, the antibody according to the present disclosure is administered at a single dose of about 25 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 antibody or antigen-binding fragment thereof 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, about 10 to about 400 mg, or about 100 mg. In certain embodiments, the initial dose is followed by administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof in an amount that is approximately 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.

[0150] 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).

[0151] The use of nanoparticles to deliver the antibodies 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] Advantageously, the pharmaceutical compositions for oral or parenteral use described above 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 antibody 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 antibody is preferably contained per dosage form in the form of injection.

[0156] Therapeutic Uses of Antibodies The antagonist antibodies of the present disclosure are useful for the treatment and / or prevention of a disease or disorder or condition associated with NPR1, and / or for ameliorating at least one symptom associated with such a disease, disorder or condition. In certain embodiments, an antibody or antigen-binding fragment thereof of the present disclosure is administered in a therapeutic dose to a patient having a disease or disorder or condition associated with NPR1.

[0157] The disorders or diseases include hypotension, and / or disorders or diseases associated with hypotension, and / or diseases or disorders, such as hypotension associated with circulatory shock, septic shock, and neurodegenerative diseases. Hypotension is a drop in systemic blood pressure below an acceptable low value. Hypotension exists as a range, and no standard low blood pressure value is recognized, but blood pressure below 90mmHg (systolic) / 60mmHg (diastolic) is recognized as hypotension. Symptoms of hypotension include, but are not limited to, lightheadedness, dizziness, fainting, chest pain, shortness of breath, arrhythmia, elevated body temperature, headache, stiff neck, severe upper back pain, cough with phlegm, diarrhea, vomiting, dysuria, acute allergic reactions, fatigue, and visual abnormalities. Complications of untreated hypotension with reduced cardiac output can be severe and ultimately fatal. In impending or fulminant shock, untreated hypotension can lead to multiple organ failure (Sharma et al., 2021 updated, Hypotension, available at https: / / www.ncbi.nlm.nih.gov / books / NBK499961 / ). In one embodiment, an antagonist anti-NPR1 antibody or antigen-binding fragment thereof according to the present disclosure is used to treat a type of hypotension symptom or sign. In another embodiment, an antagonist anti-NPR1 antibody or antigen-binding fragment thereof according to the present disclosure is used to increase blood pressure in a subject having hypotension and / or having a disease or disorder associated with hypotension.

[0158] Septic shock is characterized by refractory hypotension, which causes inadequate perfusion of tissues, and is associated with high mortality. The standard treatment for sepsis with hypotension is the administration of vasopressors, such as catecholamines or mimetics, vasopressin, or Ang II, to maintain arterial pressure and serum lactate levels in the absence of hypovolemia. However, standard treatment vasopressors have significant drawbacks, including the need for frequent titration, having a narrow therapeutic window, the need for central venous access and ICU treatment, and even the possibility of reducing capillary perfusion (causing tissue ischemia (e.g., digital necrosis) at high doses for long periods of time). Thus, there is a significant unmet need to address (refractory) hypotension. In one embodiment, the antagonist anti-NPR1 antibody or antigen-binding fragment thereof according to the present disclosure is used to treat symptoms or signs of septic shock or to treat refractory hypotension of septic shock. In another embodiment, the use of an antagonist anti-NPR1 antibody or antigen-binding fragment thereof according to the present disclosure for treating septic shock or for treating refractory hypotension in septic shock can reduce the use of vasopressors, thereby reducing the required length of intensive care unit (ICU) stay.

[0159] Neurogenic orthostatic hypotension constitutes orthostatic hypotension (blood pressure falls when standing up, causing cerebral hypoperfusion) and is generally due to defective autonomic reflexes associated with neurodegenerative diseases. It is associated with multiple diseases and significant morbidity, with almost 50% of patients being rated as severe or very severe in terms of symptom impact on daily life. Two currently approved drug therapies are only modestly effective, both are short-acting, and require administration three times a day (TID). In one embodiment, an antagonist anti-NPR1 antibody or antigen-binding fragment thereof according to the present disclosure is used to treat neurogenic orthostatic hypotension.

[0160] Disorders or diseases also include postural orthostatic tachycardia syndrome (POTS). This syndrome is characterized by tachycardia and symptoms on standing without hypotension. In POTS, an increase in heart rate from horizontal to standing (or as in a tilt table test) of at least 30 beats per minute is recorded in adults and is measured during the first 10 minutes of standing. POTS typically afflicts young women and causes significant morbidity. Symptoms occurring on standing include lightheadedness, tremors, palpitations, weakness, fatigue, blurred vision, and occasional fainting. Significantly reduced quality of life is reported by more than 80% of POTS patients. Although fludrocortisone, midodrine, and beta-blockers are commonly used, they have not shown significant treatment effects; in fact, no specific drug is currently FDA approved for treating POTS. In one embodiment, an antagonist anti-NPR1 antibody or antigen-binding fragment thereof according to the present disclosure is used to treat POTS.

[0161] In certain embodiments, the antagonist antibodies of the disclosure are useful for treating or preventing at least one symptom or sign of a disease or disorder selected from the group consisting of hypotension, circulatory shock, septic shock, neurogenic orthostatic hypotension, postural orthostatic tachycardia syndrome (POTS), heart failure, cardiogenic shock, obesity, renal failure, chronic kidney disease, macular edema, glaucoma, stroke, pulmonary disorders, pulmonary fibrosis, inflammation, asthma, skeletal growth disorders, bone fractures, diabetes, hypoglycemia, and cancer.

[0162] Also contemplated herein is the prophylactic use of one or more antibodies of the present disclosure in subjects at risk of suffering from an NPR1-related disease or disorder.

[0163] In one embodiment of the disclosure, the antibody is used for the preparation of a pharmaceutical composition or medicament for treating a patient suffering from a disease, disorder or condition disclosed herein. In another embodiment of the disclosure, the antibody is used as an adjunct therapy with any other agent or any other therapy known to one of skill in the art useful for treating or ameliorating a disease, disorder or condition disclosed herein.

[0164] Combination therapy The combination therapy may include the antagonist antibody of the present disclosure and any additional therapeutic agent that is advantageously combined with the antibody of the present disclosure or the biologically active fragment of the antibody of the present disclosure.The antibody of the present disclosure may be synergistically combined with one or more drugs or therapies used to treat NPR1-related diseases or disorders, including hypotension.In some embodiments, the antibody of the present disclosure may be combined with a second therapeutic agent or therapy to improve one or more symptoms of the disease or condition.

[0165] Depending on the disease, disorder, or condition, the antibodies of the disclosure are used in combination with one or more additional therapeutic agents, including, but not limited to, angiogenesis inhibitors, vasoconstrictors / vasopressors, immunosuppressants, ascorbic acid, calcineurin inhibitors, corticosteroids, VEGF inhibitors, decongestants, antidepressants, hormonal contraceptives, stimulants (including cardiac stimulants), caffeine, extracorporeal membrane oxygenation, ventricular assist devices, intra-aortic balloon pumps, lifestyle modifications, nutritional supplements, antibiotics, insulin, and anti-inflammatory agents.

[0166] As used herein, the term "in combination with" means that an additional therapeutically active ingredient can be administered before, simultaneously with, or after administration of an antagonist anti-NPR1 antibody of the present disclosure. The term "in combination with" also includes sequential or simultaneous administration of an anti-NPR1 antibody and a second therapeutic agent or therapy.

[0167] The additional therapeutically active component is administered to the subject prior to administration of the anti-NPR1 antibody 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 therapeutically active component is administered to the subject after administration of the anti-NPR1 antibody 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 another embodiment, the additional therapeutically active ingredient is administered to the subject at the same time as the administration of the anti-NPR1 antibody of the present disclosure. For the purposes of this disclosure, "simultaneous" administration includes administration of the anti-NPR1 antibody and the additional therapeutically active ingredient to the 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 anti-NPR1 antibody and the additional therapeutically active ingredient are administered intravenously); or each dosage form is administered via a different route (e.g., the anti-NPR1 antibody 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 by the same route, or in separate dosage forms by different routes, are all considered to be "simultaneous administration" for the purposes of this disclosure. For purposes of this disclosure, administration of an anti-NPR1 antibody "before," "concurrently," or "after" administration of an additional therapeutically active ingredient (as these terms are defined herein above) is considered administration of the anti-NPR1 antibody "in combination with" the additional therapeutically active ingredient.

[0168] The present disclosure includes pharmaceutical compositions in which the anti-NPR1 antibodies of the present disclosure are co-formulated with one or more additional therapeutically active ingredients, as described elsewhere herein.

[0169] Diagnostic Uses of Antibodies The antagonist antibodies of the present disclosure are used to detect and / or measure NPR1 in a sample, for example, for diagnostic purposes. Certain embodiments contemplate the use of one or more antibodies of the present disclosure in an assay for detecting an NPR1-related disease or disorder. An exemplary diagnostic assay for NPR1 can include, for example, contacting a sample obtained from a patient with an anti-NPR1 antibody of the present disclosure, where the anti-NPR1 antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate NPR1 from a patient sample. Alternatively, an unlabeled anti-NPR1 antibody 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 NPR1 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 NPR1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0170] The samples that can be used in the NPR1 diagnostic assay according to the present disclosure include any tissue or fluid sample that can be obtained from a patient, which contains a detectable amount of either NPR1 protein or its fragments under normal or pathological conditions.Generally, the level of NPR1 protein in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease associated with NPR1) is measured to first establish a baseline or standard level of NPR1.This baseline level of NPR1 can then be compared with the level of NPR1 measured in a sample obtained from an individual suspected of having an NPR1-associated condition or a symptom associated with such a condition.

[0171] Antibodies specific for NPR1 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 portion of the peptide is distal to the surface. EXAMPLES

[0172] 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 present disclosure. Attempts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, 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

[0173] Example 1: Generation of human antibodies against natriuretic peptide receptor 1 (NPR1) Human antibodies against the NPR1 protein, which contains DNA encoding the human immunoglobulin heavy chain variable region and the kappa light chain variable region, were generated in VELOCIMMUNE® mice, which were immunized with human NPR1 and mouse ANP DNA by hydrodynamic DNA delivery and boosted with the extracellular domain of the human NPR1 protein complexed with mouse ANP.

[0174] Antibody immune response was monitored by NPR1-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 NPR1-specific antibodies. The cell lines were used to obtain several anti-NPR1 chimeric antibodies (e.g., antibodies with human variable domains and mouse constant domains).

[0175] Anti-NPR1 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-NPR1 antibodies (i.e., antibodies with human variable and constant domains) were obtained.

[0176] Exemplary antibodies generated as disclosed above were mAb38067, mAb38072, mAb38090, and mAb22034.

[0177] The biological properties of exemplary antibodies generated according to the methods of this Example are described in detail in the Examples below. EXAMPLES

[0178] 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-NPR1 antibodies of the disclosure.

[0179] [Table 1]

[0180] The corresponding nucleic acid sequence identifiers are shown in Table 2.

[0181] [Table 2]

[0182] 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 having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a mouse IgG1 Fc can be converted to an antibody having a human IgG1 or human IgG4 Fc, etc.), but in each case the variable domains (including the CDRs) indicated by the numerical identifiers shown in Table 1 remain the same, and the antigen binding characteristics are expected to be consistent or substantially similar regardless of the nature of the Fc domain.

[0183] 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.

[0184] Exemplary antibodies mAb38067, mAb38072, mAb38090, and mAb22034, which contain a human IgG4 Fc with a serine to proline mutation (S108P) in the hinge region, were named REGN7541, REGN7544, REGN7548, and H4H22034, respectively. Table 3 shows the nucleic acid and amino acid sequence identifiers for the full-length heavy and light chain sequences of these antibodies.

[0185] [Table 3]

[0186] Control constructs used in the following examples The following control constructs (anti-NPR1 antibodies) were included in the experiments disclosed herein for comparison purposes: "Comparative 1", a monoclonal antibody against human NPR1 having the VH / VL sequence of antibody "mAb5591" from US Patent No. 20120114659 (Morphosys). EXAMPLES

[0187] Example 3: Biacore binding kinetics of anti-NPR1 monoclonal antibodies binding to various NPR1 reagents measured at 25°C and 37°C Experimental procedure Equilibrium dissociation constants (K ) for different NPR1 reagents binding to purified anti-NPR1 monoclonal antibody (mAb) D ) was determined using a real-time surface plasmon resonance (SPR)-based Biacore 8k biosensor. All binding studies were performed in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant P20, pH 7.4 (HBS-EP) running buffer at 25° C. and 37° C. The Biacore CM5 sensor chip surface was first derivatized by amine coupling with an anti-human Fc-specific antibody to capture anti-NPR1 mAb. Binding studies were performed with human NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (hNPR1-MMH; SEQ ID NO: 74), monkey NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (mfNPR1-MMH; SEQ ID NO: 75), mouse NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (mNPR1-MMH; SEQ ID NO: 76), dog NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (dog_NPR1-MMH; SEQ ID NO: 77), porcine NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (porcine_NPR1-MMH; SEQ ID NO: 78), and hNPR1-MMH+10×hANP.

[0188] Different concentrations (40 nM-0.625 nM, 4-fold serial dilutions) of hNPR1-MMH, mfNPR1-MMH, hNPR1-MMH, or a fixed concentration (40 nM) of mNPR1-MMH, dog_NPR1-MMH, or pig_NPR1-MMH were prepared in HBS-EP running buffer in the presence of 10x concentration of hANP and then injected for 150 s at a flow rate of 30 μL / min, while dissociation of the different NPR1 reagents bound to the mAbs was monitored for 30 min in HBS-EP running buffer. At the end of each cycle, the anti-NPR1 mAb capture surface was regenerated using a 12 s injection of 10 mM phosphate. Association rates (ka) and dissociation rates (kd) were determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Biacore insight evaluation software. The binding dissociation equilibrium constant (KD) and dissociation half-life (t1 / 2) were determined from the kinetic rates as follows:

number

[0189] The binding kinetic parameters for different NPR1 reagents binding to different anti-NPR1 mAbs of the present disclosure at 25° C. and 37° C. are shown in Tables 4 through 15.

[0190] result At 25°C, anti-NPR1 monoclonal antibodies have K values ​​ranging from 47.4 pM to 1.27 nM, as shown in Table 4. D The values ​​were consistent with those of hNPR1-MMH.

[0191] [Table 4]

[0192] At 37°C, anti-NPR1 monoclonal antibodies have K values ​​ranging from 57.4 pM to 1.7 nM, as shown in Table 5. D The values ​​were consistent with those of hNPR1-MMH.

[0193] [Table 5]

[0194] At 25°C, anti-NPR1 monoclonal antibodies have K values ​​ranging from 69 pM to 1.23 nM, as shown in Table 6. D values ​​bound to mfNPR1-MMH.

[0195] [Table 6]

[0196] At 37°C, anti-NPR1 monoclonal antibodies have K values ​​ranging from 53.6 pM to 1.99 nM, as shown in Table 7. D values ​​bound to mfNPR1-MMH.

[0197] [Table 7]

[0198] At 25°C, anti-NPR1 monoclonal antibodies have K values ​​ranging from 78.8 pM to 500 pM, as shown in Table 8. D It bound to hNPR1-MMH in the presence of 10-fold higher concentration of hANP.

[0199] [Table 8]

[0200] At 37°C, anti-NPR1 monoclonal antibodies have K values ​​ranging from 642 pM to 1.52 nM, as shown in Table 9. D It bound to hNPR1-MMH in the presence of 10-fold higher concentration of hANP.

[0201] [Table 9]

[0202] At 25°C, only two anti-NPR1 monoclonal antibodies had K values ​​ranging from 35.8 nM to 122 nM, as shown in Table 10.D It bound to porcine_NPR1-MMH at this value.

[0203] [Table 10]

[0204] At 37°C, only two anti-NPR1 monoclonal antibodies had K values ​​ranging from 42.1 nM to 98.1 nM, as shown in Table 11. D It bound to porcine_NPR1-MMH at this value.

[0205] [Table 11]

[0206] At 25° C. or 37° C., none of the anti-NPR1 monoclonal antibodies bound to mNPR1-MMH, as shown in Tables 12 and 13, respectively.

[0207] [Table 12]

[0208] [Table 13]

[0209] At 25° C. or 37° C., none of the anti-NPR1 monoclonal antibodies bound to canine_NPR1-MMH, as shown in Tables 14 and 15, respectively.

[0210] [Table 14]

[0211] [Table 15] EXAMPLES

[0212] Example 4: pH sensitivity of anti-NPR1 monoclonal antibody binding to NPR1 reagent measured at 37°C Experimental procedure The 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 two running buffers: (i) PBS, 0.05% v / v of surfactant Tween-20, pH 7.4 (PBS-T-pH 7.4), and (ii) PBS, 0.05% v / v of surfactant Tween-20, pH 6.0 (PBS-T-pH 6.0). The Biacore CM5 sensor chip surface was first derivatized by amine coupling with an anti-human Fc-specific antibody to capture the anti-NPR1 mAb. Different concentrations (30 nM and 10 nM) of human NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (hNPR1-MMH; SEQ ID NO: 74) or monkey NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (mfNPR1-MMH; SEQ ID NO: 75) prepared in PBS-T-pH 7.4 buffer were injected for 4 min at a flow rate of 30 μL / min, followed by dissociation of bound NPR1 reagent for 5 min in PBS-T-pH 7.4 or PBS-T-pH 6.0 running buffer.

[0213] 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

[0214] result k for different anti-NPR1 mAbs binding to hNPR1-MMH or mfNPR1-MMH in PBS-T, pH 7.4 d and t values ​​and subsequent dissociation in PBS-T-pH 7.4 of the present disclosure, or PBS-T-pH 6.0 at 37° C. are shown in Tables 16 to 19.

[0215] [Table 16]

[0216] [Table 17]

[0217] [Table 18]

[0218] [Table 19] EXAMPLES

[0219] Example 5: Octet cross-competition between different anti-NPR1 monoclonal antibodies Experimental procedure Binding competition between different anti-NPR1 monoclonal 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 shaking of the plate at a speed of 1000 rpm in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 1 mg / mL BSA, 0.02% NaN3, 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-EBT) buffer. To assess whether the two antibodies could compete with each other for binding to their respective epitopes on recombinant human NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (hNPR1-MMH; SEQ ID NO: 74), approximately 0.64 nm of hNPR1-MMH was first captured onto an Octet biosensor chip (Fortebio Inc, #18-5122) coated with anti-Penta-His antibody by immersing the biosensor chip in a well containing 40-50 μg / mL of hNPR1-MMH solution for 60 seconds.

[0220] The antigen capture biosensor chip was then saturated with a first anti-NPR1 monoclonal antibody (hereafter referred to as mAb-1) by immersion for 4 min in a well containing a 50 μg / mL solution of mAb-1. The biosensor chip was then subsequently immersed for 3 min in a well containing a 50 μg / mL solution of a second anti-NPR1 monoclonal antibody (hereafter referred to as mAb-2). The biosensor chip was washed with HBS-EBT 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.

[0221] result The response of mAb-2 binding to hNPR1-MMH precomplexed with mAb-1 was compared to the binding response when the order was reversed (mAb-2 was the first antibody bound and mAb1 was the second antibody bound) to determine the competitive / non-competitive behavior of the different anti-NPR1 monoclonal antibodies, as summarized in Table 20.

[0222] [Table 20] EXAMPLES

[0223] Example 6: Evaluation of NPR1 antagonism of antagonistic anti-NPR1 antibodies Experimental procedure To assess regulation of human natriuretic peptide receptor 1 (hNPR1), we generated a HEK293 cell line stably expressing hNPR1 (amino acids M1-G1061 of accession # NP_000897.3) with a C-terminal myc and FLAG tag and selected for high hNPR1 expressing cells. The resulting cell line was named HEK293 / hNPR1.MycDDK HS, abbreviated as HEK293 / hNPR1, and maintained in DMEM containing 10% FBS, NEAA, pen / strep / glutamine, 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 Natriuretic peptides in cardiometabolic regulation and disease. Nature Publishing Group, 11(7), 403-412). NPR1 activity was assessed using a homogeneous time-resolved fluorescence (HTRF) assay that measures cGMP levels.

[0224] Analysis of NPR1 antagonism To evaluate NPR1 antagonism, HEK293 / hNPR1 cells were seeded in 96-well half-area plates at 20,000 cells / well in complete growth medium and cultured overnight. The next day, growth medium was replaced with dilution buffer (OptiMEM with 0.1% FBS) containing anti-NPR1 or isotype control antibodies across a range of concentrations (0.017 nM to 1 µM in conditions with no antibody added) and incubated for 15 min at 37 °C. After pretreatment, HEK293 / hNPR1 cells were stimulated with ligands, ANP or BNP across a range of concentrations (ANP = 0.002 to 2 nM or BNP = 0.0039 to 4 nM in conditions with no ligand added) made in dilution buffer containing 0.2 nM ANP or dilution buffer containing 0.7 nM BNP and incubated for 30 min at 37 °C.

[0225] HTRF assays were performed using a cGMP HTRF kit (Cisbio, #62GM2PEH) according to the manufacturer's protocol. Fluorescence intensity was detected using an EnVision multilabel plate reader (Perkin Elmer) and fluorescence resonance energy transfer (FRET) ratios were calculated according to the manufacturer's instructions. FRET ratios were converted to cGMP concentrations on a logarithmic scale according to a cGMP standard curve and analyzed using a four-parameter logistic equation over a 10-point concentration-response curve to calculate the half inhibitory concentration (IC) of anti-NPR1 antibodies using GraphPad Prism8. 50 The half effective concentration (EC 50 ) values ​​were not calculated due to the limit of quantification at high concentrations. Maximum inhibition was calculated by the formula described below:

number

[0226] In this formula, [Log cGMP,M] ベースライン , [Log cGMP,M] リガンドを含む試験抗体1μM , and [Log cGMP,M] 0.2nMのANPまたは0.7nMのBNPare logarithmic scale cGMP concentration values ​​from cells treated with dilution buffer alone, the highest concentration of anti-NPR1 antibody at 1 μM with 0.2 nM ANP or 0.7 nM BNP, and 0.2 nM ANP or 0.7 nM BNP alone, respectively.

[0227] Determining the mechanism of NPR1 antagonism The mechanism of anti-NPR1 antibody inhibition of ANP-mediated NPR1 activation was assessed by Schild analysis of cGMP accumulation assay (Kenakin 1997 Pharmacologic Analysis of Drug-Receptor Interaction. 3rd ed. Philadelphia: Lippincott-Raven). Conditions were optimized such that the ANP concentration-response curve fell within the linear range of the cGMP standard curve. Briefly, HEK293 / hNPR1 cells were lifted from plates using enzyme-free cell dissociation solution. Cell suspensions were then reseeded into low-volume 96-well plates at 1,000 cells / well in dilution buffer. Cells were then pretreated with anti-NPR1 antibodies in dilution buffer at fixed concentrations of 0 nM, 50 nM, 150 nM or 450 nM for 15 min at room temperature. After pretreatment, ANP was added to the cells at various concentrations (1.0 pM to 1 μM) in dilution buffer (with additional conditions without ANP) and incubated for 5 min at room temperature.

[0228] HTRF assay, detection of fluorescence intensity and calculation of cGMP concentration were all performed as previously described. Using 96-well low volume plates, the volumes required for the cGMP standard curve and detection reagents were half that of using 96-well half area plates.

[0229] All assay parameters were analyzed using GraphPad Prism 8. EC of ANP in the presence or absence of anti-NPR1 antibody. 50 Values ​​were calculated using a four-parameter logistic equation over an 11-point concentration-response curve. Reduction of ANP maximal response by anti-NPR1 antibodies was calculated using the following formula:

number

[0230] The Schild slope was determined by Schild plot analysis derived by plotting Log(concentration ratio-1) on the Y-axis against antagonist concentration on a logarithmic scale on the X-axis. The concentration ratio represents the EC50 of ANP in the presence of different concentrations of antagonist. 50 Values ​​are the EC50 of ANP in the absence of antagonist. 50 Define it as the value divided by the value.

[0231] Analysis of anti-NPR1 antibody-mediated internalization by secondary antibody-drug conjugates in a cell-based cytotoxicity assay The internalization of anti-NPR1 antibodies was indirectly evaluated by a cytotoxicity assay using a secondary antibody conjugated with the cytotoxic payload monomethyl auristatin F (MMAF). After binding to NPR1 on cells, the anti-NPR1 antibody is internalized with the receptor, resulting in co-internalization of the anti-human Fc fab secondary antibody-drug conjugate (secondary ADC), release of the conjugated cytotoxic payload, and cell death. Thus, the degree of cytotoxicity can be used to evaluate the ability of an antibody to internalize upon target engagement.

[0232] For the assay, HEK293 / hNPR1 cells were seeded in 96-well white plates at 1,000 cells / well in complete growth medium and cultured overnight. The next day, cells were pretreated with anti-NPR1 or control antibodies across a range of concentrations (serial dilutions from 914 fM to 6 nM in dilution buffer, with additional conditions without antibody) in the presence or absence of 100 nM ANP or 100 nM BNP for 5 min at 37 °C. After pretreatment, secondary ADCs at a final concentration of 20 nM were added to HEK293 / hNPR1 cells. To assess maximum killing in the assay, digitonin at a final concentration of 48 μg / ml was added to control wells. Treated cells were incubated at 37 °C for 3 days.

[0233] To measure cell viability, Promega CellTiter-Glo was used according to the manufacturer's instructions. Luminescence intensity was detected using an EnVision multilabel plate reader and analyzed using a four-parameter logistic equation over a nine-point concentration-response curve to calculate the IC of anti-NPR1 antibody using GraphPad Prism 8. 50 The cell viability or cell death rate was calculated using the formula described below:

number

[0234] result Assessing inhibition of ligand-mediated NPR1 activation by anti-NPR1 antibodies Both ANP and BNP activated HEK293 / hNPR1 cells and stimulated cGMP accumulation in a concentration-dependent manner (Figures 1A and 1B). All anti-NPR1 antibodies significantly blocked 0.2 nM ANP or 0.7 nM BNP-induced NPR1 activation, with IC 50 Values ​​ranged from 7.0 to 51 nM with maximum inhibition ranging from 82% to 107% (Figures 1A and 1B and Table 21). Isotype control antibodies did not show significant inhibition of 0.2 nM ANP or 0.7 nM BNP-induced NPR1 activation (Figures 1A and 1B and Table 21).

[0235] [Table 21]

[0236] Taken together, H4H22034N, REGN7541, REGN7544, and REGN7548 showed significant inhibition of ligand-induced NPR1 activation as measured by a cGMP accumulation assay.

[0237] Evaluating the mechanism by which anti-NPR1 antibodies inhibit ANP-mediated NPR1 activation Schild analysis was performed to determine the mechanism of anti-NPR1 antibody inhibition of ANP-mediated NPR1 activation. In this analysis, a parallel rightward shift of the agonist concentration-response curve with a Schild slope of about 1 without changing the maximum response indicates a competitive antagonist. In contrast, a non-parallel rightward shift of the agonist concentration-response curve with a Schild slope that is not close to 1 indicates a non-competitive antagonist. Antagonists that can change the maximum response of agonists are defined as insurmountable antagonists, whereas antagonists that do not change the maximum response of agonists are defined as surmountable antagonists (Kenakin, 1997).

[0238] Using Schild analysis, H4H22034N, REGN7541, REGN7544 and REGN7548 demonstrated non-competitive inhibition of ANP agonists in the cGMP accumulation assay (Figure 2A-2E). Increasing concentrations of the four anti-NPR1 antibodies produced a non-parallel rightward shift of the ANP concentration-response curve with a Schild slope not close to 1. Furthermore, inhibition with H4H22034N did not alter the maximal response of ANP, whereas inhibition with REGN7541, REGN7544 and REGN7548 reduced the maximal response of ANP by 10-62% (Figure 2A-2E and Table 2).

[0239] [Table 22]

[0240] Taken together, H4H22034N, REGN7541, REGN7544 and REGN7548 demonstrated noncompetitive antagonism with variable effects on maximal ligand responses.

[0241] Assessment of anti-NPR1 antibody-induced NPR1 internalization We assessed the internalization of anti-NPR1 antibodies using a secondary ADC-mediated cytotoxicity assay in the presence or absence of ligand. In the absence of ligand, all anti-NPR1 antibodies induced NPR1 internalization in a concentration-dependent manner, with IC50 The IC values ​​ranged from 58 to 243 pM, with maximum killing rates ranging from 82% to 85% (Figures 3A-3C and Table 23). In the presence of 100 nM ligand, anti-NPR1 antibodies induced NPR1 internalization and IC 50 Values ​​ranged from 132 to 703 pM with maximum killing ranging from 50% to 82%. An isotype control antibody showed no significant NPR1 internalization in the presence or absence of ligand (Figures 3A-3C and Table 23).

[0242] [Table 23]

[0243] Taken together, H4H22034N, REGN7541, REGN7544 and REGN7548 demonstrated anti-NPR1 antibody internalization of NPR1 in the presence or absence of ligand as measured by secondary ADC-mediated cytotoxicity assays. EXAMPLES

[0244] Example 7: Potency and specificity of anti-NPR1 antibodies binding to NPR1 alone or in complex with ANP or BNP on the cell surface using electrochemiluminescence-based detection Experimental procedure The ability of anti-human NPR1 monoclonal antibodies to bind to human or monkey (Macaca fascicularis) NPR1 (hNPR1 or mfNPR1)-expressing cells in the presence or absence of human atrial natriuretic peptide (ANP) and human brain natriuretic peptide (BNP) ligands was determined using an electrochemiluminescence (ECL)-based immunoassay.

[0245] Briefly, HEK293 / hNPR1 expressing cells were generated by transfecting human embryonic kidney (HEK) 293 cells with the neomycin-resistant pLVX.hNPR1.myc.DDK plasmid encoding human NPR1 (amino acids M1 to G1061, UniProtKB-P16066). Similarly, HEK293 / mfNPR1 cells were generated by transfecting HEK293 cells with the neomycin-resistant pRG984 plasmid encoding full-length monkey NPR1 (amino acids M1 to G1061, accession number XP_005541809.1). A non-transfected HEK293 cell line that showed no detectable binding of the commercially available anti-hNRP1 antibody by fluorescence-activated cell sorting (FACS) was included in the experiment as a non-specific binding control.

[0246] The experiment was carried out according to the following procedure. Cultures of the above cell lines were incubated with Ca 2+ / Mg 2+ The cells were then rinsed once with 1x PBS buffer containing no Ca, and incubated at 37°C for 10 min with Enzyme Free Cell Dissociation Solution to detach the cells from the flask. 2+ / Mg 2+ The cells were washed once with 1× PBS containing 0.05% CO, resuspended, and then counted using a Cellometer™ Auto T4 cell counter (Nexcelom Bioscience, Lawrence, Mass.). Approximately 2.0×10 cells per well were counted. 4 Cells were seeded onto 96-well carbon electrode plates (Meso Scale Diagnostics, Rockville, MD) and incubated for 1 h at 37 °C. 2+ / Mg 2+Nonspecific binding sites were blocked for 1 h at room temperature using 2% BSA (w / v) in 1× PBS containing 100 nM hANP (Tocris, Minneapolis, MN), 100 nM hBNP (Tocris, Minneapolis, MN), or sample dilution buffer alone for 0.5 h at room temperature, while HEK293 cells were treated with sample dilution buffer only. Without washing, serial dilutions of anti-NPR1, comparator 1, or isotype control antibodies ranging from 1.7 pM to 100 nM, or buffer containing no antibody, were added to the cells, followed by incubation for 1 h at room temperature. Plates were then washed using an AquaMax2000 plate washer equipped with a cell wash head (MDS Analytical Technologies, Sunnyvale, CA) to remove unbound antibodies, hANP, and hBNP. Plate-bound antibodies were detected using heavy- and light-chain-specific SULFO-TAG™-conjugated polyclonal goat anti-human IgG antibodies (Jackson Immunoresearch, West Grove, PA), which were incubated with the cells for 1 hour at room temperature.

[0247] After washing, plates were developed with Read Buffer (Meso Scale Diagnostics, Rockville, MD) according to the manufacturer's recommended procedure, and luminescence signals were recorded using a SECTOR Imager 600 (Meso Scale Diagnostics, Rockville, MD). Binding signals in RLU were analyzed as a function of antibody concentration, and data were fitted to a sigmoidal (four-parameter logistic) dose-response model using the R statistical package (open source). EC 50 Values ​​were determined to indicate the binding potency of anti-NPR1 antibodies to NPR1-expressing cells with or without ANP or BNP complexed to NPR1 on the cell surface.

[0248] result The ability of anti-NPR1 monoclonal antibodies to specifically bind to the surface of HEK293 cells engineered to express human or monkey NPR1 in the presence or absence of ANP or BNP was assessed using an electrochemiluminescence-based immunoassay. The concentration dependence of antibody binding was analyzed and EC 50 Values ​​were determined and the results are summarized in Table 24.

[0249] [Table 24]

[0250] Four anti-NPR1 antibodies of the present disclosure (H4H22034, REGN7541, REGN7544, REGN7548) bound to human NPR1 engineered cells in the presence of 10 nM hANP or 100 nM hBNP. The potency of these antibodies on HEK293 / hNPR1.myc.DDK cells in the presence of 10 nM hANP or 100 nM hBNP was EC50 of 0.56 nM-2.9 nM or 0.43 nM-1.5 nM, respectively. 50 Similar binding potencies were determined for hNPR1 expressing cells in the absence of hANP and hBNP, with EC 50 The values ​​ranged from 0.34 nM to 1.7 nM. No detectable binding was observed for these antibodies on parental HEK293 cells under the same experimental conditions. These results suggest that the antibodies are NPR1-specific binders and that the human NPR1 binding potency for these antibodies is not affected by the presence of ANP or BNP.

[0251] In contrast, comparator 1 showed binding specificity to hNPR1 cells only in the presence of hANP or hBNP (10 nM hANP or 100 nM hBNP), and EC 50 The values ​​were 0.57 nM and 1.8 nM, respectively, and since no detectable binding was observed in the absence of hANP and hBNP, this is classified as a binder of only the NPR1-ANP / BNP complex.

[0252] Anti-NPR1 antibodies H4H22034, REGN7541, REGN7544, and REGN7548 also bound to monkey NPR1-expressing cells (HEK293 / mfNPR1) and increased EC 50 Similar to hNPR1 cells, the addition of 10 nM hANP or 100 nM hBNP did not affect anti-NPR1 antibody binding to HEK293 / mfNPR1 cells; EC 50 Values ​​ranged from 0.67 nM to 4.2 nM or 0.42 nM to 1.7 nM in the presence of hANP and hBNP, respectively (Table 24), with no binding detected for either antibody on parental HEK293 cells. In contrast, Comparator 1 bound specifically to HEK293 / mfNPR1 engineered cells only in the presence of 10 nM hANP or 100 nM hBNP, but the EC 50 The value could not be determined.

[0253] In this experiment, the isotype control did not bind to either HEK293 / hNPR1, HEK293 / mfNPR1, or HEK293 cells. EXAMPLES

[0254] Example 8: Telemetric Normal Blood Pressure NPR1 hu / hu Characterization of the acute effects of NPR1 antagonist mAb on systemic blood pressure following a single 25mg / kg intravenous dose in mice Experimental procedure The purpose of this study is to evaluate the efficacy and safety of telemetry-measured normotensive NPR1 hu / hu The aim of this study was to evaluate the acute effects of NPR1 antagonist antibodies on baseline systemic blood pressure in mice. hu / huMice (n=30) 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 1-5) based on body weight (Table 25). 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 (Lab Diet Standard Pellet Chow) and water were provided ad libitum.

[0255] [Table 25]

[0256] Test proteins were administered to appropriate animals by a single intravenous injection on day 0. Dose volume for each animal was based on the most recent body weight measurement.

[0257] Systolic pressure, diastolic pressure, mean arterial pressure, pulse pressure and heart rate were collected for 10 seconds every minute during the study period. Telemetry data are presented as 60 minute or 24 hour averages. All data are presented as mean ± SEM.

[0258] result In vivo evaluation demonstrated that compared to PBS controls, animals treated with NPR1 antagonist antibodies exhibited rapid and sustained increases in systemic blood pressure following a single intravenous administration of any of the antibodies evaluated (Figure 4). Pressure changes occurred almost immediately (Figure 4) and persisted through the seventh day of the study, the duration of the experiment (Figure 5). The magnitude of the rapid blood pressure increase, as assessed by the mean change from baseline in systolic blood pressure (48 hours after dosing), ranged from +7.64 ± 1.13 (REGN7541) to +9.81 ± 1.03 (H4H22034N) mmHg. The magnitude of the blood pressure increase, as assessed by the mean change from baseline in systolic blood pressure (7 days after dosing), ranged from +7.65 ± 1.03 (REGN7548) to +9.55 ± 1.16 (H4H22034N) mmHg.

[0259] Diastolic pressure, mean arterial pressure and pulse pressure (Tables 26 and 27) also changed significantly following administration of the NPR1 blocking mAb, with the magnitude and duration of effects consistent with those observed and reported for systolic blood pressure. Heart rate responses were variable (Tables 26 and 27), with rapid changes generally tending toward lower heart rates compared to baseline. These changes are consistent with the observed increases in blood pressure. Assessment of heart rate after 7 days demonstrated a significant relative increase for animals administered REGN7548.

[0260] [Table 26]

[0261] [Table 27]

[0262] NPR1 antagonist antibodies H4H22034N, REGN7541, REGN7544 and REGN7548 inhibit normotensive NPR1 hu / hu After a single subcutaneous injection into mice, it significantly and rapidly increased systemic blood pressure within hours, and the observed hemodynamic effects persisted for the duration of the 7-day experiment. EXAMPLES

[0263] Example 9: Normotensive NPR1 hu / hu Characterization of the effects of a single 1 or 25 mg / kg dose of NPR1 antagonist mAb on systemic blood pressure in mice Experimental procedure The purpose of this study is to evaluate the efficacy and safety of telemetry-measured normotensive NPR1 hu / hu The aim of this study was to evaluate the effect of NPR1 antagonist antibodies on baseline systemic blood pressure in mice. hu / huMice (n=54) 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 1-10) based on body weight and baseline systolic and pulse pressures before being assigned to groups (Table 28). 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.

[0264] [Table 28]

[0265] Test proteins were administered to appropriate animals by single subcutaneous injection on day 0. Dose volume for each animal was based on the most recent body weight measurement.

[0266] Systolic pressure, diastolic pressure, mean arterial pressure, pulse pressure and heart rate were collected for 10 seconds every 10 minutes during the study period. Telemetry data are presented as 24 hour averages. All data are presented as mean ± SEM.

[0267] result In vivo characterization of NPR1 antagonist antibodies demonstrated that NPR1 expression was significantly increased in animals treated with NPR1 antagonists compared to IgG4P isotype and PBS control treated animals. hu / huMice were demonstrated to exhibit significant and sustained increases in systemic blood pressure after a single subcutaneous dose of 1 or 25 mg / kg of any of the NPR1 antagonist antibodies evaluated (Table 29, Table 30, Figure 6, Figure 7). The magnitude of blood pressure increase after a single subcutaneous dose of 1 mg / kg, as assessed by the mean change from baseline in systolic blood pressure (after 0-14 days of dosing), ranged from +6.81 ± 0.76 (REGN7548) to +11.22 ± 0.93 (REGN7541) mmHg (Table 29 and Figure 6). The magnitude of blood pressure increase after a single subcutaneous dose of 25 mg / kg, as assessed by the mean change from baseline in systolic blood pressure (after 0-14 days of dosing), ranged from +8.82 ± 0.64 (H4H22034) to +9.76 ± 0.92 (REGN7544) mmHg (Table 29 and Figure 7).

[0268] [Table 29]

[0269] [Table 30]

[0270] The chronic effects of these NPR1 antagonist antibodies were evaluated over a 27-day period (Table 30 and Figures 6 and 7). After a single subcutaneous injection of 1 mg / kg on day 0, systemic blood pressure remained significantly elevated for three of the four NPR1 blocking mAbs evaluated for the duration of the study (Table 30 and Figure 6). The magnitude of blood pressure increase after a single subcutaneous dose of 1 mg / kg, as assessed by the mean change from baseline in systolic blood pressure (15-27 days after dosing), ranged from +4.04 ± 0.30 (H4H22034) to +9.03 ± 0.32 (REGN7541) mmHg. After a single subcutaneous injection of 25 mg / kg on day 0, systemic blood pressure remained significantly elevated for all NPR1 blocking mAbs evaluated. The magnitude of blood pressure increase after a single subcutaneous dose of 25 mg / kg, as assessed by the mean change from baseline in systolic blood pressure (15-27 days after dosing), ranged from +10.53±0.43 (H4H22034) to +14.2±0.42 (REGN7544) mmHg. Heart rate responses were variable (Tables 29 and 30), with the overall trend of heart rate reduction consistent with the observed increase in systemic blood pressure. Diastolic pressure, mean arterial pressure, and pulse pressure (Tables 29 and 30) also changed significantly after administration of NPR1 blocking mAb, with the magnitude and duration of effect consistent with that observed and reported for systolic blood pressure.

[0271] NPR1 antagonist antibodies H4H22034N, REGN7541, REGN7544 and REGN7548 inhibit normotensive NPR1 hu / hu After a single subcutaneous injection in mice, it significantly increased systemic blood pressure for up to 27 days. EXAMPLES

[0272] Example 10: Hypotension NPR1 hu / hu Characterization of the effects of ANP overexpression via hydrodynamic DNA delivery on systemic blood pressure in mice and its ability to reverse the effects following a single dose of 25 mg / kg of NPR1 antagonist mAb Experimental procedure The purpose of this study is to evaluate the remotely measured hypotensive NPR1 hu / huThe aim of this study was to evaluate the efficacy of NPR1 antagonist antibodies and their ability to reverse the blood pressure lowering effect of ANP overexpression in male NPR1 mice (approximately 12-16 weeks of age). hu / hu (n=48) 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 1-6) based on body weight (Table 31). 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 (Lab Diet Standard Pellet Chow) and water were provided ad libitum.

[0273] [Table 31]

[0274] HDD plasmid and test proteins were administered to appropriate animals by single intravenous injection on days 0 and 7, respectively. Dose volumes of test proteins were based on the most recent body weight measurements.

[0275] Systolic blood pressure, diastolic blood pressure, mean arterial pressure, pulse pressure and heart rate were collected for 10 seconds every minute during the study period. Telemetric data are presented as 24-hour average values. All data are presented as mean ± SEM.

[0276] result In vivo characterization of the NPR1 antagonist antibodies demonstrated that the NPR1 antagonist antibodies inhibited NPR1 expression in mice treated with IgG4P isotype control compared to animals treated with IgG4P isotype control. hu / huIt was demonstrated that ANP HDD can rapidly and sustainably normalize ANP overexpression-induced systemic blood pressure reduction in mice (Table 32 and Table 33). Serum NTproANP concentrations increased significantly and sustained over the study period (Table 34), indicating effective overexpression of ANP. The magnitude of blood pressure increase in ANP HDD-induced hypotensive mice after a single intravenous administration of 25 mg / kg of NPR1 blocker, assessed by the mean change from baseline in systolic blood pressure (7 to 28 days after administration), ranged from -9.27 ± 0.61 (H4H22034N) to +10.26 ± 1.48 (REGN7544) mmHg compared to before ANP HDD administration (Table 33 and Figure 8).

[0277] [Table 32]

[0278] [Table 33]

[0279] [Table 34]

[0280] Consistent with the significant and sustained reduction in systemic pressure and likely reduction in left ventricular afterload, absolute and relative heart weights were significantly reduced in mice receiving the ANP HDD (Figures 9A and 9B). Hearts from animals receiving the NPR1 blocking mAb were unchanged from normotensive controls and showed an effective increase in systemic and ventricular pressures.

[0281] NPR1 antagonist antibodies H4H22034N, REGN7541, REGN7544 and REGN7548 inhibit ANP HDD-induced hypotension NPR1 hu / hu It significantly and sustainably increased systemic blood pressure in mice for up to 28 days. EXAMPLES

[0282] Example 11: Telemetrically Measured NPR1 hu / hu NPR1 blockade for prophylactic and therapeutic administration in an LPS-induced shock model in mice Experimental procedure The aim of this study was to evaluate the efficacy and safety of telemetry-measured NPR1 in patients with hypotension caused by administration of LPS. hu / hu The aim of this study was to evaluate the efficacy of an NPR1 antagonist antibody administered prophylactically or therapeutically to male mice aged approximately 12 to 17 weeks. hu / hu (n=58) 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 1-6) based on body weight and systolic and pulse pressures before being assigned to groups (Table 35). 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-hour light / 12-hour dark cycle. Food (Lab Diet Standard Pellet Chow) and water were provided ad libitum.

[0283] [Table 35]

[0284] Animals were administered a single intraperitoneal injection of 5 mg / kg LPS or PBS on day 0. Test proteins were administered to appropriate animals by single intravenous injection approximately 24 hours before or 8 hours after LPS administration. Dose volume for each animal was based on the most recent body weight measurement.

[0285] Systolic pressure, diastolic pressure, mean arterial pressure, pulse pressure and heart rate were collected for 10 seconds every 10 minutes during the study period. Telemetric data are presented as 24 hour averages. All data are presented as mean ± SEM.

[0286] result LPS-induced hypotension NPR1 hu / huIn vivo characterization of NPR1 antagonist antibodies administered prophylactically or therapeutically to mice demonstrated significant and sustained increases in systemic blood pressure (Table 36, Table 37, Figure 10). Mice administered prophylactically presented with a rapid increase in blood pressure (Table 36). The magnitude of blood pressure increase in the prophylactic arm of the study after a single intravenous dose of 25 mg / kg, assessed by the mean change from baseline in systolic blood pressure (from 24 hours prior to LPS administration to the time of administration), ranged from +7.24 ± 1.18 (REGN7548) to +9.07 ± 1.10 (REGN7541) mmHg (Table 36 and Figure 10). The magnitude of increase in blood pressure following a single intravenous dose of 25 mg / kg administered 8 hours after LPS administration, assessed by the mean change from baseline in systolic blood pressure (8 to 48 hours after LPS administration), ranged from -1.49 ± 2.244 (REGN7548) to -1.49 ± 2.24 (REGN7544) mmHg (Table 37). Pulse pressure in therapeutically treated animals, assessed by the mean change from baseline in pulse pressure (8 to 48 hours after LPS administration), ranged from 0.27 ± 0.93 (REGN7548) to -0.40 ± 0.91 (REGN7544) mmHg (Table 37, Figure 10).

[0287] [Table 36]

[0288] [Table 37]

[0289] The NPR1 antagonist antibodies REGN7544 and REGN7548 significantly and sustainably increased systemic blood pressure when administered prophylactically or therapeutically to mice that developed LPS-induced hypotension after a single intraperitoneal administration of LPS. EXAMPLES

[0290] Example 12: Each NPR1 monomer binds one REGN7544 Fab Experimental procedure Complex formation of NPR1+REGN7544 Fab+ANP Human NPR1 extracellular domain with a C-terminal myc-myc-6xHis tag (hNPR1-mmh; SEQ ID NO: 74) was mixed with atrial natriuretic factor (ANP, Tocris) and REGN7544 Fab at a molar ratio of 1 hNPR1-mmh + 2 ANP + 1 REGN7544 Fab. The complex was incubated overnight at 4°C and then purified on a Superdex 200 increase 10 / 300GL gel filtration column equilibrated with 50 mM Tris pH 7.5, 150 mM NaCl. Peak fractions were collected and concentrated using 10 kDa MWCO centrifugal concentrators (Amicon).

[0291] Cryo-EM grid preparation and data collection The NPR1-ANP-REGN7544 complex was diluted to 0.87 mg / ml and poly(maleic anhydride-alt-1-decene) substituted with 3-(dimethylamino)propylamine (PMAL-C8; Anatrace Cat.#P5008) was added to a final concentration of 0.15%. Samples were deposited onto freshly plasma-cleaned UltrAufoil grids (Quantifoil GmbH). Excess solution was wiped off with filter paper and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Part#1086439). The grids were loaded into a Titan Krios G3i (Thermo Fisher, Part#1137337) equipped with a Bioquantum energy filter + K3 direct electron detector (Gatan Inc, Part#1147213). Movies were collected using EPU v2.7 (Thermo Fisher) at 105,000x magnification, corresponding to a pixel size of 0.86 Å. A dose rate of 15 electrons per second per pixel was used, and each movie lasted 2 s and 46 frames, with a resolution of 1 Å. 2 This corresponds to a total dose of about 40 electrons per second.

[0292] Cryo-EM data processing All cryo-EM data processing was performed using cryoSPARC v3.2.0 (Structura Biotechnology Inc.). Movies were aligned using patch motion correction and patch CTF estimation. A total of 6692 movies were collected, and then 6474 movies were selected after motion correction and patch CTF. An initial set of particles picked using a blob picker was subjected to 2D classification to generate templates for template picking. Approximately 1.6 million particles picked by template picking were subjected to multiple rounds of 2D classification, resulting in 814,655 "good" composite particles. First-principles reconstruction with six classes, followed by heterogeneous refinement, produced one "good" class containing 310,944 particles corresponding to the complete NPR1-ANP-REGN7544 Fab in the isotropic map. After non-uniform refinement of the "good" class of particles, local refinement was performed, resulting in a map with 3.15Å resolution (FSC=0.143), which was used for model construction.Into this map, we manually placed the models of NPR1 (obtained from PDB code 1T34) and two Fabs (obtained from previous Regeneron antibody structures).These models were then manually reconstructed using Coot (v0.8.2 Medical Research Council Laboratory of Molecular Biology), and real space refined against the map using Phenix (v1.15.2 The PHENIX Industrial Consortium).

[0293] Complex formation of NPR1+REGN7544 Fab Human NPR1 extracellular domain with a C-terminal myc-myc-6×His tag (hNPR1-mmh; SEQ ID NO: 74) was mixed with REGN7544 Fab at a molar ratio of 1 hNPR1-mmh + 1 REGN7544 Fab. The complex was incubated overnight at 4° C. and purified on a Superdex 200 increase 10 / 300GL gel filtration column equilibrated with 50 mM Tris pH 7.5, 150 mM NaCl. Peak fractions were collected and concentrated using 10 kDa MWCO centrifugal concentrators (Amicon).

[0294] Cryo-EM grid preparation and data collection NPR1-REGN7544 complex was diluted to 0.8mg / ml and poly(maleic anhydride-alt-1-decene) substituted with 3-(dimethylamino)propylamine (PMAL-C8; Anatrace Cat.#P5008) was added to a final concentration of 0.15%. Samples were deposited onto freshly plasma-cleaned UltrAufoil grids (Quantifoil GmbH). Excess solution was wiped off with filter paper and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Part#1086439). The grids were loaded into a Titan Krios G3i (Thermo Fisher, Part#1137337) equipped with a Bioquantum energy filter + K3 direct electron detector (Gatan Inc, Part#1147213). Movies were collected using EPU v2.7 (Thermo Fisher) at 105,000x magnification, corresponding to a pixel size of 0.86 Å. A dose rate of 15 electrons per second per pixel was used, and each movie lasted 2 s and 46 frames, with a resolution of 1 Å. 2 This corresponds to a total dose of about 40 electrons per second.

[0295] Cryo-EM data processing All cryo-EM data processing was performed using cryoSPARC v3.2.0 (Structura Biotechnology Inc.). Movies were aligned using patch motion correction and patch CTF estimation. A total of 10,378 movies were collected, and then 9660 movies were selected after motion correction and patch CTF. An initial set of particles picked using a blob picker was subjected to 2D classification to generate templates for template picking. Approximately 1.7 million particles picked by template picking were subjected to multiple rounds of 2D classification, resulting in 762,681 “good” composite particles. First-principles reconstruction with six classes, followed by heterogeneous refinement, generated one “good” class containing 256,375 particles corresponding to the complete NPR1-REGN7544 Fab in the isotropic map. Non-uniform refinement of the “good” class of particles, followed by local refinement, resulted in a map at 3.42 Å resolution (FSC=0.143), which was used for model building. Models of NPR1 (taken from PDB code 1DP4) and two Fabs (taken from previous Regeneron antibody structures) were manually placed onto this map. These models were then manually rebuilt using Coot (v0.8.2 Medical Research Council Laboratory of Molecular Biology) and real-space refined against the map using Phenix (v1.15.2 The PHENIX Industrial Consortium).

[0296] result The structures of hNPR1+ANP+REGN7544 Fab and hNPR1+REGN7544 Fab showed that each NPR1 monomer binds one REGN7544 Fab. The Fab binds to residues in the lower (C-terminal, closer to the cell membrane) lobe of the extracellular domain. Upon REGN7544 binding, the NPR1 dimer adopts an inactive conformation in the absence of ANP and an active conformation in the presence of ANP. These conformations are very similar to the observed state of antibody-free NPR1 in the presence or absence of ANP; however, these studies were performed with the soluble extracellular domain and not the fully membrane-bound form of NPR1. Both the heavy and light chains of REGN7544 interact with the NPR1 extracellular domain. The contact residues of the NPR1 extracellular domain remain the same in either the presence or absence of ANP. There are 15 contact residues in the heavy chain and 7 contact residues in the light chain. The residues in the NPR1 extracellular domain that directly interact with REGN7544 Fab are Arg143, Leu144, Glu384, Leu401, Val402, Ala103, Ser405, Gly406, Arg407, Lys408, Trp411, Leu413, Gly414, Tyr415, and Pro416. EXAMPLES

[0297] Example 13: Evaluation of the ability of vasodilators to reverse REGN7544-induced changes in blood pressure The ability of three different vasodilators with different mechanistic targets (nifedipine (Sigma-Aldrich), enalapril (Sigma-Aldrich), molsidomine (Sigma-Aldrich)) to reverse the increase in blood pressure induced by REGN7544 was evaluated in NPR1 humanized mice. The three vasodilators used, enalapril, molsidomine, and nifedipine, were administered orally. Enalapril lowers blood pressure by inhibiting angiotensin-converting enzyme (ACE), which reduces the production of angiotensin II, a vasoconstrictor, and by increasing levels of bradykinin, a peptide that increases blood vessel diameter. Molsidomine, a nitric oxide donor, and nifedipine, a calcium channel blocker, also lower blood pressure.

[0298] The dosing scheme for evaluating the reversal of REGN7544-induced effects by vasodilators in telemetered NPR1-humanized mice is summarized in the following table:

[0299] [Table 38]

[0300] Male telemetered NPR1 humanized mice aged 13-15 weeks were administered a single dose of 25 mg / kg REGN7544 (n=28) or vehicle control (i.e., PBS) (n=7) by subcutaneous (SC) injection on day 0. Beginning on day 7 (169 hours after SC administration), mice pretreated with REGN7544 began receiving daily doses of nifedipine (20 mg / kg), enalapril (25 mg / kg), molsidomine (10 mg / kg), or water (n=7 / group) by oral gavage (PO), whereas all mice pretreated with vehicle control received water. Diastolic and systolic blood pressures were recorded continuously for all animals from 72 hours prior to administration of REGN7544 or vehicle control until the end of the experiment, and these measurements were used to calculate pulse pressure. The mean change in pulse pressure normalized to baseline for each treatment group was measured i) between 149 and 221 hours after administration of REGN7544 or vehicle control, and ii) from 3 days prior to administration of REGN7544 or vehicle control to the end of the experiment.

[0301] Each of the vasodilators tested reduced pulse pressure (PP) in NPR1-humanized mice pretreated with REGN7544 ( FIG. 11 ). Indeed, administration of vasodilators was shown to reverse the pulse pressure-increasing effect of REGN7544.

[0302] Male telemetered NPR1 humanized mice aged 13–15 weeks were administered a single dose of 25 mg / kg REGN7544 (n=28) or vehicle control (i.e., PBS) (n=7) by subcutaneous (SC) injection on day 0. Beginning on day 7, mice pretreated with REGN7544 began receiving daily nifedipine (20 mg / kg), enalapril (25 mg / kg), molsidomine (10 mg / kg), or water (n=7 / group) by oral gavage (PO), whereas all mice pretreated with vehicle control received water. Pulse pressure was recorded continuously for all animals from 72 hours prior to administration of REGN7544 or vehicle control until the end of the experiment. The mean change in systolic pressure normalized to baseline for each treatment group was measured from 3 days prior to administration of REGN7544 or vehicle control until the end of the experiment.

[0303] Vasodilators reduced systolic blood pressure (SBP) in NPR1-humanized mice pretreated with REGN7544 ( FIG. 12 ). Indeed, administration of vasodilators was found to reverse the increase in systolic blood pressure induced by REGN7544.

[0304] Male telemetered NPR1 humanized mice aged 13–15 weeks were administered a single dose of 25 mg / kg REGN7544 (n=28) or vehicle control (i.e., PBS) (n=7) by subcutaneous (SC) injection on day 0. Beginning on day 7, mice pretreated with REGN7544 began receiving daily nifedipine (20 mg / kg), enalapril (25 mg / kg), molsidomine (10 mg / kg), or water (n=7 / group) by oral gavage (PO), while all mice pretreated with vehicle control received water. Pulse pressure was recorded continuously for all animals from 72 hours prior to administration of REGN7544 or vehicle control until the end of the experiment. The mean change in heart rate normalized to baseline for each treatment group was measured from 3 days prior to administration of REGN7544 or vehicle control until the end of the experiment.

[0305] No observable effect on heart rate was recorded following administration of enalapril, molsidomine, and nifedipine (data not shown). Indeed, administration of vasodilators in the presence of REGN7544 was shown to have no effect on heart rate.

[0306] Male telemetered NPR1 humanized mice aged 13-15 weeks were administered a single dose of 25 mg / kg REGN7544 (n=28) or vehicle control (i.e., PBS) (n=7) by subcutaneous (SC) injection on day 0. Beginning on day 7, mice pretreated with REGN7544 began receiving daily nifedipine (20 mg / kg), enalapril (25 mg / kg), molsidomine (10 mg / kg), or water (n=7 / group) by oral gavage (PO), while all mice pretreated with vehicle control received water. Systolic and diastolic pressures were recorded continuously for all animals from 72 hours prior to administration of REGN7544 or vehicle control until the end of the experiment, and these measurements were used to calculate pulse pressure. Mean pulse pressure for each treatment group was measured from 3 days prior to administration of REGN7544 or vehicle control until the end of the experiment. Administration of vasodilators reversed the pulse pressure-increasing effect of a single SC dose of REGN7544 (data not shown).

[0307] Male telemetered NPR1 humanized mice aged 13-15 weeks were administered a single dose of 25 mg / kg REGN7544 (n=28) or vehicle control (i.e., PBS) (n=7) by subcutaneous (SC) injection on day 0. Beginning on day 7, mice pretreated with REGN7544 began receiving daily nifedipine (20 mg / kg), enalapril (25 mg / kg), molsidomine (10 mg / kg), or water (n=7 / group) by oral gavage (PO), while all mice pretreated with vehicle control received water. Systolic pressure was recorded continuously for all animals from 72 hours prior to administration of REGN7544 or vehicle control until the end of the experiment. Mean systolic pressure for each treatment group was measured from 3 days prior to administration of REGN7544 or vehicle control until the end of the experiment. Administration of vasodilators reversed the systolic blood pressure-increasing effect of a single SC dose of REGN7544 (data not shown).

[0308] Male telemetered NPR1 humanized mice aged 13-15 weeks were administered a single dose of 25 mg / kg REGN7544 (n=28) or vehicle control (i.e., PBS) (n=7) by subcutaneous (SC) injection on day 0. Beginning on day 7, mice pretreated with REGN7544 began receiving daily nifedipine (20 mg / kg), enalapril (25 mg / kg), molsidomine (10 mg / kg), or water (n=7 / group) by oral gavage (PO), while all mice pretreated with vehicle control received water. Heart rates were recorded continuously for all animals from 72 hours prior to administration of REGN7544 or vehicle control until the end of the experiment. Mean heart rates for each treatment group were measured from 3 days prior to administration of REGN7544 or vehicle control until the end of the experiment. Administration of a vasodilator following a single dose of REGN7544 had no compensatory effect on heart rate (data not shown).

[0309] Male telemetered NPR1 humanized mice aged 13-15 weeks were administered a single dose of 25 mg / kg REGN7544 (n=28) or vehicle control (i.e., PBS) (n=7) by subcutaneous (SC) injection on day 0. Beginning on day 7, mice pretreated with REGN7544 began daily administration of nifedipine (20 mg / kg), enalapril (25 mg / kg), molsidomine (10 mg / kg), or water (n=7 / group) by oral gavage (PO), whereas all mice pretreated with vehicle control received water. Diastolic pressure was recorded continuously for all animals from 72 hours prior to administration of REGN7544 or vehicle control until the end of the experiment. Mean diastolic blood pressure, (i) normalized to baseline and (ii) unnormalized, for each treatment group was measured from 3 days prior to administration of REGN7544 or vehicle control through the end of the study. Administration of enalapril reversed the diastolic blood pressure increasing effect of a single SC dose of REGN7544 (data not shown).

[0310] Thus, the reversibility of the hemodynamic effects following SC administration of 25 mg / kg REGN7544 was tested by oral gavage of three clinical vasodilators. Three different vasodilators, nifedipine, enalapril, and molsidomine, each reversed the REGN7544-induced increase in PP, but had no observable significant effect on heart rate. The REGN7544-mediated increase in PP was separately reversed by any one of the three clinical vasodilators (nifedipine, enalapril, and molsidomine) administered by oral gavage.

[0311] The present disclosure should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the present disclosure 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. An isolated antibody or antigen-binding fragment thereof that specifically binds to natriuretic peptide receptor 1 (NPR1) protein, wherein the antibody or antigen-binding fragment thereof binds to and blocks NPR1, and the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR); and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR), wherein the HCVR / LCVR pair comprises an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 38 / 46, and 55 / 63.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a fully human monoclonal antibody.

3. The antibody (a) is a fully human monoclonal antibody; (b) has a dissociation constant (K) of less than 1.7 nM at 25°C and 37°C as measured by surface plasmon resonance assay. D (c) binds to human NPR1 with a K of less than 1.99 nM at 25° C. and 37° C. as measured by surface plasmon resonance assay; D (d) binds to monkey NPR1 with a K of less than 1.52 nM in the presence of ANP at 25°C and 37°C as measured by surface plasmon resonance assay. D (e) inhibits ligand-induced NPR1 activation as measured by a cGMP accumulation assay; (f) has an EC of less than 2.9 nM in the presence or absence of ANP or BNP as measured by an electrochemiluminescence-based immunoassay. 50 (g) binds to human NPR1 with an EC of less than 4.2 nM in the presence or absence of ANP or BNP as measured by an electrochemiluminescence-based immunoassay. 50 (h) when administered to normotensive and hypotensive mice, the increase in systemic blood pressure lasts for up to about 28 days after administration of a single dose; (i) when administered to ANP overexpression-induced hypotensive mice, the increase in systemic blood pressure lasts for up to about 28 days after administration of a single dose; and (j) when administered to LPS-induced hypotensive mice, the increase in systemic blood pressure lasts for up to about 28 days after administration of a single dose. The antibody or antigen-binding fragment thereof of claim 1 having the properties:

4. 2. The antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region (HCVR) / light chain variable region (LCVR) amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 22 / 30, 38 / 46, and 55 / 63.

5. (a) the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprise the amino acid sequences of SEQ ID NOs: 4-6-8-12-AAS-16, respectively, and the HCVR / LCVR amino acid sequence comprises SEQ ID NOs: 2 / 10; (b) the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprise the amino acid sequences of SEQ ID NOs: 24-26-28-48-AAS-32, respectively, and the HCVR / LCVR amino acid sequence comprises SEQ ID NOs: 22 / 30; (c) the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprise the amino acid sequences of SEQ ID NOs: 40-42-44-46-AAS-16, respectively, and the HCVR / LCVR amino acid sequence comprises SEQ ID NOs: 38 / 46; (d) the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains comprise the amino acid sequences of SEQ ID NOs: 57-59-61-63-GAS-69, respectively, and the HCVR / LCVR amino acid sequence comprises SEQ ID NOs: 55 / 63; The antibody or antigen-binding fragment thereof of claim 1, comprising:

6. 2. The antibody or antigen-binding fragment thereof of claim 1, comprising complementarity-determining regions (CDRs) selected from the group consisting of: (a) SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, AAS, and SEQ ID NO:16; (b) SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:12, AAS, and SEQ ID NO:32; (c) SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:48, AAS, and SEQ ID NO:16; and (d) SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:65, GAS, and SEQ ID NO:

69.

7. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain is selected from the group consisting of SEQ ID NOs: 18, 34, 51 and 71.

8. 2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain and a light chain, wherein the light chain is selected from the group consisting of SEQ ID NOs: 20, 36, 53 and 73.

9. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain / light chain pair is selected from the group consisting of SEQ ID NOs: 18 / 20, 34 / 36, 51 / 53 and 71 / 73.

10. The antibody or antigen-binding fragment thereof of claim 1, which binds to residues in the lower lobe of the extracellular domain of NPR1.

11. The antibody or antigen-binding fragment thereof described in claim 1, which interacts with at least one of NPR1 residues selected from the group consisting of Arg143, Leu144, Glu384, Leu401, Val402, Ala103, Ser405, Gly406, Arg407, Lys408, Trp411, Leu413, Gly414, Tyr415, and Pro416.

12. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 18; and the light chain comprises the amino acid sequence of SEQ ID NO:

20.

13. The heavy chain comprises the amino acid sequence of SEQ ID NO: 34; the light chain comprises the amino acid sequence of SEQ ID NO: The antibody or antigen-binding fragment thereof of claim 1, comprising the amino acid sequence of SEQ ID NO:

36.

14. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 51; and the light chain comprises the amino acid sequence of SEQ ID NO:

53.

15. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 71; and the light chain comprises the amino acid sequence of SEQ ID NO:

73.

16. An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof of any one of claims 1 to 15 for binding to natriuretic peptide receptor 1 (NPR1) protein.

17. An antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15.

18. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, and a pharmaceutically acceptable carrier or diluent.

19. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the heavy chain variable region (HCVR) of the antibody of any one of claims 1 to 15.

20. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the light chain variable region (LCVR) of the antibody of any one of claims 1 to 15.

21. A vector comprising the polynucleotide molecule of claim 19.

22. A vector comprising the polynucleotide molecule of claim 20.

23. A host cell expressing the vector of claim 21.

24. A method for producing an anti-NPR1 antibody or antigen-binding fragment thereof, comprising growing the host cell of claim 23 under conditions that allow production of the antibody or fragment, and recovering the antibody or fragment thus produced.

25. 25. The method of claim 24, further comprising formulating the antibody or antigen-binding fragment thereof as a pharmaceutical composition comprising an acceptable carrier.

26. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 15 for use in treating, preventing, or ameliorating at least one symptom or sign of an NPR1-related disease or disorder.

27. The pharmaceutical composition for use according to claim 26, wherein the NPR1-related disease or disorder is selected from the group consisting of hypotension, circulatory shock, septic shock, neurogenic orthostatic hypotension, postural orthostatic tachycardia syndrome (POTS), heart failure, cardiogenic shock, obesity, renal failure, chronic kidney disease, macular edema, glaucoma, stroke, lung damage, pulmonary fibrosis, inflammation, asthma, skeletal growth disorders, fractures, diabetes, hypoglycemia, and cancer.

28. 27. The pharmaceutical composition for use according to claim 26, wherein the pharmaceutical composition is for prophylactic or therapeutic administration.

29. 27. The pharmaceutical composition for use according to claim 26, wherein the pharmaceutical composition is in combination with a second therapeutic agent or therapy.

30. 30. The composition for use of claim 29, wherein the second therapeutic agent or therapy is selected from the group consisting of angiogenesis inhibitors, vasoconstrictors / vasopressors, immunosuppressants, ascorbic acid, calcineurin inhibitors, corticosteroids, VEGF inhibitors, decongestants, antidepressants, hormonal contraceptives, stimulants (including cardiac stimulants), caffeine, extracorporeal membrane oxygenation, ventricular assist devices, intra-aortic balloon pumps, lifestyle modifications, nutritional supplements, antibiotics, insulin, and anti-inflammatory agents.

31. 27. The pharmaceutical composition of claim 26, wherein the pharmaceutical composition is for subcutaneous, intravenous, intradermal, intraperitoneal, or intramuscular administration.

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