Anti-NPR1 antibodies and uses thereof

Abstract

To provide monoclonal antibodies that specifically bind to natriuretic peptide receptor (NPR1) protein with high affinity and activate it, and methods of use thereof.SOLUTION: Antibodies and antigen-binding fragments thereof have specific sequences and specifically bind to natriuretic peptide receptor 1 (NPR1). The antibodies are fully human antibodies that bind to NPR1. The antibodies are useful for activating NPR1 activity, thus providing 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 to U.S. Provisional Application No. 62 / 749,557, filed October 23, 2018, and U.S. Provisional Application No. 62 / 755,720, filed November 5, 2018, the disclosures of each of which are incorporated herein by reference in their entireties.

[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 these antibodies. [Background technology]

[0003] Natriuretic peptide receptor 1 (NPR1; also known as NPR-A) belongs to the cell surface family of guanylate cyclase receptors, an enzyme that catalyzes the conversion of GTP to cyclic GMP. NPR1 is highly expressed in the kidney, lung, adrenal gland, vasculature, brain, liver, endothelium, and adipose tissue, with lower levels in the heart. It is activated by binding to atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). NPR1 activation and signaling trigger numerous physiological responses involving multiple tissues. The ANP-NPR1 system has been well studied for its role in vascular relaxation, natriuresis, diuresis, endothelial permeability, and non-cardiovascular functions such as lipolysis and immune cell function (NPL 1). NPR1 activation results in natriuresis (excretion of salt by the kidney) and lower blood pressure.

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

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

[0006] [Patent Document 1] U.S. Patent No. 9,090,695 [Patent Document 2] U.S. Patent No. 20160168251 [Patent Document 3] WO2010065293 [Non-patent literature]

[0007] [Non-Patent Document 1] Potter 2011,Pharmacol.Ther.130:71-82 [Non-patent document 2] Kitano et al., 1995, Immunol. Lett. 47:215-22 Summary of the Invention

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

[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 antibodies are advantageous because they can be used to bind and activate NPR1 even when the endogenous ligand concentration varies. When administered to patients in need thereof, such antibodies can be advantageously used to avoid patient-to-patient variability in treatment (with respect to ligand concentration). Furthermore, the antibodies disclosed herein bind to NPR1 with high affinity and have improved pharmacokinetic properties (compared to standard therapeutics). At a dose of 25 mg / kg, the antibody exhibited a t½ of up to 11 days in mice. When administered to subjects in need thereof, the antibody effectively reduced blood pressure, maintaining the reduced blood pressure for as long as 28 days. A single administration of the antibody of the present invention resulted in sustained reductions in blood pressure. Such antibodies can be used to provide superior efficacy with less frequent administration in subjects with NPR1-related diseases or disorders (e.g., hypertension).

[0010] Antibodies of the invention may be full-length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and may be modified to affect functionality, for example, to increase persistence in the host or to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, antibodies 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 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 wherein 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 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 regions (HCDRs) (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDRs) (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 provides 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 antigen-binding fragment thereof, comprising an HCVR comprising the 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 comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising 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 comprising 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 antibodies or antigen-binding fragments thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence set forth in Table 1 with no more than 12 amino acid substitutions and / or the LCVR comprises an amino acid sequence set forth in Table 1 with no more than 10 amino acid substitutions. For example, the present invention provides antibodies or antigen-binding fragments thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence set forth in Table 1 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions. In another example, the present invention provides antibodies or antigen-binding fragments thereof comprising an HCVR and an LCVR, wherein the LCVR comprises an amino acid sequence set forth in Table 1 with 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 antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence set forth in Table 1, the amino acid sequence having at least one amino acid substitution, and / or the LCVR comprises an amino acid sequence set forth in Table 1, the amino acid sequence having at least one amino acid substitution.

[0019] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0020] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences set forth 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.

[0021] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences set forth 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.

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

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

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

[0025] The present invention also provides an antibody or antigen-binding fragment thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences set forth in Table 1 paired with any of the LCDR3 amino acid sequences set forth in Table 1. According to certain embodiments, the present invention provides an antibody or antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-NPR1 antibodies set forth in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 8 / 16 (e.g., mAb22033) and 72 / 80 (e.g., mAb22810).

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

[0027] The present invention also provides antibodies or antigen-binding fragments 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 present invention provides antibodies or antigen-binding fragments thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR / LCVR amino acid sequence pair defined by any of the exemplary antibodies set forth in Table 1. For example, the present invention includes antibodies or antigen-binding fragments thereof comprising the set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within the HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 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 CDRs within the HCVR and / or LCVR amino acid sequences disclosed and identified herein. Exemplary conventions that can be used to identify CDR boundaries 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 and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0029] In a specific embodiment, the present invention provides an antibody or 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 a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR), wherein the HCVRs are selected from the group consisting of: (i) SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 16 (ii) an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178; (iii) an amino acid sequence having at least 95% identity to 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 having at least 95% identity to 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. 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; and (b) 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 90% identity to 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. an amino acid sequence having the amino acid sequence of:

[0030] In certain preferred embodiments, the present invention includes antibodies that specifically bind to NPR1 in an agonistic manner, ie, enhance or induce NPR1 binding and / or activity.

[0031] The present invention includes anti-NPR1 antibodies with altered glycosylation patterns. In some embodiments, modifications to remove undesired glycosylation sites are useful; for example, antibodies lacking fucose moieties present on the oligosaccharide chains may be useful for enhancing antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be performed to alter complement-dependent cytotoxicity (CDC).

[0032] In certain embodiments, the present invention provides antibodies and antigen-binding fragments thereof that exhibit pH-dependent binding to NPR1. For example, the present 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 reduced at acidic pH).

[0033] The present invention also provides antibodies and antigen-binding fragments thereof that compete with antibodies or antigen-binding fragments thereof comprising the CDRs of an HCVR and the CDRs of an LCVR for specific binding to NPR1, wherein the HCVR and LCVR have amino acid sequences selected from the HCVR and LCVR sequences listed in Table 1, respectively.

[0034] The present invention also provides antibodies and antigen-binding fragments thereof that cross-compete with a reference antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR for binding to NPR1, wherein the HCVR and LCVR have amino acid sequences selected from the sequences of the HCVR and LCVR listed in Table 1, respectively.

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

[0036] The present invention also provides isolated antibodies and antigen-binding fragments thereof that increase or stabilize the binding of NPR1 to its ligand (e.g., ANP or BNP). In some embodiments, the antibodies or antigen-binding fragments thereof that activate the binding of NPR1 to ANP may bind to the same epitope on NPR1 as ANP, or may bind to a different epitope on NPR1 than ANP.

[0037] In certain embodiments, the antibodies or antigen-binding fragments of the present invention are bispecific, comprising a first binding specificity for a first epitope of NPR1 and a second binding specificity for a second epitope of NPR1, wherein the first and second epitopes are distinct and non-overlapping.

[0038] In certain embodiments, the present invention provides a monoclonal antibody having the following characteristics: (a) being a fully human monoclonal antibody; (b) having a dissociation constant (K) 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. D ) and binds to monomeric human NPR1; (c) binds to ANP or BN as measured by surface plasmon resonance. K<42 nM at 25 °C and 37 °C in the absence of P D (d) binds to dimeric human NPR1 with a K of less than 80 nM at 25°C and 37°C as measured by surface plasmon resonance. D (e) a K of less than 20 nM at 25°C and 37°C as measured by surface plasmon resonance. D(f) binds to human NPR1 complexed with BNP at 25°C and 37°C in the absence of ANP and / or BNP, as measured by surface plasmon resonance, with a K of less than 365 nM. D (g) binds to monomeric monkey NPR1 at 25°C and 37°C in the absence of ANP or BNP, as measured by surface plasmon resonance, with a K of less than 30 nM. D (h) binds to dimeric monkey NPR1 with a K of less than 10 nM at 25°C and 37°C as measured by surface plasmon resonance. D and binds to monkey NPR1 complexed with ANP; (i) a K of less than 10 nM at 25°C and 37°C as measured by surface plasmon resonance. D (j) binds to monkey NPR1 complexed with BNP; (k) does not bind to mouse NPR1; (k) binds to cells expressing human NPR1 (without ANP) or NPR1 complexed with ANP with an EC of less than 5 nM. 50 (l) an EC50 of less than 385 nM as measured by calcium flux cell-based bioassay. 50 (m) activates NPR1 in normal and hypertensive mice, where the reduction in systemic blood pressure and mean arterial blood pressure persists for up to 28 days after a single administration; (n) improves glucose tolerance when administered to diet-induced obese mice; and (o) comprises an HCVR comprising an amino acid sequence selected from the group consisting of the HCVR sequences listed in Table 1 and an LCVR comprising an amino acid sequence selected from the group consisting of the LCVR sequences listed in Table 1.

[0039] In a second aspect, the present invention provides nucleic acid molecules encoding anti-NPR1 antibodies or portions thereof. For example, the present invention provides nucleic acid molecules 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0040] The present invention also provides nucleic acid molecules 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

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

[0042] The present invention also provides nucleic acid molecules 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

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

[0044] The present invention also provides nucleic acid molecules 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0045] The present invention also provides nucleic acid molecules 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 thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0046] The present invention also provides nucleic acid molecules 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 thereof 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 an HCVR, wherein the 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 listed in Table 1.

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

[0049] The present invention also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences set forth in Table 1, and the LCVR comprises the amino acid sequence of 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 HCVR nucleic acid sequences set forth in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences set forth in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes an HCVR and an LCVR, wherein the HCVR and LCVR are both derived from the same anti-NPR1 antibody set forth in Table 1.

[0050] In a related aspect, the invention provides recombinant expression vectors capable of expressing a polypeptide comprising an antibody heavy and / or light chain variable region. For example, the invention includes recombinant expression vectors comprising any of the nucleic acid molecules described above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences set forth in Table 2. In certain embodiments, the invention provides a recombinant expression vector comprising: (a) a nucleic acid encoding an HCVR of an antibody that binds to NPR1; (b) an expression vector comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an HCVR of an antibody that binds to NPR1, wherein the HCVR comprises an amino acid sequence 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 an LCVR of an antibody that binds to NPR1, wherein the LCVR comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 1. Also included within the scope of the invention are host cells into which such vectors have been introduced, and methods for producing antibodies or portions thereof by culturing the host cells under conditions that permit the production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments produced. In certain embodiments, the host cells comprise 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 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 antigen-binding fragment thereof operably linked to a promoter, culturing the host cell under conditions favorable for expression of the nucleic acid sequence, and isolating the antibody or antigen-binding fragment thereof from the culture medium and / or host cell. The isolated antibody or antigen-binding fragment thereof can be purified using any method known in the 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 a related aspect, 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 an anti-NPR1 antibody. Exemplary agents that are advantageously combined with an anti-NPR1 antibody include, but are not limited to, other agents that bind to and / or activate NPR1 activity (including other antibodies or antigen-binding fragments thereof, etc.) and / or agents that do not directly bind to NPR1 but nonetheless 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 that include the anti-NPR1 antibodies of the present invention are disclosed elsewhere herein.

[0052] In a fourth aspect, the present invention provides a therapeutic method for treating an NPR1-related disease or disorder in a subject using an anti-NPR1 antibody or antigen-binding portion thereof of the present invention, the therapeutic method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof 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 enhancing NPR1 activity. In certain embodiments, the present invention provides a method for preventing or treating an NPR1-related disease or disorder, comprising administering a therapeutically effective amount of an anti-NPR1 antibody or antigen-binding fragment thereof of the present invention to a subject in need thereof. In some embodiments, the antibody or antigen-binding fragment thereof can be administered prophylactically or therapeutically to a subject having or at risk of having an NPR1-related disease or disorder. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent can be selected from the group consisting of aldosterone antagonists, alpha-adrenergic blockers, angiotensin-converting enzyme (ACE) inhibitors, arteriolar dilating agents, autonomic ganglionic vasodilators, beta-adrenergic blocking agents, catecholamine-depleting sympatholytic agents, central alpha-2 adrenergic agonists, calcium channel blockers, diuretics, renin inhibitors, anticoagulants, antiplatelet agents, cholesterol-lowering agents, vasodilators, digitalis, surgery, implantable devices, anti-tumor therapies, insulin, GLP1 agonists, metformin, dialysis, bone marrow stimulating agents, hemofiltration, lifestyle modifications, nutritional supplements, and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent is an antibody or antigen-binding fragment thereof of the invention. 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 present invention may be administered in one or more doses comprised between 10 mg and 600 mg.

[0053] The invention also includes the use of an 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 activation of NPR1 binding and / or activity.

[0054] Other embodiments will become apparent from the detailed description that follows. [Brief explanation of the drawings]

[0055] [Figure 1] Figure 3 shows the effect of select anti-NPR1 agonist antibodies on systolic blood pressure in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into groups based on body weight. Animals received a single subcutaneous injection of NPR1 agonist antibody or PBS control at 25 mg / kg as described in Table 30. All values ​​are mean ± standard error of the change from baseline on days 3-7, n=3-9 per group. Statistics—One-way ANOVA with Dunnett's method; *p<0.05 vs. PBS control. [Figure 2] Figure 3 shows the effect of anti-NPR1 antibody mAb22033 on systolic blood pressure in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into five weight-matched groups and received a single subcutaneous injection of mAb22033 at the doses listed in Table 32. IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 4-6 per group. Statistics - two-way ANOVA with Dunnett's method. [Figure 3]Figure 3 shows the effect of anti-NPR1 antibody mAb22033 on diastolic blood pressure in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into five weight-matched groups and received a single subcutaneous injection of mAb22033 at the doses listed in Table 32. IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 4-6 per group. Statistics - two-way ANOVA with Dunnett's method. [Figure 4] Figure 3 shows the effect of anti-NPR1 antibody mAb22033 on heart rate in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into five weight-matched groups and received a single subcutaneous injection of mAb22033 at the doses listed in Table 32. IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 4-6 per group. Statistics - two-way ANOVA with Dunnett's method. [Figure 5] This figure shows the effect of the anti-NPR1 antibody mAb 22033 on mean arterial blood pressure in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into five weight-matched groups and received a single subcutaneous injection of mAb 22033 at the doses listed in Table 32. An IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 4–6 per group. Statistics: Two-way ANOVA by Dunnett's method. [Figure 6] Figures 6A and 6B show the effect of the anti-NPR1 antibody mAb22033 on left ventricular function in normotensive NPR1 hu / hu mice. End-systolic volume (%) (Figure 6A) and end-diastolic volume (%) (Figure 6B) were measured in normotensive NPR1 hu / hu mice randomized into five groups based on equal telemetered body weight and administered a single subcutaneous injection of mAb22033 at the doses listed in Table 32. An IgG4 antibody was used as an isotype control. Echocardiography was performed on the short axis of anesthetized mice 28 days after administration using a high-frequency ultrasound system and probe. All values ​​are means ± standard error, n = 6–7 per group. Statistics: One-way ANOVA by Dunnett's method; *p < .05 vs. IgG4 isotype control. [Figure 7] Figures 7A and 7B show the effect of the anti-NPR1 antibody mAb22033 on left ventricular function in normotensive NPR1 hu / hu mice. Telemetered normotensive NPR1 hu / hu mice were randomized into five weight-matched groups and received a single subcutaneous injection of mAb22033 at the doses listed in Table 32. Fractional shortening (%) (Figure 7A); and ejection fraction (%) (Figure 7B) were measured. An IgG4 antibody was used as an isotype control. Echocardiography was performed on the short axis of anesthetized mice 28 days after administration using a high-frequency ultrasound system and probe. All values ​​are means ± standard error, n = 6–7 per group. Statistics: One-way analysis of variance with Dunnett's method. [Figure 8] Figure 3 shows the effect of a single dose of two anti-NPR1 antibodies on systolic blood pressure in hypertensive NPR1 hu / hu mice. Telemetered hypertensive NPR1 hu / hu mice were randomized into six groups with equal systolic blood pressure and received a single subcutaneous dose of mAb 22033 or mAb 22810 at the doses listed in Table 36. IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 4-6 per group. Statistics - Two-way ANOVA by Dunnett's method; *p<.05 mAb 22033 25 mg / kg vs. control; #p<.05 mAb 22033 5 mg / kg vs. control; !p<.05 mAb 22810 19 mg / kg vs. control. [Figure 9] Figure 3 shows the effect of a single dose of two anti-NPR1 antibodies on diastolic blood pressure in hypertensive NPR1 hu / hu mice. Telemetered hypertensive NPR1 hu / hu mice were randomized into six groups with equal systolic blood pressure and received a single subcutaneous dose of mAb 22033 or mAb 22810 at the doses listed in Table 36. IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 3–6 per group. Statistics—Two-way ANOVA with Dunnett's method; *p<.05 mAb 22033 25 mg / kg vs. control; #p<.05 mAb 22033 5 mg / kg vs. control; !p<.05 mAb 22810 19 mg / kg vs. control; %p<.05 mAb 22810 5 mg / kg vs. control. [Figure 10] Figure 3 shows the effect of a single dose of two anti-NPR1 antibodies on heart rate in hypertensive NPR1 hu / hu mice. Telemetered hypertensive NPR1 hu / hu mice were randomized into six groups with equal systolic blood pressure and received a single subcutaneous dose of mAb 22033 or mAb 22810 at the doses listed in Table 36. IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 3-6 per group. Statistics - Two-way ANOVA with Dunnett's method; *p < .05 mAb 22033 25 mg / kg vs. control; #p < .05 mAb 22033 5 mg / kg vs. control. [Figure 11] Figure 3 shows the effect of a single dose of two anti-NPR1 antibodies on mean arterial blood pressure in hypertensive NPR1 hu / hu mice. Telemetered hypertensive NPR1 hu / hu mice were randomized into six groups with equal systolic blood pressure and received a single subcutaneous dose of mAb 22033 or mAb 22810 at the doses listed in Table 36. IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 3-6 per group. Statistics - Two-way ANOVA by Dunnett's method; *p<.05 mAb 22033 25 mg / kg vs. control; #p<.05 mAb 22033 5 mg / kg vs. control; !p<.05 mAb 22810 19 mg / kg vs. control. [Figure 12] This figure shows the effect of single and repeated administration of anti-NPR1 antibodies on systolic blood pressure in hypertensive NPR1 hu / hu mice. Telemetered hypertensive NPR1 hu / hu mice were randomized into five groups with equal systolic blood pressure and administered mAb22033 at the doses listed in Table 40 either as a single subcutaneous injection or twice weekly for 3 weeks. An IgG4 antibody was used as an isotype control. All values ​​are means ± standard error, n = 3–6 per group. Arrows indicate the dose administered to mice. Statistics—Two-way ANOVA with Dunnett's method; *p<.05 mAb22033 25 mg / kg vs. control; #p<.05 mAb22033 5 mg / kg vs. control; !p<.05 mAb22033 50 mg / kg vs. control. [Figure 13]Figure 1 shows the effect of single and repeated administration of anti-NPR1 antibodies on diastolic blood pressure in hypertensive NPR1 hu / hu mice. Telemetered hypertensive NPR1 hu / hu mice were randomized into five groups with equal systolic blood pressure and administered mAb 22033 at the doses listed in Table 40 either as a single subcutaneous injection or twice weekly for 3 weeks. An IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 3-6 per group. Arrows indicate the dose administered to mice. Statistics - Dunnett's two-way ANOVA. [Figure 14] Figure 1 shows the effect of single and repeated administration of anti-NPR1 antibodies on heart rate in hypertensive NPR1 hu / hu mice. Telemetered hypertensive NPR1 hu / hu mice were randomized into five groups equal in systolic blood pressure and administered mAb 22033 at the doses listed in Table 40 either as a single subcutaneous injection or twice weekly for 3 weeks. An IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 3-6 per group. Arrows indicate the dose administered to mice. Statistics—Two-way ANOVA with Dunnett's method. [Figure 15] This figure shows the effect of single and repeated administration of anti-NPR1 antibodies on mean arterial blood pressure in hypertensive NPR1 hu / hu mice. Telemetered hypertensive NPR1 hu / hu mice were randomized into five groups with equal systolic blood pressure and administered mAb22033 at the doses listed in Table 40 either as a single subcutaneous injection or twice weekly for 3 weeks. An IgG4 antibody was used as an isotype control. All values ​​are means ± standard error, n = 3–6 per group. Arrows indicate the dose administered to mice. Statistics—Two-way ANOVA with Dunnett's method; *p<.05 mAb22033 25 mg / kg vs. control; #p<.05 mAb22033 5 mg / kg vs. control; !p<.05 mAb22033 50 mg / kg vs. control. [Figure 16]Figures 16A and 16B show the effects of single and repeated administration of anti-NPR1 antibodies on cardiac function in hypertensive NPR1 hu / hu mice. End-systolic volume % (Figure 16A); and end-systolic volume % (Figure 16B) were measured in telemetered hypertensive NPR1 hu / hu mice randomized into five groups with equal systolic blood pressure and administered mAb 22033 subcutaneously once or twice weekly for 3 weeks at the doses listed in Table 40. An IgG4 antibody was used as an isotype control. Echocardiography was performed in the short axis direction on anesthetized mice 21 days after administration using a high-frequency ultrasound device and probe. All values ​​were mean ± standard error, n = 5–6 per group. Statistics—One-way ANOVA with Dunnett's method. [Figure 17] Figures 17A and 17B show the effects of single and repeated administration of anti-NPR1 antibodies on cardiac function in hypertensive NPR1 hu / hu mice. Telemetered fractional shortening (%) (Figure 17A); and ejection fraction (%) (Figure 17B) were measured in telemetered hypertensive NPR1 hu / hu mice randomized into five groups with equal systolic blood pressure and treated with a single subcutaneous dose of mAb 22033 or twice weekly for 3 weeks at the doses listed in Table 40. An IgG4 antibody was used as an isotype control. Echocardiography was performed in the short axis of anesthetized mice 21 days after administration using a high-frequency ultrasound device and probe. All values ​​were mean ± standard error, n = 5–6 per group. Statistics—One-way ANOVA by Dunnett's method. [Figure 18]Figure 18A shows the change in body weight after administration of mAb22810 NPR1 agonist mAb, hFc.FGF21, or an isotype control mAb. Figures 18B and 18C show the total fat mass and total lean mass, respectively, measured by EchoMRI 6 weeks after administration. NPR1-humanized mice were made obese by feeding a 60% high-fat diet for 10 weeks. After this period, mice were randomized into three weight-matched groups and treated with subcutaneous injections at the doses and frequencies shown in Table 44. A human IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 10 per group. * = P < 0.05 vs. isotype control; ** = P < 0.01 vs. isotype control. Statistics were performed by two-way ANOVA with Tukey's method for body weight and one-way ANOVA with Bonferonni's method for fat mass and lean mass. [Figure 19] Figures 19A, 19B, and 19C show changes in VO2 (Figure 19A), VCO2 (Figure 19B), or energy expenditure (Figure 19C), categorized as the average value for each day / night cycle, after one week of treatment with either mAb22810 NPR1 agonist mAb, hFc.FGF21, or an isotype control mAb. One week after treatment, mice from each group were placed in a Columbia Instruments Metabolic Cage System (CLAMS) for one week to record metabolic parameters. Mice were allowed to acclimate to the cages for one week prior to measurement to minimize stress. A human IgG4 antibody was used as an isotype control. All values ​​are means ± standard error, n = 5–6 per group. ** = P < 0.01 vs. isotype control; *** = P < 0.001 vs. isotype control. One-way analysis of variance using Bonferonni's method. [Figure 20]Figure 20A shows changes in glucose tolerance as measured by an oral glucose tolerance test (2 g / kg glucose) after 2 weeks of treatment with either mAb22810 NPR1 agonist mAb, hFc.FGF21, or an isotype control mAb. Figure 20B shows blood glucose levels after an overnight fast recorded at the start of the study in A. After 2 weeks of treatment, mice from each group were fasted overnight in clean cages and given a 2 g / kg oral glucose load the following morning. Glucose was then recorded from the tail vein using a handheld glucometer at T0, T15, T30, T60, T90, and T120. A human IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 10 per group. hFc.FGF2 group: ** = P < 0.01 vs. isotype control, *** = P < 0.001 vs. isotype control, **** = P < 0.0001 vs. isotype control. mAb22810 group: ++=P<0.01 vs. isotype control. Statistics by two-way ANOVA + Bonferonni for oGTT and one-way ANOVA + Bonferonni for fasting blood glucose levels. [Figure 21] Figure 21A shows the change in body weight after administration of mAb22033 NPR1 agonist mAb, hFc.FGF21, or an isotype control mAb. Figures 21B and 21C show the total fat mass and total lean mass, respectively, measured by EchoMRI after 6 weeks of treatment. NPR1-humanized mice were made obese by feeding a 60% high-fat diet for 10 weeks. After this period, mice were randomized into three weight-matched groups and treated with subcutaneous injections at the doses and frequencies listed in Table 45. A human IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 10 per group. * = P < 0.05 vs. isotype control; **** = P < 0.0001 vs. isotype control. Statistics were performed by two-way ANOVA with Tukey's for body weight and one-way ANOVA with Bonferonni for fat mass and lean mass. [Figure 22]Figures 22A, 22B, and 22C show the changes in VO2 (Figure 22A), VCO2 (Figure 22B), or energy expenditure (Figure 22C) after one week of treatment with either mAb22033 NPR1 agonist mAb, hFc.FGF21, or an isotype control mAb, divided into average values ​​for each day / night cycle. One week after treatment, mice from each group were placed in a Columbia Instruments metabolic cage system (CLAMS) for one week to record metabolic parameters. Mice were allowed to acclimate to the cage for one week before measurements to minimize stress. A human IgG4 antibody was used as an isotype control. All values ​​are means ± standard error, n = 5–6 per group. **** = P < 0.0001 vs. isotype control. One-way ANOVA with Bonferonni analysis. [Figure 23] Figures 23A and 23B show the effect of anti-NPR1 antibody mAb22033 on glucose tolerance, as indicated by blood glucose levels (Figure 23A) and fasting blood glucose levels (Figure 23B). After 4 weeks of treatment, mice from each group were fasted overnight in clean cages and given a 2 g / kg oral glucose load the next morning. Glucose was then recorded from the tail vein using a handheld glucometer at T0, T15, T30, T60, T90, and T120. A human IgG4 antibody was used as an isotype control. All values ​​are mean ± standard error, n = 10 per group. hFc.FGF21 group: *** = P < 0.001 vs. isotype control, **** = P < 0.0001 vs. isotype control. mAb22033 group: + = P < 0.05 vs. isotype control, ++ = P < 0.01 vs. isotype control. Statistics for oGTT were performed using two-way analysis of variance + Bonferonni, and for fasting blood glucose, statistics were performed using one-way analysis of variance + Bonferonni. DETAILED DESCRIPTION OF THE INVENTION

[0056] Before describing the methods of the present invention, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety.

[0058] definition The term "NPR1," also known as "NPRA," refers to natriuretic peptide receptor 1 (also known as natriuretic peptide receptor A). NPR1 is a homodimeric transmembrane guanylate cyclase, an enzyme that catalyzes the synthesis of cGMP. NPR1 is a receptor for both atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and undergoes a conformational change in the extracellular domain upon ligand binding (Ogawa et al. 2004, J. Biol. Chem. 279:28625-31). The protein has four distinct regions, including an extracellular ligand-binding domain, a single transmembrane region, an intracellular protein kinase-like homology domain, and a guanylyl cyclase catalytic domain. The amino acid sequence of the full-length NPR1 protein is exemplified by the amino acid sequence provided in UniProtKB / Swiss-Prot under accession number P16066.1 (SEQ ID NO: 193). The term "NPR1" includes recombinant NPR1 proteins or fragments thereof. The term also encompasses, for example, NPR1 protein or a fragment thereof linked to a histidine tag, mouse or human Fc, or a signal sequence such as ROR1 (eg, SEQ ID NOs: 194-199).

[0059] As used herein, the term "antibody" refers to an immunoglobulin molecule (i.e., an "intact antibody molecule") composed of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V"). H ") and heavy chain constant region (domain C H 1. C H 2 and C H Each light chain is composed of a light chain variable region ("LCVR" or "V L ") and the light chain constant region ("C L "). V H and V LThe regions can be further divided into more conserved regions called framework regions (FR) and hypervariable regions called complementarity-determining regions (CDR) interspersed within the FR. H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present invention, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on 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 in which one or two CDRs can be omitted for binding are 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 published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have any amino acids 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 empirically and / or by molecular modeling from regions of the Kabat CDR outside the Chothia CDR, based on previous studies (e.g., residues H60-H65 of CDRH2 are often unnecessary). When a CDR or its residues are omitted, they are typically substituted with amino acids that occupy corresponding positions in other human antibody sequences or consensus consensus of such sequences. The substitution positions within the CDR and the substituted amino acids can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[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 compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, 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, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first eight amino acids of FR1 or the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a different germline sequence from the germline from which the antibody was originally derived). Furthermore, antibodies of the present invention can contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing 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 by such general methods are encompassed by the present invention.

[0063] The present 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 with one or more conservative substitutions, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. The present invention also includes anti-NPR1 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with complementary amino acid substitutions.

[0064] As used herein, the term "human antibody" or "fully human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the present 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-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" or "fully human antibody" as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. This term includes antibodies produced recombinantly in non-human mammals or in the cells of non-human mammals. This term is not intended to include antibodies isolated from or generated in a human subject.

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

[0066] The terms "specifically bind" or "specifically binds to" and the like mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10 -8 can be characterized by an equilibrium dissociation constant M or less (e.g., K D(A smaller value indicates stronger binding.) Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies that specifically bind to NPR1 have been identified by surface plasmon resonance, e.g., 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 to be "specifically binding" antibodies as used herein.

[0067] The term "high affinity" antibody refers to an antibody having an affinity of at least 10 as measured by surface plasmon resonance, e.g., BIACORE™, or solution affinity ELISA. -8 M; preferably 10 -9 M; more preferably 10 -10 M, more preferably 10 -11 K of M D The binding affinity of mAbs to NPR1 is expressed as .

[0068] The terms "slow off rate," "Koff," or "kd" refer to a slow off rate of 1×10 as determined by surface plasmon resonance, e.g., BIACORE™. -3 seconds -1 or less, preferably 1 x 10 -4 seconds -1 It means an antibody that dissociates from NPR1 with a rate constant equal to or less than that.

[0069] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like 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 of the antibodies that retain the ability to bind to the NPR1 protein. This means the above fragment.

[0070] In certain embodiments, the antibodies or antibody fragments of the invention may be conjugated to a moiety such as a ligand or therapeutic moiety (an "immunoconjugate"), a second anti-NPR1 antibody, or other therapeutic moiety useful for treating an NPR1-associated disease or disorder.

[0071] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that specifically binds NPR1 or a fragment thereof is substantially free of Abs that specifically bind antigens other than NPR1).

[0072] As used herein, an "activating antibody" or "agonist antibody" (i.e., an "antibody that increases or enhances NPR1 activity" or an "antibody that stabilizes the activated conformation") refers 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 reduce systemic blood pressure when administered to a subject in need thereof.

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

[0074] As used herein, the term "K D " refers 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 the paratope. An antigen may have more than one epitope. Therefore, different antibodies may bind to different regions of the antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics.

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

[0077] The terms "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, there is at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or GAP, as described below. 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 term "substantial similarity" or "substantially similar," as applied to polypeptides, means that two peptide sequences, when optimally aligned using, for example, the programs GAP or BESTFIT with default gap weights, share at least 90% sequence identity, more preferably at least 95%, 98%, or 99% sequence identity. Preferably, residue positions are not identical but differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-33, 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 substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is a change that has a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnetet et al. (1992) Science 256:1443 45, incorporated herein by reference, and a "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0079] Sequence similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutein. See, for example, GCG Version 6.1. Polypeptide sequences can also be analyzed using default or recommended parameters in FASTA;GCG Comparison can be performed using the program BLAST version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the best overlapping regions between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention with 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, each of which is incorporated herein by reference.

[0080] By "therapeutically effective amount" is meant the amount that produces the desired effect for which it is administered. The exact amount will vary depending on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., 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 also includes human subjects having or at risk of having such a disease or disorder.

[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 administering a therapeutic agent, such as an antibody of the present invention, to a subject in need thereof. The term includes inhibiting the progression of the disease or worsening of symptoms / signs. The term also includes that administering a therapeutic agent, such as an antibody of the present invention, may improve the prognosis of the disease, i.e., the subject may become disease-free or have a reduced disease. The therapeutic agent may be administered to the subject in a therapeutic dose.

[0083] The terms "prevent," "preventing," or "prevention" refer to suppressing the onset of an NPR1-associated 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 fragments of antibodies As used herein, the term "antibody" is understood to encompass not only antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., "intact antibody molecules"), but also antigen-binding fragments thereof, unless otherwise specified. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like include naturally occurring, enzymatically obtained, 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 an NPR1 protein. Antibody fragments include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion, or recombinant genetic engineering techniques, involving the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and / or can be obtained, for example, from commercial sources, DNA libraries (e.g., phage). The DNA can be readily obtained from libraries (including antibody libraries) or synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques to, for example, place one or more variable and / or constant domains in the 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) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of antibodies (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the term "antigen-binding fragment" as used herein.

[0086] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR, which is adjacent to or in frame with one or more framework sequences. L V related to domain H In the antigen-binding fragment containing the V H Domains and V L The domains can be positioned in any suitable arrangement relative to each other. For example, the variable region is a dimer, and the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H or V L It may also include a domain.

[0087] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any configuration of 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 comprised of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, resulting in a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may be obtained by combining any of the above variable and constant domain configurations with each other and / or with one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of domains non-covalently associated (eg, by disulfide bonds).

[0088] Like intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats described above, can be adapted for use in connection with antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0089] Preparation of human antibodies Methods for preparing human antibodies in transgenic mice are known in the art, and any such known method can be used in the context of the present invention to prepare human antibodies that specifically bind to NPR1.

[0090] Antibodies against the NPR1 protein can be produced using an immunogen comprising any one of the following: In certain embodiments, antibodies of the present invention are obtained from mice 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 can be produced using standard biochemical techniques, modified, and used as an immunogen.

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

[0092] Using VELOCIMMUNE® technology (see, e.g., US Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or other known methods for generating monoclonal antibodies, high-affinity chimeric antibodies against NPR1 with human variable regions and mouse constant regions are first isolated. VELOCIMMUNE® technology involves generating transgenic mice whose genomes contain human heavy and light chain variable regions operably linked to endogenous mouse constant region sites, so that the mice produce antibodies containing human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding human heavy and light chain constant regions. This DNA is then expressed in cells capable of expressing fully human antibodies.

[0093] Generally, VELOCIMMUNE® mice are exposed to an antigen of interest, and lymphoid cells (such as B cells) are collected from the mice that express antibodies. These lymphoid cells are fused with a myeloma cell line to prepare immortalized hybridoma cell lines, which can then be screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions may be isolated and linked to constant regions of the desired heavy and light chain isotypes. Such antibody proteins may be produced in cells such as CHO cells. Alternatively, DNA encoding antigen-specific chimeric antibodies or the light and heavy chain variable regions may be isolated directly from antigen-specific lymphocytes.

[0094] First, high-affinity chimeric antibodies having human variable regions and mouse constant regions are isolated. As described in the experimental section below, the antibodies are characterized and selected for desirable properties, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant regions to produce fully human antibodies of the invention, such as wild-type or modified IgG1 or IgG4.

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

[0096] Bioequivalents The anti-NPR1 antibodies and antibody fragments of the present invention include proteins having amino acid sequences that vary from those of the described antibodies but retain the ability to bind to the NPR1 protein. Such mutant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit essentially the same biological activity as the described antibody. Similarly, DNA sequences encoding the antibodies of the present invention include sequences that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but 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 bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes that do not exhibit significant differences in the rate and extent of absorption when administered at the same molar dose under similar experimental conditions, for example, either single or multiple doses. Some antibodies may be considered equivalents or pharmaceutical substitutes if they exhibit a similar extent of absorption but not a similar rate of absorption, but may still be considered bioequivalent because such differences in absorption rate are intentional, reflected in the labeling, are not essential to achieving effective body drug concentrations, for example, during chronic use, and are not considered medically significant for the particular pharmaceutical product being studied.

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

[0099] In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch between the reference product and the biological product one or more times without an expected increase in the risk of side effects, including clinically significant changes in immunogenicity, or a decrease in efficacy, compared to continuing treatment without such a switch.

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

[0101] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Measures of bioequivalence include, for example, (a) in vivo tests in humans or other mammals that measure the concentration of an antibody or its metabolites in blood, plasma, serum, or other biological fluid as a function of time; (b) in vitro tests that correlate with and are reasonably predictive of human in vivo bioavailability data; (c) in vivo tests in humans or other mammals that measure the relevant acute pharmacological effect of the antibody (or its target) as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the 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 by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bonds during renaturation. In other contexts, biologically equivalent antibodies can include antibody variants containing amino acid changes that alter the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0103] Anti-NPR1 antibodies containing Fc variants According to certain embodiments of the present invention, anti-NPR1 antibodies are provided that comprise an Fc domain containing one or more mutations that enhance or decrease binding of the antibody to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present invention provides an anti-NPR1 antibody that comprises an Fc domain containing one or more mutations that enhance or decrease binding of the antibody to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HThe present invention also includes anti-NPR1 antibodies containing mutations in three regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., within endosomes, where the pH ranges from about 5.5 to about 6.0). Such mutations may result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and / or 308 modification (e.g., 308F or 308P). In yet another embodiment, the modifications include a 265A (e.g., D265A) and / or a 297A (e.g., N297A) modification.

[0104] For example, the present invention includes anti-NPR1 antibodies comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations and other mutations in the antibody variable domains disclosed herein are contemplated within the scope of the present invention.

[0105] The present invention also provides a chimeric heavy chain constant (C H ) region, H The region is C of more than one immunoglobulin isotype. H For example, the antibodies of the present invention may comprise a C region derived from a human IgG1, human IgG2, or human IgG4 molecule. H Part or all of the 2 domain and C from a human IgG1, human IgG2, or human IgG4 molecule H Chimera C consisting of a combination of part or all of the three domains H According to a particular embodiment, the antibody of the present invention may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may combine an "upper hinge" amino acid sequence (amino acid residues from positions 216 to 227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region with a "lower hinge" sequence (amino acid residues from positions 228 to 236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. The chimeric C described herein H Antibodies comprising the F region may, in certain embodiments, be modified without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. c effector function (see, e.g., 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 and enhancing the activity of NPR1 protein. For example, the present invention includes antibodies that bind to monomeric human NPR1 protein in the absence of either ANP or BNP (e.g., at 25°C or 37°C) and have a K of less than 690 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 has a K of 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, e.g., using an assay format as defined in Example 3 herein or a substantially similar assay. D It binds to NPR1.

[0107] The present invention also provides a method for assaying dimeric human NPR1 protein in the absence of either ANP or BNP (e.g., at 25°C or 37°C) to determine 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 In certain embodiments, the antibody or antigen-binding fragment thereof has 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, e.g., using an assay format as defined in Example 3 herein or a substantially similar assay. D It binds to NPR1.

[0108] The present invention also includes a method for detecting 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, e.g., using an assay format as defined in Example 3 herein. D In certain embodiments, the antibody or antigen-binding fragment thereof has a K of less than about 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, e.g., using an assay format as defined in Example 3 herein or a substantially similar assay. D It binds to NPR1.

[0109] The present invention also includes a method for determining whether human NPR1 protein complexed with BNP has a K of less than 20 nM (e.g., at 25°C or 35°C), as measured by surface plasmon resonance, using, for example, an assay format as defined in Example 3 herein. DIn certain embodiments, the antibody or antigen-binding fragment thereof has 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, e.g., using an assay format as defined in Example 3 herein or a substantially similar assay. D It binds to NPR1.

[0110] The present invention also includes a method for assaying a 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, e.g., using an assay format as defined in Example 3 herein. D In certain embodiments, the antibodies or antigen-binding fragments thereof can be detected by, for example, the assay format defined in Example 3 herein or a substantially similar assay. 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 plasmon resonance D It binds to NPR1.

[0111] The present invention also includes a method for determining whether dimeric monkey NPR1 protein has 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 Also included are antibodies and antigen-binding fragments thereof that bind 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, e.g., using an assay format as defined in Example 3 herein or a substantially similar assay. DIt binds to NPR1.

[0112] The present invention also includes compounds having a K of less than 10 nM, as measured by surface plasmon resonance, for example, using an assay format as defined in Example 3 herein. D Also included are antibodies and antigen-binding fragments of antibodies that bind to monkey NPR1 protein complexed with ANP (e.g., at 25°C or 37°C) at 25°C or 37°C. In certain embodiments, the antibodies or antigen-binding fragments thereof have 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, e.g., using an assay format as defined in Example 3 herein or a substantially similar assay. D It binds to NPR1.

[0113] The present invention also includes a method for detecting dimeric monkey NPR1 protein complexed with BNP (e.g., at 25°C or 37°C) that has a K of less than 10 nM, as measured by surface plasmon resonance, using, for example, an assay format as defined in Example 3 herein. D Also included are antibodies and antigen-binding fragments of antibodies that bind to NPR1 with a KD 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, e.g., using an assay format as defined in Example 3 herein or a substantially similar assay.

[0114] The present invention also includes compounds that have an EC50 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 in cells expressing human NPR1 with or without ANP, as measured, for example, using an assay format as described in Example 5 herein or a substantially similar assay. 50 Also included are antibodies and antigen-binding fragments thereof that bind to the antibody.

[0115] The present invention also provides compounds having an EC of less than 385 nM, as measured by a calcium flux cell-based bioassay, for example, using an assay format as defined in Example 6 herein. 50 In certain embodiments, the antibody or antigen-binding fragment thereof has 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, for example, an assay format as defined in Example 6 herein, or a substantially similar assay. 50 activates NPR1.

[0116] The present invention also includes antibodies and antigen-binding fragments of antibodies that bind to NPR1 and, upon a single administration to a subject in need thereof, reduce systemic blood pressure in the subject for more than 28 days, as shown, for example, in Example 8 herein.

[0117] The present invention also includes antibodies and antigen-binding fragments of antibodies that bind to NPR1 and reduce fasting blood glucose levels when administered to a subject in need thereof, for example, as shown in Example 11 herein.

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

[0119] Antibodies of the invention may have one or more of the aforementioned biological properties, or a combination thereof. Other biological properties of the antibodies of the invention will be apparent to those skilled in the art from consideration of this 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 consist of a single, contiguous 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 consist of multiple, non-contiguous amino acids (or amino acid sequences) located within either or both of the aforementioned domains of the protein molecule (e.g., a conformational epitope).

[0121] A variety of techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, those described in Antibodies, Harlow and L. ane(Cold Spring Harbor Press,Cold Spring These include routine cross-blocking assays, such as those described in [End Page 111] (Harbor, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide truncation analysis, crystallographic studies, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange, as detected by mass spectrometry. Generally, hydrogen / deuterium exchange involves deuterium-labeling the protein of interest and then binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, where exchangeable protons in amino acids protected by the antibody complex undergo deuterium-hydrogen back exchange at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein-antibody interface will retain deuterium and therefore exhibit a relatively higher mass than amino acids that are not included in the interface. After dissociation of the antibody, cleavage of the target protein with a protease and mass spectrometry will reveal deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

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

[0123] Modification-Assisted Profiling (MAP), also known as Antigen Structure-Based Antibody Profiling (ASAP), is a method for classifying multiple monoclonal antibodies (mAbs) directed against the same antigen according to the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces (see US 2004 / 0101920, specifically incorporated herein by reference in its entirety). Each category may reflect unique epitopes that are distinct or partially overlapping with those represented in other categories. This technique allows for rapid filtering of genetically identical antibodies and allows for focused characterization of genetically distinct antibodies. When applied to hybridoma screening, MAP 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 present invention includes anti-NPR1 antibodies and antigen-binding fragments thereof that interact with one or more epitopes found within the extracellular domain of NPR1. An epitope may consist of one or more contiguous sequences of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids located within the extracellular domain of NPR1. Alternatively, an epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within NPR1.

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

[0126] Whether an antibody binds to the same epitope as a reference anti-NPR1 antibody or competes for binding can be easily determined using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference anti-NPR1 antibody of the present invention, the reference antibody is bound to an NPR1 protein or peptide under saturation conditions. The ability of the test antibody to bind to an NPR1 protein molecule is then evaluated. If the test antibody can bind to NPR1 after saturation binding with the reference anti-NPR1 antibody, it can be concluded that the test antibody binds to a different epitope from the reference anti-NPR1 antibody. On the other hand, if the test antibody cannot bind to NPR1 protein after saturation binding with the reference anti-NPR1 antibody, the test antibody may bind to the same epitope as the reference anti-NPR1 antibody of the present invention.

[0127] To determine whether an antibody competes for binding with a reference anti-NPR1 antibody, the above-mentioned binding method is carried out in two ways. In the first way, the reference antibody is allowed to bind to NPR1 protein under saturating conditions, and then the binding of the test antibody to the NPR1 molecule is evaluated. In the second way, the test antibody is allowed to bind to the NPR1 molecule under saturating conditions, and then the binding of the reference antibody to the NPR1 molecule is evaluated. In both ways, if only the first (saturating) antibody 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 for binding with a reference antibody does not necessarily bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

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

[0129] Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to determine whether the observed lack of binding of the test antibody is in fact because it binds to the same epitope as the reference antibody, or whether the observed lack of binding is due to steric blocking (or another phenomenon). These types of experiments can be performed 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 present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to natriuretic peptide receptor 1 (NPR1) protein, wherein the antibody or antigen-binding fragment thereof comprises one or more sequences contained within the extracellular domain of NPR1 (amino acids 29-347 of SEQ ID NO: 194) as determined by hydrogen / deuterium exchange. and (e) amino acids 70-81 of SEQ ID NO: 194. In one embodiment, the antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof interacts in the presence of ANP 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 one embodiment, the antibody or antigen-binding fragment thereof interacts in the presence of ANP 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 one embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to NPR1 protein in the presence of ANP, wherein the antibody or antigen-binding fragment thereof 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, but not with amino acids 70-81 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 ANP; and / or (ii) binds to and activates NPR1.

[0131] immune complex The present invention encompasses human anti-NPR1 monoclonal antibodies ("immunoconjugates") conjugated to a therapeutic moiety for treating diseases or disorders associated with NPR1 (e.g., hypertension). As used herein, the term "immunoconjugate" refers to an antibody chemically or biologically conjugated to a radiopharmaceutical, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antibody can be conjugated to the radiopharmaceutical, cytokine, interferon, target or reporter moiety, enzyme, peptide, therapeutic agent, etc. at any position along the molecule, as long as it is capable of binding to the target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In some embodiments, the agent may be a second, different antibody directed against the NPR1 protein. The type of therapeutic moiety 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 immunoconjugates are known in the art; see, for example, WO 05 / 103081.

[0132] multispecific antibodies The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single 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 produced using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques), as known to those skilled in the art. ) can be constructed using

[0134] In some embodiments, NPR1-specific antibodies are produced 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. Properly designed bispecifics can enhance both specificity and avidity, thereby increasing the overall inhibitory effect of the NPR1 protein. Variable regions that have specificity for individual domains (e.g., segments of the N-terminal domain) or that can bind to different regions within a single domain are paired on a structural scaffold that allows each region to simultaneously bind to separate epitopes or to different regions within a single domain. In one example of a bispecific, a heavy chain variable region (V) of a binder with specificity for one domain is linked together with a heavy chain variable region (V) of a binder with specificity for one domain. H ) with the light chain variable region (V) of a series of binders with specificity for the second domain. L ) and V H Without losing the original specificity of V H Non-cogate V that can be paired with L Identify your partner. In this way, you can identify a single V L Segments (e.g., V L 1) Two different V H Domain (e.g., V H 1 and V H 2) to form two bond "arms" (V H 1-V L 1 and V H 2-V L 1) can be generated. L The use of segments reduces the complexity of the system, thereby simplifying and improving the efficiency of the cloning, expression, and purification processes used to generate bispecifics (see, e.g., US2011 / 0195454 and US2010 / 0331527).

[0135] Alternatively, antibodies that bind more than one domain and a second target, such as, but not limited to, a second, different anti-NPR1 antibody, can be prepared in a bispecific format using the techniques described herein or other techniques known to those skilled in the art. Antibody variable regions that bind to different regions can be linked together, for example, with variable regions that bind to related sites in the extracellular domain of NPR1, to confer dual antigen specificity within a single binding molecule. A properly designed bispecific antibody of this nature performs dual functions. A variable region with specificity for the extracellular domain is combined with a variable region with specificity for the outside of the extracellular domain, paired on a structural scaffold that allows each variable region to bind to a different antigen.

[0136] An exemplary bispecific antibody format that can be used in the context of the present invention is H 3 domain and second Ig C H the first and second Ig C domains, H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody without the amino acid difference. H The 3 domain binds protein A and the second Ig C H The 3 domain contains mutations that reduce or eliminate Protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further comprise a Y96F modification (by IMGT; Y436F by EU). HFurther modifications that may be found within 3 include, for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU); for IgG2 antibodies, N44S, K52N, and V82I (by IMGT; N384S, K392N, and V422I by EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU). Variations on 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 antibody bispecific formats, 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 acting Fab (DAF)-IgG, and Mab 2 Bispecific formats (for a review of the aforementioned formats, see, e.g., Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugates, e.g., using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that can then self-assemble into multimeric complexes with predetermined composition, valency, and shape (see, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0138] Therapeutic Administration and Formulations The present invention provides therapeutic compositions comprising the anti-NPR1 antibody or antigen-binding fragment thereof of the present invention. The therapeutic compositions of the present invention are administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Numerous suitable formulations can be found in formularies known to all pharmaceutical chemists, such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and carbowax-containing semi-solid mixtures. See also Powell et al., "Compendium of Excipients for Parenteral Formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0139] The dose of the antibody may vary depending on the age and physique of the subject to be administered, the target disease, condition, route of administration, etc. When the antibody of the present invention is used to treat a disease or disorder in an adult patient or to prevent such a disease, it is generally advantageous to administer the antibody of the present invention at a single dose of about 0.1 to about 100 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention 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 an initial dose, a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof can be administered in an amount that is about the same as or less than that of the initial dose, with the subsequent doses being 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, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions of the present invention (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (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. Pharmaceutical compositions can also be delivered in vesicles, particularly liposomes (see, e.g., Langer (1990) Science 249:1527-1533).

[0141] The use of nanoparticles for delivering the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods for their 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, page 24, doi:10.1155 / 2009 / 439389), which is incorporated herein by reference. Nanoparticles can also be developed and conjugated to antibodies contained in pharmaceutical compositions for target cells. Nanoparticles for drug delivery are also described, for example, in US8257740 or US8246995, each of which is incorporated herein in its entirety.

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

[0143] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by known methods.

[0144] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device is easily adapted to deliver the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. Disposable pen delivery devices do not have a replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is empty, the entire device is discarded.

[0145] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in a unit dosage form suitable for the dosage of the active ingredient. Examples of such unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained per unit dosage form is generally about 5 to about 500 mg, preferably about 5 to about 300 mg for injections and about 10 to about 300 mg for other dosage forms.

[0146] Therapeutic Uses of Antibodies The antibodies of the invention are useful for treating and / or preventing diseases or disorders or conditions associated with NPR1 and / or ameliorating at least one symptom associated with such diseases, disorders or conditions. In certain embodiments, the antibodies of the invention or their antigen binding The synthetic fragments may be administered in therapeutic doses to patients with 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-associated disease or disorder selected from the group consisting of hypertension, heart failure, obesity, renal failure, chronic kidney disease, macular edema, glaucoma, stroke, lung damage, 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 subjects at risk of developing an NPR1-associated 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 medicament for treating a patient suffering from a disease, disorder, or condition disclosed herein. In another embodiment of the invention, the antibodies are used as an adjunct therapy with any other drug or therapy known to those of skill in the art that is useful for treating or ameliorating a disease, disorder, or condition disclosed herein.

[0150] Combination therapy Combination therapy may include an antibody of the invention and any additional therapeutic agent that may be advantageously combined with an antibody of the invention or a biologically active fragment of an antibody of the invention. The antibody of the invention may be synergistically combined with one or more drugs or therapies used to treat an NPR1-related disease or disorder. In some embodiments, the antibody of the invention may be combined with a second therapeutic agent to ameliorate one or more symptoms of the disease or condition.

[0151] Depending on the disease, disorder, or condition, the antibodies of the invention may 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, laminin), and the like. mipril, trandolapril), arterial vasodilators (e.g., hydrazine, minoxidil), autonomic ganglionic vasodilators (e.g., mecamylamine), beta-adrenergic blocking agents (acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, carteolol, esmolol, labetrol, metoprolol, nadolol, penbuterol, pindolol, propranolol, timolol), catecholamine-depleting sympatholytics (e.g., deserpidine, reserpine), central alpha-2 adrenergic agonists (e.g., clonidine, glucan), Anabenz, guanfacine, methyldopa), calcium channel blockers (diltiazem, verapamil, amlodipine, felodipine, isradipine, nicadipine, nifedipine, nisoldipine), diuretics (e.g., bumetanide, ethaclinic acid, furosemide, torsemide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide, chlorthalidone, indapamide, metolazone), renin inhibitors (e.g., aliskiren), anticoagulants (e.g., coumadan, dabigatran, apixaban), antiplatelet agents (e.g., aspirin, steroids, clopidogrel), cholesterol-lowering agents (e.g., statins, PCSK9 inhibitors such as alirocumab), vasodilators (e.g., minoxidil, hydrazine, nitrates), digitalis, surgery (e.g., angioplasty, coronary artery bypass grafting, heart transplant), implantable devices (e.g., valve replacement, defibrillator, left ventricular assist device, pacemaker), anti-tumor therapy (e.g., chemotherapy, surgery, radiation, PD-1 inhibitors), insulin, GLP1 agonists (e.g., exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide), methotrexate (e.g., methotrexate ... Formin, dialysis, bone marrow stimulants, hemofiltration, lifestyle modifications, and nutritional supplements.

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

[0153] An additional therapeutically active ingredient may be administered to a subject prior to administration of an anti-NPR1 antibody of the present invention. For example, if a first ingredient is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less than 30 minutes before administration of the second ingredient, the first ingredient may be considered to be administered "before" the second ingredient. In other embodiments, an additional therapeutically active ingredient may be administered to a subject after administration of an anti-NPR1 antibody of the present invention. For example, if a first ingredient is administered 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or more after administration of the second ingredient, the first ingredient may be considered to be administered "after" the second ingredient. In yet other embodiments, the additional therapeutically active ingredient may be administered to the subject simultaneously with the administration of the anti-NPR1 antibody of the present invention. For purposes of the present invention, "simultaneous" administration includes, for example, administering the anti-NPR1 antibody and the additional therapeutically active ingredient to the subject in a single dosage form or in separate dosage forms that are 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 therapeutically active ingredient may be administered intravenously), 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 therapeutically active ingredient may be administered orally). In any case, for purposes of the present disclosure, administration of the ingredients in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered "simultaneous administration." For purposes of this disclosure, administration of an anti-NPR1 antibody "before," "concurrently with," or "after" administration of an additional therapeutically active ingredient (as these terms are defined herein) is considered administration of the anti-NPR1 antibody "in combination with" the additional therapeutically active ingredient.

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

[0155] Diagnostic Uses of Antibodies The antibodies of the present invention may be used to detect and / or measure NPR1 in a sample, for example, for diagnostic purposes. In some embodiments, it is contemplated that one or more antibodies of the present invention are used in assays for detecting diseases or disorders associated with NPR1. An exemplary diagnostic assay for NPR1 involves, for example, contacting a sample obtained from a patient with an anti-NPR1 antibody of the present invention, where the anti-NPR1 antibody is labeled with a detectable label or reporter molecule or is used as a capture ligand to selectively isolate NPR1 from the patient sample. Alternatively, an unlabeled anti-NPR1 antibody can be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125 Fluorescent or chemiluminescent moieties such as fluorescein isothiocyanate or rhodamine; or enzymes such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Certain 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 assays of the present invention include any tissue or fluid sample obtained from a patient, which contains detectable amounts of NPR1 protein or fragments thereof under normal or pathological conditions. Generally, the level of NPR1 protein in a particular sample obtained from a healthy patient (e.g., a patient not suffering from an NPR1-related disease) is measured to first establish a baseline or standard level of NPR1. This baseline NPR1 level can then be compared with the NPR1 level measured in a sample obtained from an individual suspected of having an NPR1-related condition or symptoms associated with such a condition.

[0157] Antibodies specific to NPR1 protein may contain no additional label or moiety, or may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if any) may determine the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, a peptide containing biotin at the N-terminus will be oriented so that the C-terminal portion of the peptide is distal to the surface.

[0158] Example The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation must be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric.

[0159] Example 1: Generation of human antibodies against natriuretic peptide receptor 1 (NPR1) Human antibodies against the NPR1 protein were generated in VELOCIMMUNE™ mice containing DNA encoding the 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 immunoassay. When the desired immune response was obtained, spleen cells were collected 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 produced NPR1-specific antibodies. Using these cell lines, several anti-NPR1 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) were obtained.

[0161] Anti-NPR1 antibodies have also been isolated directly from antigen-positive mouse B cells without fusion with myeloma cells, as described in U.S. Patent No. 7,582,298, the entire contents of which are incorporated herein by reference. Using this method, several fully human anti-NPR1 antibodies (i.e., antibodies with human variable and constant domains) have been obtained.

[0162] Exemplary antibodies produced as disclosed above are 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 methods of this example are described in detail in the Examples 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 corresponding nucleic acid sequence identifiers are shown in Table 2.

[0167] [Table 2]

[0168] Antibodies referred to herein typically have fully human variable regions but may also have human or mouse constant regions. As will be appreciated by those skilled in the art, an antibody with a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a murine IgG1 Fc can be converted to an antibody with a human IgG4 Fc), but in either case, the variable domains (including CDRs) indicated by the numerical identifiers in Table 2 remain the same, and the antigen-binding characteristics are expected to be the same or substantially similar regardless of the nature of the Fc domain. In certain embodiments, a selected antibody with a murine IgG1 Fc is converted to an antibody with 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 serine to proline mutation in the hinge region (S108P) to promote dimer stabilization. Unless otherwise indicated, all antibodies used in the following examples have the human IgG4 isotype.

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

[0170] Example 3: Antibody Binding to NPR1 by Surface Plasmon Resonance Experimental procedure The equilibrium dissociation constants (K ) of different NPR1 reagents binding to purified anti-NPR1 monoclonal antibody (mAb) D) was measured using a real-time surface plasmon resonance-based Biacore 4000 biosensor. All binding experiments 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 with either a mouse anti-human Fc-specific mAb (GE Healthcare, #BR100839) or a goat anti-human Fcγ-specific polyclonal antibody (Jackson ImmunoResearch Laboratories, #BR-1008-39) by amine coupling to capture the anti-NPR1 mAb. Binding tests were performed using the human NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (hNPR1-MMH) (SEQ ID NO: 194), the monkey NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (mfNPR1-MMH) (SEQ ID NO: 195), the mouse NPR1 extracellular domain expressed with C-terminal myc-myc-hexahistidine (mNPR1-MMH) (SEQ ID NO: 196), and the human NPR1 extracellular domain expressed with C-terminal mouse IgG2a (hNPR The experiments were carried out using monkey NPR1 extracellular domain (mfNPR1-mFc) (SEQ ID NO: 197), monkey NPR1 extracellular domain expressed in C-terminal mouse IgG2a (mfNPR1-mFc) (SEQ ID NO: 198), mouse NPR1 extracellular domain expressed in C-terminal mouse IgG2a (mNPR1-mFc) (SEQ ID NO: 199), hNPR1-mFc + hANP, hNPR1-mFc + hBNP, mfNPR1-mFc + hANP, mfNPR1-mFc + hBNP, mNPR1-mFc + mANP, and mNPR1-mFc + mBNP.hNPR1-MMH, mfNPR1-MMH at different concentrations (100 nM to 3.7 nM, 3-fold serial dilutions or 100 nM to 6.25 nM, 4-fold serial dilutions); hNPR1-mFc, mfNPR1.mFc (100 nM to 1.56 nM, 4-fold serial dilutions or 100 nM to 3.7 nM, 3-fold serial dilutions); mNPR1.mmh (100 nM), hNPR1-mFc or mfNPR1-mFc complexed with 10x concentrations of hANP or hBNP (100 nM, 25 nM, 6.25 nM or 100 nM to 3.7 nM, 3-fold serial dilutions); and mFNPR1-mFc complexed with 10x concentrations of mANP or mBNP, prepared in HBS-ET running buffer. mNPR1.mFc (100 nM, 25 nM, 100 nM-3.7 nM, 3-fold serial dilutions) or hNPR1-hFc complexed with 10x concentrations of hANP or hNPR1-hFc (100 nM-6.25 nM, 4-fold serial dilutions) were injected at a flow rate of 30 μL / min for 4 min, while dissociation of mAb bound to different NPR1 reagents was monitored for 10 min in HBS-ET running buffer. At the end of each cycle, the NPR1 mAb capture surface was regenerated by a 10-s injection of 20 mM phosphate for mouse anti-human Fc-specific mAb surfaces, a 40-s injection of 10 mM glycine, HCl, pH 1.5 for goat anti-human Fcγ-specific polyclonal antibodies, or two 1-min injections of 10 mM glycine, pH 1.5. The binding rate (k a ) and dissociation rate (k d The binding-dissociation equilibrium constant (K) was determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve-fitting software. D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rates as follows:

[0171]

number

[0172] result The binding kinetic parameters of different NPR1 proteins that bind to selected anti-NPR1 antibodies of the present invention at 25°C and 37°C are shown in Tables 3-26.

[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 to 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] No antibodies bound to mouse NPR1, except that mAb25502 bound to mNPR1 in the presence of ANP / BNP (Tables 21-26).

[0200] Example 4: Cross-competition between 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 bio-layer interferometry assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25°C with plate shaking at 1000 rpm in 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 assess whether two antibodies compete with each other for binding to their respective epitopes, 100 nM of recombinant human NPR1 (hNPR1-mFc; SEQ ID NO: 453) expressed as a C-terminal mouse IgG2a antibody was first incubated with 2 μM human ANP for at least 2 hours. First, an Octet biosensor chip (Fortebio Inc, #18-5090) was coated with anti-mouse Fc antibody. Approximately 0.3-0.5 nm of recombinant hNPR1-mFc / hANP complexes were captured by immersing the biosensor chip in a well containing the hNPR1-mFc / hANP complex for 45 seconds. The captured biosensor chip was then saturated with a 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. The biosensor chip was then immersed in a well containing a 50 μg / mL solution of a second anti-NPR1 monoclonal antibody (referred to as mAb-2) for 3 minutes. The biosensor chip was washed with HBS-EBT buffer between each step of the experiment. Real-time binding responses were monitored throughout the entire experiment, and the binding responses at the end of each step were recorded. The response of mAb-2 to binding to hNPR1-mFc / hANP pre-complexed with mAb-1 was compared to determine the competitive / non-competitive behavior of the different anti-NPR1 monoclonal antibodies.

[0201] result [Table 27-1] [Table 27-2]

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

[0203] Example 5: Antibody binding to cells expressing NPR1 Experimental procedure The ability of anti-human (h)NPR1 monoclonal antibodies to bind to cells expressing human NPR1 (hNPR1) with or without one of its ligands, human ANP (hANP), was determined using electrochemiluminescence (ECL)-based detection.

[0204] Briefly, HEK293 / hNPR1.myc.DKK-expressing cells were engineered 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 showed no detectable NPR1 expression by fluorescence-activated cell sorting (FACS) using a commercially available a-hNRP1 antibody and were included as a nonspecific binding control.

[0205] The experiment was carried out according to the following procedure. 2+ / Mg 2+ The cells were washed once with 1x PBS buffer without Ca and then incubated with enzyme-free cell dissociation solution at 37°C for 10 minutes to detach the cells from the flask. 2+ / Mg 2+ The cells were washed once with 1x PBS containing 0.05% CO₂ and counted using a Cellometer™ Auto T4 cell counter (Nexcelom Bioscience). Approximately 2.0x10 4HEK293 / hNPR1.myc.DDK or HEK293 cells were cultured on a 96-well carbon electrode plate (MULTI-ARRAY high bind plate, Meso The nonspecific binding sites were plated separately on Scale Discovery (MSD, Rockville, MD) and incubated at 37°C for 1 hour. 2+ / Mg 2+ Plates were blocked with 2% BSA (w / v) in 1x PBS containing 1% CI for 1 h at room temperature (RT). HEK293 / hNPR1.myc.DDK cells were incubated in sample dilution buffer with or without 10 nM human ANP (Tocris, Minneapolis, MN) for 0.5 h at room temperature, while HEK293 cells were incubated in sample dilution buffer alone under the same conditions. Without washing, serial dilutions of anti-NPR1, COMP1, or isotype control antibodies ranging from 1.7 pM to 100 nM, or buffer without antibody, were added to the plate-bound cells for 1 h at room temperature. The plate was then washed to remove unbound antibody and / or hANP using an AquaMax2000 plate washer equipped with a cell wash head (MDS Analytical Technologies, Sunnyvale, CA). Plate-bound antibodies were detected with heavy- and light-chain-specific SULFO-TAG™-conjugated goat polyclonal anti-human IgG antibodies (Jackson Immunoresearch, West Grove, PA) for 1 hour at room temperature.

[0206] After washing, plates were developed with Read Buffer (MSD, Rockville, MD) according to the manufacturer's recommended procedure, and luminescence signals were recorded using a SECTOR Imager 600 (MSD, Rockville, MD). Luminescence intensity, measured in relative light units (RLU), was recorded to indicate binding strength across a range of antibody concentrations. The ratio of the signal detected with 3.7 nM antibody binding to NPR1-modified cells with or without 10 nM hANP compared to the same concentration of antibody binding to parental cells without hANP was reported as an indicator of NPR1 binding specificity. Antibodies with a binding ratio of 3 or greater were considered specific binders, while 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 sigmoidal (four-parameter logistic) dose-response model using the R statistical package (open source). 50 The EC value was defined as the antibody concentration at which 50% of the maximum binding signal was detected and was determined as representing the binding capacity to NPR1 gene-modified cells with or without 10 nM hANP. 50 Values ​​are reported only for specific binders, and antibodies with ratios below 3 are marked with a (-) in Table 28.

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

[0209] [Table 28]

[0210] As the results in Table 28 show, all anti-NPR1 antibodies specifically bound to hNPR1 modified cells in the presence of 10 nM hANP at a ratio of ≥ 2. The potency of these antibodies in HEK293 / hNPR1.myc.DDK cells in the presence of 10 nM hANP was EC 50The values ​​ranged from 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 were classified as peptide-independent NPR1 binders. The potency of these antibodies in HEK293 / hNPR1.myc.DDK cells without added hANP was measured using EC 50 The values ​​ranged from 0.49 nM to 4.6 nM.

[0211] Example 6: Activation of NPR1 by agonistic anti-NPR1 monoclonal antibodies Experimental procedure To assess the transcriptional activation of 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. CNGA2 is a calcium channel activated by cGMP and can therefore 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 produced (Zois et al., 2014, PMID: 24820868), CNGA2 is activated and fluorescent Ca ++ Calcium influx can be measured using an indicator. Cell lines highly expressing hNPR1, HEK293 / hNPR1.MycDDK / CNGA2.Myc HS, or the abbreviated form 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 black clear-bottom PDL plates in DMEM containing 10% FBS, NEAA, pen / strep / glutinin, and incubated overnight at 37°C and 5% CO2. The following morning, the medium was removed, and the cells were loaded with 80 μl of FLUO4-NW Assay Buffer (Thermo Scientific) with probenecid for 30 minutes at 37°C. Purified hNPR1 antibodies or isotype control antibodies were serially diluted 1:3 from approximately 0.4 nM to 1 μM (8-point series), or ANP was serially diluted 1:3 from approximately 0.3 pM to 2 nM (10-point series), and transferred to the assay plate using a FLIPR TETRA® (Molecular Devices). Baseline and response images were taken. Dose-response curves were determined based on max-min or area under the curve (as shown here), and the results were analyzed using nonlinear regression (four-parameter logistic) in Prism™ 6 software (GraphPad) to determine EC 50 Values ​​were obtained. The % activation of the antibody was calculated by the maximum range of RFU achieved by the antibody over the maximum range of RFU achieved by ANP.

[0213] result 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 calcium flux activity of HEK293 / CNGA2 / hNPR1 in two experiments (Experiments I and II) (Table 29). As shown in Table 29 (Experiment I), 11 purified NPR1 antibodies demonstrated Ca2+ flux activation, with maximal activation ranging from 55 to 130%, and EC 50The EC values ​​ranged from 83.7 to 383.9 nM. Nineteen antibodies showed weak activation, with maximal activation less than 31% (data not shown). In Experiment II, nine anti-NPR1 antibodies had EC values ​​ranging from 41.6 to >200 nM. 50 ANP showed a maximum activation of 57-129% at EC 50 The activation activity of the control hIgG4 isotype antibody was not observed.

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

[0217] 10-20 week old male NPR1 hu / hu (n=50) mice were implanted with PA-C10 telemetry devices (DSI, St. Paul, Minneapolis, 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) and relative humidity 30%–70%) and maintained on a 12-hour light / 12-hour dark cycle. Food (Research Diets Standard pellet chow) and water were available ad libitum.

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

[0221] Systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate were collected for 10 seconds every minute during the study. Acute efficacy assessments were performed using data collected from study day 3 to study day 7. Chronic efficacy of the NPR1 agonist antibody was assessed by collecting data 24 hours a day, averaging over 28 days. All data are presented as mean ± standard error.

[0222] result In our initial in vivo screening of NPR1 agonist antibodies, we demonstrated that nine antibodies (mAb22033, mAb25479, mAb25497, mAb22805, mAb25545, mAb22809, mAb25491, mAb25502, and mAb22810) significantly reduced systemic blood pressure compared to PBS-treated control animals from days 3 to 7 after treatment, while one antibody (mAb22035) had no effect (Figure 1). The magnitude of blood pressure reduction, assessed by the mean change from baseline in systolic blood pressure from days 3 to 7 after treatment, ranged from -3.2 ± 0.2 (mAb25497) to -11.5 ± 0.8 (mAb22810) mmHg.

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

[0224] [Table 31]

[0225] Normotensive NPR1 hu / hu In mice, one antibody (mAb22035) significantly increased blood pressure by approximately 4-7 mmHg, while the remaining antibodies decreased systemic blood pressure by 2-11 mmHg after a single subcutaneous injection on day 0. Heart rate responses to increases and decreases in systemic blood pressure were variable, increasing in some groups and decreasing in others.

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

[0227] [Table 32]

[0228] Animals were housed individually under standard conditions (temperature 64°F–84°F (18°C–29°C) and relative humidity 30%–70%) and maintained on a 12-hour light / 12-hour dark cycle. Food (Research Diets Standard pellet chow) and water were available ad libitum.

[0229] Test proteins were administered to appropriate animals via subcutaneous injection once on day 0. Dose volumes for each animal were based on the most recent body weight measurement. Urine was collected on day 28, and blood samples were collected on day 14 and at the end of the study for urinary and serum biomarker assessment. Echocardiograms were performed on day 28, prior to diuresis.

[0230] Systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate were collected for 10 seconds every minute during the study. The telemetry data shown was obtained from animals with a vital signal during the survival portion of the study.

[0231] result Blood pressure (Figures 2, 3 and 5) was measured using normotensive NPR1 hu / hu After a single dose of mAb 22033 in mice, blood pressure was reduced by 10–15 mmHg for up to 4 weeks. Peak blood pressure reductions were similar at all doses, and the duration of the blood pressure effect ranged from approximately 10 days at 1 mg / kg to more than 28 days at 50 mg / kg.

[0232] [Table 33]

[0233] Urinary cGMP concentrations (Table 34) were significantly increased at day 28 in the 50 mg / kg group and were at levels similar to controls at all other doses.

[0234] [Table 34]

[0235] Relative heart weight (Table 35) was lower in the 50 mg / kg group and tended to decrease in the 1, 5, and 25 mg / kg groups. No significant effects were observed on body weight (Table 35), absolute heart weight (Table 35), normal 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) chemistries.

[0236] [Table 35]

[0237] After administration of mAb22033, cardiac function (Figs. 6 and 7A) improved, with a statistically significant decrease in end-systolic volume in the 25 mg / kg dose group (Fig. 7A) and a trend toward increases in both fractional shortening (Fig. 7C) and ejection fraction (Fig. 7B), most evident in the 25 and 50 mg / kg dose groups.

[0238] Importantly, the NPR1 agonist mAb, mAb22033, significantly reduced systolic and mean arterial blood pressure, sustained for up to 28 days. A compensatory increase in heart rate was initially observed in all groups, with a persistent, minor increase in heart rate in the 50 mg / kg mAb22033 dose group compared with the isotype control mAb.

[0239] Example 9: Hypertensive NPR1 hu / huEffects of a single dose of agonist anti-NPR1 monoclonal antibody on systemic blood pressure in mice Experimental procedure The purpose of this study was to investigate the effects of telemetry on angiotensin II (Ang II)-induced hypertension using NPR1 hu / hu The purpose of this study was to evaluate the effect of a single administration of NPR1 agonist antibodies (mAb22033 or mAb22810) on systemic blood pressure in male NPR1 mice approximately 13 weeks of age. hu / hu (n=36) Mice were implanted with PA-C10 telemetry devices (DSI, St. Paul, MN) and allowed to recover for 7 days before being assigned to groups (Groups 1-6) (Table 36).

[0240] [Table 36]

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

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

[0243] Systolic blood pressure, diastolic blood pressure, mean arterial pressure, and heart rate were collected for 10 seconds every minute during the study period. Urine samples were collected on days 14 and 20.

[0244] result A single dose of NPR1 agonist mAb (mAb22033 or mAb22810) suppresses angiotensin II-induced hypertension. hu / hu When administered to mice, mAb22033 significantly reduced systemic blood pressure (Figures 8, 9, and 11). The 23-day mean blood pressure (Table 37) was all 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] Heart rate effects (Figure 10) showed significant increases acutely and more modest increases chronically. Mean 23-day heart rates (Table 37) were significantly higher in all test article-treated groups compared with isotype control animals. Urinary cGMP levels tended to be higher in most groups treated with either mAb22033 or mAb22810, with animals receiving 25 mg / kg mAb22033 showing significantly increased urinary cGMP levels compared with IgG isotype control animals on days 14 and 20 (Table 38). No effects on body weight, absolute, or relative organ weights were seen (Table 39).

[0249] Example 10: NPR1 in hypertension hu / hu Effect of repeated administration of agonist anti-NPR1 monoclonal antibody on systemic blood pressure in mice Experimental procedure The purpose of this study was to investigate the effect of telemetry on angiotensin II (Ang II)-induced hypertension (NPR1h). u / huThe purpose of this study was to evaluate the effect of repeated administration of the NPR1 agonist antibody mAb22033 on systemic blood pressure in male NPR1 mice approximately 26 weeks of age. hu / hu (n=30) Mice were implanted with PA-C10 telemetry devices (DSI, St. Paul, MN), allowed to recover for 7 days, and then assigned to groups (Groups 1-5) (Table 40).

[0250] [Table 40]

[0251] The animals were then implanted with osmotic minipumps (Alzet Micro-Osmotic Pump; Model 1004; Lot 10335-14). The minipumps were filled with angiotensin II acetate (Bachem; Lot #1066804) and set at an average pumping rate of 0.11 μL / h to deliver 1.5 mg / kg / day of angiotensin II. The minipumps were implanted subcutaneously over the scapulae 7 days before the start of treatment. The animals were housed individually under standard conditions (temperature 64°F–84°F (18°C–29°C) and relative humidity 30%–70%) and maintained on a 12-hour light / 12-hour dark cycle. Food (Research Diets Standard pellet chow) and water were available ad libitum.

[0252] Animals were stratified into groups based on systolic blood pressure. Test proteins were administered to appropriate animals by subcutaneous injection once on day 0 (Group 5) or twice weekly for 3 weeks starting on day 6 (Groups 1-4). Dose volume for each animal was based on the most recent body weight measurement.

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

[0254] result Single or repeated administration of the NPR1 agonist mAb 22033 inhibits angiotensin I NPR1 I-induced hypertension hu / huIn mice, systemic blood pressure (Figures 12, 13, and 15) was reduced to near-normal levels. Repeated administration of 1, 5, or 25 mg / kg of mAb22033 dose-dependently reduced systolic blood pressure, with maximum reductions from baseline of 11, 19, and 39 mmHg in each group after 3 weeks of twice-weekly administration. A single dose of 50 mg / kg reduced systolic blood pressure by 31 mmHg by day 7, followed by a gradual return to more hypertensive levels. 21-day mean blood pressure (Table 41) was all significantly lower in animals receiving either a single or repeated dose of mAb22033 compared with the IgG4 isotype control.

[0255] [Table 41]

[0256] [Table 42]

[0257] [Table 43]

[0258] The acute effects of mAb 22033 were analyzed, with a 10-20 mmHg reduction in blood pressure within 24 hours of the first dose. Heart rate effects (Figure 14) were variable, with higher and lower values ​​observed during the 21-day treatment period. The 21-day mean heart rate (Table 41) was significantly lower in the 5 and 25 mg / kg repeat-dose groups and tended to be higher in the 50 mg / kg single-dose group. Urinary cGMP levels were significantly elevated in the 25 mg / kg repeat-dose group, with all other groups showing a statistically insignificant trend toward elevation compared to animals treated with the IgG4 isotype control mAb (Table 43). No effects were observed on body weight, absolute or relative organ weights, standard serum or urine chemistries, or cardiac function (Table 42 and Figures 16-17).

[0259] Example 11: Effects of anti-NPR1 agonist antibodies on body weight, metabolic rate, and glucose homeostasis in diet-induced obese (DIO) mice Experiment 1 In this experiment, we tested the effects of mAb22810 NPR1 agonist mAb on body weight, metabolic rate, and glucose homeostasis in diet-induced obese (DIO) mice.

[0260] 30 male NPR1 hu / hu Mice were fed a 60% high-fat diet for 10 weeks and then randomized to one of three groups (n=10 per group): an isotype control (human IgG4) antibody, the NPR1 agonist antibody mAb22810, or hFc.FGF21. FGF21 is a molecule that has been shown to improve glucose tolerance, increase energy expenditure, and reduce body weight in obese mouse models (Veniant MM, Endocrinology, 2012). 012, PMID: 22798348). It was used as a positive endpoint control in this study. Treatments were administered by subcutaneous injection (SC) in saline vehicle either weekly (for control antibody and mAb22810) or twice weekly (for hFc.FGF21). Table 44 shows the groups, number of animals, and doses in the study.

[0261] [Table 44]

[0262] Individual body weights were recorded before treatment and twice weekly thereafter. During the second week of the study, mice were placed in metabolic cages to assess energy expenditure. During the third week of the study, oral glucose tolerance was assessed, and body composition was measured by EchoMRI after 6 weeks of treatment.

[0263] Figure 18A shows the change in body weight after administration of mAb22810 NPR1 agonist mAb, hFc.FGF21, or isotype control mAb. Figures 18B and 18C show total fat mass and total lean mass, respectively, measured by EchoMRI after 6 weeks of treatment. An important finding is that the hFc.FGF21 molecule resulted in 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 V(O2) (A), V(C) (B), or energy expenditure (C) after one week of treatment with either mAb22810 NPR1 agonist mAb, hFc.FGF21, or an isotype control mAb, divided as the average value for each day-night cycle. A key finding is that the hFc.FGF21 molecule resulted in significant increases in V(O2), V(C) and energy expenditure during the treatment period, whereas the mAb22810 NPR1 agonist antibody did not.

[0265] Figure 20A shows the change in glucose tolerance as measured by an oral glucose tolerance test (2 g / kg glucose) after two weeks of treatment with either mAb22810 NPR1 agonist mAb, hFc.FGF21, or an isotype control mAb. Figure 20B shows blood glucose levels after an overnight fast recorded at the start of the study in A. An important finding is that both mAb22810 and hFc.FGF21 molecules resulted in significant improvements in glucose tolerance after two weeks. Furthermore, the improvement in glucose tolerance by mAb22810 was independent of changes in body weight or energy expenditure (as shown in Figures 18 and 19).

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

[0267] 30 male NPR1 hu / huMice were fed a 60% high-fat diet for 10 weeks and then randomized to three groups (n=10 per group): isotype control (human IgG4) antibody, NPR1 agonist antibody mAb22033, or hFc.FGF21 as a positive control. Treatments were administered by subcutaneous injection (SC) in saline vehicle either once a week (for control antibody and mAb22033) or twice a week (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 before and twice weekly thereafter. During the second week of the study, mice were placed in metabolic cages to assess energy expenditure. During the fourth week of the study, oral glucose tolerance was assessed, and body composition was measured by EchoMRI after 6 weeks of treatment.

[0270] Figure 21A shows the change in body weight after administration of mAb22033 NPR1 agonist mAb, hFc.FGF21, or isotype control mAb. Figures 21B and 21C show total fat mass and total lean mass, respectively, after 6 weeks of treatment, as measured by EchoMRI. An important finding is that the hFc.FGF21 molecule resulted in 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 V(O2) (A), V(C) (B), or energy expenditure (C) after one week of treatment with either the mAb22033 NPR1 agonist mAb, hFc.FGF21, or an isotype control mAb, divided as the average value for each day-night cycle. A key finding is that the hFc.FGF21 molecule resulted in significant increases in V(O2), V(C) and energy expenditure during the treatment period, whereas the mAb22033 NPR1 agonist antibody did not.

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

[0273] Example 12: HDX epitope mapping To determine the epitope of human NPR1 recognized by the anti-NPR1 antibodies, we performed hydrogen-deuterium exchange (HDX) studies on mAb22033 and mAb22810, respectively. Previous in-house experiments have shown that the presence of the ANP peptide is required for NPR1 binding to mAb22810. Therefore, in addition to conventional HDX experiments using the NPR1 / mAb22033 complex, we also performed HDX experiments on the NPR1 / ANP / mAb22033 complex and the NPR1 / ANP / mAb22810 complex.

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

[0275] Deuteration buffer was prepared in DO containing 137 mM NaCl, 2.7 mM KCl, 8 mM NaHPO, and 2 mM KHPO (pD = 7.4). For either "antigen-on" or "complex-on" experiments, 30 μL antibody bead slurry (equivalent to 15 μL beads) was mixed with hNPR1.mmh or hNPR1.mmh / ANP complex. The mixture was incubated at room temperature with gentle rotation. Deuteration was quenched with 0.075% ice-cold TFA during elution of hNPR1.mmh from the antibody beads. The quenched sample was immediately injected into a Waters HDX Manager and subjected to online pepsin digestion (Waters Enzymate BEH Pepsin Column, 2.1 x 30 mm). Digested peptides were captured on an ACQUITY UPLC BEH C18 1.7 μm, 2.1 x 5 mm VanGuard precolumn at 0 °C 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 to a cone voltage of 37 V, a scan time of 0.5 s, and a mass / charge range of 50-1700 Th.

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

[0277] The second experiment is referred to as the "conjugate-on" format (HDX of conjugated antigen / antibody beads). In this experiment, hNPR.mmh was first bound to mAb22033 beads in regular PBS (pH = 7.4) for 2 min. The conjugate was then deuterated by incubation in PBS-D (pH = 7.4) for 5 or 10 min (in separate preliminary experiments). The following steps (elution, injection, pepsin digestion, and MS analysis) were performed as described in the previous "antigen-on" procedure.

[0278] LC-MS from undeuterated hNPR1.mmh for peptide identification and deuterium incorporation measurements EThe data were 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 by two criteria: 1) a minimum product per amino acid of 0.3, and 2) a replicate file threshold of 2. DynamX software then allowed us to determine the deuterium uptake of each peptide from the "antigen-on" and "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] Centroid values ​​or average mass-to-charge ratios (m / z) of all detected peptides were calculated and analyzed for the two time points in the "antigen-on" and "complex" experiments. Peptides that showed increased mass after deuteration in the "antigen-on" compared to after deuteration in the "complex-on" contained amino acids that were protected from deuterium exchange as a result of antibody binding, and thus represent the binding epitope region.

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

[0281] [Table 46]

[0282] Because two peptides, amino acids 46–54 and 328–335, showed no difference in deuterium incorporation between the "antigen-on" and "complex-on" procedures, the regions protected from deuterium exchange in peptides 29–50 and 328–347 were reduced to residues 29–45 and 336–347. Thus, the two segments containing amino acids 29–45 and 336–347 are identified as the epitopes of antibody mAb22033, which binds to the hNPR1.mmh protein.

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

[0284] [Table 47]

[0285] Another peptide, amino acids 46–54, showed no difference in deuterium incorporation between the "antigen-on" and "complex-on" procedures, so the region protected from deuterium exchange in the 29–50 peptide was reduced to residues 29–45. Thus, two segments encompassing amino acids 29–45 and 331–347 are identified as the epitopes of antibody mAb22033, which 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, representing 70% sequence coverage. Of these peptides, 10 peptides covering amino acids 29-50, 70-81, and 331-347 showed significant mass increases after deuterium exchange in the "antigen-on" compared to the "complex-on" deuterium exchange, as shown in Table 48.

[0287] [Table 48]

[0288] Because three peptides, amino acids 46–54, 336–347, and 337–347, showed no difference in deuterium incorporation between the "antigen-on" and "complex-on" procedures, the regions protected from deuterium exchange for peptides 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 epitopes of antibody mAb22810, which binds to the hNPR1.mmh / ANP protein complex.

[0289] Example 13: Reduction of intraocular pressure by intravitreal injection of NPR1 antibody 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] Methods: Humanized NPR1 mice (NPR1 hu / hu ) was produced 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 of Abs, mice were injected with an AAV2 vector expressing NPR1 antibody or eGFP, and intraocular pressure was monitored over a 7-week period.

[0291] Result: NPR1 hu / huIntravitreal injection of 40 μg of mAb22033 into mice significantly reduced intraocular pressure from day 1 to day 3 compared with control antibody. The mean change in intraocular pressure was 5 mmHg. However, there was no intraocular pressure-lowering effect in WT mice. In a dose-response study, intravitreal injection of 40 or 12.6 μg of mAb22033 produced similar intraocular pressure-lowering effects, but the effect of 40 μg of mAb22033 lasted longer than that of 12.6 μg. Intravitreal injection of 4 μg of NPR1 antibody did not reduce intraocular pressure. No intravitreal effect on intraocular pressure was observed after intravitreal injection of AAV2-GFP or AAV2-NPR1 antibody at any experimental time point. This may be due to the low expression of the NPR1 antibody, i.e., only 10 ng was detected in the whole eye lysate.

[0292] Conclusion: Intravitreal administration of human NPR1 antibody (mAb22033) in humanized NPR1 mice significantly reduced intraocular pressure, indicating the potential of agonistic anti-NPR1 antibodies for reducing intraocular pressure in glaucoma disease.

[0293] Example 14: Structural analysis of antibody-NPR1 complexes by electron microscopy method Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) Titration Several titration series of human NPR1 extracellular domain (hNPR1-mmh; SEQ ID NO: 194) with a C-terminal myc-myc-6xHis tag were prepared in complex with various antibodies at different molar ratios. 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 a 2-fold molar excess of atrial natriuretic factor (ANP, Tocris) relative to hNPR1-mmh. After overnight incubation in PBS at 4°C, the complexes were injected into a SEC-MALS system. This system was equipped with an AKTA microphone. The SEC column consisted of a Superdex 200 Increase 10 / 300 GI column on a SEC system (GE Healthcare Life Sciences), followed by a miniDAWN Treos and Optilab T-rEX (Wyatt Technology Corporation). Because phosphate buffer is incompatible with negative staining in electron microscopy, the SEC column was equilibrated with a running buffer of 50 mM Tris pH 7.5, 150 mM NaCl, and all large complexes prepared below were in this buffer. Size-exclusion chromatography data were evaluated using Unicorn (Version 5.20 General Electric Company), and MALS data were evaluated using ASTRA (Version 7.0.0.69 Wyatt). The evaluation was carried out using the Technology.

[0294] Negative stain electron microscopy sample preparation For use in negative staining electron microscopy, hNPR1 complexes were prepared on a larger scale. Five samples were prepared: Sample 1 = hNPR1-mmh (SEQ ID NO: 194) alone; Sample 2 = hNPR1-Fc (SEQ ID NO: 197) alone; Sample 3 = hNPR1-mmh + ANP (1:2 molar ratio); Sample 4 = hNPR1-mmh + REGN5308 (1:1.5 molar ratio); and Sample 5 = hNPR1-mmh + ANP + REGN5308 (1:2:1.5 molar ratio). Samples 1, 3, 4, and 5 were purified by size exclusion chromatography using the same method as for the SEC-MALS experiments. Peak fractions were collected, frozen at -80°C, and sent to NanoImaging Services, Inc. for EM analysis. Sample 2 was taken directly from a 2.62 mg / ml stock solution 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 shipped with the other samples.

[0295] Negative stain electron microscopy data collection and processing Negatively stained EM grids were prepared for the five protein samples using standard methods with uranyl formate (NanoImaging Services). The grids comprised a thin layer of continuous carbon placed on a C-flat holey carbon grid. TEM images were collected at room temperature using a Tecnai T12 electron microscope (FEI / Thermo Fisher) operated at 120 keV with an FEI Eagle 4k x 4k CCD camera. Images were collected at various nominal magnifications, primarily 67,000x and 110,000x. The collected images were further processed in-house.

[0296] NanoImaging micrographs were visually inspected, and images with poor contrast due to contamination, approximately one-fifth of the total image, were removed. The remaining images were divided by magnification. The 110,000x image was not very useful for further analysis due to the low particle count per image. Therefore, all subsequent processing steps were performed using the 67,000x image. All images were CTF corrected using CTFFIND4.

[0297] EM particle picking and 2D class averaging The particle distributions for all five negatively stained samples were very good, with uniform particle size, minimal clumping, and good particle density. For samples 4 and 5, particles were picked using Relion with an automated picking template derived from initial manual picking and 2D class averaging. For sample 4 (hNPR1 + REGN5308), 19,184 good particles were selected from a total of 75 micrographs. For sample 5 (hNPR1 + ANP + REGN5308), 20,318 good particles were initially selected from a total of 88 micrographs. Initial 2D class averaging using Relion revealed substantial heterogeneity in both datasets, with a significant number of classes representing only REGN5308 Fab or only NPR1.

[0298] The 2D class average for sample 5 was further refined by eliminating particles corresponding to only Fab or only NPR1. A new 2D class average was calculated using the remaining 9219 particles, which provided a better picture of the NPR1 + REGN5308 complexes, revealing that a minority of complexes contained only one Fab bound to an NPR1 dimer, while the majority of complexes contained two Fabs. Removal of single-Fab complexes further reduced the particle set to 6728 particles.

[0299] 3D image reconstruction from negative stain EM data An initial 3D model of the NPR1-ANP-REGN5308 complex was constructed in Relion using the stochastic gradient descent "3D initial model" procedure, limited to a resolution of 40 Å. This model was then low-pass filtered to 60 Å, limiting the predicted resolution to 25 Å, and used as the basis for 3D classification of the 6,728 particles in Relion. The best 3D class was then further refined in Relion until convergence was achieved, with a final resolution of 22 Å, as measured by the "gold standard" FSC. Note that two-fold symmetry was not considered during 3D classification or refinement. The resulting density map of the 3D reconstruction showed two Fabs bound to one side of a square particle, consistent with the crystal structure of ANP-bound NPR1 (PDB code 1T34).

[0300] Cryo-EM sample preparation and data collection Samples of the NPR1-ANP-REGN5308 complex were prepared for cryo-electron microscopy (cryoEM) in a similar manner to 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 techniques with UltrAuFoil grids (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 K2 direct electron detector in counting mode and a GIF energy filter (Gatan, Inc.). The magnification was 130,000x (1.04 Å / pixel), with a defocus range of -0.5 to -1.5 microns, and a total dose of 45.44 e. - / Å 2 A total of 1409 videos were collected using Leginon software.

[0301] CryoEM data processing and structure determination All cryoEM movies were motion-corrected, dose-weighted, and CTF-corrected using the cisTEM package. Images were then manually inspected to remove images with thick ice, poor CTF parameters (fit resolution worse than 6 Å), missing particles, or contaminants. After this filtering, 1,172 images remained, which were then used for non-template particle picking in cisTEM, yielding 872,915 particle locations. After 2D classification to remove bad particles, 3D automatic refinement using cisTEM with the initial generated starting 3D reference volume contained 686,709 particles. 3D refinement converged to a single solution at a resolution of 2.8 Å, estimated from the Fourier-shell correlation curve.

[0302] This 3D map was then used for structural refinement, starting from the model incorporated into the negative-stain EM 3D map described above. The N- and C-terminal domains of both NPR1 molecules were refined in real space as rigid bodies in the EM map, followed by manual reconstruction in some locations where the model did not match the EM density. The homology model of REGN5308 was manually placed into the EM density. Careful observation of the CDR regions allowed us to determine the orientation of the heavy chain relative to the light chain. The CDR regions of this model required extensive reconstruction to match the EM density. Finally, the real-space positions were refined using Phenix to create an updated structural model of the complex.

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

[0304] We then simplified the system by considering the Fab fragment of mAb22033, REGN5308. The complex of hNPR1-mmh and REGN5308 exhibited a significantly different SEC profile due to the bound ANP compared to the same complex without ANP. The NPR1-REGN5308 complex had a molecular weight of approximately 155 kDa, consistent with one Fab bound per NPR1 dimer. In the presence of ANP, the molecular weight of the NPR1-ANP-REGN5308 complex increased by approximately 50 kDa, consistent with two Fabs bound per NPR1 dimer. We propose that the previously described conformational change in NPR1 upon ANP binding (Ogawa, H. et al., 2004) allows for the binding of a second Fab, and that the growth of the paper doll polymer observed with intact IgG mAb22033 requires a complex of two Fabs, two NPR1s, and ANP (see below for further discussion).

[0305] Titrations with hNPR1-mmh and mAb22810 were also performed. SEC-MALS analysis indicated that a small portion of the mAb22810 sample was dimeric, with a molecular weight of approximately 315 kDa, compared with the molecular weight of 150 kDa for standard IgG. SEC-MALS titrations with the NPR1-mAb22810-ANP complex showed a heterogeneous mixture of some kind in the 430–700 kDa range. This profile was too complex for reliable interpretation, likely due to the presence of a specific molecule in the mAb22810 protein. This is due to an IgG dimer impurity present in the NPR1 complex. SEC-MALS titrations with REGN5314, the Fab fragment of mAb22810, showed that in the absence of ANP, binding of REGN5314 to NPR1 was too weak or transient to generate a complex species that could be isolated by SEC. In the presence of ANP, a single NPR1-REGN5314-ANP complex was formed with a molecular weight of approximately 170 kDa, consistent with one Fab bound per NPR1 dimer.

[0306] Negative staining 2D class averages The hNPR1+REGN5308 and hNPR1+ANP+REGN5308 complexes were further analyzed by negative-stain electron microscopy. 2D classification and averaging of the complex particles revealed substantial heterogeneity in the samples. The majority of particles on the EM grid could be classified as either hNPR1 alone or REGN5308 Fab alone. Because the protein samples submitted for imaging were purified homogeneous complexes, these complexes likely dissociated into their components during the process of creating the negative-stain grids. However, a significant portion of the complexes remained intact and could be used for analysis.

[0307] The negative-stained 2D class average of hNPR1 + REGN5308 shows a "one-armed" complex, in which only a single Fab is seen bound to the hNPR1 dimer, consistent with the SEC-MALS results. In contrast, the negative-stained 2D class average of hNPR1 + ANP + REGN5308 shows a "two-armed" complex, in which two Fabs are bound to the dimer. Different averages represent different projections of the actual 3D complex. In one class average, the hNPR1 dimer is viewed from the side, with two lobes of density, one corresponding to the overlapping N-terminal domains of the two monomers, and the other corresponding to the overlapping C-terminal domains. In this orientation, the two bound Fabs appear as "bunny ears" on top of the NPR1 density. Alternatively, all four domains of hNPR1 can be seen as four dense clusters forming a square, with the two REGN5308 Fabs on top of the square crossing each other to form an inverted V. The class average of hNPR1+ANP+REGN5308 also showed "one-arm" complexes, likely due to dissociation of the same complex that generated free Fab and free NPR1.

[0308] The 2D class averages calculated from the hNPR1+ANP+REGN5308 cryoEM data show a different view of the complex compared to the negative-stain data, notably the absence of the "rabbit ear" orientation. However, other cryoEM 2D class averages can be closely matched with the corresponding averages from the negative-stain data. The cryoEM data show less evidence of complex dissociation, likely due to a more homogeneous starting sample and the low-temperature freezing conditions better preserving the intrinsic conformation of the complex in solution.

[0309] 3D image reconstruction Using the 2D class averages as a starting point, a 3D map of the cryoEM density of hNPR1 + ANP + REGN5308 was constructed. Because 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, the resulting cryoEM map is not biased by expectations of how the antibody-target complex should form. The known hNPR1 + ANP crystal structure, along with a model of the REGN5308 Fab generated by homology with the known Fab structure, was then aligned and refined onto this EM density map. The NPR1 and Fab structures were manually aligned in approximate positions and then refined as rigid bodies in the correct positions using Phenix. The resolution of the cryoEM map is sufficient to manually reconstruct residues in the NPR1:antibody contact interface, particularly those in the REGN5308 complementarity-determining regions (CDRs), which cannot be accurately modeled by homology. The current structural model places all antibody CDR residues and NPR1 residues in contact. The more distal regions of the model (the C-terminal domain of NPR1 and the constant domains of the antibody Fabs) were modeled using a combination of the current cryoEM map, the previously determined X-ray crystal structure of NPR1 (PDB code 1T34), and the isolated antibody structure.

[0310] mAb22033 epitope on NPR1Inspection of the hNPR1+ANP+REGN5308 structure revealed which residues of NPR1 contact the REGN5308 Fab (and thus the parental IgG mAb22033). This epitope is composed of four distinct stretches of NPR1 amino acids: residues 2-4, 41-45, 47, 73-79, 332, 336-344, and 347 (numbered according to SEQ ID NO:194), which combine to form a three-dimensionally continuous surface. While previous hydrogen / deuterium exchange (HDX) mass spectrometry experiments identified several of these residues as important (see Example 12), the cryo-EM structure provides more detail about the epitope.

[0311] Structural mechanism of action of mAb22033 The two Fabs in this model of the NPR1-antibody complex are within approximately 10 Å of each other, near the "elbow" between the Fab variable and constant domains. The C-termini of the two Fabs are far enough apart, approximately 100 Å, that they cannot represent the two arms of a single IgG molecule. The Fabs are also not particularly close to the modeled ANP peptide (closest approach is approximately 30 Å), so a direct interaction between the Fabs and ANP is unlikely.

[0312] Assuming a fixed position and relative orientation of the Fab with respect to the binding site on the N-terminal domain of NPR1, an explanation for the ANP-dependent binding of the REGN5308 Fab becomes clear. Upon ANP binding, NPR1 has been shown to undergo a conformational change in which one NPR1 monomer rotates relative to the other NPR1 monomer while remaining dimerized. Applying this rotation to one half of the two NPR1 + two Fab complexes results in a model in which the NPR1 dimer resembles the crystal structure of NPR1 without ANP. However, in this case, one of the REGN5308 Fabs rotates into a position that sterically clashes with the other Fab, a physically impossible situation. This steric interference explains 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, binding of the first Fab blocks binding of the second Fab.

[0313] Conversely, the binding of two Fabs to an ANP-containing NPR1 dimer prevents the complex from relaxing back to an ANP-free structure as long as both Fabs remain bound. Assuming the effect observed here is maintained at the cell surface, at equilibrium, this would increase the proportion of NPR1 molecules in an active state capable of downstream signaling. Another possible effect of antibody binding is the formation of oligomeric clusters of 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 ceases at a much smaller species containing only a maximum of one IgG with an NPR1 dimer bound to each Fab arm. Both of these effects, receptor clustering and the extension of the receptor's active state, could 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. Indeed, 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 accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to natriuretic peptide receptor 1 (NPR1) protein, wherein the antibody or antigen-binding fragment thereof 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 and activates NPR1.

2. 2. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or 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.

3. 3. The isolated antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof 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.

4. 3. The isolated antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof 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.

5. 3. The isolated antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof 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.

6. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to natriuretic peptide receptor 1 (NPR1) protein in the presence of atrial natriuretic peptide (ANP), wherein the antibody or antigen-binding fragment thereof 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 331-335 of SEQ ID NO: 194, but not with amino acids 70-81 of SEQ ID NO: 194, as determined by hydrogen / deuterium exchange, and wherein the antibody or antigen-binding fragment thereof (i) binds to cells expressing human NPR1 in the presence or absence of ANP; and / or (ii) binds to and activates NPR1.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is a fully human monoclonal antibody.

8. The antibody (a) is a fully human monoclonal antibody; (b) has a dissociation constant (K) 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. Ｄ ) binds to monomeric human NPR1; (c) has 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. Ｄ and binds to dimeric human NPR1 (d) a K of less than 80 nM at 25°C and 37°C as measured by surface plasmon resonance; Ｄ (e) a K of less than 20 nM at 25° C. and 37° C. as measured by surface plasmon resonance. Ｄ (f) binds to human NPR1 complexed with BNP at a 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. Ｄ (g) binds to monomeric monkey NPR1 with a 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. Ｄ (h) binds to dimeric monkey NPR1 with a K of less than 10 nM at 25°C and 37°C as measured by surface plasmon resonance. Ｄ (i) binds to monkey NPR1 complexed with ANP with a K of less than 10 nM at 25°C and 37°C as measured by surface plasmon resonance. Ｄ (j) binds to monkey NPR1 complexed with BNP; (k) does not bind to mouse NPR1; and (k) has an EC50 of less than 5 nM on cells expressing human NPR1 (without ANP) or NPR1 complexed with ANP. ５０ (l) an EC50 of less than 385 nM as measured by a calcium flux cell-based bioassay; ５０ (m) activates NPR1 at 2400 nm; (m) reduces systemic blood pressure when administered to normotensive and hypertensive mice, and the reduction in systemic blood pressure and mean arterial blood pressure persists for up to 28 days after administration of a single dose; and (n) improves glucose tolerance when administered to diet-induced obese mice.

9. 9. The antibody or antigen-binding fragment of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR); and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR), wherein the HCVRs have an amino acid sequence selected from the group consisting of the HCVR sequences set forth in Table 1.

10. 10. The antibody or antigen-binding fragment thereof according to claim 9, comprising an LCVR having an amino acid sequence selected from the group consisting of the LCVR sequences set forth in Table 1.

11. (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, and 180; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, and 182; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, and 184; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, and 188; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, and 190; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, and 192. The antibody or antigen-binding fragment thereof according to claim 9 or 10, comprising:

12. The antibody or antigen-binding fragment thereof described in claim 11, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, and 178 / 186.

13. The antibody or antigen-binding fragment thereof of claim 12, comprising CDRs selected from the group consisting of: (a) SEQ ID NOs: 4, 6, 8, 12, 14, and 16; and (b) SEQ ID NOs: 68, 70, 72, 76, 78, and 80.

14. The antibody or antigen-binding fragment thereof according to claim 13, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, and 66 / 74.

15. An antibody or antigen-binding fragment thereof that binds to a natriuretic peptide receptor 1 (NPR1) protein, wherein the antibody or antigen-binding fragment comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within an HCVR; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR, wherein the HCVR is (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 to 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 to 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 LCVR is (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 to 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 to 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. An antibody or antigen-binding fragment thereof comprising:

16. The antibody or antigen-binding fragment thereof of claim 15, comprising an HCVR having 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.

17. The antibody or antigen-binding fragment thereof of claim 15, comprising an LCVR having 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.

18. The antibody or antigen-binding fragment thereof according to any one of claims 15 to 17, comprising three CDRs contained within an HCVR selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, and 178; and three CDRs contained within an LCVR selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and 186.

19. The antibody or antigen-binding fragment of any one of claims 15 to 18, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, and 178 / 186.

20. (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, and 180; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, and 182; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, and 184; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, and 188; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, and 190; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, and 192. The antibody or antigen-binding fragment thereof of claim 15, comprising:

21. The antibody or antigen-binding fragment thereof of claim 20, comprising CDRs selected from the group consisting of: (a) SEQ ID NOs: 4, 6, 8, 12, 14, and 16; and (b) SEQ ID NOs: 68, 70, 72, 76, 78, and 80.

22. 22. The antibody or antigen-binding fragment thereof of claim 21, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, and 66 / 74.

23. An isolated monoclonal antibody or antigen-binding fragment thereof that activates natriuretic peptide receptor 1 (NPR1) protein, comprising three CDRs of HCVR, wherein the HCVR has 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; and three CDRs of LCVR, wherein the LCVR has 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.

24. An antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof of any one of claims 1 to 23 for binding to natriuretic peptide receptor 1 (NPR1) protein.

25. An antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody or antigen-binding fragment thereof according to any one of claims 1 to 23.

26. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that binds to the natriuretic peptide receptor 1 (NPR1) protein of any one of claims 1 to 25, and a pharmaceutically acceptable carrier or diluent.

27. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of the antibody of any one of claims 1 to 25.

28. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the LCVR of the antibody of any one of claims 1 to 25.

29. 29. A vector comprising the polynucleotide sequence of claim 27 and / or the polynucleotide sequence of claim 28.

30. A host cell expressing the vector of claim 29.

31. A method for producing an anti-NPR1 antibody or its antigen-binding fragment, comprising culturing the host cell described in claim 30 under conditions that allow the production of the antibody or fragment, and recovering the antibody or fragment thus produced.

32. 32. The method of claim 31, further comprising formulating the antibody or antigen-binding fragment thereof as a pharmaceutical composition comprising an acceptable carrier.

33. A method for treating, preventing, or ameliorating at least one symptom or sign of an NPR1-related disease or disorder, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 25.

34. The method of claim 33, wherein the NPR1-related disease or disorder is selected from the group consisting of hypertension, heart failure, obesity, renal failure, chronic kidney disease, macular edema, glaucoma, stroke, lung damage, pulmonary fibrosis, inflammation, asthma, skeletal growth disorders, fractures, diabetes, and cancer.

35. 35. The method of claim 33 or 34, wherein the pharmaceutical composition is administered prophylactically or therapeutically to a subject in need thereof.

36. The method of any one of claims 33 to 35, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

37. 37. The method of claim 36, wherein the second therapeutic agent is selected from the group consisting of aldosterone antagonists, alpha-adrenergic blockers, angiotensin-converting enzyme (ACE) inhibitors, arterial vasodilators, autonomic ganglionic vasodilators, beta-adrenergic blockers, catecholamine-depleting sympathomimetic agents, central alpha-2 adrenergic agonists, calcium channel blockers, diuretics, renin inhibitors, anticoagulants, antiplatelet agents, cholesterol-lowering agents, vasodilators, digitalis, surgery, implantable devices, anti-tumor therapy, insulin, GLP1 agonists, metformin, dialysis, bone marrow stimulants, hemofiltration, lifestyle modification, and nutritional supplements.

38. The method of any one of claims 33 to 37, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly.

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