Anti-NPR1 Antibody and Its Use

Abstract

To provide monoclonal antibodies that bind to natriuretic peptide receptor (NPR1) protein, and to provide methods of use thereof.SOLUTION: Provided is an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the heavy chain variable region (HCVR) and / or light chain variable region (LCVR) of an antibody or antigen-binding fragment thereof that specifically binds to NPR1 protein, the antibody or antigen-binding fragment thereof comprising: three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) comprised in a HCVR containing specific amino acid sequences; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) comprised in a LCVR containing specific amino acid sequences. The antibody of the present invention is useful for activating NPR1 activity, thus providing a means of treating or preventing a disease, disorder or condition associated with NPR1 in humans.SELECTED DRAWING: None

Description

Technical Field

[0001] This application was filed as a PCT international patent application on October 18, 2019, and claims the benefit of priority of U.S. Provisional Application No. 62 / 749,557, filed on October 23, 2018, and No. 62 / 755,720, filed on November 5, 2018, the entire disclosure of each of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to antibodies and antigen-binding fragments of antibodies that specifically bind to natriuretic peptide receptor 1 (NPR1), and to therapeutic and diagnostic methods using such antibodies.

Background Art

[0003] Natriuretic peptide receptor 1 (NPR1; also known as NPR-A) belongs to the cell surface family of guanylyl cyclase receptors, which are enzymes that catalyze the conversion of GTP to cyclic GMP. NPR1 is highly expressed in the kidney, lung, adrenal gland, vascular system, brain, liver, endothelium, and adipose tissue, and is present at low levels in the heart. It is activated by binding to atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). NPR1 activation and signaling trigger many physiological responses involving many tissues. The ANP-NPR1 system has been well studied for its role in vasorelaxation, natriuresis, diuresis, endothelial permeability, and non-cardiovascular functions such as lipolysis and immune cell function (Non-Patent Document 1). When NPR1 is activated, natriuresis (excretion of salts by the kidney) occurs and blood pressure decreases.

[0004] Monoclonal antibodies against NPR1 were first described by Kitano et al. in Non-Patent Document 2 in 1995. Activating or agonist anti-NPR1 antibodies are disclosed, for example, in Patent Document 1, Patent Document 2, and Patent Document 3.

[0005] Fully human antibodies that specifically bind to the NPR1 protein with high affinity and activate it may play an important role in the prevention and treatment of, for example, hypertension, obesity, heart failure, etc.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0008] The present invention provides antibodies that specifically bind to the natriuretic peptide receptor 1 (NPR1) protein and antigen-binding fragments thereof. In certain embodiments, the anti-NPR1 antibody is a fully human antibody that binds to NPR1 with high affinity and activates NPR1 or stabilizes the activated conformation. The antibodies of the present invention are particularly useful for activating or increasing the activity of the NPR1 protein. In certain embodiments, the antibody is a subject It is useful for preventing, treating, or ameliorating at least one symptom or sign of an NPR1-related disease or disorder. In certain embodiments, the antibody can be administered prophylactically or therapeutically to a subject having or at risk of an NPR1-related disease or disorder. In certain embodiments, the antibody is used to reduce the systemic blood pressure of a subject suffering from hypertension. Such an antibody can be used as a treatment for disorders such as heart failure when administered to a subject in need thereof.

[0009] In certain embodiments, the antibody binds to NPR1 in the presence or absence of atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP), i.e., the antibody is a "peptide-independent binder". Such an antibody is advantageous because it can be used to bind and activate NPR1 even when the concentration of the endogenous ligand is different. Such an antibody can be advantageously used to avoid inter-patient variability (with respect to ligand concentration) in treatment when administered to a patient in need thereof. Furthermore, the antibodies disclosed herein bind to NPR1 with high affinity and have improved pharmacokinetic properties (compared to standard therapeutic agents). This antibody showed a t1 / 2 of up to 11 days in mice at a dose of 25 mg / kg. This antibody has the effect of reducing blood pressure and maintaining the reduced blood pressure for up to 28 days when administered to a subject in need thereof. A single administration of the antibody of the present invention resulted in a sustained reduction in blood pressure. Such an antibody can be used to provide excellent efficacy at a lower dosing frequency in subjects having an NPR1-related disease or disorder (e.g., hypertension).

[0010] The antibody of the present invention may be a full-length (e.g., IgG1 or IgG4 antibody) or may contain only an antigen-binding portion (e.g., Fab, F(ab’)2 or scFv fragment), and may be modified, for example, to enhance persistence in a host or to eliminate residual effector functions, in order to affect functionality (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, the antibody may be bispecific.

[0011] In a first aspect, the present invention provides an isolated recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to NPR1.

[0012] In some embodiments, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to a natriuretic peptide receptor 1 (NPR1) protein, wherein the antibody or antigen-binding fragment thereof interacts with one or more amino acids contained within the extracellular domain of NPR1 (amino acids 29-347 of SEQ ID NO: 194) as determined by hydrogen / deuterium exchange, and the antibody or antigen-binding fragment thereof (i) binds to cells expressing human NPR1 in the presence or absence of atrial natriuretic peptide (ANP) and / or (ii) binds to NPR1 and activates NPR1.

[0013] In some embodiments, the antibody is a fully human monoclonal antibody.

[0014] Exemplary anti-NPR1 antibodies of the invention are set forth in Tables 1 and 2 herein. Table 1 provides amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining region (HCDR) (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining region (LCDR) (LCDR1, LCDR2, and LCDR3) of exemplary antibodies. Table 2 provides nucleic acid sequence identifiers for the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary antibodies.

[0015] The present invention relates to an amino acid sequence selected from any of the amino acid sequences of HCVRs listed in Table 1. The present invention provides an antibody, or an antigen-binding fragment thereof, comprising an HCVR comprising an amino acid sequence, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0016] The present invention also provides an antibody, or an antigen-binding fragment thereof, comprising an LCVR comprising an amino acid sequence selected from any of the amino acid sequences of the LCVRs 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 invention also provides antibodies, or antigen-binding fragments thereof, that comprise an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) that comprises any of the HCVR amino acid sequences set forth in Table 1 paired with any of the LCVR amino acid sequences set forth in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, that comprise an HCVR / LCVR amino acid sequence pair included in any of the exemplary anti-NPR1 antibodies set forth 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., mAb22033), and 66 / 74 (e.g., mAb22810).

[0018] The present invention also provides an antibody or an antigen-binding fragment thereof that includes an HCVR and an LCVR, wherein the HCVR includes the amino acid sequence set forth in Table 1 and has no more than 12 amino acid substitutions, and / or the LCVR includes the amino acid sequence set forth in Table 1 and has no more than 10 amino acid substitutions. For example, the present invention provides an antibody or an antigen-binding fragment thereof that includes an HCVR and an LCVR, wherein the HCVR includes the amino acid sequence set forth in Table 1, and the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions. In another example, the present invention provides an antibody or an antigen-binding fragment thereof that includes an HCVR and an LCVR, wherein the LCVR includes the amino acid sequence set forth in Table 1, and the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In one embodiment, the present invention provides an anti-NPR1 antibody or an antigen-binding fragment thereof that includes an HCVR and an LCVR, wherein the HCVR includes the amino acid sequence set forth in Table 1, the amino acid sequence has at least one amino acid substitution, and / or the LCVR includes the amino acid sequence set forth in Table 1, and the amino acid sequence has at least one amino acid substitution.

[0019] The present invention also provides an antibody or an antigen-binding fragment thereof that includes a heavy chain complementarity-determining region 1 (HCDR1) that includes an amino acid sequence selected from any of the HCDR1 amino acid sequences set forth in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0020] The present invention also provides an antibody or an antigen-binding fragment thereof that includes a heavy chain complementarity-determining region 2 (HCDR2) that includes an amino acid sequence selected from any of the HCDR2 amino acid sequences set forth in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0021] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences set forth in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0022] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light-chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences set forth in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0023] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light-chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences set forth in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0024] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light-chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences set forth in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0025] Furthermore, the present invention provides an antibody or an 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 a particular embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising the HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-NPR1 antibodies listed in Table 1. In a particular embodiment, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 8 / 16 (e.g., mAb22033), and 72 / 80 (e.g., mAb22810).

[0026] The present invention also provides an antibody or an antigen-binding fragment thereof that includes an HCVR and an LCVR, wherein the HCVR includes an HCDR1 having an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by only one amino acid, an HCDR2 having an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by only one amino acid, and an HCDR3 having an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by only one amino acid. In certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that includes an HCVR and an LCVR, wherein the LCVR includes an LCDR1 having an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by only one amino acid, an LCDR2 having an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by only one amino acid, and an LCDR3 having an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by only one amino acid. For example, the present invention provides an antibody or an antigen-binding fragment thereof that includes an HCVR and an LCVR, wherein the HCVR includes an HCDR1 having the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that differs from SEQ ID NO: 4 by only one amino acid, an HCDR2 having the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence that differs from SEQ ID NO: 6 by only one amino acid, and an HCDR3 having the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by only one amino acid. In another exemplary embodiment, the present invention provides an antibody or an antigen-binding fragment thereof that includes an HCVR and an LCVR, wherein the LCVR includes an LCDR1 having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that differs from SEQ ID NO: 12 by only one amino acid, an LCDR2 having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that differs from SEQ ID NO: 14 by only one amino acid, and an LCDR3 having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that differs from SEQ ID NO: 16 by only one amino acid.

[0027] In addition, the present invention provides an antibody or an antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary antibodies set forth 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 NOs: 4-6-8-12-14-16 (e.g., mAb22033), and 68-70-72-76-78-80 (e.g., mAb22810).

[0028] In related embodiments, the invention provides an antibody or an antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair defined by any of the exemplary antibodies set forth in Table 1. For example, the invention provides an antibody or an antigen-binding fragment thereof comprising an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 (e.g., mAb22033), and 66 / 74 (e.g., mAb22810). 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 the CDRs within the HCVR and / or LCVR amino acid sequences disclosed and specified herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence diversity, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat approach and the Chothia approach. See, e.g., 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.

[0029] In certain embodiments, the invention provides an antibody or an antigen-binding fragment thereof that specifically binds to NPR1, wherein 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), and the HCVR comprises: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178; (ii) an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178; (iii) an amino acid sequence having at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178; or (iv) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178, wherein the amino acid sequence has no more than 12 amino acid substitutions; and the LCVR comprises: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and 186; (b) an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and 186; (c) an amino acid sequence having at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and 186; or (d) an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and 186, wherein the amino acid sequence has no more than 10 amino acid substitutions and includes an antibody or an antigen-binding fragment thereof.

[0030] In certain preferred embodiments, the invention includes antibodies that specifically bind to NPR1 in an agonist-like manner, i.e., antibodies that enhance or induce the binding and / or activity of NPR1.

[0031] The invention includes anti-NPR1 antibodies having an altered glycosylation pattern. In some embodiments, modifications for removing undesirable glycosylation sites are useful, i.e., for example, antibodies lacking fucose moieties present on the oligosaccharide chain may be useful for enhancing antibody-dependent cell cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modifications of galactosylation can be made to modify complement-dependent cytotoxicity (CDC).

[0032] In certain embodiments, the invention provides antibodies and antigen-binding fragments thereof that exhibit pH-dependent binding to NPR1. For example, the invention includes antibodies and antigen-binding fragments thereof that bind to NPR1 with higher affinity at neutral pH than at acidic pH (i.e., binding is decreased at acidic pH).

[0033] The invention also provides antibodies and antigen-binding fragments thereof that compete with an antibody or antigen-binding fragment thereof that includes the CDRs of the HCVR and the CDRs of the LCVR with respect to specific binding to NPR1, wherein the HCVR and the LCVR each have an amino acid sequence selected from the sequences of the HCVR and LCVR set forth in Table 1.

[0034] The invention also provides antibodies and antigen-binding fragments thereof that cross-compete with a reference antibody or antigen-binding fragment thereof that includes the CDRs of the HCVR and the CDRs of the LCVR with respect to binding to NPR1, wherein the HCVR and the LCVR each have an amino acid sequence selected from the sequences of the HCVR and LCVR set forth in Table 1.

[0035] The present invention also provides an antibody and an antigen-binding fragment thereof that bind to the same epitope as a reference antibody or an antigen-binding fragment thereof that includes three CDRs of HCVR and three CDRs of LCVR, wherein the HCVR and LCVR each have an amino acid sequence selected from the sequences of HCVR and LCVR set forth in Table 1.

[0036] The present invention also provides an isolated antibody and an antigen-binding fragment thereof that increase or stabilize the binding of NPR1 to its ligand (e.g., ANP or BNP). In some embodiments, an antibody or an antigen-binding fragment thereof that activates the binding of NPR1 to ANP may bind to the same epitope on NPR1 as ANP or may bind to an epitope on NPR1 that is different from that of ANP.

[0037] In certain embodiments, the antibody or antigen-binding fragment of the present invention is bispecific and includes a first binding specificity for a first epitope of NPR1 and a second binding specificity for a second epitope of NPR1, wherein the first epitope and the second epitope are distinct and non-overlapping.

[0038] In certain embodiments, the present invention has the following characteristics: (a) it is a fully human monoclonal antibody; (b) it binds to monomeric human NPR1 with a dissociation constant (K D ) of less than 690 nM at 25° C. and 37° C. in the absence of ANP and / or BNP, as measured by surface plasmon resonance; (c) it binds to dimeric human NPR1 with a K of less than 42 nM at 25° C. and 37° C. in the absence of ANP or BN D P, as measured by surface plasmon resonance; (d) it binds to human NPR1 complexed with ANP with a K D of less than 80 nM at 25° C. and 37° C., as measured by surface plasmon resonance; (e) it binds to human NPR1 complexed with BNP with a K Dand binds to human NPR1 complexed with BNP; (f) less than 365 nM K at 25° C. and 37° C. in the absence of ANP and / or BNP, as measured by surface plasmon resonance D and binds to monomeric monkey NPR1; (g) less than 30 nM K at 25° C. and 37° C. in the absence of ANP or BNP, as measured by surface plasmon resonance D and binds to dimeric monkey NPR1; (h) less than 10 nM K at 25° C. and 37° C., as measured by surface plasmon resonance D and binds to monkey NPR1 complexed with ANP; (i) less than 10 nM K at 25° C. and 37° C., as measured by surface plasmon resonance D and binds to monkey NPR1 complexed with BNP; (j) does not bind to mouse NPR1; (k) less than 5 nM EC binds to cells expressing human NPR1 (without ANP) or NPR1 complexed with ANP 50 ; (l) less than 385 nM EC activates NPR1, as measured by a calcium flux cell-based bioassay 50 ; (m) when administered to normal and hypertensive mice, reduces systemic blood pressure, and the reduction in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single administration; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; provided is an isolated antibody or an antigen-binding fragment thereof having one or more of the foregoing

[0039] In a second aspect, the present invention provides a nucleic acid molecule encoding an anti-NPR1 antibody or a portion thereof. For example, the present invention provides a nucleic acid molecule encoding any of the HCVR amino acid sequences set forth in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences set forth in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0040] The present invention also provides a nucleic acid molecule encoding any of the LCVR amino acid sequences set forth in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences set forth in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0041] The present invention also provides a nucleic acid molecule encoding any of the HCDR1 amino acid sequences set forth in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences set forth in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0042] The present invention also provides a nucleic acid molecule encoding any of the HCDR2 amino acid sequences set forth in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences set forth in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0043] The present invention also provides a nucleic acid molecule encoding any of the HCDR3 amino acid sequences set forth in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences set forth in Table 2 It comprises a polynucleotide sequence selected from any of the nucleic acid sequences, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0044] The present invention also provides a nucleic acid molecule encoding any of the LCDR1 amino acid sequences set forth in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences set forth in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0045] The present invention also provides a nucleic acid molecule encoding any of the LCDR2 amino acid sequences set forth in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences set forth in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0046] The present invention also provides a nucleic acid molecule encoding any of the LCDR3 amino acid sequences set forth in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences set forth in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0047] The present invention also provides a nucleic acid molecule encoding HCVR, wherein HCVR comprises a set of three CDRs (i.e., HCDR1 - HCDR2 - HCDR3), and the amino acid sequence set of HCDR1 - HCDR2 - HCDR3 is as defined by any of the exemplary antibodies set forth in Table 1.

[0048] In addition, the present invention provides a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), and the set of amino acid sequences of LCDR1-LCDR2-LCDR3 is as defined by any of the exemplary antibodies described in Table 1.

[0049] In addition, the present invention provides a nucleic acid molecule encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences described in Table 1, and the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences described in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences described in Table 2, or a substantially similar sequence 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 described in Table 1, or a substantially similar sequence 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 invention, the nucleic acid molecule encodes an HCVR and an LCVR, and both the HCVR and the LCVR are derived from the same anti-NPR1 antibody described in Table 1.

[0050] In a related aspect, the present invention provides a recombinant expression vector capable of expressing a polypeptide comprising the heavy and / or light chain variable regions of an antibody. For example, the present invention includes a recombinant expression vector comprising any of the nucleic acid molecules described above, i.e., a nucleic acid molecule encoding any of the HCVR, LCVR, and / or CDR sequences described in Table 2. In certain embodiments, the present invention provides (a) a nucleic acid encoding the HCVR of an antibody that binds to NPR1 A nucleic acid molecule comprising an array, the nucleic acid molecule comprising an amino acid sequence in which the HCVR is selected from the group consisting of the sequences set forth in Table 1, and / or (b) a nucleic acid molecule comprising a nucleic acid sequence encoding the LCVR of an antibody that binds to NPR1, the nucleic acid molecule comprising an amino acid sequence in which the LCVR is selected from the group consisting of the sequences set forth in Table 1, is provided. Also included within the scope of the present invention are host cells into which such vectors have been introduced, and a method of culturing the host cells under conditions that permit the production of an antibody or antibody fragment, producing an antibody or a portion thereof, and recovering the produced antibody and antibody fragment. In certain embodiments, the host cells include mammalian cells or prokaryotic cells. In certain embodiments, the host cells are Chinese hamster ovary (CHO) cells or Escherichia coli (E. coli) cells. In certain embodiments, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof of the present invention, the method comprising introducing into a host cell an expression vector comprising a nucleic acid sequence encoding the HCVR and / or LCVR of the antibody or an antigen-binding fragment thereof of the present invention operably linked to a promoter, culturing the host cells under conditions favorable for the expression of the nucleic acid sequence, and isolating the antibody or an antigen-binding fragment thereof from the culture medium and / or the host cells. The isolated antibody or antigen-binding fragment can be purified using any method known in the prior art.

[0051] In a third aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to NPR1, and a pharmaceutically acceptable carrier. In related aspects, the present invention features a composition that is a combination of an anti-NPR1 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-NPR1 antibody. Exemplary agents that are advantageously combined with the anti-NPR1 antibody include, but are not limited to, other agents that bind to NPR1 and / or activate NPR1 activity (including other antibodies or antigen-binding fragments thereof), and / or agents that do not bind directly to NPR1 but nevertheless treat or ameliorate at least one symptom or sign of an NPR1-related disease or disorder (disclosed elsewhere herein). Additional combination therapies and co-formulations comprising the anti-NPR1 antibody of the present invention are disclosed elsewhere herein.

[0052] In a fourth aspect, the present invention provides a method of treating 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 invention, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the antibody or an antigen-binding fragment of the antibody of the present invention. The disorder to be treated is any disease or condition (e.g., hypertension) that is improved, ameliorated, inhibited, or prevented by enhancement of NPR1 activity. In certain embodiments, the present invention provides a method of preventing or treating an NPR1-related disease or disorder comprising administering to a subject in need thereof a therapeutically effective amount of an anti-NPR1 antibody or an antigen-binding fragment thereof of the present invention. In some embodiments, the antibody or an antigen-binding fragment thereof can be administered prophylactically or therapeutically to a subject having or at risk of an NPR1-related disease or disorder. In certain embodiments, the antibody or an antigen-binding fragment thereof of the present invention is administered in combination with a second therapeutic agent to a subject in need thereof. The second therapeutic agent can be selected from the group consisting of an aldosterone antagonist, an alpha-adrenergic blocker, an angiotensin-converting enzyme (ACE) inhibitor, an arteriolar dilator, an autonomic ganglionic vasodilator, a beta-adrenergic blocker, a catecholamine-depleting sympathetic blocker, a central alpha-2 adrenergic agonist, a calcium channel blocker, a diuretic, a renin inhibitor, an anticoagulant, an antiplatelet agent, a cholesterol-lowering agent, a vasodilator, digitalis, surgery, an implantable device, an antitumor therapy, insulin, a GLP1 agonist, metformin, dialysis, a bone marrow stimulant, hemofiltration, lifestyle improvement, nutritional supplements, and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent is the antibody or an antigen-binding fragment thereof of the present invention It may also be an agent useful for counteracting or reducing side effects that may be associated with tolvaptan. The antibody or fragment thereof may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, or intramuscularly. The antibody or fragment thereof may be administered at a dose of about 0.1 mg / kg to about 100 mg / kg of the subject's body weight. In certain embodiments, the antibody of the invention may be administered at one or more doses that are comprised between 10 mg and 600 mg.

[0053] The invention also includes the use of the anti-NPR1 antibody or antigen-binding fragment thereof of the invention in the manufacture of a medicament for the treatment of a disease or disorder that benefits from the binding and / or activation of NPR1.

[0054] Other embodiments will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0055]

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Mode for Carrying Out the Invention

[0056] Before describing the method of the present invention, it is to be understood that the present invention is not limited to the specific methods and experimental conditions described, and such methods and conditions may be varied. 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 present invention is limited only by the appended claims.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference in their entirety.

[0058] Definitions The term "NPR1", also known as "NPRA", refers to natriuretic peptide receptor 1 (also called natriuretic peptide receptor A). NPR1 is a homodimeric transmembrane guanylate cyclase, an enzyme that catalyzes the synthesis of cGMP. NPR1 is a receptor for both atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and upon ligand binding, it undergoes a conformational change in its extracellular domain (Ogawa et al 2004, J. Biol. Chem. 279:28625-31). This protein has four distinct regions: 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 as accession number P16066.1 (SEQ ID NO: 193) in UniProtKB / Swiss-Prot. The term "NPR1" includes recombinant NPR1 protein or fragments thereof. Additionally, this term encompasses NPR1 protein or fragments thereof conjugated to signal sequences such as, for example, a histidine tag, mouse or human Fc, or ROR1 (e.g., SEQ ID NOs: 194-199).

[0059] As used herein, the term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds (i.e., a "complete antibody molecule"), as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain is composed of a heavy chain variable region ("HCVR" or "V H ") and a heavy chain constant region (composed of domains C H 1, C H 2, and C H 3). Each light chain is composed of a light chain variable region ("LCVR" or "V L ") and a light chain constant region ("C L "). V H and V LThe region can be further divided into a more conserved region, also called the framework region (FR), and a region of hypervariability called the complementarity determining region (CDR) that is scattered within that region. Each V H and V L is composed of three CDRs and four FRs, and is arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FR of the antibody (or antigen-binding fragment thereof) may be identical to the sequence of the human germline, or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0060] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies that can omit one or two CDRs for binding have been described in the scientific literature. Padlan et al. (1995 FASEB J. 9:133-13 9) analyzed the contact regions between an antibody and its antigen based on the published crystal structure and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also discovered many antibodies that do not have amino acids in one or two CDRs that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0061] CDR residues that do not contact the antigen can be identified by molecular modeling and / or empirically from the region of the Kabat CDR outside the Chothia CDR, based on prior studies (e.g., residues H60-H65 of CDRH2 are often not required). When a CDR or its residue is omitted, it is usually replaced with an amino acid that occupies the corresponding position in other human antibody sequences or in the consensus recognition of such sequences. The substitution positions and the amino acids to substitute within the CDR can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[0062] 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 as compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein, for example, to germline sequences available from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions have been mutated to the corresponding residues of the germline from which the antibody is derived, or to the corresponding residues of another human germline, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate a number of antibodies and antigen-binding fragments that contain one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LAll of the framework residues and / or CDR residues within the domain mutate back to the residues found in the original germline from which the antibody is derived. In other embodiments, only certain residues mutate back to the original germline, such as only the mutated residues found within the first 8 amino acids of FR1, or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework residues and / or CDR residues mutate to the corresponding residues of a different germline (i.e., a germline different from the germline from which the antibody originally derived). Additionally, the antibodies of the invention can include any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues mutate to the corresponding residues of a particular germline sequence while certain other residues that differ from the sequence of the original germline are maintained or mutate to the corresponding residues of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that include one or more germline mutations can be readily tested for one or more desired properties such as improved binding specificity, improved binding affinity, improved or enhanced antagonistic biological properties, reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in such a general manner are encompassed by the invention.

[0063] The invention also includes fully human anti-NPR1 monoclonal antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the invention includes anti-NPR1 antibodies having HCVR, LCVR, and / or CDR amino acid sequences having conservative amino acid substitutions, such as, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0064] As used herein, the terms "human antibody" or "fully human antibody" are intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs of the invention may include, for example, in the CDRs, particularly CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the terms "human antibody" or "fully human antibody" as used herein are not intended to include mAbs in which CDR sequences derived from the germline of other mammalian species (e.g., mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or in cells of non-human mammals. The term is not intended to include antibodies isolated from or generated in a human subject.

[0065] As used herein, the term "recombinant" means an antibody or antigen-binding fragment thereof of the invention made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA techniques, including, for example, DNA splicing and transgenic expression. The term refers to an antibody expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice) or cell (e.g., CHO cells) expression system, or an antibody isolated from a recombinant combinatorial human antibody library.

[0066] Terms such as "specifically binds" or "binds specifically to" mean that an antibody or antigen-binding fragment thereof forms a relatively stable complex with an antigen under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1×10 -8 M or less (e.g., K D(indicating that the smaller the value, the stronger the binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies that specifically bind to NPR1 have been identified by surface plasmon resonance, such as BIACORE™. Furthermore, multispecific antibodies that bind to one domain of NPR1 and one or more additional antigens, or bispecific antibodies that bind to two different regions of NPR1, are still considered "specifically binding" antibodies as used herein.

[0067] The term "high affinity" antibody means an mAb having a binding affinity for NPR1, as measured by surface plasmon resonance, such as BIACORE™ or solution affinity ELISA, of at least 10 -8 M; preferably 10 -9 M; more preferably 10 -10 M, even more preferably 10 -11 M of K D as represented.

[0068] The terms "slow off rate", "Koff" or "kd" mean an antibody that dissociates from NPR1 with a rate constant of 1×10 -3 seconds -1 or less, preferably 1×10 -4 seconds -1 or less as determined by surface plasmon resonance, such as BIACORE™.

[0069] As used herein, terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. As used herein, the term "antigen-binding fragment" or "antibody fragment" of an antibody means one or more fragments of an antibody that retain the ability to bind to the NPR1 protein.

[0070] In certain embodiments, the antibodies or antibody fragments of the invention may be conjugated to a ligand or therapeutic moiety (“immunoconjugate”), a second anti-NPR1 antibody, or other therapeutic moieties useful for treating NPR1-related diseases or disorders, etc.

[0071] As used herein, the term “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 that specifically binds NPR1 or a fragment thereof is substantially free of Abs that specifically bind antigens other than NPR1).

[0072] As used herein, the term “activating antibody” or “agonist antibody” (i.e., “antibody that increases or enhances NPR1 activity” or “antibody that stabilizes the activated conformation”) shall refer to an antibody whose binding to NPR1 results in activation of at least one biological activity of NPR1. For example, an antibody of the invention can lower systemic blood pressure when administered to a subject in need thereof.

[0073] “Surface plasmon resonance” refers to an optical phenomenon by which real-time biomolecular interactions can be analyzed by detecting changes in protein concentration within a biosensor matrix, for example, using a BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0074] As used herein, the term “K D ” shall refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0075] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. An antigen can potentially have more than one epitope. Thus, different antibodies may bind to different regions of the antigen and may have different biological effects. Also, the term "epitope" refers to the site on an antigen to which B cells and / or T cells respond. It is also the region of the antigen to which an antibody binds. An epitope can be defined structurally or functionally. A functional epitope is generally a subset of a structural epitope and has residues that directly contribute to the affinity of the interaction. Also, an epitope may be composed of conformational, i.e., non-linear, amino acids. In certain embodiments, an epitope may include a determinant that is a chemically active surface group of a molecule such as an amino acid, sugar side chain, phosphoryl group, or sulfonyl group, and in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0076] As used herein, the term "cross-competition" means that an antibody or its antigen-binding fragment binds to an antigen and inhibits or blocks the binding of another antibody or its antigen-binding fragment. This term also includes competition in both directions between two antibodies, i.e., a first antibody that binds to a second antibody and inhibits its binding, and vice versa. In certain embodiments, the first antibody and the second antibody may bind to the same epitope. Alternatively, the first antibody and the second antibody may bind to different but overlapping epitopes such that the binding of one antibody inhibits or blocks the binding of the second antibody via steric hindrance. Cross-competition between antibodies can be determined by methods known in the art, such as real-time, label-free bio-layer interferometry assay It can be measured by an assay). The cross-competition between the two antibodies may be expressed as the binding of a second antibody that is less than the background signal due to self-self binding (where the first and second antibodies are the same antibody). The cross-competition between the two antibodies may be expressed, for example, as the binding % of a second antibody that is less than the background binding due to baseline self-self binding (where the first and second antibodies are the same antibody).

[0077] The terms "substantially identical" or "substantially the same" when referring to a nucleic acid or a fragment thereof, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, are measured by any well-known algorithm for sequence identity such as FASTA, BLAST or GAP described below, and indicate that there is nucleotide sequence identity of at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0078] The terms "substantial similarity" or "substantially similar" as applied to polypeptides means that two peptide sequences share at least 90% sequence identity, more preferably at least 95%, 98% or 99% sequence identity when optimally aligned using a program such as GAP or BESTFIT with the default gap weights. Preferably, the positions of the residues differ not by identity but 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 alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward for correction of the conservative nature of the substitution. Means for making such adjustments are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-33, which is 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: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is a change having a positive value in the PAM250 log-likelihood matrix disclosed by Gonnet et al. (1992) Science 256:1443-45, which is incorporated herein by reference. Also, a "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0079] The sequence similarity of polypeptides is usually measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between related polypeptides such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant. See, for example, GCG Version 6.1. Also, polypeptide sequences can be compared using the FASTA;GC programs of G Version 6.1 using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides the alignment of the region that overlaps best between the query sequence and the search sequence and the percentage of sequence identity (Pearson (2000) supra). Another preferred algorithm when comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, which are hereby incorporated by reference in their entirety.

[0080] "A therapeutically effective amount" means an amount that produces the desired effect for which it is administered. The exact amount will vary depending on the purpose of the treatment and can be ascertained by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0081] 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 hypertension. The term includes human subjects having or at risk of such diseases or disorders.

[0082] As used herein, the terms "treat", "treating", or "treatment" refer to the reduction or amelioration of the severity of at least one symptom or sign of an NPR1-related disease or disorder by administration of a therapeutic agent, such as an antibody of the invention, to a subject in need thereof. The term includes suppression of the progression of the disease or worsening of the symptoms / signs. The term also includes the improvement of the prognosis of the disease by administration of a therapeutic agent, such as an antibody of the invention, i.e., the disease in the subject may be eliminated or the disease may be reduced. The therapeutic agent may be administered to the subject in a therapeutically effective amount.

[0083] The terms "prevent", "preventing", or "prevention" refer to the suppression of the development of an NPR1-related disease or disorder, or any symptom or sign of such a disease or disorder, by administration of an antibody of the invention.

[0084] Antigen-binding fragment of an antibody As used herein, the term "antibody" is understood to include not only antibody molecules (i.e., "complete antibody molecules") comprising two immunoglobulin heavy chains and two immunoglobulin light chains, unless otherwise indicated, but also antigen-binding fragments thereof. As used herein, terms such as "antigen-binding portion of an antibody", "antigen-binding fragment of an antibody" include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind 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 the 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 an antibody can be derived from complete antibody molecules using any suitable standard techniques, such as proteolysis, or recombinant genetic engineering techniques that involve manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques to, for example, arrange one or more variable and / or constant domains in an appropriate configuration, or to introduce codons, create cysteine residues, modify, add, or delete amino acids.

[0085] 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) the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides) or minimal recognition units consisting of amino acid residues mimicking a constrained FR3-CDR3-FR4 peptide. Other modified molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0086] An antigen-binding fragment of an antibody typically comprises at least one variable domain. The variable domain can be of any size or amino acid composition and generally comprises at least one CDR, which is adjacent to or within a framework having one or more framework sequences. The V L domain associated with the V H domain in an antigen-binding fragment having the V H domain and the V L domain can be positioned in any suitable arrangement relative to each other. For example, the variable region can be dimeric and can comprise a dimer of V H -V H , V H -V L or V L -V L . Alternatively, an antigen-binding fragment of an antibody can comprise a monomeric V H or V L domain.

[0087] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently attached to at least one constant domain. Non-limiting and exemplary configurations of variable and constant domains that may be found within the antigen-binding fragments of the antibodies of the invention 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 Lis included. In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may be composed of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible linkage being formed between adjacent variable and / or constant domains within a single polypeptide molecule. Further, the antigen-binding fragment of the antibody of the present invention may be such that any of the above variable domain configurations and constant domain configurations are non-covalently bound to each other and / or to one or more monomeric V H or V L domains (e.g., by disulfide bonds) and may include homodimers or heterodimers (or other multimers).

[0088] Similar to a complete antibody molecule, the antigen-binding fragment may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically include at least two different variable domains, each of which can specifically bind to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art.

[0089] Preparation of Human Antibodies Methods for preparing human antibodies in transgenic mice are known in the art. Any of such known methods can be used in connection with the present invention to prepare human antibodies that specifically bind to NPR1.

[0090] An antibody against the NPR1 protein can be produced using an immunogen comprising any one of the following. In certain embodiments, the antibody of the invention is obtained from a mouse immunized with the full-length native NPR1 protein (see, e.g., UniProtKB / Swiss-Prot accession number P16066.1), or DNA encoding the protein or a fragment thereof. Alternatively, the protein or a fragment thereof may be produced using standard biochemical techniques and modified for use as an immunogen.

[0091] In some embodiments, the immunogen may be a recombinant NPR1 protein or a fragment thereof expressed in Escherichia coli (E. coli) or any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells (e.g., SEQ ID NOs: 194-199).

[0092] A high-affinity chimeric antibody against NPR1 having human variable regions and mouse constant regions is first isolated using the VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or other known methods for generating monoclonal antibodies. The VELOCIMMUNE® technology involves generating a transgenic mouse having a genome that includes human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces antibodies that contain human variable regions and mouse constant regions in response to antigen stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding the constant regions of the human heavy and light chains. This DNA is then expressed in cells capable of expressing a fully human antibody.

[0093] Generally, VELOCIMMUNE (registered trademark) mice are exposed to the antigen of interest, and lymphocytes (such as B cells) are recovered from the mice that express the antibody. These lymphoid cells are fused with a myeloma cell line to prepare an immortalized hybridoma cell line, and such hybridoma cell lines are screened and selected to identify a hybridoma cell line that produces an antibody specific to the antigen of interest. DNA encoding the variable regions of the heavy and light chains may be isolated and ligated to the constant regions of the desired isotype of the heavy and light chains. Such antibody proteins may be produced in cells such as CHO cells. Alternatively, DNA encoding an antigen-specific chimeric antibody or the variable regions of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

[0094] First, a high-affinity chimeric antibody having a human variable region and a mouse constant region is isolated. As in the following experimental section, the antibody is characterized and selected for desired properties including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to produce a fully human antibody of the present invention, for example, wild-type or modified IgG1 or IgG4.

[0095] The selected constant region may vary depending on the particular application, but the properties of high-affinity antigen binding and target specificity reside in the variable region.

[0096] Biological equivalents The anti-NPR1 antibodies and antibody fragments of the present invention include proteins having an amino acid sequence that varies from that of the described antibodies but retains the ability to bind to the NPR1 protein. Such variant antibodies and antibody fragments include one or more additions, deletions, or substitutions of amino acids compared to the parental sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Similarly, the DNA sequences encoding the antibodies of the present invention include one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but include sequences that encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the present invention.

[0097] Two antigen-binding proteins, i.e., antibodies, are considered to be biologically equivalent if, for example, when administered at the same molar dose under similar experimental conditions, either single-dose or multiple-dose, they do not show a significant difference in the rate and extent of absorption, i.e., are pharmaceutical equivalents or pharmaceutical alternatives. Some antibodies may be considered equivalents or pharmaceutical alternatives when the extent of absorption is the same but the rate of absorption is not, yet such differences in absorption rate are intentional, reflected in the labeling, and not considered medically important for the particular pharmaceutical being studied, for example, not essential to achieve an effective in vivo drug concentration in chronic use, and may be considered biologically equivalent.

[0098] In one embodiment, two antigen-binding proteins are biologically equivalent if there are no clinically meaningful differences in their safety, purity, or potency.

[0099] In one embodiment, two antigen-binding proteins are biologically equivalent if a patient can switch between a reference product and a biological product one or more times and there is no expected increase in the risk of side effects, including a clinically significant change in immunogenicity, or decrease in efficacy, compared to continuing treatment without such a switch.

[0100] In one embodiment, two antigen-binding proteins are biologically equivalent if both act by one or more common mechanisms of action with respect to one or more conditions of use, to the extent such mechanisms are known.

[0101] Biological equivalence can be demonstrated by in vivo and / or in vitro methods. Measures of biological equivalence include, for example, (a) in vivo tests in humans or other mammals that measure the concentration of an antibody or its metabolite in blood, plasma, serum, or other biological fluids as a function of time; (b) in vitro tests that correlate with and are reasonably predictive of in vivo human bioavailability data; (c) in vivo tests in humans or other mammals that measure the appropriate acute pharmacological effects of an antibody (or its target) as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or biological equivalence of an antibody.

[0102] Biologically equivalent variants of the antibodies of the present invention can be constructed, for example, by making various substitutions of residues or sequences, or 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 can be deleted or substituted with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bonds during regeneration. In other contexts, biologically equivalent antibodies can include antibody variants that contain amino acid changes that alter the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

[0103] Anti-NPR1 antibody comprising an Fc variant According to certain embodiments of the present invention, there are provided anti-NPR1 antibodies comprising an Fc domain that contains one or more mutations that enhance or decrease the binding of the antibody to the FcRn receptor, for example, at acidic pH as compared to neutral pH. For example, the present invention relates to the C of the Fc domain H 2 or C HAn anti-NPR1 antibody containing a mutation in three regions, the mutation increasing the affinity of the Fc domain for FcRn in an acidic environment (e.g., within an endosome where the pH is in the range of about 5.5 to about 6.0). Such a mutation may result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 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 position 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 position 250 and / or 428; or modifications at position 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.

[0104] For example, the present invention 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), and includes an anti-NPR1 antibody comprising an Fc domain containing such mutations. All possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are contemplated within the scope of the present invention.

[0105] In addition, the present invention also provides an anti-NPR1 antibody comprising a chimeric heavy chain constant (C H ) region, wherein the chimeric C H region comprises segments derived from the C H regions of more than one immunoglobulin isotype. For example, the antibody of the present invention may comprise a chimeric C H region formed by combining part or all of the C H 2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule and part or all of the C H 3 domain derived from a human IgG1, human IgG2 or human IgG4 molecule. According to certain embodiments, the antibody of the present invention has a chimeric hinge region and a chimeric C Hincludes a region. For example, a chimeric hinge may be a combination of an "upper hinge" amino acid sequence (amino acid residues from position 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2 or human IgG4 hinge region and a "lower hinge" sequence (amino acid residues from position 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 includes amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. The chimeric C H region-containing antibodies, in certain embodiments, can exhibit modified F c effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, U.S. Patent Application Publication No. 2014 / 0243504, the disclosure of which is incorporated herein by reference in its entirety).

[0106] Biological properties of the antibodies of the present invention Generally, the antibodies of the present invention function by binding to the NPR1 protein and enhancing its activity. For example, in the present invention, monomeric human NPR1 protein in the absence of either ANP or BNP (e.g., at 25°C or 37°C) is measured by surface plasmon resonance using an assay format as defined in Example 3 herein, with a K D less than 690 nM. Antibodies and antigen-binding fragments of the antibodies that bind are included. In certain embodiments, the antibody or its antigen-binding fragment binds to NPR1 with a K D less than about 650 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 50 nM, or less than about 25 nM, as measured by surface plasmon resonance using an assay format as defined in Example 3 herein or a substantially similar assay.

[0107] Also, the present invention provides, in the absence of either ANP or BNP (e.g., at 25°C or 37°C), a dimer human NPR1 protein having a K of less than 42 nM as measured by surface plasmon resonance using an assay format such as that defined in Example 3 herein. D Antibodies and antigen-binding fragments thereof that bind to NPR1 with a K of less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, or less than about 0.5 nM as measured by surface plasmon resonance using an assay format such as that defined in Example 3 herein or an assay substantially similar thereto are included. D bind to NPR1 with a K of less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, or less than about 0.5 nM as measured by surface plasmon resonance using an assay format such as that defined in Example 3 herein or an assay substantially similar thereto.

[0108] Also, the present invention provides antibodies and antigen-binding fragments of the antibodies that bind to a human NPR1 protein complexed with ANP (e.g., at 25°C or 37°C) with a K of less than 80 nM as measured by surface plasmon resonance using an assay format such as that defined in Example 3 herein. D In certain embodiments, the antibody or antigen-binding fragment thereof binds to NPR1 with a K of less than about 70 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, or less than about 0.5 nM as measured by surface plasmon resonance using an assay format such as that defined in Example 3 herein or an assay substantially similar thereto. D bind to NPR1 with a K of less than about 70 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, or less than about 0.5 nM as measured by surface plasmon resonance using an assay format such as that defined in Example 3 herein or an assay substantially similar thereto.

[0109] Also, the present invention provides a human NPR1 protein complexed with BNP (e.g., at 25°C or 35°C) having a K of less than 20 nM as measured by surface plasmon resonance using an assay format such as that defined in Example 3 herein. DIt includes antibodies and antigen-binding fragments of antibodies that bind with []. In certain embodiments, the antibody or its antigen-binding fragment binds to NPR1 with a K of less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, or less than about 0.5 nM as measured by surface plasmon resonance using, for example, an assay format as defined in Example 3 herein or a substantially similar assay. D that binds to NPR1 with [].

[0110] In addition, the present invention also includes antibodies and their antigen-binding fragments that bind to monomeric monkey NPR1 protein with a K of less than 365 nM in the absence of either ANP or BNP (e.g., at 25 °C or 37 °C) as measured by surface plasmon resonance using, for example, an assay format as defined in Example 3 herein. D It also includes antibodies and their antigen-binding fragments that bind with []. In certain embodiments, the antibody or its antigen-binding fragment binds to NPR1 with a K of less than about 360 nM, less than about 300 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, or less than about 0.5 nM as measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein or a substantially similar assay. that binds to NPR1 with []. D that binds to NPR1 with [].

[0111] In addition, the present invention also includes antibodies and their antigen-binding fragments that bind to dimeric monkey NPR1 protein with a K of less than 30 nM in the absence of either ANP or BNP (e.g., at 25 °C or 37 °C) as measured by surface plasmon resonance using, for example, an assay format as defined in Example 3 herein. D It also includes antibodies and their antigen-binding fragments that bind with []. In certain embodiments, the antibody or its antigen-binding fragment binds to NPR1 with a K of less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, or less than about 0.5 nM as measured by surface plasmon resonance using, for example, an assay format as defined in Example 3 herein or a substantially similar assay. DIt binds to NPR1.

[0112] In addition, the present invention also provides, for example, an antibody and an antigen-binding fragment of the antibody that bind to the monkey NPR1 protein complexed with ANP (e.g., at 25°C or 37°C) with a K of less than 10 nM as measured by surface plasmon resonance using an assay format as defined in Example 3 of the present specification. D D D It binds to NPR1 with a K of less than 10 nM as measured by surface plasmon resonance using an assay format as defined in Example 3 of the present specification. In certain embodiments, the antibody or its antigen-binding fragment binds to NPR1 with a K of less than about 9 nM, less than about 8 nM, less than about 5 nM, less than about 1 nM, or less than about 0.5 nM as measured by surface plasmon resonance using an assay format as defined in Example 3 of the present specification or an assay substantially similar thereto.

[0113] In addition, the present invention also provides an antibody and an antigen-binding fragment of the antibody that bind to the dimeric monkey NPR1 protein complexed with BNP (e.g., at 25°C or 37°C) with a K of less than 10 nM as measured by surface plasmon resonance using an assay format as defined in Example 3 of the present specification. D 50

[0114] In certain embodiments, the antibody or its antigen-binding fragment binds to NPR1 with a K of less than about 9 nM, less than about 8 nM, less than about 5 nM, less than about 1 nM, or less than about 0.5 nM as measured by surface plasmon resonance using an assay format as defined in Example 3 of the present specification or an assay substantially similar thereto. 50 The present invention also provides an antibody and its antigen-binding fragment that bind to human NPR1, with or without ANP, with an EC of less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM as measured using an assay format as described in Example 5 of the present specification or an assay substantially similar thereto.

[0115] In addition, the present invention provides an antibody and an antigen-binding fragment thereof that activate NPR1 with an EC of less than 385 nM as measured by a calcium flux cell-based bioassay using an assay format as defined in Example 6 of the present specification, for example. 50 The antibody and antigen-binding fragment thereof that activate NPR1 with an EC of less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 50 nM, less than about 10 nM, or less than about 1 nM as measured by a calcium flux cell-based bioassay using an assay format as defined in Example 6 of the present specification, for example, or a substantially similar assay. 50 activate NPR1.

[0116] In addition, the present invention provides an antibody and an antigen-binding fragment thereof that bind to NPR1 and reduce the systemic blood pressure of a subject for more than 28 days when administered as a single dose to a subject in need thereof, as shown in Example 8 of the present specification, for example.

[0117] In addition, the present invention provides an antibody and an antigen-binding fragment thereof that bind to NPR1 and reduce the fasting blood glucose level when administered to a subject in need thereof, as shown in Example 11 of the present specification, for example.

[0118] In one embodiment, the present invention provides an isolated recombinant antibody or an antigen-binding fragment thereof that specifically binds to NPR1 protein in the presence or absence of ANP or BNP and increases the activity of NPR1, and the antibody or fragment thereof has the following characteristics: (a) a fully human monoclonal antibody; (b) a dissociation constant (K D ) of less than 690 nM at 25 °C and 37 °C in the absence of ANP and / or BNP and binds to monomeric human NPR1 as measured by surface plasmon resonance; (c) a K of less than 42 nM at 25 °C and 37 °C in the absence of ANP or BNP as measured by surface plasmon resonance. Dbinds to dimeric human NPR1; (d) K of less than 80 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to human NPR1 complexed with ANP; (e) K of less than 20 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to human NPR1 complexed with BNP; (f) K of less than 365 nM at 25° C. and 37° C. in the absence of ANP and / or BNP, as measured by surface plasmon resonance, binds to monomeric monkey NPR1; (g) K of less than 30 nM at 25° C. and 37° C. in the absence of ANP or BNP, as measured by surface plasmon resonance, binds to dimeric monkey NPR1; (h) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with ANP; (i) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with BNP; (j) does not bind to mouse NPR1; (k) binds with an EC of less than 5 nM to cells expressing human NPR1 (in the absence of ANP) or NPR1 complexed with ANP; (l) activates NPR1 with an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; indicating one or more of D binds to human NPR1 complexed with ANP; (e) K of less than 20 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to human NPR1 complexed with BNP; (f) K of less than 365 nM at 25° C. and 37° C. in the absence of ANP and / or BNP, as measured by surface plasmon resonance, binds to monomeric monkey NPR1; (g) K of less than 30 nM at 25° C. and 37° C. in the absence of ANP or BNP, as measured by surface plasmon resonance, binds to dimeric monkey NPR1; (h) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with ANP; (i) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with BNP; (j) does not bind to mouse NPR1; (k) binds with an EC of less than 5 nM to cells expressing human NPR1 (in the absence of ANP) or NPR1 complexed with ANP; (l) activates NPR1 with an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; D binds to human NPR1 complexed with BNP; (f) K of less than 365 nM at 25° C. and 37° C. in the absence of ANP and / or BNP, as measured by surface plasmon resonance, binds to monomeric monkey NPR1; (g) K of less than 30 nM at 25° C. and 37° C. in the absence of ANP or BNP, as measured by surface plasmon resonance, binds to dimeric monkey NPR1; (h) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with ANP; (i) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with BNP; (j) does not bind to mouse NPR1; (k) binds with an EC of less than 5 nM to cells expressing human NPR1 (in the absence of ANP) or NPR1 complexed with ANP; (l) activates NPR1 with an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; D binds to monomeric monkey NPR1; (g) K of less than 30 nM at 25° C. and 37° C. in the absence of ANP or BNP, as measured by surface plasmon resonance, binds to dimeric monkey NPR1; (h) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with ANP; (i) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with BNP; (j) does not bind to mouse NPR1; (k) binds with an EC of less than 5 nM to cells expressing human NPR1 (in the absence of ANP) or NPR1 complexed with ANP; (l) activates NPR1 with an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; D binds to dimeric monkey NPR1; (h) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with ANP; (i) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with BNP; (j) does not bind to mouse NPR1; (k) binds with an EC of less than 5 nM to cells expressing human NPR1 (in the absence of ANP) or NPR1 complexed with ANP; (l) activates NPR1 with an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; D binds to monkey NPR1 complexed with ANP; (i) K of less than 10 nM at 25° C. and 37° C., as measured by surface plasmon resonance, binds to monkey NPR1 complexed with BNP; (j) does not bind to mouse NPR1; (k) binds with an EC of less than 5 nM to cells expressing human NPR1 (in the absence of ANP) or NPR1 complexed with ANP; (l) activates NPR1 with an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; D binds to monkey NPR1 complexed with BNP; (j) does not bind to mouse NPR1; (k) binds with an EC of less than 5 nM to cells expressing human NPR1 (in the absence of ANP) or NPR1 complexed with ANP; (l) activates NPR1 with an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; (j) does not bind to mouse NPR1; (k) binds with an EC of less than 5 nM to cells expressing human NPR1 (in the absence of ANP) or NPR1 complexed with ANP; (l) activates NPR1 with an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; 50 binds with an EC of less than 5 nM to cells expressing human NPR1 (in the absence of ANP) or NPR1 complexed with ANP; (l) activates NPR1 with an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; 50 activates NPR1 with an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; (m) when administered to normal and hypertensive mice, decreases systemic blood pressure, and the decrease in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose; (n) when administered to diet-induced obese mice, improves glucose tolerance; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1; indicating one or more of

[0119] The antibody of the present invention may have one or more of the aforementioned biological properties, or a combination thereof. Other biological properties of the antibody of the present invention will be apparent to those skilled in the art from a consideration of the present disclosure, including the examples herein.

[0120] Epitope mapping and related techniques The present invention includes anti-NPR1 antibodies that interact with one or more amino acids found within one or more regions of the NPR1 protein molecule. The epitope to which the antibody binds may be composed 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 within any of the aforementioned domains of the NPR1 protein molecule (e.g., a linear epitope within a domain). Alternatively, the epitope may be composed of a plurality of non-contiguous amino acids (or amino acid sequences) located within any or both of the aforementioned domains of the protein molecule (e.g., a conformational epitope).

[0121] To determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein, various techniques known to those skilled in the art can be used. Exemplary techniques include, for example, Antibodies, Harlow and L ane (Cold Spring Harbor Press, Cold Spring Included are conventional cross-blocking assays as described in Harbor, NY). Other methods include alanine scanning mutagenesis analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystallographic studies, NMR analysis, etc. Further, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally, in the hydrogen / deuterium exchange method, it involves labeling the protein of interest with deuterium and then binding an antibody to the deuterium-labeled protein. Next, when the protein / antibody complex is transferred into water, the exchangeable protons within the amino acids protected by the antibody complex will undergo deuterium-hydrogen back-exchange at a slower rate than the exchangeable protons within the amino acids that are not at the interface. As a result, the amino acids forming the interface between the protein and the antibody will retain deuterium and thus exhibit a relatively higher mass compared to the amino acids not included in the interface. After dissociation of the antibody, the target protein is cleaved with protease and mass spectrometry is performed, revealing the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0122] The term "epitope" refers to the site on an antigen to which B cells and / or T cells react. B cell epitopes can be formed from both contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from contiguous amino acids are retained even when exposed to a denaturing solvent, whereas epitopes formed by tertiary folding are typically lost when treated with a denaturing solvent. Epitopes usually consist of at least 3, more commonly at least 5 or 8 - 10 amino acids forming a unique spatial conformation.

[0123] Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP), is a method of classifying multiple monoclonal antibodies (mAbs) directed against the same antigen according to the similarity of the binding profiles of each antibody to a chemically or enzymatically modified antigen surface (see US2004 / 0101920, which is specifically incorporated herein by reference in its entirety). Each category may reflect unique epitopes that are clearly distinct from or partially overlapping with epitopes represented by other categories. This technique allows for the rapid filtering of genetically identical antibodies and enables the characterization of genetically distinct antibodies. Applying MAP to the screening of hybridomas can facilitate the identification of rare hybridoma clones that produce mAbs with desired properties. MAP can be used to classify the antibodies of the present invention into groups of antibodies that bind different epitopes.

[0124] In certain embodiments, the invention includes anti-NPR1 antibodies and antigen-binding fragments thereof that interact with one or more epitopes found within the extracellular domain of NPR1. The epitope may be composed 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, the epitope may be composed of multiple non-contiguous amino acids (or amino acid sequences) located within NPR1.

[0125] The invention includes anti-NPR1 antibodies that bind to the same epitope, or a portion of the epitope, as any of the specific exemplary antibodies listed in Table 1. Similarly, the invention includes anti-NPR1 antibodies that compete with the binding of any of the specific exemplary antibodies listed in Table 1 to the NPR1 protein or fragments thereof. For example, the invention includes anti-NPR1 antibodies that cross-compete the binding to the NPR1 protein with one or more of the antibodies listed in Table 1.

[0126] Whether an antibody binds to the same epitope as, or competes with, a reference anti-NPR1 antibody can be readily determined using conventional methods known in the art. For example, to determine whether a test antibody binds to the same epitope as the reference anti-NPR1 antibody of the invention, the reference antibody is bound to the NPR1 protein or peptide in a saturated state. Next, the ability of the test antibody to bind to the NPR1 protein molecule is evaluated. If the test antibody can bind to NPR1 after saturated binding with the reference anti-NPR1 antibody, it can be concluded that the test antibody binds to an epitope different from that of the reference anti-NPR1 antibody. On the other hand, if the test antibody cannot bind to the NPR1 protein after saturated binding with the reference anti-NPR1 antibody, the test antibody may bind to the same epitope as the epitope to which the reference anti-NPR1 antibody of the invention binds.

[0127] To determine whether an antibody competes with the binding of a reference anti-NPR1 antibody, the binding method described above is carried out in two directions. In the first direction, after binding the reference antibody to the NPR1 protein in a saturated state, the binding of the test antibody to the NPR1 molecule is evaluated. In the second direction, after binding the test antibody to the NPR1 molecule in a saturated state, the binding of the reference antibody to the NPR1 molecule is evaluated. In both directions, if only the first (saturated) antibody can bind to the NPR1 molecule, 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 the binding of a reference antibody does not necessarily bind to the same epitope as the reference antibody, and can sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0128] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antibody to the antigen. That is, an excess of one antibody at 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold, when measured in a competitive binding assay, inhibits the binding of the other by at least 50%, preferably 75%, 90%, or even 99% (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if an amino acid mutation in the antigen that reduces or eliminates the binding of one antibody also reduces or eliminates the binding of the other antibody, the two antibodies have the same epitope. If several amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, the two antibodies have overlapping epitopes.

[0129] Next, additional routine experiments (such as peptide mutagenesis and binding analysis) can be performed to confirm whether the lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether the lack of binding is due to steric hindrance (or another phenomenon). This type of experiment can be carried out using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art.

[0130] In certain embodiments, the invention is 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 comprises one contained within the extracellular domain of NPR1 (amino acids 29-347 of SEQ ID NO: 194) as determined by hydrogen / deuterium exchange interacts with one or more amino acids, and the antibody or its antigen-binding fragment binds to cells expressing human NPR1 in the presence or absence of atrial natriuretic peptide (ANP); and / or (ii) binds to NPR1 and activates NPR1, to provide an isolated antibody or its antigen-binding fragment. In one embodiment, the antibody or its antigen-binding fragment interacts with an amino acid sequence selected from the group consisting of: (a) amino acids 29-45 of SEQ ID NO: 194; (b) amino acids 331-347 of SEQ ID NO: 194; (c) amino acids 336-347 of SEQ ID NO: 194; (d) amino acids 331-335 of SEQ ID NO: 194; and (e) amino acids 70-81 of SEQ ID NO: 194. In one embodiment, the antibody or its antigen-binding fragment interacts with an amino acid sequence selected from the group consisting of: (a) amino acids 29-45 of SEQ ID NO: 194; and (b) amino acids 336-347 of SEQ ID NO: 194. In one embodiment, the antibody or its antigen-binding fragment interacts with an amino acid sequence selected from the group consisting of: (a) amino acids 29-45 of SEQ ID NO: 194; and (b) amino acids 331-347 of SEQ ID NO: 194 in the presence of ANP. In one embodiment, the antibody or its antigen-binding fragment interacts with an amino acid sequence selected from the group consisting of: (a) amino acids 29-45 of SEQ ID NO: 194; (b) amino acids 70-81 of SEQ ID NO: 194; and (c) amino acids 331-335 of SEQ ID NO: 194 in the presence of ANP. In one embodiment, the present invention provides an isolated antibody or its antigen-binding fragment that specifically binds to the NPR1 protein in the presence of ANP, wherein the antibody or its antigen-binding fragment interacts with an amino acid sequence selected from the group consisting of: (a) amino acids 29-45 of SEQ ID NO: 194; and (b) amino acids 331-347 of SEQ ID NO: 194 as determined by hydrogen / deuterium exchange, but does not interact with amino acids 70-81 of SEQ ID NO: 194, and the antibody or its antigen-binding fragment (i) binds to cells expressing human NPR1 in the presence or absence of ANP; and / or (ii) binds to NPR1 and activates NPR1; to provide an isolated antibody or its antigen-binding fragment.

[0131] Immune complex The present invention encompasses a human anti-NPR1 monoclonal antibody (an "immune complex") conjugated to a therapeutic moiety for treating NPR1-related diseases or disorders (e.g., hypertension). As used herein, the term "immune complex" refers to an antibody that is chemically or biologically conjugated to a radiopharmaceutical, cytokine, interferon, target or reporter site, enzyme, peptide or protein, or therapeutic agent. The antibody can be conjugated at any position along the molecule with a radiopharmaceutical, cytokine, interferon, target or reporter site, enzyme, peptide, therapeutic agent, etc., as long as it can bind to the target. Examples of immune complexes include antibody-drug conjugates and antibody-toxin fusion proteins. In certain embodiments, the agent may be a second, different antibody to the NPR1 protein. The type of therapeutic site that can be conjugated to the anti-NPR1 antibody will depend on the condition to be treated and the desired therapeutic effect to be achieved. Examples of agents suitable for forming immune complexes are known in the art; see, for example, WO05 / 103081.

[0132] Multispecific antibody The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for 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.

[0133] Any of the multispecific antigen-binding molecules of the present invention, or variants thereof, can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques ) known to those of skill in the art.

[0134] In some embodiments, the NPR1-specific antibodies are generated in a bispecific format (“bispecific”) in which variable regions that bind to different domains of the NPR1 protein are linked together to confer dual domain specificity within a single binding molecule. Appropriately designed bispecificity can enhance the inhibitory effect on the overall NPR1 protein by enhancing both specificity and binding affinity. Variable regions that have specificity for individual domains (e.g., segments of the N-terminal domain) or can bind to different regions within one domain are paired on a structural scaffold that allows each region to simultaneously bind to separate epitopes or to different regions within one domain. In one example of bispecificity, the heavy chain variable region (V H ) of a binder having specificity for one domain is recombined with the light chain variable region (V L ) of a series of binders having specificity for a second domain to identify a non-cogate V H partner that can pair with V H without sacrificing the original specificity of V L . In this way, a single V L segment (e.g., V L 1) is combined with two different V H domains (e.g., V H 1 and V H 2) to generate bispecificity that includes two binding “arms” (V H 1-V L 1 and V H 2-V L 1). Using a single V L segment reduces the complexity of the system, thereby simplifying the cloning, expression, and purification processes used to generate bispecificity and improving efficiency (see, e.g., US2011 / 0195454 and US2010 / 0331527).

[0135] Alternatively, antibodies that bind more than one domain and a second target, e.g., but not limited to, a second different anti-NPR1 antibody, may be prepared in a bispecific format using the techniques described herein or other techniques known to those of skill in the art. Antibody variable regions that bind to different regions may be linked together, e.g., with a variable region that binds to a relevant site of the extracellular domain of NPR1, to confer bispecific antigenicity within a single binding molecule. Appropriately designed bispecificity of this nature serves a dual function. The variable region having specificity for the extracellular domain is paired with a variable region having specificity for outside the extracellular domain on a structural backbone where each variable region can bind to a separate antigen.

[0136] Exemplary bispecific antibody formats that can be used in the context of the present invention include a first immunoglobulin (Ig) C H 3 domain and a second Ig C H 3 domain, wherein the first and second Ig C H 3 domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces the binding of the bispecific antibody to protein A as compared to a bispecific antibody having no amino acid differences. In one embodiment, the first Ig C H 3 domain binds protein A, and the second Ig C H 3 domain contains a mutation that reduces or abolishes protein A binding, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering). The second C H 3 may further include the Y96F modification (by IMGT; Y436F by EU). The second C HAdditional modifications that may be found within 3 include, for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); for IgG2 antibodies, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I according to EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU). Variations of the bispecific antibody formats described above are contemplated within the scope of the present invention.

[0137] Other exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or bispecific formats of bispecific antibodies, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2 Bispecific formats (for a review of the formats described above, see, for example, Klein et al., 2012, mAbs 4:6, 1-11, and the references cited therein). Also, bispecific antibodies can be constructed using peptide / nucleic acid conjugates, for example, using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates, which can then self-organize into multimeric complexes having a predetermined composition, valency, and shape (see, for example, Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0138] Therapeutic Administration and Formulations The present invention provides a therapeutic composition comprising the anti-NPR1 antibody of the present invention or an antigen-binding fragment thereof. The therapeutic composition according to the present invention is administered together with a suitable carrier, excipient, and other agents incorporated into the formulation to provide improved delivery, delivery, resistance, etc. A number of suitable formulations can be found in the formularies known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) - containing vesicles (e.g., LIPOFECTIN (trademark)), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and carbowax-containing semi-solid mixtures. Also refer to "Compendium of excipients for parenteral formulations" by Powell et al., PDA (1998) J Pharm Sci Technol 52:238 - 311.

[0139] The dosage of the antibody may vary depending on the age and physical build of the subject to be administered, the target disease, condition, route of administration, and the like. When the antibody of the present invention is used for the treatment of diseases or disorders in adult patients, or for the prevention of such diseases, it is usually advantageous to administer the antibody of the present invention as a single dose of about 0.1 to about 100 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In certain embodiments, the antibody of the present invention or its antigen-binding fragment can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 500 mg, or about 10 to about 400 mg. In certain embodiments, following the initial dose, subsequent doses of the antibody or its antigen-binding fragment can be administered in an amount substantially the same as or less than that of the initial dose, and 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.

[0140] Various delivery systems are known, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-dependent endocytosis, and can be used to administer the pharmaceutical composition of the present invention (see, for example, Wu et al. (1987) J. Biol. Chem. 262: 4429-4432). Introduction Routes of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or skin mucosa (e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can also be delivered in vesicles, particularly liposomes (see, for example, Langer (1990) Science 249: 1527-1533).

[0141] Also contemplated herein is the use of nanoparticles for delivering the antibodies of the present invention. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles, as well as methods of preparation and use, are described in detail in Arruebo, M. et al., 2009 (“Antibody-conjugated nanoparticles for biomedical applications” J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi:10.1155 / 2009 / 439389), which is incorporated herein by reference. Nanoparticles can be developed and conjugated to the antibodies included in the pharmaceutical composition for target cells. Also, nanoparticles for drug delivery are described, for example, in US8257740, or US8246995, each of which is incorporated herein by reference in its entirety.

[0142] In certain circumstances, the pharmaceutical composition can be delivered with a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled release system can be placed in proximity to the target of the composition, such that only a small fraction of the systemic dose is required.

[0143] Injectable formulations may include dosage forms for intravenous, subcutaneous, intracranial, intraperitoneal and intramuscular injection, infusion, etc. These injectable formulations may be prepared by known methods.

[0144] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. Further, with respect to subcutaneous delivery, pen-type delivery devices are easily applicable for the delivery of the pharmaceutical composition of the present invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. When all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. Thereafter, the pen-type delivery device can be reused. In disposable pen-type delivery devices, there are no replaceable cartridges. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the reservoir is empty, the entire device is discarded.

[0145] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared in a unit dosage form suitable for adapting to the dosage of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like. The amount of the antibody contained is generally about 5 to about 500 mg per unit dosage form, preferably about 5 to about 300 mg in the form of injections and about 10 to about 300 mg in other dosage forms.

[0146] Therapeutic Use of Antibodies The antibodies of the present invention are useful for the treatment, and / or prevention, of a disease or disorder or condition associated with NPR1, and / or for the amelioration of at least one symptom associated with such a disease, disorder or condition. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention may be administered at a therapeutic dose to a patient having a disease or disorder or condition associated with NPR1.

[0147] In certain embodiments, the antibodies of the invention are useful for treating or preventing at least one symptom or sign of an NPR1-related disease or disorder selected from the group consisting of hypertension, heart failure, obesity, renal failure, chronic kidney disease, macular edema, glaucoma, stroke, lung disorders, pulmonary fibrosis, inflammation, asthma, skeletal growth disorders, fractures, diabetes, and cancer.

[0148] Also contemplated herein is the prophylactic use of one or more antibodies of the invention in a subject at risk of developing an NPR1-related disease or disorder.

[0149] In one embodiment of the invention, the antibodies of the invention are used in the preparation of a pharmaceutical composition or agent for treating a patient suffering from a disease, disorder or condition disclosed herein. In another embodiment of the invention, the antibodies are used as adjuvant therapy with any other agent or any other therapy known to those skilled in the art useful for treating or ameliorating a disease, disorder or condition disclosed herein.

[0150] Combination therapy Combination therapy may include the antibody of the invention and any additional therapeutic agent that can be advantageously combined with the antibody of the invention or a biologically active fragment of the antibody of the invention. The antibodies of the invention may be used in a synergistic combination with one or more drugs or therapies used to treat NPR1-related diseases or disorders. In some embodiments, the antibodies of the invention may be combined with a second therapeutic agent to improve one or more symptoms of a disease or condition.

[0151] Depending on the disease, disorder or condition, the antibodies of the invention can be used in combination with one or more additional therapeutic agents including, but not limited to: aldosterone antagonists (e.g., eplerenone, spironolactone), alpha - adrenergic blockers (e.g., doxazosin, phenoxybenzamine, phentolamine, prazosin, terazosin), angiotensin - converting enzyme (ACE) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril), arterial vasodilators (e.g., hydralazine, minoxidil), ganglionic vasodilators (e.g., mecamylamine), beta - adrenergic blockers (acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, carteolol, esmolol, labetalol, metoprolol, nadolol, penbutolol, pindolol, propranolol, timolol), catecholamine - depleting sympathetic blockers (e.g., debrisoquine, reserpine), central alpha - 2 adrenergic agonists (e.g., clonidine, guanabenz, guanfacine, methyldopa), calcium antagonists (diltiazem, verapamil, amlodipine, felodipine, isradipine, nicardipine, nifedipine, nisoldipine), diuretics (e.g., bumetanide, ethacrynic acid, furosemide, torsemide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide, chlorthalidone, indapamide, metolazone), renin inhibitors (e.g., aliskiren), anticoagulants (e.g., coumadin, dabigatran, apixaban), anti - platelet agents (e.g., aspirin, clopidogrel), cholesterol - lowering agents (e.g., statins, PCSK9 inhibitors such as alirocumab), vasodilators (e.g., minoxidil, hydralazine, nitrates), digitalis, surgery (e.g., angioplasty, coronary artery bypass, heart transplant), implantable devices (e.g., valve replacement, defibrillator, left ventricular assist device, pacemaker), anti - tumor therapies (e.g., chemotherapeutic agents, surgery, radiation, PD - 1 inhibitors), insulin, GLP1 agonists (e.g., exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide), meth Formin, dialysis, bone marrow stimulants, hemofiltration, improvement of lifestyle habits, and dietary supplements, etc.

[0152] As used herein, the term "in combination with" means that an additional therapeutically active ingredient may be administered before, simultaneously with, or after the administration of the anti-NPR1 antibody of the present invention. Further, the term "in combination with" includes sequential or simultaneous administration of the anti-NPR1 antibody and the second therapeutic agent.

[0153] The additional therapeutic active ingredient may be administered to the subject before administration of the anti-NPR1 antibody of the present invention. For example, if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, or less than 30 minutes before the administration of the second component, the first component may be considered to be administered "before" the second component. In other embodiments, an additional therapeutic active ingredient may be administered to the subject after administration of the anti-NPR1 antibody of the present invention. For example, if the first component is administered 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after, or later than the administration of the second component, the first component may be considered to be administered "after" the second component. In still other embodiments, an additional therapeutic active ingredient may be administered to the subject simultaneously with the administration of the anti-NPR1 antibody of the present invention. "Simultaneous" administration for the purposes of the present invention includes, for example, administering the anti-NPR1 antibody and the additional therapeutic active ingredient to the subject in a single dosage form, or in separate dosage forms administered to the subject within about 30 minutes or less of each other. When administered in separate dosage forms, each dosage form may be administered by the same route (e.g., both the anti-NPR1 antibody and the additional therapeutic active ingredient may be administered intravenously, etc.), or alternatively, each dosage form may be administered by a different route (e.g., the anti-NPR1 antibody may be administered intravenously and the additional therapeutic active ingredient may be administered orally, etc.). In any case, for the purposes of the present disclosure, administering the components in a single dosage form, administering the components in separate dosage forms by the same route, or administering the components in separate dosage forms by different routes are all considered to be "simultaneous administration". For the purposes of the present disclosure, administration of the anti-NPR1 antibody "before", "simultaneously with", or "after" administration of the additional therapeutic active ingredient (as these terms are defined herein) is considered to be administration of the anti-NPR1 antibody "in combination with" the additional therapeutic active ingredient.

[0154] The present invention includes a pharmaceutical composition in which the anti-NPR1 antibody of the present invention is co-formulated with one or more additional therapeutic active ingredients as described elsewhere herein.

[0155] Diagnostic Use of the Antibody The antibodies of the present invention may be used, for example, for diagnostic purposes to detect and / or measure NPR1 in a sample. In some embodiments, it is contemplated to use one or more of the antibodies of the present invention in an assay for detecting a disease or disorder associated with NPR1. Exemplary diagnostic assays for NPR1 include, for example, contacting a sample obtained from a patient with an anti-NPR1 antibody of the present invention, wherein the anti-NPR1 antibody is labeled with a detectable label or reporter molecule or is used as a capture ligand for selectively isolating NPR1 from the patient sample. Alternatively, an unlabeled anti-NPR1 antibody can be used for diagnostic purposes in combination with a secondary antibody labeled with a detectable label per se. Detectable labels or reporter molecules are 3 H, 14 C, 32 P, 35 S, or 125 radioisotopes such as I; fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine; or enzymes 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).

[0156] Samples that can be used in the NPR1 diagnostic assay according to the present invention include any tissue or liquid sample obtained from a patient, and these samples contain a detectable amount of NPR1 protein or a fragment thereof in a normal or pathological state. Generally, the level of NPR1 protein in a specific sample obtained from a healthy patient (e.g., a patient not suffering from a disease related to NPR1) is measured to first establish a baseline or standard level of NPR1. Next, this baseline NPR1 level can be compared with the NPR1 level measured in a sample obtained from a person suspected of having a condition related to NPR1 or symptoms associated with such a condition.

[0157] Antibodies specific for the NPR1 protein may not contain additional labels or sites, or may contain N-terminal or C-terminal labels or sites. In one embodiment, the label or site is biotin. In a binding assay, the orientation of the peptide with respect to the surface to which the peptide is bound may be determined by the position of the label (if any). For example, when the surface is coated with avidin, a peptide containing biotin at the N-terminus is oriented such that the C-terminal portion of the peptide is distal to the surface.

[0158] Examples The following examples are presented to provide those skilled in the art with a complete disclosure and description of the methods and compositions of the present invention and how to make and use them, and are not intended to limit the scope that the inventors regard as their invention. Although efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, room temperature is about 25°C, and pressures are at or near atmospheric pressure.

[0159] Example 1: Production of Human Antibodies Against Natriuretic Peptide Receptor 1 (NPR1) Human antibodies against the NPR1 protein were produced in VELOCIMMUNE™ mice that contain DNA encoding human immunoglobulin heavy and kappa light chain variable regions. The mice 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.

[0160] The antibody immune response was monitored by NPR1-specific immunoassays. When the desired immune response was obtained, spleen cells were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. These hybridoma cell lines were screened to select cell lines that produce NPR1-specific antibodies. Using these cell lines, several anti-NPR1 chimeric antibodies (i.e., antibodies having human variable domains and mouse constant domains) were obtained.

[0161] Alternatively, anti-NPR1 antibodies were isolated directly from antigen-positive mouse B cells without fusion to myeloma cells, as described in U.S. Patent No. 7,582,298, which is hereby incorporated by reference in its entirety. Using this method, several fully human anti-NPR1 antibodies (i.e., antibodies having human variable domains and human constant domains) were obtained.

[0162] Exemplary antibodies produced as disclosed above were designated as mAb22033, mAb22035, mAb22805, mAb22809, mAb22810, mAb25479, mAb25491, mAb25497, mAb25498, mAb25502, mAb25508, and mAb25545.

[0163] The biological properties of exemplary antibodies produced according to the method of this example are described in detail in the examples shown below.

[0164] Example 2: Amino Acid and Nucleotide Sequences of Heavy and Light Chain Variable Regions Table 1 shows the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-NPR1 antibodies of the present invention.

[0165] [Table 1]

[0166] The identifiers of the corresponding nucleic acid sequences are shown in Table 2.

[0167] [Table 2]

[0168] The antibodies referred to herein typically have fully human variable regions, but may have human or mouse constant regions. As will be understood by those skilled in the art, antibodies having a particular Fc isotype can be converted to antibodies having a different Fc isotype (e.g., an antibody having a mouse IgG1 Fc can be converted to an antibody having a human IgG4, etc.). In any case, the variable domains (including CDRs) indicated by the numerical identifiers shown in Table 2 remain the same, and the binding properties to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain. In certain embodiments, the selected antibodies having a mouse IgG1 Fc are converted to antibodies having a human IgG4 Fc. In one embodiment, the IgG4 Fc domain contains two or more amino acid changes as disclosed in U.S. Patent No. 20100331527. In one embodiment, the human IgG4 Fc contains a mutation from serine to proline in the hinge region (S108P) to promote dimer stabilization. Unless otherwise indicated, all antibodies used in the following examples include the human IgG4 isotype.

[0169] The control constructs used in the following examples The experiments disclosed in this specification include, for comparison purposes, the following control constructs (anti-NPR1 antibodies): "Comparator 1", a monoclonal antibody (Morphosys) against human NPR1 having the VH / VL sequences of the antibody "mAb5591" according to U.S. Patent No. 20120114659.

[0170] Example 3: Binding of Antibodies to NPR1 by Surface Plasmon Resonance Experimental Procedure Equilibrium dissociation constants (K of different NPR1 reagents binding to purified anti-NPR1 monoclonal antibodies (mAbs) D) was measured using a real-time surface plasmon resonance-based Biacore 4000 biosensor. All binding tests were performed at 25 °C and 37 °C using a running buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v / v Surfactant Tween-20, pH 7.4 (HBS-ET). The Biacore CM5 sensor chip surface was first derivatized by amine coupling with either mouse anti-human Fc-specific mAb (GE Healthcare, #BR100839) or goat anti-human Fcγ-specific polyclonal antibody (Jackson ImmunoResearch Laboratories, #BR-1008-39) to capture the anti-NPR1 mAb. Binding tests were performed with human NPR1 extracellular domain (hNPR1-MMH) (SEQ ID NO: 194) expressed with a C-terminal myc-myc-hexahistidine, simian NPR1 extracellular domain (mfNPR1-MMH) (SEQ ID NO: 195) expressed with a C-terminal myc-myc-hexahistidine, mouse NPR1 extracellular domain (mNPR1-MMH) (SEQ ID NO: 196) expressed with a C-terminal myc-myc-hexahistidine, human NPR1 extracellular domain (hNPR1-mFc) (SEQ ID NO: 197) expressed with a C-terminal mouse IgG2a, simian NPR1 extracellular domain (mfNPR1-mFc) (SEQ ID NO: 198) expressed with a C-terminal mouse IgG2a, mouse NPR1 extracellular domain (mNPR1-mFc) (SEQ ID NO: 199) expressed with a C-terminal mouse IgG2a, hNPR1-mFc + hANP, hNPR1-mFc + hBNP, mfNPR1-mFc + hANP, mfNPR1-mFc + hBNP, mNPR1-mFc + mANP, mNPR1-mFc + mBNP.hNPR1-MMH, mfNPR1-MMH at different concentrations (100 nM to 3.7 nM, 3-fold serial dilution or 100 nM to 6.25 nM, 4-fold serial dilution); hNPR1-mFc, mfNPR1.mFc (100 nM to 1.56 nM, 4-fold serial dilution, or 100 nM to 3.7 nM, 3-fold serial dilution); mNPR1.mmh (100 nM), hNPR1-mFc or mfNPR1-mFc (100 nM, 25 nM, 6.25 nM or 100 nM to 3.7 nM, 3-fold serial dilution) complexed with 10-fold concentration of hANP or hBNP; complexed with 10-fold concentration of mANP or mBNP prepared in HBS-ET running buffer. Inject mNPR1.mFc (100 nM, 25 nM, 100 nM - 3.7 nM, 3-fold serial dilution) or hNPR1-hFc or hNPR1-hFc (100 nM to 6.25 nM, 4-fold serial dilution) complexed with 10-fold concentration of hANP at a flow rate of 30 μL / min for 4 minutes, while monitoring the dissociation of mAb conjugated with different NPR1 reagents in HBS-ET running buffer for 10 minutes. At the end of each cycle, inject 20 mM phosphoric acid for 10 seconds in the case of mouse anti-human Fc specific mAb surface, or inject 10 mM glycine, HCl, pH 1.5 for 40 seconds in the case of goat anti-human Fcγ specific polyclonal antibody, or inject 10 mM glycine pH 1.5 for 1 minute twice to regenerate the NPR1 mAb capture surface. Association rate (k a ) and dissociation rate (k d ) were determined by fitting the real-time binding sensorgram to a mass transport-limited 1:1 binding model using Scrubber 2.0c curve fitting software. Binding dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rates as follows.

[0171]

Equation

[0172] Results Table 3 to 26 show the binding kinetic parameters of different NPR1 proteins that bind to the selected anti-NPR1 antibodies of the present invention at 25°C and 37°C.

[0173]

Table 3

[0174]

Table 4

[0175]

Table 5

[0176]

Table 6

[0177]

Table 7

[0178]

Table 8

[0179]

Table 9

[0180]

Table 10

[0181]

Table 11

[0182]

Table 12

[0183]

Table 13

[0184]

Table 14

[0185]

Table 15

[0186]

Table 16

[0187]

Table 17

[0188]

Table 18

[0189]

Table 19

[0190]

Table 20

[0191]

Table 21

[0192]

Table 22

[0193]

Table 23

[0194]

Table 24

[0195]

Table 25

[0196]

Table 26

[0197] As shown in Tables 3 - 6, the selected anti - NPR1 antibodies bound to monomeric human and monkey NPR1.

[0198] As shown in Tables 9 - 20, the antibodies bound to human and monkey NPR1 dimers both in the presence and absence of ANP or BNP.

[0199] With the exception that mAb25502 bound to mNPR1 in the presence of ANP / BNP, the antibodies did not bind to mouse NPR1 (Tables 21 - 26).

[0200] Example 4: Cross - competition among selected anti - NPR1 agonist monoclonal antibodies Experimental procedure Binding competition within a panel of anti-NPR1 monoclonal antibodies was determined using a real-time, label-free biolayer interferometry assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25 °C with the plate shaken at 1000 rpm using 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v / v Surfactant Tween-20, 1 mg / mL, BSA, pH 7.4 (HBS-EBT) buffer. To evaluate whether two antibodies compete with each other to bind to their respective epitopes, 100 nM recombinant human NPR1 (hNPR1-mFc; SEQ ID NO: 453) expressing C-terminal mouse IgG2a was first incubated with 2 μM human ANP for at least 2 hours. First, an Octet biosensor tip (Fortebio Inc, #18-5090) coated with anti-mouse Fc antibody was immersed in a well containing the hNPR1-mFc / hANP complex for 45 seconds to capture approximately 0.3 - 0.5 nm of the recombinant hNPR1-mFc / hANP complex. Next, the biosensor tip capturing the antigen was saturated with the first anti-NPR1 monoclonal antibody (referred to as mAb-1) by immersing it in a well containing a 50 μg / mL solution of mAb-1 for 5 minutes. Next, the biosensor tip was immersed in a well containing a 50 μg / mL solution of the second anti-NPR1 monoclonal antibody (called mAb-2) for 3 minutes. The biosensor tip was washed with HBS-EBT buffer between each step of the experiment. The binding response in real time was monitored throughout the experiment, and the binding response at the end of each step was recorded. The response of mAb-2 binding to pre-complexed hNPR1-mFc / hANP formed with mAb-1 was compared to determine the competitive / non-competitive behavior of different anti-NPR1 monoclonal antibodies.

[0201] Results

Table 27-1

Table 27-2

[0202] Table 27 shows the cross-competition among the selected anti-NPR1 antibodies.

[0203] Example 5: Binding of Antibodies to Cells Expressing NPR1 Experimental Procedure Using or without using one of the ligands, human ANP (hANP), the ability of anti-human (h) NPR1 monoclonal antibodies to bind to cells expressing human NPR1 (hNPR1) was determined using detection based on electrochemiluminescence (ECL).

[0204] Briefly, HEK293 / hNPR1.myc.DKK-expressing cells were designed by transfecting human embryonic kidney (HEK) 293 cells with the neomycin-resistant pLVX.hNPR1.myc.DDK expression plasmid encoding human NPR1 (amino acids M1-G1061, UniProtKB-P16066). Non-transfected HEK293 cells, in which the expression of NPR1 was not detected by fluorescence-activated cell sorting (FACS) using a commercially available a-hNRP1 antibody, were included as a non-specific binding control.

[0205] The experiment was performed according to the following procedure. The cells of the above-mentioned strain were washed once with 1xPBS buffer without Ca 2+ / Mg 2+ , incubated at 37 °C for 10 minutes using an enzyme-free cell dissociation solution to detach the cells from the flask. All cells were washed once with 1xPBS containing Ca 2+ / Mg 2+ and counted using a Cellometer (trademark) Auto T4 cell counter (Nexcelom Bioscience). Approximately 2.0x10 4Individual HEK293 / hNPR1.myc.DDK or HEK293 cells were seeded separately into 96-well carbon electrode plates (MULTI-ARRAY high bind plate, Meso Scale Discovery (MSD, Rockville, Maryland)) and incubated at 37 °C for 1 hour (h). Nonspecific binding sites were blocked with 2% BSA (w / v) in 1xPBS containing Ca 2+ / Mg 2+ for 1 hour at room temperature (RT). HEK293 / hNPR1.myc.DDK cells were incubated for 0.5 hour at room temperature in sample dilution buffer with or without 10 nM human ANP (Tocris, Minneapolis, Minnesota), and HEK293 cells were incubated under the same conditions with sample dilution buffer only. Without washing, serial dilutions of anti-NPR1, COMP1, or isotype control antibodies in the range of 1.7 pM to 100 nM, or buffer without antibody, were added to the cells bound to the plate for 1 hour at room temperature. The plate was then washed, and unbound antibody and / or hANP were removed using an AquaMax2000 plate washer (MDS Analytical Technologies, Sunnyvale, California) equipped with a cell washing head. Antibodies bound to the plate were detected for 1 hour at room temperature using a SULFO-TAG™ conjugate goat polyclonal anti-human IgG antibody (Jackson Immunoresearch, West Grove, Pennsylvania) specific for the heavy and light chains.

[0206] After washing, the plates were developed in a Read Buffer (MSD, Rockville, MD) according to the manufacturer's recommended procedure, and the luminescence signals were recorded with a SECTOR Imager 600 (MSD, Rockville, MD). The luminescence intensity measured in relative light units (RLU) was recorded to show the binding strength in the concentration range of each antibody. The ratio of the signal detected with a 3.7 nM antibody that binds to NPR1 gene-modified cells with or without 10 nM hANP to the same concentration of antibody that binds to parental cells without hANP was reported as an indicator of the specificity of NPR1 binding. Antibodies with a binding ratio of 3 or more were considered specific binders, and antibodies with a binding ratio of less than 3 were considered non-binders.

[0207] Furthermore, the direct binding signal (RLU) was analyzed as a function of antibody concentration, and the data were fitted with a sigmoid (4-parameter logistic) dose-response model using the R statistical package (open source). EC 50 values were defined as the antibody concentration at which 50% of the maximum binding signal was detected and were determined to indicate the binding affinity for NPR1 gene-modified cells with or without 10 nM hANP. EC 50 values were reported only for specific binders, and antibodies with a ratio below 3 in Table 28 were marked with (-).

[0208] Results Table 28 shows the binding of selected anti-NPR1 antibodies to cells designed to express human NPR1 or the NPR1-ANP complex.

[0209]

Table 28

[0210] As shown by the results in Table 28, all anti-NPR1 antibodies specifically bound to hNPR1-modified cells with a ratio ≥ 2 in the presence of 10 nM hANP. The potencies of these antibodies in HEK293 / hNPR1.myc.DDK cells in the presence of 10 nM hANP were EC 50The value was in the range of 0.15 nM to 3.7 nM. Five antibodies and Comparator 1 specifically bound to NPR1-modified cells only in the presence of 10 nM hANP and were classified as peptide-dependent NPR1 binders. The other seven antibodies bound to hNPR1-modified cells with and without 10 nM hANP, and these antibodies were classified as peptide-independent NPR1 binders. The potencies of these antibodies in HEK293 / hNPR1.myc.DDK cells without added hANP were EC 50 The value was in the range of 0.49 nM to 4.6 nM.

[0211] Example 6: Activation of NPR1 by Agonist Anti-NPR1 Monoclonal Antibodies Experimental Procedure To evaluate the transcriptional activation of the human natriuretic peptide receptor 1 (hNPR1), a stable cell line was created by stably expressing cyclic nucleotide-gated channel alpha 2 (CNGA2) and hNPR1 in HEK293. Since CNGA2 is a calcium channel activated by cGMP, it can be used as a sensor for cGMP generation (Wunder et al., 2005, PMID: 157667 16). When NPR1 is activated by a ligand and cGMP is generated (Zois et al., 2014, PMID: 24820868), CNGA2 is activated and calcium influx can be measured using a fluorescent Ca ++ indicator. Cell lines highly expressing hNPR1, HEK293 / hNPR1.MycDDK / CNGA2.Myc HS, or the abbreviation HEK293 / CNGA2 / hNPR1 were selected and maintained in DMEM containing 10% FBS, NEAA, pen / strep / glut, 100 μg / mL hygromycin, and 500 μg / mL G418 sulfate.

[0212] A bioassay was performed to measure the effect of anti-hNPR1 antibodies on human NPR1 signaling in the absence of ANP. For the bioassay, HEK293 / CNGA2 / hNPR1 cells were seeded at 30,000 cells / well in a black clear-bottom PDL plate in DMEM containing 10% FBS, NEAA, pen / strep / glut and incubated overnight at 37 °C and 5% CO2. The next morning, the medium was removed and the cells were loaded with 80 μl of FLUO4-NW assay buffer (Thermo Scientific) containing probenecid for 30 minutes at 37 °C. Purified hNPR1 antibody or isotype control antibody was serially diluted 1:3 to approximately 0.4 nM - 1 μM (8 points), or ANP was serially diluted 1:3 to approximately 0.3 pM - 2 nM (10 points) and transferred to the assay plate using FLIPR TETRA® (Molecular Devices). Baseline and reaction images were taken. The dose-response curve was determined based on maximum-minimum or area under the curve (shown here), and the results were analyzed using non-linear regression (4-parameter logistic) of Prism® 6 software (GraphPad) to obtain the EC 50 values. The % activation of the antibody was calculated as the maximum range of RFU achieved by the antibody that exceeded the maximum range of RFU achieved by ANP.

[0213] Results Table 29 shows the activation of human NPR1 by anti-NPR1 antibodies.

[0214]

Table 29

[0215] Activation of hNPR1 by selected anti-NPR1 antibodies of the present invention was tested by measuring the calcium flux activity of HEK293 / CNGA2 / hNPR1 in two experiments (Experiment I and II) (Table 29). As shown in Table 29 (Experiment I), 11 purified NPR1 antibodies showed activation of Ca2+ flux, with maximum activation in the range of 55 - 130%, and EC 50was in the range of 83.7 - 383.9 nM. Nineteen antibodies showed weak activation with a maximum activation of less than 31% (data not shown). In Experiment II, nine anti-NPR1 antibodies showed a maximum activation of 57 - 129% at an EC 50 of 41.6 - >200 nM (Table 29). ANP had an EC 50 of 60.5 - 98.8 pM. No activation was seen with the control hIgG4 isotype antibody.

[0216] Example 7: Effect of a single administration of an agonist anti-NPR1 monoclonal antibody on systemic blood pressure in normotensive NPR1 hu / hu mice Experimental procedure The purpose of this study was to evaluate the effect of selected NPR1 agonist antibodies on baseline systemic blood pressure in hu / hu remotely measured normotensive NPR1 mice.

[0217] Male NPR1 hu / hu (n = 50) mice were implanted with a PA-C10 telemetry device (DSI, St. Paul, MN) and allowed to recover for 7 days before being assigned to groups (Groups 1 - 1 2) (Table 30).

[0218] [Table 30]

[0219] 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 (Research Diets Standard pellet chow) and water were provided ad libitum.

[0220] The test protein or phosphate-buffered saline (PBS) was administered as a single subcutaneous injection to the appropriate animals on Day 0. The dose volume for each animal was determined based on the most recent body weight measurement.

[0221] During the test period, systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate were collected every 1 minute for 10 seconds. The acute evaluation of efficacy was performed using the data aggregated from day 3 to day 7 of the test. The chronic effects of the NPR1 agonist antibody were evaluated by collecting data for 24 hours every day and averaging over 28 days. All data were presented as mean ± standard error.

[0222] Results In the first in vivo screening of the NPR1 agonist antibody, compared with the PBS-administered control animals from day 3 to day 7 after administration, nine antibodies (mAb22033, mAb25479, mAb25497, mAb22805, mAb25545, mAb22809, mAb25491, mAb25502, and mAb22810) showed a significant decrease in systemic blood pressure, and one antibody (mAb22035) had no effect (Figure 1). The magnitude of blood pressure decrease evaluated by the average change from the baseline of systolic blood pressure from day 3 to day 7 after administration ranged from -3.2 ± 0.2 (mAb25497N) to -11.5 ± 0.8 (mAb22810) mmHg.

[0223] The chronic effects of the NPR1 agonist antibody were evaluated over 28 days (Table 31).

[0224]

Table 31

[0225] Normal blood pressure NPR1 hu / hu In normal blood pressure NPR1 mice, when a single subcutaneous injection was given on day 0, one antibody (mAb22035) showed a significant increase in blood pressure by about 4 - 7 mmHg, and the remaining antibodies showed a decrease in systemic blood pressure by 2 - 11 mmHg. The response of heart rate to the increase and decrease in systemic blood pressure was variable, increasing in some groups and decreasing in others.

[0226] Example 8: Normal blood pressure NPR1 hu / hu Dose - effect of agonist anti - NPR1 monoclonal antibody on systemic blood pressure in mice Experimental procedure The purpose of this study was to evaluate the dose response of the NPR1 agonist antibody mAb22033 on systemic blood pressure in hu / hu normotensive NPR1 mice. Male NPR1 mice (n = 30) approximately 20 weeks of age were implanted with a PA-C10 hu / hu telemetry device (DSI, St. Paul, Minnesota) and allowed to recover for 7 days before being assigned to groups (Groups 1-5) (Table 32).

[0227]

Table 32

[0228] Animals were individually housed 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 (Research Diets Standard pellet chow) and water were provided ad libitum.

[0229] The test protein was administered to the appropriate animals by subcutaneous injection once on Day 0. The dose volume for each animal was based on the most recent body weight measurement. Urine was collected on Day 28 for urine and serum biomarker evaluation, and blood samples were collected on Day 14 of the study and at the end of the study. Echocardiograms were performed on Day 28 prior to diuresis.

[0230] During the study period, systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate were collected for 10 seconds every minute. The illustrated telemetry data were obtained from animals with viable signals during the survival portion of the study.

[0231] Results Blood pressure (Figures 2, 3, and 5) decreased by up to 10 - 15 mmHg for up to 4 weeks after single administration of mAb22033 to normotensive NPR1 mice. Peak blood pressure decreases were similar at all doses, and the duration of the blood pressure effect was approximately 10 days at 1 mg / kg and longer than 28 days at 50 mg / kg. hu / hu

[0232]

Table 33

[0233] The cGMP concentration in urine (Table 34) significantly increased on day 28 in the 50 mg / kg group and was at the same level as the control in all other doses.

[0234]

Table 34

[0235] The relative heart weight (Table 35) was lower in the 50 mg / kg group and showed a decreasing trend in the 1, 5, and 25 mg / kg groups. No significant effects were observed on the chemical properties of body weight (Table 35), absolute heart weight (Table 35), standard serum (ALT, AMYLAS, AST, CHOL, CKNAC, CREA, DHDL, IP, TRIG, TP, UA, UN, MG, NEFA) or urine (ALB, GLUH, CREA, PRO, CA, IP, MG, UN, AMYLAS, UA, NA, K, CL).

[0236]

Table 35

[0237] After administration of mAb22033, cardiac function (Figures 6 and 7A) improved, the end-systolic volume decreased statistically significantly in the 25 mg / kg dose group (Figure 7A), and there was a tendency to increase in both the shortening fraction (Figure 7C) and ejection fraction (Figure 7B), most clearly in the 25 and 50 mg / kg dose groups.

[0238] As an important finding, the NPR1 agonist mAb, mAb22033, significantly decreased systolic blood pressure and mean arterial blood pressure, which lasted up to 28 days. A compensatory increase in heart rate was initially observed in all groups, and there was a persistent and mild increase in the 50 mg / kg mAb22033 dose group compared to the isotype control mAb.

[0239] Example 9: Hypertension NPR1 hu / huEffect of single administration of agonist anti-NPR1 monoclonal antibody on systemic blood pressure in mice Experimental procedure The purpose of this study was to evaluate the effect of single administration of NPR1 agonist antibody (mAb22033 or mAb22810) on systemic blood pressure in angiotensin II-(Ang II)-induced hypertensive NPR1 hu / hu mice. Male NPR1 mice (n = 36) approximately 13 weeks old were implanted with a PA-C10 telemetry device (DSI, St. Paul, Minnesota) and allowed to recover for 7 days before being assigned to groups (groups 1-6) (Table 36). hu / hu (n = 36) mice were implanted with a PA-C10 telemetry device (DSI, St. Paul, Minnesota) and allowed to recover for 7 days before being assigned to groups (groups 1-6) (Table 36).

[0240]

Table 36

[0241] Next, an osmotic minipump (Alzet Micro-Osmotic Pump; Model 1004; Lot 10335-14) was implanted in the animals. The minipump was filled with angiotensin II acetate (Bachem; Lot #1066804), and the average pumping rate was set to 0.11 μL / h to deliver 1.5 mg / kg / day of angiotensin II. The minipump was implanted subcutaneously in the scapular region 3 days before the start of administration. The animals were individually housed under standard conditions (temperature 64°F to 84°F (18°C to 29°C), relative humidity 30% to 70%) and maintained on a 12-hour light / 12-hour dark cycle. Food (Research Diets Standard pellet chow) and water were provided ad libitum.

[0242] The test protein was administered subcutaneously by subcutaneous injection once on day 3 to the appropriate animals. The dose volume for each animal was based on the most recent body weight measurement.

[0243] During the test period, systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate were collected for 10 seconds every minute. Urine was collected on days 14 and 20.

[0244] Results Single administration of the NPR1 agonist mAb (mAb22033 or mAb22810) significantly reduced the systemic blood pressure in angiotensin-II-induced hypertensive NPR1 hu / hu mice when administered (Figures 8, 9, and 11). The mean blood pressure over 23 days (Table 37) was significantly lower in animals administered either mAb22033 or mAb22810 compared to the IgG4 isotype control.

[0245]

Table 37

[0246]

Table 38

[0247]

Table 39

[0248] The heart rate effect (Figure 10) increased significantly acutely and showed a more gradual increase chronically. The mean heart rate over 23 days (Table 37) was significantly higher in all test substance-administered groups compared to the isotype control animals. Urinary cGMP levels tended to be higher in most groups administered either mAb22033 or mAb22810, and in animals with 25 mg / kg mAb22033, urinary cGMP levels increased significantly on days 14 and 20 compared to IgG isotype control animals (Table 38). No effects on body weight, absolute or relative organ weights were observed (Table 39).

[0249] Example 10: NPR1 in Hypertension hu / hu Effect of repeated administration of an agonist anti-NPR1 monoclonal antibody on systemic blood pressure in mice Experimental Procedure The purpose of this study was to determine the effect of a remotely measured angiotensin II-(Ang II)-induced hypertensive NPR1h u / huTo evaluate the effect of repeated administration of the NPR1 agonist antibody mAb22033 on the systemic blood pressure of mice. Male NPR1 mice hu / hu (n = 30) were implanted with a PA-C10 telemetry device (DSI, St. Paul, Minnesota) and allowed to recover for 7 days before being assigned to groups (Groups 1 - 5) (Table 40).

[0250]

Table 40

[0251] Subsequently, an osmotic minipump (Alzet Micro - Osmotic Pump; Model 1004; Lot 10335 - 14) was implanted into the animals. The minipump was filled with angiotensin II acetate (Bachem; Lot #1066804), and the average pumping rate was set to 0.11 μL / hour to deliver 1.5 mg / kg / day of angiotensin II. The minipump was implanted subcutaneously in the scapular region 7 days prior to the start of dosing. The animals were individually housed 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 (Research Diets Standard pellet chow) and water were provided ad libitum.

[0252] Animals were stratified into groups based on systolic blood pressure. The test protein was administered by subcutaneous injection, once on Day 0 (Group 5), or starting on Day 6 for 3 weeks, twice a week (Groups 1 - 4), to the appropriate animals. The dose volume for each animal was based on the most recent body weight measurement.

[0253] During the test period, systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate were measured for 10 seconds every minute.

[0254] Results Single or repeated administration of the NPR1 agonist mAb22033 did not affect angiotensin - I I - induced hypertension in NPR1 hu / huIn mice, systemic blood pressure (Figures 12, 13, and 15) was reduced to levels close to normal blood pressure. Repeated administration of 1, 5, or 25 mg / kg of mAb22033 dose-dependently decreased systolic blood pressure, and after administration twice a week for 3 weeks, the maximum decreases relative to the baseline of each group were 11, 19, and 39 mmHg, respectively. With a single administration of 50 mg / kg, systolic blood pressure decreased by 31 mmHg by day 7 and then gradually returned to higher blood pressure levels. The 21-day mean blood pressure (Table 41) was significantly lower in animals that received either a single or repeated administration of mAb22033 compared to the IgG4 isotype control.

[0255]

Table 41

[0256]

Table 42

[0257]

Table 43

[0258] Analysis of the acute effects of mAb22033 showed a blood pressure decrease of 10 - 20 mmHg within 24 hours of the first administration. The heart rate effect (Figure 14) fluctuated, with higher and lower values observed during the 21-day dosing period. The 21-day mean heart rate (Table 41) was significantly lower in the repeated administration groups of 5 and 25 mg / kg and trended higher in the single administration group of 50 mg / kg. Urinary cGMP levels were significantly increased in the 25 mg / kg repeated administration group and showed a statistically non-significant increasing trend in all other groups compared to IgG4 isotype control mAb-administered animals (Table 43). No effects were observed on body weight, absolute or relative organ weights, standard serum or urine chemistry, or cardiac function (Table 42 and Figures 16 - 17).

[0259] Example 11: Effect of an anti-NPR1 agonist antibody on body weight, metabolic rate, and glucose homeostasis in diet-induced obesity (DIO) mice Experiment 1 In this experiment, the effect of the mAb22810 NPR1 agonist mAb on body weight, metabolic rate, and glucose homeostasis in diet-induced obesity (DIO) mice was tested.

[0260] Thirty male NPR1 hu / hu mice were fed a 60% high-fat diet for 10 weeks and then randomized into 3 groups (n = 10 per group): isotype control (human IgG4) antibody, NPR1 agonist antibody mAb22810, or hFc.FGF21. FGF21 is a molecule that has been shown to improve glucose tolerance, increase energy consumption, and reduce body weight in obese mouse models (Veniant MM, Endocrinology, 2 012, PMID: 22798348). In this study, it was used as a positive control for the endpoint. Treatments were administered by subcutaneous injection (S.C.) either weekly (for control antibody and mAb22810) or twice weekly (for hFc.FGF21) in saline vehicle. Table 44 shows the groups, number of animals, and doses in the study.

[0261]

Table 44

[0262] Individual body weights were recorded before dosing and then twice weekly thereafter. During the second week of the study, mice were placed in metabolic cages to evaluate energy consumption. Oral glucose tolerance was evaluated during the third week of the study, and body composition was measured by EchoMRI after 6 weeks of treatment.

[0263] Figure 18A shows the changes in body weight after administration of mAb22810 NPR1 agonist mAb, hFc.FGF21, or isotype control mAb. Figures 18B and 18C show the total fat mass and total fat-free mass, respectively, measured by EchoMRI 6 weeks after treatment. The key finding is that the hFc.FGF21 molecule brought about a significant decrease in body weight and fat accumulation during the treatment period, whereas the mAb22810 NPR1 agonist antibody did not.

[0264] Figures 19A - C show the changes in VO2 (A), VCO2 (B), or energy consumption (C) after treatment for 1 week with either mAb22810 NPR1 agonist mAb, hFc.FGF21, or isotype control mAb, presented as the average value for each day-night cycle. The key finding is that the hFc.FGF21 molecule brought about a significant increase in VO2, VCO2, and energy consumption during the treatment period, whereas the mAb22810 NPR1 agonist antibody did not.

[0265] Figure 20A shows the changes in glucose tolerance measured by an oral glucose tolerance test (2 g / kg glucose) after treatment for 2 weeks with either mAb22810 NPR1 agonist mAb, hFc.FGF21, or isotype control mAb. Figure 20B shows the blood glucose level after an overnight fast recorded at the start of the test in A. The key finding is that both molecules, mAb22810 and hFc.FGF21, brought about a significant improvement in glucose tolerance after 2 weeks. Furthermore, the improvement in glucose tolerance by mAb22810 was independent of changes in body weight or energy consumption (as shown in Figures 18 and 19).

[0266] Experiment 2 This experiment explains the effect of mAb22033 NPR1 agonist mAb on body weight, metabolic rate, and glucose homeostasis in diet-induced obesity (DIO) mice.

[0267] 30 male NPR1 hu / huAfter feeding mice a 60% high-fat diet for 10 weeks, they were randomized into 3 groups: isotype control (human IgG4) antibody, NPR1 agonist antibody mAb22033, or hFc.FGF21 as a positive control (n = 10 per group). Treatments were administered by subcutaneous injection (S.C.) in physiological saline vehicle either once weekly (for control antibody and mAb22033) or twice weekly (for hFc.FGF21). Table 45 shows the groups, number of animals, and doses in the study.

[0268]

Table 45

[0269] Individual body weights were recorded twice weekly before and after dosing. During the second week of the study, mice were placed in metabolic cages to evaluate energy consumption. Oral glucose tolerance was evaluated during the fourth week of the study, and body composition was measured by EchoMRI after 6 weeks of treatment.

[0270] Figure 21A shows the changes in body weight after administration of mAb22033 NPR1 agonist mAb, hFc.FGF21, or isotype control mAb. Figures 21B and 21C show the total fat mass and total fat-free mass, respectively, measured by EchoMRI after 6 weeks of treatment. The key finding is that the hFc.FGF21 molecule brought about a significant decrease in body weight and fat accumulation over the treatment period, whereas the mAb22033 NPR1 agonist antibody did not.

[0271] Figures 22A-C show the changes in VO2 (A), VCO2 (B), or energy consumption (C) after treatment with mAb22033 NPR1 agonist mAb, hFc.FGF21, or isotype control mAb for 1 week, divided as the mean value for each circadian cycle. The key finding is that the hFc.FGF21 molecule brought about a significant increase in VO2, VCO2, and energy consumption during the treatment period, whereas the mAb22033 NPR1 agonist antibody did not.

[0272] Figure 23A shows the changes in glucose tolerance measured by an oral glucose tolerance test (2 g / kg glucose) after 4 weeks of treatment with either the mAb22033 NPR1 agonist mAb, hFc.FGF21, or an isotype control mAb. Figure 23B shows the blood glucose levels after an overnight fast recorded at the start of the test in A. The key finding is that both molecules, mAb22033 and hFc.FGF21, brought about a significant improvement in glucose tolerance after 2 weeks. Furthermore, the improvement in glucose tolerance by mAb22033 was independent of changes in body weight or energy consumption (as shown in Figures 21 and 22).

[0273] Example 12: HDX Epitope Mapping To determine the epitopes of human NPR1 recognized by anti-NPR1 antibodies, hydrogen-deuterium exchange (HDX) studies were performed on mAb22033 and mAb22810, respectively. In prior in-house experiments, it has been shown that the presence of the ANP peptide is required for the binding of NPR1 to mAb22810. Therefore, in addition to the conventional HDX experiments using the NPR1 / mAb22033 complex, HDX experiments were also performed on the NPR1 / ANP / mAb22033 complex and the NPR1 / ANP / mAb22810 complex.

[0274] For this study, anti-NPR1 antibodies (mAb22033 and mAb22810) were covalently attached to N-hydroxysuccinimide (NHS) agarose beads (GE Lifescience, cat #17-0906-01) according to the manufacturer's protocol. The recombinant human NPR1 protein expressed in CHO cells contains the extracellular domain of the human NPR1 protein (Uniprot accession #P16066) with a C-terminal myc-myc-hexahistidine tag (SEQ ID NO: 194). The ANP pept ide was purchased from TOCRIS (cat #1906).

[0275] The deuterated buffer was prepared in D2O containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4 (pD = 7.4). For either the "antigen-on" or "complex-on" experiment, 30 μL of antibody bead slurry (equivalent to 15 μL of beads) was mixed with hNPR1.mmh or the hNPR1.mmh / ANP complex. The mixture was incubated with gentle rotation at room temperature. Deuteration was quenched using 0.075% ice-cold TFA while eluting hNPR1.mmh from the antibody beads. The quenched sample was immediately injected into a Waters HDX Manager for on-line pepsin digestion (Waters Enzymate BEH pepsin column, 2.1 x 30 mm). The digested peptides were trapped at 0 °C on an ACQUITY UPLC BEH C18 1.7 μm, 2.1 x 5 mm VanGuard pre-column and eluted onto an ACQUITY UPLC BEH C18 1.7 μm, 2.1 x 50 mm column using an 8-minute gradient separation of 1% - 30% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was set with a cone voltage of 37 V, a scan time of 0.5 seconds, and a mass / charge range of 50 - 1700 Th.

[0276] To map the hNPR.mmh binding epitope recognized by mAb22033, two sets of H / D exchange experiments were performed. In the first experiment, an “antigen-on” format (HDX of antigen only followed by binding to antibody beads) was used. In the “antigen-on” experiment, hNPR1.mmh was deuterated in phosphate buffered saline (PBS-D, pD = 7.4) prepared with D2O at room temperature for 3 and 8 minutes (in two separate preliminary experiments). Subsequently, the deuterated hNPR1.mmh was added to mAb22033 beads washed with PBS-D and incubated at room temperature for 2 minutes, resulting in total deuteration times of 5 and 10 minutes, respectively. Thereafter, the bound hNPR.mmh was eluted from the beads using an ice-cold aqueous solution of 0.075% trifluoroacetic acid (TFA). The eluted hNPR.mmh was immediately injected into a Waters HDX management system for on-line pepsin digestion followed by digestion peptide mass measurement.

[0277] The second experiment is called the “complex-on” format (HDX of complexed antigen / antibody beads). In this experiment, first hNPR.mmh was bound to mAb22033 beads in normal PBS (pH = 7.4) for 2 minutes. Subsequently, the complex was incubated and deuterated in PBS-D (pD = 7.4) for 5 or 10 minutes (in separate preliminary experiments). The next steps (elution, injection, pepsin digestion, and MS analysis) were carried out as described in the previous “antigen-on” procedure.

[0278] For peptide identification and deuterium incorporation measurement, LC-MS from non-deuterated hNPR1.mmh EThe data was first processed and searched against a database containing hNPR1.mmh via Waters ProteinLynx Global Server (PLGS) software. The identified peptides were imported into DynamX software and filtered based on two criteria: 1) minimum product per amino acid: 0.3, and 2) replicate file threshold: 2. Next, DynamX software was able to determine the deuterium incorporation of each peptide from the "antigen-on", "complex-on" experiments based on the retention time and mass accuracy (<30 ppm) of each peptide at two time points. All identified peptides were manually inspected and screened to minimize false positive hits.

[0279] The centroid values or average mass-to-charge ratio (m / z) of all detected peptides were calculated and compared between the "antigen-on" experiment and the "complex- -on" experiment at two time points. Peptides that showed an increase in mass after deuteration in the "antigen-on" compared to after deuteration in the "complex-on" contained amino acids protected from deuterium exchange as a result of antibody binding and thus indicated the binding epitope region.

[0280] In the NPR1 / mAb22033 HDX experiment, a total of 101 peptides were identified from hNPR1.mmh, showing a sequence coverage of 74%. Of these peptides, 10 peptides containing amino acids 29 - 50 and 328 - 347 showed a significant increase in mass after deuteration in the "antigen-on" compared to deuteration in the "complex-on", as shown in Table 46.

[0281]

Table 46

[0282] Two peptides, amino acids 46 - 54 and 328 - 335, did not show a difference in deuterium incorporation between the "antigen - on" and "complex - on" procedures. Therefore, the regions protected from deuterium exchange in the peptides of 29 - 50 and 328 - 347 were reduced to residues 29 - 45 and 336 - 347. Thus, two segments containing amino acids 29 - 45 and 336 - 347 were identified as the epitope of antibody mAb22033 that binds to the hNPR1.mmh protein.

[0283] In the NPR1 / ANP / mAb22033 HDX experiment, a total of 95 peptides were identified from hNPR.mmh, showing a 68% sequence coverage. Among these peptides, nine peptides containing amino acids spanning 29 - 50 and 331 - 347 showed a significant increase in mass after deuterium exchange in the "antigen - on" compared to the "complex - on" deuterium exchange, as shown in Table 47.

[0284] [Table 47]

[0285] Another peptide, amino acids 46 - 54, did not show a difference in deuterium incorporation between the "antigen - on" and "complex - on" procedures. Therefore, the region protected from deuterium exchange in the 29 - 50 peptide was reduced to residues 29 - 45. Thus, two segments containing amino acids 29 - 45 and 331 - 347 were identified as the epitope of antibody mAb22033 that binds to the hNPR1.mmh / ANP protein complex.

[0286] In the NPR1 / ANP / mAb22810 HDX experiment, a total of 93 peptides were identified from hNPR.mmh, showing a 70% sequence coverage. Among these peptides, ten peptides covering amino acids spanning 29 - 50, 70 - 81 and 331 - 347 showed a significant increase in mass after deuterium exchange in the "antigen - on" compared to the "complex - on" deuterium exchange, as shown in Table 48.

[0287]

Table 48

[0288] The three peptides, amino acids 46 - 54, 336 - 347, 337 - 347, did not show a difference in deuterium incorporation between the "antigen-on" and "complex-on" procedures. Therefore, the regions protected from deuterium exchange of the peptides of 29 - 50 and 331 - 347 are reduced to residues 29 - 45 and 331 - 335. Thus, three segments containing amino acids 29 - 45, 70 - 81, and 331 - 335 are identified as the epitope of the antibody mAb22810 that binds to the hNPR1.mmh / ANP protein complex.

[0289] Example 13: Intraocular injection of an NPR1 antibody reduces intraocular pressure in humanized NPR1 mice This example describes the effect of intravitreal injection (IVT) of an exemplary human NPR1 antibody, mAb22033, on intraocular pressure (IOP) in humanized NPR1 mice.

[0290] Method: Humanized NPR1 mice (NPR1 hu / hu ) were generated using VelociGene technology (Regeneron). Humanized NPR1 or wild-type (WT) mice received a single intravitreal injection of 40 μg mAb22033 or control Ab. Intraocular pressure was measured daily for 4 days after injection. In a second experiment, to examine the dose response, 40, 12.6, or 4 μg of mAb22033 or 40 μg of control Ab were injected intravitreally and intraocular pressure was monitored daily. In another experiment, to examine the effect of long-term delivery Ab, an AAV2 vector expressing an NPR1 antibody or eGFP was injected and intraocular pressure was followed over 7 weeks.

[0291] Results: NPR1 hu / huIntravitreal injection of 40 μg of mAb22033 into mice significantly reduced intraocular pressure from day 1 to day 3 compared to the control antibody. The average change in intraocular pressure was 5 mmHg. However, there was no intraocular pressure-lowering effect in WT mice. In the dose-response test, similar intraocular pressure-lowering effects were obtained with intravitreal injection of 40 or 12.6 μg of mAb22033, but the effect of 40 μg of mAb22033 lasted longer than that of 12.6 μg. Intravitreal injection of 4 μg of the NPR1 antibody did not lower intraocular pressure. No intraocular pressure effect was observed at all experimental time points after intravitreal injection of AAV2-GFP or AAV2-NPR1 antibody. This is thought to be due to the low expression of the NPR1 antibody, that is, only 10 ng was detected in the whole eye lysate. This is thought to be due to the low expression of the NPR1 antibody, that is, only 10 ng was detected in the whole eye lysate.

[0292] Conclusion: Intravitreal administration of a human NPR1 antibody (mAb22033) to humanized NPR1 mice significantly reduced intraocular pressure, indicating the potential of an agonist anti-NPR1 antibody for intraocular pressure reduction in glaucoma disease.

[0293] Example 14: Structural analysis of the antibody-NPR1 complex by electron microscopy Method Size-exclusion chromatography multi-angle light scattering (SEC-MALS) titration Several titration series of the human NPR1 extracellular domain (hNPR1-mmh; SEQ ID NO: 194) with a C-terminal myc-myc-6xHis tag complexed with various antibodies at different molar ratios were prepared. The antibodies tested were mAb22033, REGN5308 (Fab fragment of mAb22033), mAb22810, and REGN5314 (Fab fragment of mAb22810). All titration series were performed both with and without using a 2-fold molar excess of atrial natriuretic factor (ANP, Tocris) relative to hNPR1-mmh. After incubation overnight at 4°C in PBS, the complexes were injected into the SEC-MALS system. This system is an AKTA micro It consists of a Superdex 200 Increase 10 / 300 Gl column on a ROS system (GE Healthcare Life Sciences), followed by miniDAWN Treos and Optilab T-rEX (Wyatt Technology Corporation). Since the phosphate buffer is not compatible with negative staining of electron microscopy, the SEC column was equilibrated with a running buffer of 50 mM Tris pH 7.5, 150 mM NaCl, and all the large-scale complexes prepared below were in this buffer. The size exclusion chromatography data was evaluated using Unicorn (Version 5.20 General Electric Company), and the MALS data was evaluated using ASTRA (Version 7.0.0.69 Wyatt Technology).

[0294] Negative staining electron microscopy sample preparation For use in negative staining electron microscopy, the hNPR1 complex was prepared on a larger scale. Five samples were prepared as follows: Sample 1 = hNPR1-mmh (SEQ ID NO: 194) alone; Sample 2 = hNPR1-Fc (SEQ ID NO: 197) alone; Sample 3 = hNPR1-mmh + ANP, molar ratio 1:2; Sample 4 = hNPR1-mmh + REGN5308; molar ratio 1:1.5; Sample 5 = hNPR1-mmh + ANP + REGN5308, molar ratio 1:2:1.5. Samples 1, 3, 4, and 5 were purified by size exclusion chromatography in the same manner as the SEC-MALS experiment. The peak fractions were collected, frozen at -80 °C, and sent to NanoImaging Services, Inc. for EM analysis. Sample 2 was taken directly from a stock solution of 2.62 mg / ml dissolved in PBS, buffer-exchanged into 50 mM Tris, pH 7.5 + 150 mM NaCl, diluted to 1.5 mg / ml, frozen at -80 °C, and sent together with the other samples.

[0295] Collection and processing of negative staining electron microscopy data Five protein samples were used to prepare negative stain EM grids in a standard manner using uranyl formate (NanoImaging Services). The grids contained a thin layer of continuous carbon placed on a C-flat holey carbon grid. TEM images were acquired at 120 keV using a Tecnai T12 electron microscope (FEI / Thermo Fisher) and an FEI Eagle 4k x 4k CCD camera at room temperature. Images were collected at various nominal magnifications, mainly 67,000x and 110,000x. The collected images were further processed in-house.

[0296] NanoImaging micrographs were visually inspected by eye, and approximately 1 / 5 of the images with poor contrast due to dirt were removed. The remaining images were separated by magnification. The 110,000x images had too few particles per image to be very useful for further analysis. Therefore, all subsequent processing steps were performed using the 67,000x images. All images were subjected to CTF correction using CTFFIND4.

[0297] EM Particle Picking and 2D Class Averaging The particle distributions for all five negative stain samples were very good, with uniform particle sizes, very little aggregation, and good particle density. For Samples 4 and 5, particles were picked using Relion with an autopicking template obtained from initial manual picking and 2D class averaging. For Sample 4 (hNPR1+REGN5308), a total of 19,184 good particles were selected from 75 micrographs. For Sample 5 (hNPR1+ANP+REGN5308), a total of 20,318 good particles were initially selected from 88 micrographs. Initial 2D class averaging using Relion showed substantial heterogeneity in both datasets, with a significant number of classes showing only REGN5308 Fab or only NPR1.

[0298] The 2D class averaging of Sample 5 was further refined by excluding particles corresponding to Fab only or NPR1 only. Using the remaining 9,219 particles, a new 2D class average was calculated, which showed a better distribution map of the NPR1+REGN5308 complex. It was revealed that a small number of complexes contained only one Fab bound to the NPR1 dimer, while the majority of complexes contained two Fabs. Removing the complexes with one Fab reduced the particle set to 6,728 particles.

[0299] 3D Image Reconstruction from Negative Staining EM Data The initial 3D model of the NPR1-ANP-REGN5308 complex was constructed in Relion using the "3D initial model" procedure of stochastic gradient descent, with the resolution limited to 40 Å. Next, this model was further low-pass filtered to 60 Å, limiting the resolution at the prediction stage to 25 Å, and used as a reference when performing 3D class classification of the 6,728 particles in Relion. Subsequently, the best 3D class was further refined in Relion until convergence, with a final resolution of 22 Å measured by the "gold standard" FSC. Note that two-fold symmetry was not considered during 3D class classification or refinement. The density map obtained from the 3D reconstruction showed two Fabs bound to one side of a square particle, which was consistent with the crystal structure of ANP-bound NPR1 (PDB code 1T34).

[0300] Preparation and Data Collection of Cryo-Electron Microscopy Samples Samples of the NPR1-ANP-REGN5308 complex were prepared for cryo-electron microscopy (cryoEM) in the same manner as the negative stain EM samples described above. The final complex concentration was 0.8 mg / ml in 50 mM Tris, pH 7.5, 150 mM NaCl. CryoEM samples were prepared using standard procedures with an UltrAuFoil grid (Quantifoil Micro Tools GmbH) and a Vitrobot (FEI / Thermo Fisher). Data collection was performed on a Titan Krios electron microscope (FEI / Thermo Fisher) operating at 300 kV, using a counting mode K2 direct electron detector and a GIF energy filter (Gatan, Inc). Movies were collected at a magnification of 130,000x (1.04 Å / pixel), a defocus range of -0.5 to -1.5 µm, and a total dose of 45.44 e - / Å 2 yielding 1409 movies. Data collection was controlled using the Leginon software.

[0301] CryoEM Data Processing and Structure Determination All cryoEM movies were motion corrected, dose-weighted, and CTF corrected using the cisTEM package. Subsequently, the images were inspected manually to remove those with thick ice, poor CTF parameters (fit resolution worse than 6 Å), no particles, contamination, etc. After this filtering, 1172 images remained, which were then used for non-template particle picking in cisTEM to obtain 872915 particle positions. After removing bad particles by 2D class classification, 686709 particles were included in the 3D auto-refinement using cisTEM with the initially generated starting 3D reference volume. 3D refinement converged to a single solution with a resolution of 2.8 Å estimated from the Fourier shell correlation curve.

[0302] Next, this 3D map was used for structure refinement starting from the model incorporated into the above-mentioned negative stain EM 3D map. The N-terminal and C-terminal domains of both NPR1 molecules were subjected to real-space refinement as rigid bodies in the EM map, and then rebuilt manually at several locations where the model did not match the EM density. The homology model of REGN5308 was manually placed into the EM density. By carefully observing the CDR regions, the orientation of the heavy chain relative to the light chain could be determined. The CDR regions of this model required extensive rebuilding to fit the EM density. Finally, a structural model of the latest complex was created by real-space position refinement using Phenix.

[0303] Results / Discussion Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS) Titration Using Multi-Angle Light Scattering In the interaction between hNPR1-mmh and mAb22033, hNPR1-mmh itself behaved as a dimer with a molecular weight of approximately 110 kDa in the presence and absence of ANP, and the molecular weight increased slightly when ANP bound to NPR1. No peak for free monomer was observed for hNPR1-mmh itself. Titration with mAb22033 in the absence of ANP showed two major species of the complex: a species with a molecular weight corresponding to one IgG bound to one NPR1 dimer, and a species with a molecular weight corresponding to one IgG bound to two NPR1 dimers. However, when mAb22033 was added to NPR1 in the presence of ANP, higher molecular weight species that might represent a "paper doll" polymer of NPR1 and IgG were formed.

[0304] Subsequently, the system was simplified considering the Fab fragment of mAb22033, REGN5308. In the complex of hNPR1-mmh and REGN5308, the bound ANP shows a very different SEC profile compared to the same complex without ANP. The NPR1-REGN5308 complex has a molecular weight of approximately 155 kDa, consistent with one Fab binding per NPR1 dimer. In the presence of ANP, the molecular weight of the NPR1-ANP-REGN5308 complex increases by approximately 50 kDa, consistent with two Fabs binding per NPR1 dimer. The inventors have proposed that the previously described structural change of NPR1 due to ANP binding (Ogawa, H et al., 2004) enables the binding of the second Fab, and that a 2Fab+2NPR1+ANP complex is required for the growth of the paper doll polymer observed with the complete IgG mAb22033 (see further discussion below).

[0305] Titration using hNPR1-mmh and mAb22810 was also performed. In SEC-MALS analysis, a small portion of the mAb22810 sample was found to be a dimer, with a molecular weight of approximately 315 kDa compared to the 150 kDa of standard IgG. SEC-MALS titration using the NPR1-mAb22810-ANP complex showed a heterogeneous mixture of species in the range of 430 - 700 kDa. This profile was too complex for reliable interpretation, probably due to the presence of IgG dimer impurities contained in the mAb22810 protein. SEC-MALS titration using the Fab fragment of mAb22810, REGN5314, showed that the binding of REGN5314 to NPR1 in the absence of ANP is too weak or transient to produce complex species isolable by SEC. In the presence of ANP, a single NPR1-REGN5314-ANP complex is formed with a molecular weight of approximately 170 kDa, consistent with one Fab binding per NPR1 dimer.

[0306] Negative stain 2D class average The hNPR1+REGN5308 and hNPR1+ANP+REGN5308 complexes were further analyzed by negative stain electron microscopy. Substantial heterogeneity of the samples was revealed by 2D class classification and averaging of the complex particles. Most of the particles on the EM grid could be classified into either hNPR1 only or REGN5308 Fab only. Since the protein samples submitted for image processing were purified homogeneous complexes, these complexes are thought to dissociate into components during the preparation of the negative stain grid. However, a significant portion of the complexes remained intact and could be used for analysis.

[0307] The negative stain 2D class average of hNPR1+REGN5308 shows a "one armed" complex where only a single Fab is bound to the hNPR1 dimer, consistent with the results of SEC-MALS. In contrast, the negative stain 2D class average of hNPR1+ANP+REGN5308 shows a "two armed" complex with two Fabs bound to the dimer. The different averages represent different projections of the actual three-dimensional complex. In one class average, the hNPR1 dimer is seen from the side with two lobes of density, one corresponding to the overlapping N-terminal domains of the two monomers and the other lobe corresponding to the two overlapping C-terminal domains. In this orientation, the two bound Fabs appear like "rabbit ears" at the top of the density of NPR1. Viewed another way, all four domains of hNPR1 can be seen as four blocks of density forming a square, and the two REGN5308 Fabs on top of the square cross each other to form an inverted V. In the class average of hNPR1+ANP+REGN5308, "one armed" complexes were also seen, but these are thought to be due to the same dissociation of the complex that produced free Fab and free NPR1.

[0308] The 2D class averages calculated from the cryoEM data of hNPR1+ANP+REGN5308 show different views of the complex compared to the negative stain data, and in particular, the "rabbit ear" orientation is absent. However, other cryoEM 2D class averages can be closely matched to the corresponding averages of the negative stain data. There is little evidence of complex dissociation in the cryoEM data, which is probably due to the starting sample being more homogeneous and the cryogenic conditions better preserving the native conformation of the complex in solution.

[0309] 3D Image Reconstruction Using the 2D class averages as a starting point, a three-dimensional map of the cryoEM density of hNPR1+ANP+REGN5308 was constructed. This reconstruction procedure does not require any prior information about the expected shape or size of the complex beyond a rough estimate of the complex diameter, so the resulting cryoEM map is not biased by expectations of how the antibody-target complex should be formed. Next, the known crystal structure of hNPR1+ANP was placed and refined into this EM density map together with a model of the Fab of REGN5308 generated by homology to the known Fab structure. The structures of NPR1 and the Fab were placed by hand at approximate positions and then refined as rigid bodies to the correct positions using Phenix. The resolution of the cryoEM map is sufficient to manually reconstruct the residues at the NPR1:antibody contact interface, especially the residues in the complementarity-determining regions (CDRs) of REGN5308 that cannot be accurately modeled by homology. In the current structural model, all the CDR residues of the antibody and the residues of NPR1 are placed in contact. The more distant regions of the model (the C-terminal domain of NPR1 and the constant domains of the antibody Fabs) are modeled by combining the current cryoEM map with previously determined X-ray crystal structure information of NPR1 (PDB code 1T34) and the isolated antibody structure.

[0310] mAb22033 epitope on NPR1: Examination of the hNPR1+ANP+REGN5308 structure revealed which residues of NPR1 are in contact with REGN5308 Fab (and thus the parental IgG mAb22033). This epitope consists of four independent regions of NPR1 amino acids: residues 2-4, 41-45, 47, 73-79, 332, 336-344, 347 (numbered according to SEQ ID NO: 194), and they combine to form a three-dimensionally continuous surface. In previous hydrogen / deuterium exchange (HDX) mass spectrometry experiments, some of these residues were identified as important (see Example 12), but the cryoEM structure provides more details of the epitope.

[0311] Structural mechanism of action of mAb22033 In this model of the NPR1 antibody complex, the two Fabs are within approximately 10 Å of each other at a point near the "elbow" between the Fab variable domain and the Fab constant domain. Since the C-termini of these two Fabs are quite separated at approximately 100 Å, they cannot be the two arms of one IgG molecule. Also, the Fabs do not approach particularly closely to the modeled ANP peptide (the closest distance is approximately 30 Å), and no direct interaction between the Fab and ANP is considered likely.

[0312] Assuming the fixed positions and relative orientations of the Fabs with respect to the binding sites on the N-terminal domain of NPR1, the explanation for the ANP-dependent binding of REGN5308 Fab becomes clear. NPR1 has been shown to undergo a structural change upon ANP binding, where one NPR1 monomer rotates relative to the other while remaining dimerized. Applying this rotation to half of the two NPR1 + two Fab complexes results in a model similar to the crystal structure of NPR1 without ANP. However, here, one of the REGN5308 Fabs rotates to a position where it sterically collides with the other Fab, creating a physically impossible situation. This steric interference is the reason why only one REGN5308 Fab can bind to the NPR1 dimer in the absence of ANP. Although both antibody-binding sites on the two monomers are equally accessible, the binding of the first Fab blocks the binding of the second Fab.

[0313] Considering this effect in reverse, if two Fabs are bound to the NPR1 dimer containing ANP, as long as both Fabs are bound, this complex can be prevented from relaxing back to the ANP-free structure. Assuming that the effects seen here are maintained at the cell surface, at equilibrium, the proportion of NPR1 molecules in the active state capable of downstream signaling would increase. Another possible effect of antibody binding is the formation of oligomeric clusters of the antibody and NPR1 + ANP, as described above. This effect is only possible if each NPR1 dimer can bind two Fab arms from two separate IgG molecules. In the absence of ANP, only one Fab arm can bind to each NPR1 dimer, and complex formation stops at a much smaller species containing at most one IgG together with the NPR1 dimers bound to each Fab arm. Both of these effects, receptor clustering and extension of the receptor active state, can explain the activating effect of mAb22033 on the NPR1 receptor.

[0314] The present invention is not limited in scope by the specific embodiments described herein. In fact, various modifications of the invention 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 included within the scope of the appended claims.

Claims

1. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR) of an antibody or an antigen-binding fragment thereof that specifically binds to a natriuretic peptide receptor 1 (NPR1) protein, wherein the antibody or the antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the HCVR comprising the amino acid sequence of SEQ ID NO: 2; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the LCVR comprising the amino acid sequence of SEQ ID NO:

10.

2. The isolated polynucleotide molecule according to claim 1, wherein the antibody or the antigen-binding fragment thereof comprises an HCVR having at least 90% identity to the amino acid sequence of SEQ ID NO:

2.

3. The isolated polynucleotide molecule according to claim 1, wherein the antibody or the antigen-binding fragment thereof comprises an LCVR having at least 90% identity to the amino acid sequence of SEQ ID NO:

10.

4. The antibody or the antigen-binding fragment thereof is (a) HCDR1 having the amino acid sequence of SEQ ID NO: 4; (b) HCDR2 having the amino acid sequence of SEQ ID NO: 6; (c) HCDR3 having the amino acid sequence of SEQ ID NO: 8; (d) LCDR1 having the amino acid sequence of SEQ ID NO: 12; (e) LCDR2 having the amino acid sequence of SEQ ID NO: 14; and (f) LCDR3 having the amino acid sequence of SEQ ID NO: 16 The isolated polynucleotide molecule according to claim 1.

5. The isolated polynucleotide molecule according to claim 1, wherein the antibody or the antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 2 / 10.

6. The antibody or the antigen-binding fragment thereof interacts with one or more amino acids contained within the extracellular domain of NPR1 (amino acids 29 to 347 of SEQ ID NO: 194) as determined by hydrogen / deuterium exchange, and the antibody or the antigen-binding fragment thereof (i) binds to cells expressing human NPR1 in the presence or absence of atrial natriuretic peptide (ANP); and / or (ii) activates NPR1. The isolated polynucleotide molecule according to claim 1.

7. The isolated polynucleotide molecule according to claim 1, wherein the antibody or an antigen-binding fragment thereof interacts with an amino acid sequence selected from the group consisting of (a) amino acids 29 to 45 of SEQ ID NO: 194; and (b) amino acids 336 to 347 of SEQ ID NO:

194.

8. The isolated polynucleotide molecule according to claim 1, wherein the antibody or an antigen-binding fragment thereof interacts with an amino acid sequence selected from the group consisting of (a) amino acids 29 to 45 of SEQ ID NO: 194; and (b) amino acids 331 to 347 of SEQ ID NO: 194 in the presence of ANP.

9. The isolated polynucleotide molecule according to claim 1, wherein the antibody is a fully human monoclonal antibody.

10. The antibody is: (a) a fully human monoclonal antibody; (b) binds to monomeric human NPR1 with a dissociation constant (K D ) of less than 690 nM at 25°C and 37°C in the absence of ANP and / or brain natriuretic peptide (BNP), as measured by surface plasmon resonance; (c) binds to dimeric human NPR1 with a K D of less than 42 nM at 25°C and 37°C in the absence of ANP or BNP, as measured by surface plasmon resonance; (d) binds to human NPR1 complexed with ANP with a K D of less than 80 nM at 25°C and 37°C, as measured by surface plasmon resonance; (e) binds to human NPR1 complexed with BNP with a K D of less than 20 nM at 25°C and 37°C, as measured by surface plasmon resonance; (f) binds to monomeric monkey NPR1 with a K D of less than 365 nM at 25°C and 37°C in the absence of ANP and / or BNP, as measured by surface plasmon resonance; (g) binds to dimeric monkey NPR1 with a K D of less than 30 nM at 25°C and 37°C in the absence of ANP or BNP, as measured by surface plasmon resonance; (h) binds to monkey NPR1 complexed with ANP with a K D of less than 10 nM at 25°C and 37°C, as measured by surface plasmon resonance; (i) binds to monkey NPR1 complexed with BNP with a K D of less than 10 nM at 25°C and 37°C, as measured by surface plasmon resonance; (j) does not bind to mouse NPR1; (k) binds to cells expressing human NPR1 (without ANP) or NPR1 complexed with ANP with an EC 50 of less than 5 nM; (l) has an EC 50 of less than 385 nM as measured by a calcium flux cell-based bioassayactivates NPR1; (m) when administered to normal and hypertensive mice, reduces systemic blood pressure, and the reduction in systemic blood pressure and mean arterial blood pressure persists for up to 28 days with a single dose administration; and (n) when administered to diet-induced obese mice, improves glucose tolerance; an isolated polynucleotide molecule according to claim 1, having one or more properties selected from the group consisting of.

11. A vector comprising the polynucleotide sequence according to claim 1.

12. A host cell expressing the vector according to claim 11.

13. A method for producing an anti-NPR1 antibody or an antigen-binding fragment thereof, comprising culturing the host cell according to claim 1 2 under conditions that allow the production of the antibody or fragment, and recovering the antibody or fragment so produced.

14. The method according to claim 13, further comprising formulating the antibody or an antigen-binding fragment thereof as a pharmaceutical composition comprising an acceptable carrier.

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