EA046104B1 - ANTI-NPR1 ANTIBODIES AND THEIR APPLICATIONS - Google Patents

Description

This invention was filed on October 18, 2019 as a PCT international patent application and claims priority to U.S. Provisional Application No. 62/749,557 filed on October 23, 2018; and 62/755,720, filed November 5, 2018, the disclosures of each of which are incorporated herein by reference in their entirety.

Field of technology to which the invention relates

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

State of the art

Natriuretic peptide receptor 1 (NPR1; also known as NPR-A) belongs to the family of cell surface guanylyl cyclase receptors, enzymes that catalyze the conversion of GTP to cyclic GMP. NPR1 has high expression levels in the kidney, lung, adrenal gland, vasculature, brain, liver, endothelial and adipose tissues and lower levels in the heart. It is activated by binding to atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP). NPR1 activation and signaling stimulate many physiological responses that involve many tissues. The role of the ANP-NPR1 system in vasorelaxation, natriuresis, diuresis, endothelial permeability and non-cardiovascular functions such as lipolysis and immune cell functions has been well studied (Potter, 2011, Pharmacol. Ther. 130:71-82). Activation of NPR1 leads to natriuresis (salt excretion by the kidneys) and lowers blood pressure.

Monoclonal antibodies to NPR1 were first described by Kitano et al. in 1995 in Immunol. Lett. 47:215-22. Activating antibodies or NPR1 agonist antibodies are disclosed, for example, in US patents/publications No. 9090695 and 20160168251 and in WO 2010/065293.

Fully human antibodies that specifically bind to and activate the NPR1 protein with high affinity may prove important for the prevention and treatment of, for example, hypertension, obesity and heart failure.

The essence of the invention

The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to the natriuretic peptide receptor 1 (NPR1) protein. In some embodiments, anti-NoK.1 antibodies are fully human antibodies that bind to NPR1 with high affinity and activate NPR1 or stabilize the activated conformation. The antibodies of the present invention are useful, among other things, for activating or enhancing the activity of the NPR1 protein. In some embodiments, the antibodies are useful for preventing, treating, or alleviating at least one symptom or sign of an NPR1-associated disease or disorder in a subject. In some embodiments, antibodies can be administered for the purpose of preventing or treating a subject having or at risk of developing an NPR1-associated disease or disorder. In specific embodiments, the antibodies are used to lower systemic blood pressure in a subject suffering from high blood pressure. Such antibodies can be used as a therapy for a disorder such as heart failure by administration to a subject in need thereof.

In some specific embodiments, the antibodies bind to NPR1 in the presence or absence of atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP), i.e. antibodies are peptide-independent binders. Such antibodies have the advantage that they can be used to bind and activate NPR1 independent of varying concentrations of endogenous ligand. Such antibodies, when administered to a patient in need thereof, can be advantageously used to avoid variability (with respect to ligand concentration) between patients during treatment. In addition, the antibodies disclosed herein bind to NPR1 with high affinity and have improved pharmacokinetic properties (compared to standard treatment drugs). Antibodies showed t1/2 reaching 11 days in mice at a dose of 25 mg/kg. The antibodies are effective in lowering blood pressure and maintaining low blood pressure for 28 days when administered to a subject in need thereof. A single dose of the antibody of the present invention provided a sustained reduction in blood pressure. Such antibodies can be used to achieve higher efficacy along with less frequent dosing in a subject suffering from an NPR1-associated disease or disorder (eg, hypertension).

Antibodies of the invention may be full length (eg an IgG1 or IgG4 antibody) or may contain only an antigen binding portion (eg a Fab fragment, F(ab') ₂ or scFv) and may be modified to affect functionality, for example to increase resistance in in the host or to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933). In some embodiments, the antibodies may be bispecific.

In a first aspect of the present invention, isolated recombinant monoclonal antibodies or antigen binding fragments thereof are provided that specifically bind NPR1.

In some embodiments, the present invention provides an isolated antibody

- 1 046104 or an antigen-binding fragment thereof, which specifically binds to the natriuretic peptide receptor protein 1 (NPR1), wherein the antibody or antigen-binding fragment thereof interacts with one or more amino acids contained in the extracellular domain of NPR1 (amino acids 29-347 SEQ ID NO: 194), defined by hydrogen/deuterium substitution, 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 NPR1 and activates NPR1.

In some embodiments, the antibodies are fully human monoclonal antibodies.

Examples of anti-NPR1 antibodies of the present invention are shown in table. 1 and 2 in the present description. In table Figure 1 shows the amino acid sequence identifiers of the heavy chain variable regions (HCVR), light chain variable regions (LCVR), heavy chain complementarity determining regions (HCDR) (HCDR1, HCDR2 and HCDR3) and light chain complementarity determining regions (LCDR) (LCDR1, LCDR2 and LCDR3) illustrative antibodies. In table 2 shows the nucleic acid sequence identifiers HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of illustrative antibodies.

The present invention relates to antibodies or antigen-binding fragments thereof containing HCVR containing an amino acid sequence selected from any of the HCVR amino acid sequences listed in table. 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof containing an LCVR containing an amino acid sequence selected from any of the LCVR amino acid sequences listed in table. 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof containing a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), containing any of the amino acid sequences HCVR listed in table. 1, paired with any of the LCVR amino acid sequences listed in table. 1. In accordance with some embodiments, the present invention provides antibodies or antigen binding fragments thereof comprising the HCVR/LCVR amino acid sequence pair contained in any of the exemplary anti-NPR1 antibodies listed in Table. 1. In some embodiments, the HCVR/LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 2/10 (eg, mAb22033) and 66/74 (eg, mAb22810).

The present invention also relates to antibodies or antigen-binding fragments thereof containing HCVR and LCVR, wherein said HCVR contains the one specified in table. 1 amino acid sequence having no more than twelve amino acid substitutions, and/or the specified LCVR contains the one specified in table. 1 amino acid sequence containing no more than ten amino acid substitutions. For example, the present invention relates to antibodies or antigen-binding fragments thereof containing HCVR and LCVR, wherein said HCVR contains the one specified in table. 1 amino acid sequence, wherein said amino acid sequence has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acid substitutions. In another example, the present invention relates to antibodies or antigen-binding fragments thereof containing HCVR and LCVR, and the specified LCVR contains specified in the table. 1 amino acid sequence, wherein said amino acid sequence has one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions. In one embodiment, the present invention provides anti-NPR1 antibodies or antigen binding fragments thereof comprising HCVR and LCVR, said HCVR comprising the amino acid sequence set forth in Table. 1, wherein said amino acid sequence has at least one amino acid substitution, and/or said LCVR contains the amino acid sequence shown in table. 1, wherein said amino acid sequence has at least one amino acid substitution.

The present invention also provides antibodies or antigen binding fragments 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.

The present invention also provides antibodies or antigen binding fragments thereof comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1. 1, or a sequence substantially similar thereto, having at least 90%, at least 95%, of

- 2 046104 at least 98% or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof containing a heavy chain CDR3 (HCDR3) containing an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in table. 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof containing a light chain CDR1 (LCDR1) containing 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.

The present invention also provides antibodies or antigen-binding fragments 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. 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to antibodies or antigen-binding fragments thereof containing a light chain CDR3 (LCDR3) containing 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.

The present invention also relates to antibodies or antigen-binding fragments thereof containing a pair of amino acid sequences HCDR3 and LCDR3 (HCDR3/LCDR3), containing any of the amino acid sequences of HCDR3 listed in table. 1, paired with any of the LCDR3 amino acid sequences listed in table. 1. In accordance with some embodiments, the present invention provides antibodies or antigen binding fragments thereof comprising the HCDR3/LCDR3 amino acid sequence pair contained in any of the exemplary anti-No.K.1 antibodies listed in Table. 1. In some embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 8/16 (eg, mAb22033) and 72/80 (eg, mAb22810).

The present invention also relates to antibodies or antigen-binding fragments thereof containing HCVR and LCVR, wherein said HCVR comprises HCDR1 containing an amino acid sequence different from the amino acid sequence shown in table. 1, one amino acid, HCDR2, containing an amino acid sequence different from the amino acid sequence listed in table. 1, one amino acid, and HCDR3 containing an amino acid sequence different from the amino acid sequence shown in table. 1, one amino acid. In some embodiments, the present invention provides antibodies or antigen binding fragments thereof comprising HCVR and LCVR, wherein said LCVR comprises LCDR1 containing an amino acid sequence different from the amino acid sequence listed in Table. 1, one amino acid, LCDR2, containing an amino acid sequence different from the amino acid sequence listed in table. 1, one amino acid, and LCDR3 containing an amino acid sequence different from the amino acid sequence listed in table. 1, one amino acid. For example, the present invention relates to antibodies or antigen binding fragments thereof comprising HCVR and LCVR, wherein said HCVR comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence differing from SEQ ID NO: 4 by one amino acid, HCDR2 containing the amino acid sequence SEQ ID NO: 6 or an amino acid sequence different from SEQ ID NO: 6 by one amino acid, and HCDR3 containing the amino acid sequence SEQ ID NO: 8 or an amino acid sequence different from SEQ ID NO: 8 by one amino acid. In another illustrative embodiment, the present invention provides antibodies or antigen binding fragments thereof comprising HCVR and LCVR, wherein said LCVR comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by one amino acid; LCDR2 containing the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by one amino acid; and LCDR3 containing the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by one amino acid.

The present invention also provides antibodies or antigen binding fragments thereof comprising a set of six CDRs (ie, HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary antibodies listed in table. 1. In some embodiments, the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is selected

- 3 046104 from the group consisting of SEQ ID NO: 4-6-8-12-14-16 (for example, mAb22033) and 68-70-72-76-78-80 (for example, mAb22810).

In a related embodiment, 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 in the HCVR/LCVR amino acid sequence pair defined for any of illustrative antibodies listed in table. 1. For example, the present invention includes antibodies or antigen binding fragments thereof comprising a set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1LCDR2-LCDR3 contained in a pair of amino acid sequences HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/10 ( eg mAb22033) and 66/74 (eg mAb22810). Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in specific HCVR and/or LCVR amino acid sequences disclosed herein. Examples of conventions that may be used to identify CDR boundaries include, for example, the Kabat definition, the Chotia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chotia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chotia 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 to identify CDR sequences in an antibody.

In some embodiments, the present invention includes 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 in the light chain variable region (LCVR), wherein HCVR contains (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162 and 178; (ii) an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162 and 178 ; (iii) an amino acid sequence having at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 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 said amino acid sequence has no more than 12 amino acids replacements; and LCVR contains (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170 and 186; (b) an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, and 186; (c) an amino acid sequence having at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 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 said amino acid sequence has no more than 10 amino acids replacement

In some preferred embodiments, the present invention includes antibodies that specifically bind to NPR1 in an agonistic manner, i.e. potentiate or induce NPR1 binding and/or activity.

The present invention includes anti-NPR1 antibodies having a modified glycosylation pattern. In some embodiments, modification may be useful to remove undesired glycosylation sites or to produce an antibody lacking a fucose moiety present in the oligosaccharide chain, for example, to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) function (see Shield et al. (2002), JBC 277 :26733). In other applications, modification of galactosylation may be performed to alter complement dependent cytotoxicity (CDC).

In some 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 (ie, have a lower level of binding at acidic pH).

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

- 4 046104

The present invention also provides antibodies and antigen binding fragments thereof that cross-compete for binding to NPR1 with a reference antibody or antigen binding fragment thereof comprising a HCVR CDR and an LCVR CDR, wherein each of the HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences, listed in table. 1.

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 HCVR CDRs and three LCVR CDRs, each of the HCVR and LCVR having an amino acid sequence selected from the HCVR sequences and LCVR listed in table. 1.

The present invention also provides isolated antibodies and antigen binding fragments thereof that increase or stabilize the binding of NPR1 to a ligand (eg, ANP or BNP). In some embodiments, an antibody or antigen-binding fragment thereof that activates NPR1 binding to ANP may bind to the same epitope on NPR1 as ANP, or may bind to a different epitope on NPR1 as ANP.

In some embodiments, the antibodies or antigen binding fragments of the present invention are bispecific, including a first binding specificity to a first NPR1 epitope and a second binding specificity to a second NPR1 epitope, wherein the first and second epitopes are different and non-overlapping.

In some embodiments, the present invention provides an isolated antibody or antigen binding fragment thereof that has one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to monomeric human NPR1 in the absence of ANP and/or BNP at 25 and 37°C with a dissociation constant (KD) of less than 690 nM measured in a surface plasmon resonance assay; (c) binds to dimeric human NPR1 in the absence of ANP or BNP at 25 and 37°C with a KD of less than 42 nM measured in a surface plasmon resonance assay; (d) binds to human NPR1 in complex with ANP at 25 and 37°C with a KD of less than 80 nM measured in a surface plasmon resonance assay; (e) binds to human NPR1 complexed with BNP at 25 and 37°C with a KD of less than 20 nM measured in a surface plasmon resonance assay; (f) binds to monomeric monkey NPR1 in the absence of ANP and/or BNP at 25 and 37°C with a KD of less than 365 nM measured in a surface plasmon resonance assay; (g) binds to dimeric monkey NPR1 in the absence of ANP or BNP at 25 and 37°C with a KD of less than 30 nM measured in a surface plasmon resonance assay; (h) binds to monkey NPR1 in complex with ANP at 25 and 37°C with a KD of less than 10 nM measured in a surface plasmon resonance assay; (i) binds to monkey NPR1 in complex with BNP at 25 and 37°C with a KD of less than 10 nM measured in a surface plasmon resonance assay; (j) does not bind to mouse NPR1; (k) binds to cells expressing human NPR1 (without ANP) or NPR1 complexed with ANP, with an EC ₅₀ of less than 5 nM; (l) activates NPR1 with an EC ₅₀ of less than 385 nM measured in a cellular calcium flux bioassay; (m) reduces systemic blood pressure when administered to normotensive and hypertensive mice, with reductions in systemic and mean arterial pressure lasting up to 28 days after a single dose; (n) improves glucose tolerance when administered to diet-induced obese mice; and (o) contains an HCVR containing an amino acid sequence selected from the group consisting of the HCVR sequences listed in table. 1, and LCVR containing an amino acid sequence selected from the group consisting of LCVR sequences listed in table. 1.

In a second aspect, the present invention relates to nucleic acid molecules encoding anti-NaK1 antibodies or portions thereof. For example, the present invention relates to nucleic acid molecules encoding any of the HCVR amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCVR amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table. 2, or a sequence substantially similar thereto, having at least 90%, at least 95%,

- 5 046104 at least 98% or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table. 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in table. 1; in some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides nucleic acid molecules encoding HCVR, wherein HCVR includes a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the set of amino acid sequences HCDR1-HCDR2-HCDR3 is as defined for any of the exemplary antibodies , listed in table. 1.

The present invention also provides nucleic acid molecules encoding LCVR, wherein LCVR includes a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the set of amino acid sequences LCDR1-LCDR2-LCDR3 is as defined for any of the exemplary antibodies , listed in table. 1.

The present invention also relates to nucleic acid molecules encoding both HCVR and LCVR, where HCVR contains the amino acid sequence of any of the HCVR amino acid sequences listed in table. 1, and where LCVR contains the amino acid sequence of any of the LCVR amino acid sequences listed in table. 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table. 2, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, according to this aspect of the invention, the nucleic acid molecule encodes HCVR and LCVR, where HCVR and LCVR are derived from the same anti-NPR1 antibody listed in table. 1.

In a related aspect, the present invention provides recombinant expression vectors capable of expressing a polypeptide comprising an antibody heavy and/or light chain variable region. For example, the present invention includes recombinant expression vectors containing any of the nucleic acid molecules mentioned above, i.e. nucleic acid molecules encoding any of the HCVR, LCVR and/or CDR sequences listed in table. 2. In some embodiments, the present invention provides expression vectors comprising (a) a nucleic acid molecule comprising a nucleic acid sequence encoding an antibody HCVR that binds to NPR1, wherein the HCVR comprises an amino acid sequence selected from the group consisting of the sequences listed in table 1; and/or

- 6 046104 (b) a nucleic acid molecule containing a nucleic acid sequence encoding an LCVR antibody that binds to NPR1, wherein the LCVR contains an amino acid sequence selected from the group consisting of the sequences listed in table. 1. Also included within the scope of the present invention are host cells into which such vectors are introduced, and methods for producing antibodies or parts thereof by culturing the host cells under conditions allowing the production of antibodies or antibody fragments and isolating the antibodies and antibody fragments so produced . In some embodiments, the host cells comprise a mammalian cell or a prokaryotic cell. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell or an Escherichia coli (E. coli) cell. In some embodiments, the present invention provides methods for producing an antibody or an antigen binding fragment thereof of the present invention, the methods comprising introducing into a host cell an expression vector containing a nucleic acid sequence encoding the HCVR and/or LCVR of an antibody or antigen binding fragment thereof of the invention, functionally linked to the 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 by any method known in the art.

In a third aspect, the 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 NPR1, and a pharmaceutically acceptable carrier. In a related aspect, the invention provides 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. Examples of agents that can be effectively combined with an anti-NPR1 antibody include, but are not limited to, other agents that bind to NPR1 and/or activate NPR1 (including other antibodies or antigen-binding fragments thereof, etc.), and/or agents that which do not bind directly to NPR1, but nevertheless treat or ameliorate at least one symptom or symptom of an NPR1-associated disease or disorder (disclosed elsewhere herein). Additional combination therapies and co-formulations comprising the anti-NPR1 antibodies of the present invention are disclosed elsewhere herein.

In a fourth aspect, the invention provides therapeutic methods for treating an NPR1-associated disease or disorder in a subject with an anti-NPR1 antibody or an antigen binding moiety of an antibody of the invention, wherein the therapeutic methods comprise administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antigen binding moiety antibodies according to the invention. A treatable disorder is any disease or condition that can be ameliorated, attenuated, suppressed, or prevented by enhancing NPR1 activity (eg, hypertension). In some embodiments, the invention provides methods for preventing or treating an NPR1-associated disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-NPR1 antibody or antigen binding fragment thereof of the invention. In some embodiments, an antibody or antigen-binding fragment thereof can be administered for the purpose of prophylaxis or therapy to a subject suffering from or at risk of developing an NPR1-associated disease or disorder. In some embodiments, an antibody or antigen binding fragment thereof of the invention is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent may be selected from the group consisting of an aldosterone antagonist, an alpha-blocker, an angiotensin-converting enzyme (ACE) inhibitor, an arteriolar vasodilator, an autonomic ganglia vasodilator, a beta-blocker, a sympatholytic, a catecholamine depleting agent, a central alpha-2 adrenergic agonist, calcium channel blocker, diuretic, renin inhibitor, anticoagulant, antiplatelet agent, cholesterol-lowering agent, vasodilator, digitalis, surgery, implantable device, anticancer therapy, insulin, GLP1 agonist, metformin, dialysis, bone marrow stimulator, hemofiltration, lifestyle modification , dietary supplement and any other drug or therapeutic agent known in the art. In some embodiments, the second therapeutic agent may be an agent that helps counteract or reduce any potential side effect(s) associated with an antibody or antigen binding fragment thereof of the invention if such side effect(s) are likely to occur. The antibody or fragment thereof can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, or intramuscularly. The antibody or fragment thereof can be administered at a dose of from about 0.1 to about 100 mg/kg of the subject's body weight. In some embodiments, the antibody of the present invention can be administered in one or more doses ranging from 10 to 600 mg.

- 7 046104

The present invention also includes the use of an anti-NPRl antibody or an antigen binding fragment thereof of the present invention in the manufacture of a medicament for the treatment of a disease or disorder that would be effective against activation of NPR1 binding and/or NPR1 activity.

Other embodiments will become apparent from the following detailed description.

Brief description of the drawings

In fig. Figure 1 shows the effect of selected NPR1 agonist antibodies on systolic blood pressure in normotensive NPR1 ^hu/hu mice. Telemetry-controlled normotensive NPR1 ^hu/hu mice were divided into groups depending on body weight. Animals were given a single subcutaneous injection of 25 mg/kg NPR1 agonist antibody or PBS control as shown in Table 1. 30. All values represent mean change from baseline over 3–7 days ± standard error of the mean (SEM), n=3–9 per group. Statistical analysis was performed using one-way ANOVA with Dunnett's test; *p<0.05 compared to PBS control.

In fig. Figure 2 shows the effect of the anti-NPR1 antibody mAb22033 on systolic blood pressure in normotensive NPR1 ^hu/hu mice. Telemetry-controlled normotensive NPR1 ^hu/hu mice were randomly divided into five equal body weight groups and given a single subcutaneous injection of mAb22033 at the doses listed in Table 1. 32. IgG4 antibody was used as an isotype control. All values represent mean ± SEM, n=4-6 per group. Statistical analysis was performed using two-way ANOVA with Dunnett's test.

In fig. Figure 3 shows the effect of anti-NaK4 antibody mAb22033 on diastolic blood pressure in normotensive NPR1 ^hu/hu mice. Telemetry-controlled normotensive NPR1 ^hu/hu mice were randomly divided into five equal body weight groups and given a single subcutaneous injection of mAb22033 at the doses listed in Table 1. 32. IgG4 antibody was used as an isotype control. All values represent mean ± SEM, n=4-6 per group. Statistical analysis was performed using two-way ANOVA with Dunnett's test.

In fig. Figure 4 shows the effect of the anti-NPR1 antibody mAb22033 on heart rate in normotensive NPR1 ^hu/hu mice. Telemetry-controlled normotensive NPR1 ^hu/hu mice were randomly divided into five equal body weight groups and given a single subcutaneous injection of mAb22033 at the doses listed in Table 1. 32. IgG4 antibody was used as an isotype control. All values represent mean ± SEM, n=4-6 per group. Statistical analysis was performed using two-way ANOVA with Dunnett's test.

In fig. 5 shows the effect of the anti-NPR1 antibody mAb22033 on mean arterial blood pressure in normotensive NPR1 ^hu/hu mice. Telemetry-controlled normotensive NPR1 ^hu/hu mice were randomly divided into five equal body weight groups and given a single subcutaneous injection of mAb22033 at the doses listed in Table 1. 32. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=4-6 per group. Statistical analysis was performed using two-way ANOVA with Dunnett's test.

In fig. 6A and 6B show the effect of the anti-NPR1 antibody mAb22033 on left ventricular function in normotensive NPR1 ^hu/hu mice. Shown (Fig. 6A) are end-systolic volume (%); and (Fig. 6B) end-diastolic volume (%) in telemetry-controlled normotensive NPR1 ^hu/hu mice allocated to five equal body weight groups that received a single subcutaneous injection of mAb22033 at the doses listed in Table 1. 32. IgG4 antibody was used as an isotype control. Echocardiography was performed on short-axis anesthetized mice on day 28 post-dose using a high-frequency ultrasound system and probe. All values represent mean ± SEM, n=6-7 per group. Statistical analysis was performed using one-way ANOVA with Dunnett's test; *p<0.05 compared with IgG4 isotype control.

In fig. 7A and 7B show the effect of the anti-NPR1 antibody mAb22033 on left ventricular function in normotensive NPR1 ^hu/hu mice. Shown (Fig. 7A) are the shortening fraction (%); and (Fig. 7B) ejection fraction (%) in telemetry-controlled normotensive NPR1 ^hu/hu mice allocated to five groups of equal body weight, which received a single subcutaneous injection of mAb22033 at the doses listed in table. 32. IgG4 antibody was used as an isotype control. Echocardiography was performed on short-axis anesthetized mice on day 28 post-dose using a high-frequency ultrasound system and probe. All values represent mean ± SEM, n=6-7 per group. Statistical analysis was performed using one-way ANOVA with Dunnett's test.

In fig. Figure 8 shows the effect of a single dose of 2 anti-NPR1 antibodies on systolic blood pressure in hypertensive NPR1 ^hu/hu mice. Telemetry-controlled hypertensive NPR1 ^hu/hu mice were randomly divided into six systolic blood pressure equalized groups and given a single subcutaneous injection of mAb22033 or mAb22810 at the doses listed in Table 1 . 36. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=4-6 per group. Statistical analysis was performed using two-way ANOVA

- 8 046104 with Dunnett’s criterion; *p<0.05 25 mg/kg mAb22033 compared to control; #p<0.05 5 mg/kg mAb22033 compared to control; !p<0.05 19 mg/kg mAb22810 compared to control.

In fig. Figure 9 shows the effect of a single dose of 2 anti-NPR1 antibodies on diastolic blood pressure in hypertensive NPR1 ^hu/hu mice. Telemetry-controlled hypertensive NPR1 ^hu/hu mice were randomly divided into six systolic blood pressure equalized groups and given a single subcutaneous injection of mAb22033 or mAb22810 at the doses listed in Table 1 . 36. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=3-6 per group. Statistical analysis was performed using two-way ANOVA with Dunnett's test; *p<0.05 25 mg/kg mAb22033 compared to control; #p<0.05 5 mg/kg mAb22033 compared to control; !p<0.05 19 mg/kg mAb22810 compared to control; %p<0.05 5 mg/kg mAb22810 compared to control.

In fig. 10 shows the effect of one dose of 2 anti-NPR1 antibodies on heart rate in hypertensive NPR1 ^hu/hu mice. Telemetry-controlled hypertensive NPR1 ^hu/hu mice were randomly divided into six systolic blood pressure equalized groups and administered a single subcutaneous dose of mAb22033 or mAb22810 at the doses listed in Table 1 . 36. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=3-6 per group. Statistical analysis was performed using two-way ANOVA with Dunnett's test; *p<0.05 25 mg/kg mAb22033 compared to control; #p<0.05 5 mg/kg mAb22033 compared to control.

In fig. 11 shows the effect of one dose of 2 anti-Na.1 antibodies on mean arterial blood pressure in hypertensive NPR1 ^hu/hu mice. Telemetry-controlled hypertensive NPR1 ^hu/hu mice were randomly divided into six systolic blood pressure equalized groups and given a single subcutaneous injection of mAb22033 or mAb22810 at the doses listed in Table 1 . 36. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=3-6 per group. Statistical analysis was performed using two-way ANOVA with Dunnett's test; *p<0.05 25 mg/kg mAb22033 compared to control; #p<0.05 5 mg/kg mAb22033 compared to control; !p<0.05 19 mg/kg mAb22810 compared to control.

In fig. 12 shows the effect of single and repeated doses of anti-NPR1 antibody on systolic blood pressure in hypertensive NPR1 ^hu/hu mice. Telemetry-monitored hypertensive NPR1 ^hu/hu mice were randomly divided into five equal systolic blood pressure groups and administered a subcutaneous dose of mAb22033 either once or twice weekly for 3 weeks at the doses listed in Table 1 . 40. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=3-6 per group. Arrows indicate doses administered to mice. Statistical analysis was performed using two-way ANOVA with Dunnett's test; *p<0.05 25 mg/kg mAb22033 compared to control; #p<0.05 5 mg/kg mAb22033 compared to control; !p<0.05 50 mg/kg mAb22033 compared to control.

In fig. 13 shows the effect of single and repeated doses of anti-NPR1 antibody on diastolic blood pressure in hypertensive NPR1 ^hu/hu mice. Telemetry-monitored hypertensive NPR1 ^hu/hu mice were randomly divided into five equal systolic blood pressure groups and administered a subcutaneous dose of mAb22033 either once or twice weekly for 3 weeks at the doses listed in Table 1 . 40. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=3-6 per group. Arrows indicate doses administered to mice. Statistical analysis was performed using two-way ANOVA with Dunnett's test.

In fig. 14 shows the effect of single and repeated doses of anti-NPR1 antibody on heart rate in hypertensive NPR1 ^hu/hu mice. Telemetry-monitored hypertensive NPR1 ^hu/hu mice were randomly divided into five equal systolic blood pressure groups and administered a subcutaneous dose of mAb22033 either once or twice weekly for 3 weeks at the doses listed in Table 1 . 40. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=3-6 per group. Arrows indicate doses administered to mice. Statistical analysis was performed using two-way ANOVA with Dunnett's test.

In fig. 15 shows the effect of single and repeated doses of anti-NPR1 antibody on mean arterial blood pressure in hypertensive NPR1 ^hu/hu mice. Telemetry-monitored hypertensive NPR1 ^hu/hu mice were randomly divided into five equal systolic blood pressure groups and administered a subcutaneous dose of mAb22033 either once or twice weekly for 3 weeks at the doses listed in Table 1 . 40. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=3-6 per group. Arrows indicate doses administered to mice. Statistical analysis was performed using two-way ANOVA with Dunnett's test; *p<0.05 25 mg/kg mAb22033 compared to control; #p<0.05 5 mg/kg mAb22033 compared to control; !p<0.05 50 mg/kg mAb22033 compared to control.

- 9 046104

In fig. 16A and 16B show the effects of single and repeated doses of anti-NPRl antibody on cardiac function in hypertensive NPR1 ^hu/hu mice. Shown (FIG. 16A) are % end-systolic volume and (FIG. 16B) % end-diastolic volume in telemetry-controlled hypertensive NPR1 ^hu/hu mice assigned to five equal systolic blood pressure groups that received either a subcutaneous injection of mAb22033 alone once or twice a week for 3 weeks in the doses listed in table. 40. IgG4 antibody was used as isotype control. Echocardiography was performed on short-axis anesthetized mice at 21 days post-dose using a high-frequency ultrasound system and probe. All values represent mean ± SEM, n=5-6 per group. Statistical analysis was performed using one-way ANOVA with Dunnett's test.

In fig. 17A and 17B show the effects of single and repeated doses of anti-NaK1 antibody on cardiac function in hypertensive NPR1 ^hu/hu mice. Shown (FIG. 17A) are the shortening fraction (%) and (FIG. 17B) ejection fraction (%) of telemetry-controlled hypertensive NPR1 ^hu/hu mice allocated to five groups with equal systolic blood pressure, which received a subcutaneous injection of mAb22033 or one once or twice a week for 3 weeks in the doses listed in table. 40. IgG4 antibody was used as isotype control. Echocardiography was performed on short-axis anesthetized mice at 21 days post-dose using a high-frequency ultrasound system and probe. All values represent mean ± SEM, n=5-6 per group. Statistical analysis was performed using one-way ANOVA with Dunnett's test.

In fig. 18A shows changes in body weight following administration of the NPR1 agonist mAb mAb22810, hFc.FGF21, or isotype control mAb. In fig. 18B and FIG. 18C shows total fat and total muscle mass, respectively, after six weeks of treatment, measured using EchoMRI. Humanized NPR1 mice were rendered obese by a 10-week 60% high-fat diet. After this period, mice were randomly divided into three groups of equal body weight and given subcutaneous injections of therapeutic drugs at the doses and frequencies listed in Table 1. 44. Human IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=10 per group. *P<0.05 compared with isotype control; **P<0.01 compared with isotype control. Statistical analysis was performed using two-way ANOVA + Tukey for body weight, one-way ANOVA + Bonferonni for fat and muscle mass.

In fig. 19A-19C show changes in VO ₂ (FIG. 19A), VCO ₂ (FIG. 19B), or energy expenditure (FIG. 19C) by average for each day/night cycle after one week of treatment with either the NPR1 agonist mAb mAb22810, hFc.FGF21 or isotype control mAb. After one week of treatment, mice from each group were placed in the Columbia Instruments Cage Metabolic System (CLAMS) for one week to record metabolic parameters. Mice were allowed to acclimate to their cages for one week before measurements began to minimize stress. Human IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=5-6 per group. **P<0.01 compared to isotype control, ***P<0.001 compared to isotype control. Statistical analysis was performed using one-way ANOVA + Bonferonni.

In fig. 20A shows the change in glucose tolerance measured by an oral glucose tolerance test (2 g/kg glucose) after two weeks of treatment with either the NPR1 agonist mAb mAb22810, hFc.FGF21, or an isotype control mAb.

In fig. 20B shows overnight fasted glucose levels recorded at the start of the study in A. After two weeks of treatment, mice from each group were fasted overnight in clean cages and given an oral glucose load of 2 g/kg the following morning. Glucose levels were then recorded in the tail vein using a hand-held glucometer at T0, T15, T30, T60, T90 and T120. Human IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=10 per group. hFc.FGF21 group: **P<0.01 compared to isotype control, ***P<0.001 compared to isotype control, ****p<0.0001 compared to isotype control. mAb22810 group: ++P<0.01 compared with isotype control. Statistical analysis was performed using two-way ANOVA + Bonferonni for oGTT, one-way ANOVA + Bonferonni for fasting glucose.

In fig. 21A shows the change in body weight following administration of the NPR1 agonist mAb mAb22033, hFc.FGF21, or isotype control mAb.

In fig. 21B and 21C show total fat and total muscle mass, respectively, after six weeks of treatment, measured using EchoMRI. Humanized NPR1 mice were rendered obese by a 10-week 60% high-fat diet. After this period, mice were randomly divided into three groups of equal body weight and given subcutaneous injections of therapeutic drugs at the doses and frequencies listed in Table 1. 45. Human IgG4 antibody

- 10 046104 lo was used as an isotype control. All values represent mean ± SEM, n=10 per group. *P<0.05 compared to isotype control, ****P<0.0001 compared to isotype control. Statistical analysis was performed using two-way ANOVA + Tukey for body weight, one-way ANOVA + Bonferonni for fat and muscle mass.

In fig. 22A-22C show changes in VO ₂ (FIG. 22A), VCO ₂ (FIG. 22B), or energy expenditure (FIG. 22C) by average for each day/night cycle after one week of treatment with the NPR1 agonist mAb mAb22033, hFc. FGF21 or isotype control mAb. After one week of treatment, mice from each group were placed in the Columbia Instruments Cage Metabolic System (CLAMS) for one week to record metabolic parameters. Mice were allowed to acclimate to the cages for one week before measurements were taken to minimize stress. Human IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=5-6 per group. ****P<0.0001 compared with isotype control. Statistical analysis was performed using one-way ANOVA+Bonferonni.

In fig. 23A and 23B show the effect of the anti-NaK1 antibody mAb22033 on glucose tolerance as measured by blood glucose (FIG. 23A) and fasting glucose (FIG. 23B). After four weeks of treatment, mice from each group were fasted overnight in clean cages and given an oral glucose load of 2 g/kg the next morning. Glucose levels were then recorded in the tail vein using a hand-held glucometer at T0, T15, T30, T60, T90 and T120. Human IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=10 per group. hFc.FGF21 group: ***P<0.001 compared with isotype control, ****P<0.0001 compared with isotype control. mAb22033 group: +P<0.05 compared to isotype control, ++P<0.01 compared to isotype control. Statistical analysis was performed using two-way ANOVA + Bonferonni for oGTT, one-way ANOVA + Bonferonni for fasting glucose.

Detailed Description of the Invention

Before describing the methods of the present invention, it should be understood that this invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

Unless otherwise specified, all technical and scientific terms used in this specification have the same meanings as commonly understood by one skilled in the art to which the present invention relates. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are described below. All publications referred to herein are incorporated by reference in their entirety.

Definitions

The term NPR1, also referred to as NPRA, refers to natriuretic peptide receptor 1 (also known as natriuretic peptide A receptor). NPR1 is a homodimeric transmembrane guanylate cyclase, an enzyme that catalyzes the synthesis of cGMP. NPR1 is a receptor for both atrial (ANP) and brain (BNP) natriuretic peptides 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 helical 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 represented by the amino acid sequence given in UniProtKB/Swiss-Prot under accession number P16066.1 (SEQ ID NO: 193). The term NPR1 includes recombinant NPR1 protein or a fragment thereof. The term also includes the NPR1 protein or fragment thereof linked to, for example, a histidine tag, a mouse or human Fc, or a signal sequence such as ROR1 (eg, SEQ ID NO: 194-199).

As used herein, the term antibody is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds (i.e., full-length antibody molecules), as well as their multimers (for example, IgM) or their antigen-binding fragments. Each heavy chain consists of a heavy chain variable region (HCVR or VH) and a heavy chain constant region (consisting of CH1, CH2 and CH3 domains). Each light chain consists of a light chain variable region (LCVR or _VL ) and a light chain constant region ( _CL ). The VH and _VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with regions that are more conserved called

- 11 046104 frame areas (FR). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FR antibody (or antigen binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on analysis of two or more CDRs.

It is also possible to replace one or more CDR residues or omit one or more CDRs. The scientific literature describes antibodies in which binding is possible without one or two CDRs. Padlan et al. (1995, FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only one-fifth to one-third of the CDR residues actually contact the antigen. Padlan has also found a large number of antibodies in which one or two CDRs do not contain antigen-contacting amino acids (see also Vajdos et al., 2002, J. Mol. Biol. 320:415-428).

Non-antigen contact CDR residues can be identified based on previous studies (eg, residues H60-H65 in CDRH2 are often not required) of Kabat CDR regions outside the Chotia CDR, molecular modeling, and/or empirically. If a CDR or residue(s) thereof is omitted, it is typically replaced by an amino acid occupying the corresponding position in another human antibody sequence or consensus sequences of such sequences. The positions to be replaced in the CDR and the amino acids to be replaced can also be selected empirically. Empirical substitutions may be conservative or non-conservative.

The fully human monoclonal αntu-NPR1 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 relative to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention includes antibodies and antigen binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding germline sequence residue(s), from which the antibody was produced, or to the corresponding residue(s) of another human germline sequence, or have a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein generically as germline mutations). One skilled in the art, starting with the heavy and light chain variable region sequences disclosed herein, can readily produce numerous antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In some embodiments, all framework region residues and/or CDRs in the V _H and/or V _L domains are mutated back to residues 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, such as only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework residues and/or CDRs are mutated to the corresponding residue(s) of another germline sequence (ie, a germline sequence that is different from the germline sequence from which the antibody was originally derived). In addition, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, for example, in which certain individual residues are mutated to the corresponding residue of a particular germline sequence, while certain other residues that differ from the original germline sequence, are retained, or are mutated to the corresponding residue of another germline sequence. Once produced, 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, increased binding affinity, improved or enhanced antagonistic biological properties, reduced immunogenicity, etc. . Antibodies and antigen binding fragments obtained in this general manner are included in the present invention.

The present invention also includes fully human monoclonal anti-NPR1 antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes αntu-NPR1 antibodies having amino acid sequences HCVR, LCVR and/or CDR, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. canner

- 12 046104 ative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein.

The term human antibody or fully human antibody as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), for example, in the CDR and, in particular, in CDR3. However, as used herein, the term human antibody or fully human antibody is not intended to include monoclonal antibodies in which the human FR sequence is grafted with CDR sequences derived from the germ line of another mammalian species (eg, mouse). The term includes antibodies that are recombinantly produced in a non-human mammal or in cells of a non-human mammal. The term is not intended to include antibodies isolated or produced in humans.

The term recombinant as used herein refers to antibodies of the invention or antigen binding fragments thereof generated, expressed, isolated or produced using technologies or methods known in the art as recombinant DNA techniques, which include, for example, DNA splicing and transgene expression. The term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, such as transgenic mice) or in a cell expression system (eg, CHO cells) or isolated from a human recombinant combinatorial antibody library.

The term specifically binds to or specifically binds to, etc. means that the antibody or antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10 ^-8 M or less (eg, a lower KD value indicates stronger binding). Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. Antibodies disclosed herein that specifically bind to NPR1 have been identified using surface plasmon resonance, such as BIACORE™. Moreover, multispecific antibodies that bind to one domain in NPR1 and one or more additional antigens, or bispecific antibodies that bind to two different regions of NPR1, are also considered as antibodies that bind specifically as used herein.

The term high-affinity antibody refers to those mAbs that have a binding affinity for NPR1, expressed as K _D , of at least 10 ^-8 M; preferably 10 ^-9 M; more preferably 10 ^-10 M, even more preferably 10 ^-11 M, measured by surface plasmon resonance, eg BIACORE™ or ELISA to determine affinity in solution.

The term slow dissociation rate, K _off or kd means an antibody that dissociates from the complex with NPR1 with a rate constant of 1x 10 ^-3 s ^-1 or less, preferably 1x 10 ^-4 s ^-1 or less, determined by a surface plasmon resonance method, such as BIACORE ™.

The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, etc. as used herein include any natural, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The terms antigen binding antibody fragment or antibody fragment as used herein refer to one or more antibody fragments that retain the ability to bind the NPR1 protein.

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

The term isolated antibody as used herein means an antibody that is substantially free of other antibodies (Abs) having a different antigen specificity (e.g., an isolated antibody that specifically binds to NPR1, or a fragment thereof, substantially free of Abs that specifically binds to antigens other than NPR1).

The term activating antibody or agonist antibody as used herein (or an antibody that increases or enhances the activity of NPR1 or an antibody that stabilizes the activated conformation) means an antibody that, when bound to NPR1, results in activation of at least one biological activity of NPR1. For example, an antibody of the invention may reduce systemic blood pressure when administered to a subject in need thereof.

- 13 046104

The term surface plasmon resonance as used herein refers to an optical phenomenon that allows the analysis of biomolecular interactions in real time by detecting changes in protein concentrations in a biosensor matrix, for example, using the BIACORE™ system. (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term KD as used herein denotes the equilibrium dissociation constant of a particular antibody-antigen interaction.

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. One antigen can have more than one epitope. Thus, different antibodies can bind to different sites on the antigen and have different biological effects. The term epitope also refers to the region of an antigen to which B and/or T cells respond. It also refers to the region of an antigen that binds to an antibody. Epitopes can be defined as structural or functional. Functional epitopes are usually a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e. consist of nonlinear amino acids. In some embodiments, epitopes may include determinants that are reactive surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have certain three-dimensional structural characteristics and/or specific charge characteristics.

The term cross-competition as used herein means that an antibody or antigen binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen binding fragment thereof. The term also includes competition between two antibodies in both directions, i.e. a first antibody that binds to and blocks the binding of a second antibody, and vice versa. In some embodiments, the first antibody and the second antibody 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, for example, as a result of steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, such as real-time label-free biolayer interferometry. Cross competition between two antibodies can be expressed as binding of a second antibody that is less than the background signal due to binding to the same antibody (where the first and second antibodies are the same antibody). Cross-competition between two antibodies can be expressed, for example, as % binding of the second antibody that is less than the initial background binding to the same antibody (where the first and second antibodies are the same antibody).

The term substantially identical or substantially identical, when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with corresponding nucleotide insertions or deletions with another nucleic acid (or its complementary strand), the nucleotide sequence identity is at least approximately 90%, and more preferably at least about 95, 96, 97, 98, or 99% of the nucleotide bases, as measured by any known sequence identity algorithm, such as FASTA, BLAST, or GAP, as described below. A nucleic acid molecule having a significant degree of identity with a reference nucleic acid molecule may, in some cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

When applied to polypeptides, the term highly similar or substantially similar means that two peptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs, which use gap penalties by default, have at least 90% sequence identity, even more preferably at least 95, 98 or 99% sequence identity. Preferably, the positions of residues that are not identical are distinguished by conservative amino acid substitutions. Conservative amino acid substitution means a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). Typically, a conservative amino acid substitution does not significantly change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted to correct for the conservative nature of the substitution. The means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994), Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids that have 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) side chains containing amide:

- 14 046104 asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) main side chains: lysine, arginine and histidine; 6) acid side chains: aspartate and glutamate and 7) sulfur side chains: cysteine and methionine. Preferred groups of conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamate-aspartate and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992), Science, 256:1443 45, incorporated herein by reference. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity of polypeptides is usually measured using sequence analysis software. Protein analysis software allows you to match similar sequences using similarity measures related to various substitutions, deletions and other modifications, including conserved amino acid substitutions. For example, the GCG software package contains programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and its mutant variant. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA using default or recommended parameters; programs in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides the alignment and percent sequence identity of the regions with the best overlap between the query sequence and the search sequence (Pearson (2000), supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the BLAST computer program, 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.

The phrase a therapeutically effective amount means an amount that provides the desired effect for which it is administered. The exact amount will depend on the purpose of treatment and can be determined by one skilled in the art using known methods (see, for example, Lloyd (1999), The Art, Science and Technology of Pharmaceutical Compounding).

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-associated disease or disorder, such as hypertension. The term includes people who have or are at risk of developing such a disease or disorder.

As used herein, the terms treat, treating, or treatment refer to reducing or alleviating the severity of at least one symptom or sign of an NPR1-associated disease or disorder by administering to a subject in need thereof a therapeutic agent, such as an antibody of the present invention. These terms include inhibiting the progression of a disease or worsening of a symptom/sign. The terms also include a positive prognosis of the disease, i.e. the subject may not have the disease, or the disease may be ameliorated upon administration of a therapeutic agent, such as an antibody of the present invention. The therapeutic agent can be administered to a subject at a therapeutic dose.

The terms prevent, prevent or prophylaxis refer to inhibiting the occurrence of an NPR1-associated disease or disorder, or any symptoms or signs of such a disease or disorder, upon administration of an antibody of the present invention.

Antigen-binding fragments of antibodies.

Unless specifically stated otherwise, the term antibody as used herein should be understood to include antibody molecules containing two immunoglobulin heavy chains and two immunoglobulin light chains (ie, full-length antibody molecules), as well as antigen-binding fragments thereof. The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, etc. as used herein, include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The terms antigen binding antibody fragment or antibody fragment as used herein refer to one or more antibody fragments that retain the ability to specifically bind to the NPR1 protein. The antibody fragment may include a Fab fragment, an F(ab')2 fragment, an Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In some embodiments, the term antigen binding fragment refers to a polypeptide fragment of a polyspecific antigen binding molecule. Antigen-binding antibody fragments can be produced, for example, from full-length antibody molecules using any suitable standard methods, such as proteolytic digestion, or recombinant genetic engineering techniques, including manipulation and excision.

- 15 046104 compression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and/or available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA may be sequenced and subjected to chemical or molecular biology manipulation, for example, to place one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc. .

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 molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, a dedicated complementarity determining region (CDR) such as the CDR3 peptide), or a FR3-CDR3-FR4 peptide with limited conformational freedom. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, mini-bodies, nanobodies (e.g. monovalent nanobodies, divalent nanobodies, etc. .), small modular immunopharmaceuticals (SMTPs) and shark IgNAR variable domains are also encompassed by the expression antigen binding fragment as used herein.

The antigen binding fragment of an antibody typically contains at least one variable domain. The variable domain can be of any size or amino acid composition and typically contains at least one CDR that is adjacent to or in reading frame with one or more framework sequences. In antigen binding fragments having a VH domain linked to a VL domain, the VH and VL domains may be arranged relative to each other in any suitable order. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen binding fragment of the antibody may comprise a monomeric _VH or _VL domain.

In some embodiments, the 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 are possible in an antigen binding fragment of an antibody of the present invention include^(i)vh^-ch^1; (ii)^vh^-ch^2; (w) VH-CH3; (iv)^vh^-Ch^1-ch^{2; (}v)^vh^-ch^1-Ch^2-ch^3; (vi)^vh^-ch^2-ch^3; (vii)^vh C.L.^{; (viii) V}L^-CH^{1; (ix)} VL-CH2;^(x)VL^-CH^3; (xi)^VL^-CH^1-CH^{2; (xii)V}L^-CH^1-CH^2-CH^{3; (xiii) V}L^-CH^2-CH^{3 and}(xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can either be directly linked to each other or can be linked through a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, which provides a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, the antigen binding fragment of an antibody of the present invention may include a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric V_H or V_L domains (eg via disulfide bond(s)).

As with full-length antibody molecules, antigen-binding fragments can be monospecific or polyspecific (eg, bispecific). A polyspecific antigen-binding antibody fragment typically contains at least two different variable domains, where each variable domain is capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using standard techniques available in the art.

Obtaining human antibodies.

Methods for producing human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to obtain human antibodies that specifically bind to NPR1.

An immunogen containing any of the following can be used to generate antibodies to the NPR1 protein. In some embodiments, antibodies of the present invention are produced using mice immunized with full-length native NPR1 protein (see, for example, UniProtKB/Swiss-Prot accession number P16066.1) or DNA encoding the protein or fragment thereof. Alternatively, the protein or fragment thereof can be produced using standard biochemical methods, modified and used as an immunogen.

In some embodiments, the immunogen may be a recombinant NPR1 protein or fragment thereof expressed in E. coli or any other eukaryotic cells or

- 16 046104 mammalian cells, such as Chinese hamster ovary (CHO) cells (eg SEQ ID NO: 194-199).

Using VELOCIMMUNE® technology (see, for example, US 6596541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for producing monoclonal antibodies, chimeric antibodies with high affinity for NPR1 having a human variable region and a murine constant region are first isolated. VELOCIMMUNE® technology involves the creation of a transgenic mouse whose genome contains human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that, in response to antigenic stimulation, the mouse produces an antibody containing the human variable region and the mouse constant region. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, a VELOCIMMUNE® mouse is challenged with the antigen of interest and lymphatic cells (such as B cells) are isolated from the antibody-expressing mice. Lymph cells can be fused with a myeloma cell line to produce immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the desired heavy chain and light chain isotype constant regions. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

Initially, high-affinity chimeric antibodies having a human variable region and a murine constant region are isolated. As in the experimental section below, antibodies are characterized and selected for the presence of desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant region to produce a fully human antibody of the invention, for example, wild type or modified IgG1 or IgG4. Although the selected constant region may vary depending on the specific application, the characteristics of high affinity antigen binding and target specificity are located in the variable region.

Bioequivalents.

Anti-NPRI antibodies and antibody fragments of the present invention include proteins having amino acid sequences that differ from those of the described antibodies, but which retain the ability to bind to the NPR1 protein. Such variant antibodies and antibody fragments contain one or more additions, deletions or substitutions of amino acids from the original sequence, but exhibit biological activity that is substantially equivalent to that of the described antibodies. Likewise, DNA sequences encoding an antibody of the present invention include sequences that contain one or more additions, deletions or substitutions of nucleotides relative to the disclosed sequence, but which encode an antibody or antibody fragment that is substantially bioequivalent to an antibody or antibody fragment of the invention .

Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives that do not differ significantly in the rate and extent of absorption when administered at the same molar dose under similar experimental conditions, either as a single dose or as multiple doses. doses Some antibodies may be considered equivalents or pharmaceutical alternatives if they are equivalent in extent of absorption but not in rate of absorption, and may still be considered bioequivalent as long as such differences in rate of absorption are intentional and reflected in the labeling, and to achieve effective drug concentrations in body are not important, for example, during long-term use, and are considered medically insignificant for the specific drug being studied.

In one embodiment, two antigen binding proteins are bioequivalent if they have no clinically significant differences in their safety, purity or potency.

In one embodiment, two antigen binding proteins are bioequivalent if the patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity or reduction in efficacy, compared to continuing therapy without such switching.

In one embodiment, two antigen binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for a condition or

- 17 046104 conditions of use to the extent such mechanisms are known.

Bioequivalence can be demonstrated using in vivo and/or in vitro methods. Measures of bioequivalence include, for example, (a) an in vivo test in humans or other mammals in which the concentration of an antibody or its metabolites is measured in blood, plasma, serum or other biological fluid as a function of time; (b) an in vitro test that has been correlated with in vivo bioavailability data in humans and can reasonably predict such bioavailability; (c) an in vivo test in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical study that establishes the safety, efficacy, bioavailability or bioequivalence of the antibody.

Bioequivalent variants of the antibodies of the invention can be constructed, for example, by introducing various substitutions of residues or sequences or removing terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or irregular intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants containing amino acid changes that modify the glycosylation characteristics of the antibodies, such as mutations that eliminate or remove glycosylation.

Anti-NPRI antibodies containing Fc variants.

In some embodiments, the present invention provides anti-NoK.1 antibodies comprising an Fc domain containing one or more mutations that increase or decrease the binding of the antibody to the FcRn receptor, for example, at an acidic pH compared to a neutral pH. For example, the present invention includes anti-NaK.1 antibodies containing a mutation in the CH2 or CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain for FcRn in an acidic environment (for example, in the endosome, where the pH is range from about 5.5 to about 6.0). Such mutations may result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc fragment modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (for example, L/Y/F/W or T), 254 (for example, S or T) and 256 (for example, S/R/Q/E/D or T); or modification at position 428, and/or 433 (for example, H/L/R/S/P/Q or K), and/or 434 (for example, A, W, H, F or Y [N434A, N434W, N434H , N434F or N434Y]); or modification at position 250 and/or 428; or modification at position 307 or 308 (eg, 308F, V308F) and 434. In one embodiment, the modification includes modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259I (for example, V259I) and 308F (for example, V308F); modification 433K (for example, H433K) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L) and modification 307 and/or 308 (for example, 308F or 308P). In yet another embodiment, the modification includes modification 265A (eg, D265A) and/or 297A (eg, N297A).

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

The present invention also includes anti-NPR1 antibodies containing a chimeric heavy chain constant region ( _CH ), wherein the chimeric _CH region includes segments derived from the _CH regions of more than one immunoglobulin isotype. For example, antibodies of the invention may comprise a chimeric _CH region comprising part or all of a _CH 2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule in combination with part or all of a CH3 domain derived from a human IgG1, human IgG2 molecule or human IgG4. In some embodiments, the antibodies of the invention comprise a chimeric _CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an upper hinge amino acid sequence (amino acid residues 216 to 227 according to EU numbering) derived from the hinge region of human IgG1, human IgG2 or human IgG4, in combination with a lower hinge sequence (amino acid residues 228 to 236 according to EU numbering) derived from the hinge region of human IgG1, human IgG2 or human IgG4. In some embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge region of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge

- 18 046104 no region of human IgG2. An antibody containing a chimeric CH region as described herein, in some embodiments, can exhibit the effector functions of a modified Fc fragment without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, US Patent Application Publication 2014/0243504, the disclosure of which is incorporated herein by reference in its entirety.)

Biological characteristics of antibodies.

Typically, the antibodies of the present invention act by binding to the NPR1 protein and increasing its activity. For example, the present invention includes antibodies and antigen-binding antibody fragments that bind to human monomeric NPR1 protein in the absence of ANP or BNP (for example, at 25 or 37°C) with a KD of less than 690 nM, measured by surface plasmon resonance, for example, in an assay format , defined in example 3 of this description. In some embodiments, antibodies or antigen-binding fragments thereof bind to NPR1 with a KD 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 about 25 nM, measured by surface plasmon resonance, for example, in the assay format defined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-binding antibody fragments that bind to human NPR1 dimeric protein in the absence of ANP or BNP (e.g., at 25 or 37°C) with a KD of less than 42 nM, as measured by surface plasmon resonance, e.g., in an assay format defined in example 3 of this description. In some embodiments, antibodies or antigen-binding fragments thereof bind to NPR1 with a KD 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 , measured by surface plasmon resonance, for example, in the assay format defined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-binding antibody fragments that bind to human NPR1 protein in complex with ANP (for example, at 25 or 37°C) with a K _D of less than 80 nM, measured by surface plasmon resonance, for example, in the assay format defined in example 3 of this description. In some embodiments, antibodies or antigen-binding fragments thereof bind to NPR1 with a KD 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 , measured by surface plasmon resonance, for example, in the assay format defined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-binding antibody fragments that bind to human NPR1 protein in complex with BNP (for example, at 25 or 37°C) with a KD of less than 20 nM, measured by surface plasmon resonance, for example, in the assay format defined in Example 3 of this description. In some embodiments, antibodies or antigen-binding fragments thereof bind to NPR1 with a KD 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. format of the analysis defined in Example 3 of the present description, or a substantially similar analysis.

The present invention also includes antibodies and antigen-binding antibody fragments that bind to monomeric monkey NPR1 protein in the absence of ANP or BNP (e.g., at 25 or 37°C) with a KD of less than 365 nM, as measured by surface plasmon resonance, e.g., in an assay format defined in example 3 of this description. In some embodiments, antibodies or antigen binding fragments thereof bind to NPR1 with a KD 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 about 5 nM, less than about 1 nM, or less than about 0.5 nM, measured by surface plasmon resonance, for example, in the assay format defined in Example 3 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-binding antibody fragments that bind to monkey NPR1 dimeric protein in the absence of ANP or BNP (e.g., at 25 or 37°C) with a KD of less than 30 nM as measured by surface plasmon resonance, e.g., in an assay format defined in example 3 of this description. In some embodiments, antibodies or antigen-binding fragments thereof bind to NPR1 with a KD 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. format of the analysis defined in Example 3 of the present description, or a substantially similar analysis.

The present invention also includes antibodies and antigen-binding antibody fragments that bind to simian NPR1 protein in complex with ANP (for example, at 25 or 37°C) with a KD of less than 10 nM, measured by surface plasmon resonance, for example, in the assay format defined in Example 3 of this description. In some embodiments, antibodies or

- 19 046104 antigen binding fragments bind to NPR1 with a K _D 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, measured by surface plasmon resonance, for example, in an assay format, defined in Example 3 of the present description, or a substantially similar analysis.

The present invention also includes antibodies and antigen-binding antibody fragments that bind to the monkey NPR1 dimeric protein complexed with BNP (e.g., at 25 or 37°C) with a K _D of less than 10 nM, as measured by surface plasmon resonance, e.g., in an assay format defined in example 3 of this description. In some embodiments, antibodies or antigen-binding fragments thereof 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. format of the analysis defined in Example 3 of the present description, or a substantially similar analysis.

The present invention also includes antibodies and antigen binding fragments thereof that bind to cells expressing human NPR1 with or without ANP at 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 , measured, for example, in the assay format described below in Example 5, or a substantially similar assay.

The present invention also includes antibodies and antigen binding fragments thereof that activate NPR1 with an EC ₅₀ of less than 385 nM as measured by a cellular calcium flux bioassay, for example, in the assay format defined in Example 6 herein. In some embodiments, antibodies or antigen-binding fragments thereof activate NPR1 with an EC ₅₀ 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 bioassay based on cellular calcium flux, for example, in the assay format defined in Example 6 herein, or a substantially similar assay.

The present invention also includes antibodies and antigen-binding antibody fragments that bind to NPR1 and reduce systemic blood pressure in a subject for more than 28 days when administered to a subject in need thereof as a single dose, for example, as described below in Example 8.

The present invention also includes antibodies and antigen-binding antibody fragments that bind to NPR1 and reduce fasting blood glucose levels when administered to a subject in need thereof, for example, as described below in Example 11.

In one embodiment, the present invention provides an isolated recombinant antibody or antigen binding fragment thereof that specifically binds to NPR1 protein in the presence or absence of ANP or BNP and enhances NPR1 activity, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: ( a) is a fully human monoclonal antibody; (b) binds to human monomeric NPR1 in the absence of ANP and/or BNP at 25 and 37°C with a dissociation constant (K _D ) less than 690 nM, measured in a surface plasmon resonance assay; (c) binds to human dimeric NPR1 in the absence of ANP or BNP at 25 and 37°C with a K _D of less than 42 nM measured in a surface plasmon resonance assay; (d) binds to human NPR1 complexed with ANP at 25 and 37°C with a K _D of less than 80 nM measured in a surface plasmon resonance assay; (e) binds to human NPR1 complexed with BNP at 25 and 37°C with a K _D of less than 20 nM measured in a surface plasmon resonance assay; (f) binds to monomeric monkey NPR1 in the absence of ANP and/or BNP at 25 and 37°C with a K _D of less than 365 nM measured in a surface plasmon resonance assay; (g) binds to dimeric monkey NPR1 in the absence of ANP or BNP at 25 and 37°C with a K _D of less than 30 nM measured in a surface plasmon resonance assay; (h) binds to monkey NPR1 in complex with ANP at 25 and 37°C with a K _D of less than 10 nM measured in a surface plasmon resonance assay; (i) binds to monkey NPR1 in complex with BNP at 25 and 37°C with a K _D of less than 10 nM measured in a surface plasmon resonance assay; (j) does not bind to mouse NPR1; (k) binds to cells expressing human NPR1 (in the absence of ANP) or the NPR1-ANP complex with an EC ₅₀ of less than 5 nM; (l) activates NPR1 with an EC ₅₀ of less than 385 nM measured in a cellular calcium flux bioassay; (m) reduces systemic blood pressure when administered to normotensive and hypertensive mice, with reductions in systemic and mean arterial pressure lasting up to 28 days after a single dose; (n) improves glucose tolerance when administered to diet-induced obese mice; and (o) contains HCVR containing an amino acid sequence selected from the group consisting of the HCVR sequence shown in table. 1, and LCVR containing an amino acid sequence selected from the group consisting of LCVR sequences given in table. 1.

Antibodies of the present invention may have one or more of the above biological characteristics or any combinations thereof. Other biological characteristics of the antibodies of the present invention will be apparent to one skilled in the art from a review of the present invention, including the working examples provided herein.

- 20 046104

Epitope mapping and related technologies.

The present invention includes anti-NPR1 antibodies that react with one or more amino acids present in one or more regions of the NPR1 protein molecule. The epitope to which antibodies bind may consist of one contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located in any of the above domains of the NPR1 protein molecule (eg, a linear epitope within the domain). Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within one or both of the above domains of the protein molecule (eg, a conformational epitope).

To determine whether an antibody reacts with one or more amino acids in a polypeptide or protein, various methods known to those skilled in the art can be used. Examples of methods include, for example, routine cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004), Methods Mol. Biol. 248:443-63), peptide bond cleavage analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope removal, epitope isolation, 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 reacts is hydrogen/deuterium exchange, detected by mass spectrometry. In general terms, the hydrogen-deuterium exchange method involves labeling a protein of interest with deuterium, followed by binding of an antibody to the deuterium-labeled protein. The protein/antibody complex is then transferred to water, and the exchanged protons in the amino acids that are protected by the antibody complex undergo back-deuterium-to-hydrogen exchange at a lower rate than the exchanged protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface can retain deuterium and will therefore have a relatively higher mass compared to amino acids not included in the interface. Once the antibody is dissociated, the target protein is subjected to protease digestion and mass spectrometric analysis, thereby identifying deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999), Analytical Biochemistry, 267:252-259; Engen and Smith (2001), Anal. Chem. 73:256A-265A.

The term epitope refers to the region of an antigen to which B and/or T cells respond. B cell epitopes can be formed from either contiguous amino acids or non-contiguous amino acids superimposed on each other due to tertiary protein folding. Epitopes formed from contiguous amino acids are usually retained when exposed to denaturing solvents, whereas epitopes formed from tertiary stacking are usually lost when exposed to denaturing solvents. An epitope usually includes at least 3, and more often at least 5 or 8-10 amino acids in a unique spatial conformation.

Modification-by-modification profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method that allows the classification of large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to profile similarity binding each antibody to chemically or enzymatically modified antigen surfaces (see US 2004/0101920, specifically incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is either clearly different from or partially overlaps with the epitope represented by another category. This technology allows for rapid filtering of genetically identical antibodies so that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs that have desired characteristics. MAP can be used to sort the antibodies of the invention into groups of antibodies that bind to different epitopes.

In some embodiments, the present invention includes hhtu-NPRI antibodies and antigen binding fragments thereof that react with one or more epitopes found in the extracellular domain of NPR1. The epitope(s) may consist of one or more contiguous sequences of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20 or more) amino acids located in the extracellular domain of NPR1. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within NPR1.

The present invention includes hhtu-NPR1 antibodies that bind to the same epitope or part of an epitope as any of the specific exemplary antibodies listed in table. 1. Likewise, the present invention also includes anti-NaK.1 antibodies that compete for binding to the NPR1 protein or a fragment thereof with any of the specific exemplary antibodies listed in Table 1. 1. For example, the present invention includes anti-NPR! antibodies that cross-compete for binding to the NPR1 protein with one or more of the antibodies listed in table. 1.

- 21 046104

Using standard methods known in the art, it is easy to determine whether an antibody binds to or competes for binding to the same epitope as the reference anti-NaK1 antibody. For example, to determine whether a test antibody binds to the same epitope as a reference anti-NPR1 antibody of the invention, allow the reference antibody to bind to the NPR1 protein or peptide under saturation conditions. The ability of the test antibody to bind to the NPR1 protein molecule is then assessed. If the test antibody is able to bind to NPR1 after saturation of binding to the reference hhtu-NPRI antibody, it can be concluded that the test antibody binds to a different epitope than that of the reference anti-N.K.1 antibody. On the other hand, if the test antibody is unable to bind to the NPR1 protein after saturation of binding to the reference anti-NPR1 antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-NPR1 antibody of the invention.

To determine whether an antibody competes for binding with a reference anti-NPR1 antibody, the binding method described above is performed in two ways: in the first case, allowing the reference antibody to bind to the NPR1 protein under saturation conditions, followed by assessing the binding of the test antibody to the NPR1 molecule. In the second case, the test antibody is allowed to bind to the NPR1 molecule under saturation conditions, followed by an assessment of the binding of the reference antibody to the NPR1 molecule. If in both directions only the first (saturating) antibody is able to bind to the NPR1 molecule, then it is concluded that the test antibody and the reference antibody are competing for binding to NPR1. As one skilled in the art will appreciate, an antibody that competes for binding to a reference antibody may not necessarily bind to an epitope identical to the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the other antibody from binding to the antigen. That is, a 1-, 5-, 10-, 20-, 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 by a competitive binding assay ( see, for example, Junghans et al., Cancer Res. 1990, 50:1495-1502). Alternatively, two antibodies have the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if certain amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

Additional standard experiments (eg, peptide mutation assays and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope to which the reference antibody also binds, or whether the lack of observed binding is due to steric blocking (or other phenomenon). Experiments of this nature can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

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 said antibody or antigen-binding fragment thereof interacts with one or more amino acids contained in the extracellular domain of NPR1 (amino acids 29-347 SEQ ID NO: 194) defined by hydrogen/deuterium exchange, and wherein the antibody or antigen binding fragment (i) binds to cells expressing human NPR1 in the presence or absence of atrial natriuretic peptide (ANP); and/or (ii) binds to NPR1 and activates NPR1. In one embodiment, the antibody or antigen binding fragment thereof reacts with an amino acid sequence selected from the group consisting of (a) amino acids 29-45 SEQ ID NO: 194; (b) amino acids 331-347 SEQ ID NO: 194; (c) amino acids 336-347 SEQ ID NO: 194; (d) amino acids 331-335 SEQ ID NO: 194 and (e) amino acids 70-81 SEQ ID NO: 194. In one embodiment, the antibody or antigen binding fragment thereof reacts with an amino acid sequence selected from the group consisting of (a) amino acids 29-45 SEQ ID NO: 194 and (b) amino acids 336-347 SEQ ID NO: 194. In one embodiment, the antibody or antigen binding fragment thereof reacts with an amino acid sequence selected from the group consisting of (a) amino acids 29- 45 SEQ ID NO: 194 and (b) amino acids 331-347 SEQ ID NO: 194, in the presence of ANP. In one embodiment, the antibody or antigen binding fragment thereof reacts with an amino acid sequence selected from the group consisting of (a) amino acids 29-45 SEQ ID NO: 194; (b) amino acids 70-81 SEQ ID NO: 194; and (c) amino acids 331-335 SEQ ID NO: 194, in the presence of ANP. In one embodiment, the present invention provides an isolated antibody or antigen binding fragment thereof that specifically binds to the NPR1 protein in the presence of ANP, wherein the antibody or antigen binding fragment thereof reacts 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

- 22 046104

SEQ ID NO: 194, but not with amino acids 70-81 SEQ ID NO: 194, as determined by hydrogen/deuterium exchange, and wherein the antibody or antigen binding fragment (i) binds to cells expressing human NPR1 in the presence or in absence of ANP; and/or (ii) binds to NPR1 and activates NPR1.

Immunoconjugates.

The invention includes a human monoclonal anti-NPR1 antibody conjugated to a therapeutic moiety (immunoconjugate) for the treatment of an NPR1-associated disease or disorder (eg, hypertension). As used herein, the term immunoconjugate refers to an antibody that is chemically or biologically linked to a radioactive agent, cytokine, interferon, target or reporter fragment, enzyme, peptide or protein, or therapeutic agent. An antibody may be associated with a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide, or a therapeutic agent anywhere in the molecule as long as it is capable of binding to its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second, different antibody to the NPR1 protein. The type of therapeutic moiety that can be conjugated to the anti-NaK4 antibody is selected based on the condition being treated and the desired therapeutic effect to be achieved. Examples of suitable agents for the formation of immunoconjugates are known in the art; see, for example, WO 05/103081.

Polyspecific antibodies.

Antibodies of the present invention may be monospecific, bispecific or polyspecific. Polyspecific 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.

Any of the multispecific antigen binding molecules of the invention or variants thereof can be constructed using standard molecular biology techniques (eg, recombinant DNA and protein expression technology) known to one of ordinary skill in the art.

In some embodiments, NPR1-specific antibodies are generated in a bispecific format (bispecific), in which the variable regions that bind to individual domains of the NPR1 protein are linked together, providing dual-domain specificity within a single binding molecule. Properly designed bispecific drugs can improve the overall efficacy of NPR1 protein inhibition by increasing both binding specificity and avidity. Variable regions with specificity for individual domains (for example, segments of the N-terminal domain) or that can bind to different regions within the same domain are paired in a structural framework, allowing each region to bind at the same time to separate epitopes, or in different regions within the same domain. In one example, to obtain bispecificity, heavy chain variable regions (VH) from a binder with specificity for one domain are recombined with light chain variable regions (VL) from a series of binders with specificity for a second domain to identify unrelated V partners _L , which can be paired with the original _VH without violating the original specificity for this VH. Thus, one VL segment (eg, VL1) can be combined with two different VH domains (eg, VH1 and VH2) to create a bispecific molecule consisting of two binding arms (VH1-VL1 and VH2-VL1). The use of a single VL segment reduces the complexity of the system and thus simplifies and increases the efficiency of the cloning, expression and purification processes used to create a bispecific antibody (see, for example, US 2011/0195454 and US 2010/0331527).

Alternatively, antibodies that bind to more than one domain and a second target, such as, but not limited to, for example, a second other anti-NPR1 antibody, can be produced in a bispecific format using the methods presented herein or other methods known to those skilled in the art. this area. Antibody variable regions that bind to different regions can be linked together with variable regions that bind to corresponding regions, for example on the extracellular domain of NPR1, to impart dual antigen specificity within a single binding molecule. Properly designed bispecificities of this nature perform a dual function. Variable regions with extracellular domain specificity are combined with a variable region with extracellular domain specificity and paired on a structural framework that allows each variable region to bind to distinct antigens.

An exemplary bispecific antibody format that can be used in the context of the present invention includes the use of a first immunoglobulin (Ig) CH3 domain and a second Ig C _H 3 domain, wherein the first and second Ig C _H 3 domains differ from each other by at least one

- 23 046104 amino acid, and wherein at least a difference in one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody that does not have such a difference in amino acid. In one embodiment, the first CH3 Ig domain binds to protein A, and the second CH3 Ig domain contains a mutation that reduces or abolishes protein A binding, such as modification H95R (IMGT exon numbering; H435R EU numbering). The second CH3 may additionally contain the modification Y96F (according to IMGT; Y436F according to EU). Additional modifications that may be present in the second CH3 include D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of IgG1 antibodies; N44S, K52N and V82I (by IMGT; N384S, K392N and V422I by EU) in the case of IgG2 and Q15R antibodies, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and EU V422I) in the case of IgG4 antibodies. Variants of the bispecific antibody format described above are contemplated within the scope of the present invention.

Other exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, bispecific formats based on scFv or diabodies, IgG-scFv fusions, double variable domain (DVD)-Ig, quads, peak-valve structures, conventional light chains (e.g. common light chain with peak-to-valley structures, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, Fab in two-in-one format (DAF)-IgG and Mab (see, for example, Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein for an overview of the above formats). Bispecific antibodies can also be constructed by peptide/nucleic acid conjugation, for example, where unnatural amino acids with orthogonal chemical reactivity are used to create site-specific antibody-oligonucleotide conjugates, which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

Therapeutic administration and formulations.

The invention relates to therapeutic compositions containing anti-NPR1 antibodies or antigen-binding fragments thereof according to the present invention. Therapeutic compositions in accordance with the invention can be administered with suitable carriers, excipients and other agents that are formulated to enhance transport, delivery, tolerance and the like. Many suitable formulations can be found in a formulary known to all pharmaceutical chemists: 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 (eg, LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions and water-in-oil, Carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing Carbowax. See also Powell et al. Compendium of excipients for parenteral formulations, PDA (1998), J. Pharm. Sci. Technol. 52:238-311.

The dose of the antibody may vary depending on the age and size of the subject to be administered, the target disease, conditions, route of administration, and the like. When an 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 preferable to administer the antibody of the present invention generally in a single dose of about 0.1 to about 100 mg/kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In some embodiments, an antibody or antigen binding fragment thereof of the invention may be administered at an initial dose of at least about 0.1 to about 800 mg, about 1 to about 600 mg, about 5 to about 500 mg, or about 10 to about 500 mg. approximately 400 mg. In some embodiments, the initial dose may be followed by a second or more subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be about the same or less than the amount of the initial dose, with subsequent doses separated by at least 1-3 days; at least one 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.

Various delivery systems are known that can be used to administer the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) , J Biol Chem 262:4429–4432). Routes of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example by infusion or bolus injection, absorption through epithelial or mucocutaneous membranes (for example, oral mucosa, rectal mucosa, etc.), and can also be administered together with other biological active agents. Administration may be systemic or local. The pharmaceutical composition can also be delivered to

- 24 046104 vesicle, in particular in liposomes (see, for example, Langer (1990), Science, 249:1527-1533).

The present description also discusses the use of nanoparticles for delivering antibodies of the present invention. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic purposes. Antibody-conjugated nanoparticles and methods for their preparation and use are described in detail by Arruebo, M., et al. 2009 (Antibody-conjugated nanoparticles for biomedical applications in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporated herein by reference. Nanoparticles can be formulated and conjugated with antibodies contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery are also described, for example, in US 8257740 or US 8246995, each of which is included in the present description in its entirety.

In certain situations, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials may be used. In yet another embodiment, the controlled release system can be placed in close proximity to the target to which the composition is directed, requiring only a fraction of the systemic dose.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injection preparations can be prepared by generally known methods.

The pharmaceutical composition of the present invention can be administered subcutaneously or intravenously using a standard needle and syringe. In addition, with regard to subcutaneous delivery, a delivery pen can be used when delivering the pharmaceutical composition of the present invention. The delivery device, such as a pen, can be reusable or disposable. A pen-type reusable delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. Once the entire amount of the pharmaceutical composition contained in the cartridge has been administered and the cartridge has been emptied, 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. The pen is a disposable delivery device and does not have a replaceable cartridge. More specifically, the pen in the form of a disposable delivery device comes with a pre-filled pharmaceutical composition contained in a reservoir within the device. Once the pharmaceutical composition in the reservoir has been completely used up, the device itself is discarded.

Advantageously, the pharmaceutical compositions described above for oral or parenteral administration are prepared in unit dose dosage forms suitable for the prescribed dosage of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained is typically from about 5 to about 500 mg per dosage form per unit dose; In particular, in the case of an injectable dosage form, it is preferred that the antibody be contained in an amount of from about 5 to about 300 mg and other dosage forms are contained in an amount of from about 10 to about 300 mg.

Therapeutic use of antibodies.

The antibodies of the present invention are useful for treating and/or preventing a disease or disorder or condition associated with NPR1, and/or for alleviating at least one symptom associated with such a disease, disorder or condition. In some embodiments, an antibody or antigen-binding fragment thereof of the invention may be administered at a therapeutic dose to a patient with a disease, disorder, or condition associated with NPR1.

In some embodiments, the antibodies of the present 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 disease, pulmonary fibrosis, inflammation, asthma, skeletal growth disorders, bone fractures, diabetes and cancer.

The present description also provides for the use of one or more antibodies of the present invention for prophylactic purposes in subjects at risk of developing an NPR1-associated disease or disorder.

In one embodiment of the present invention, the antibodies of the present invention are used for the manufacture of a pharmaceutical composition or medicament for treating patients suffering from a disease, disorder or condition disclosed herein. In another embodiment, the antibodies of the present invention are used as adjunctive therapy with any other agent or any other therapy known to those skilled in the art to be useful for treating or alleviating a disease, disorder or condition disclosed herein.

- 25 046104

Combination therapy.

The combination therapy may include an antibody of the invention and any additional therapeutic agent that can be effectively combined with an antibody of the invention or a biologically active fragment of an antibody of the invention. Antibodies of the present invention can be synergistically combined with one or more drugs or therapies used to treat an NPR1-associated disease or disorder. In some embodiments, the antibodies of the invention can be combined with a second therapeutic agent to alleviate one or more symptoms of a specified disease or condition.

Depending on the disease, disorder, or condition, the antibodies of the present invention may be used in combination with one or more additional therapeutic agents, including, but not limited to, an aldosterone antagonist (e.g., eplerenone, spironolactone), an alpha blocker (e.g., doxazosin, phenoxybenzamine, phentolamine, prazosin , terazosin), angiotensin-converting enzyme (ACE) inhibitor (eg, benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, traprindopril), arteriolar vasodilator (eg, hydralazine, minoxidil), autonomic ganglion vasodilator (eg, mecamylamine), beta-blocker (acebutolol, atenolol, betaxolol, bisoprolol, carvedilol, carteolol, esmolol, labetolol, metaprolol, nadolol, penbutyrol, pindolol, propranolol, timolol), sympatholytic catecholamine depleting agent (for example, deserpidine, reserpine), central alpha-2 adrenergic agonist (eg, clonidine, guanabenz, guanfacine, methyldopa), calcium channel blocker (diltiazem, verapamil, amlodipine, felodipine, isradipine, nicadipine, nifedipine, nisoldipine), diuretic (eg, bumetanide, ethacrynic acid, furoseamide, torsemide, chlorothiazide, hydrochlorothiazide, hydroflumthiazide, methyclothiazide, polythiazide, chlorthalidone, indapamide, metolazone), renin inhibitor (eg, aliskiren), anticoagulant (eg, coumardin, dabigatran, apixaban), antiplatelet agent (eg, aspirin, clopidogrel), agent, cholesterol lowering agent (eg, statin, PCSK9 inhibitor such as alirocumab), vasodilator (eg, minoxidil, hydralazine, nitrates), digitalis, surgery (eg, angioplasty, coronary artery bypass grafting, heart transplant), implantable device (eg, valve replacement, defibrillator, left ventricular assist device, pacemaker), antineoplastic therapy (eg, chemotherapy, surgery, radiation, PD-1 inhibitor), insulin, GLP1 agonist (eg, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide), metformin , dialysis, bone marrow stimulator, hemofiltration, lifestyle modification and nutritional supplement.

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

Additional therapeutically active component(s) can be administered to the subject prior to administration of the anti-NPRI antibody of the present invention. For example, the first component may be considered administered before the second component if the first component is administered in 1 week, 72, 60, 48, 36, 24, 12, 6, 5, 4, 3, 2, 1 hour, 30 minutes, or less than 30 min before introducing the second component. In other embodiments, additional therapeutically active component(s) may be administered to a subject following administration of an anti-NPR1 antibody of the present invention. For example, the first component may be considered administered after the second component if the first component is administered 30 minutes, 1, 2, 3, 4, 5, 6, 12, 24, 36, 48, 60, 72 hours or more after administration of the second component. In other embodiments, additional therapeutically active component(s) may be administered to a subject concurrently with administration of the anti-NPR1 antibody of the present invention. Concurrent administration for purposes of the present invention includes, for example, administering an anti-No.P.1 antibody and an additional therapeutically active component to a subject in a single dosage form or in separate dosage forms administered to the subject within about 30 minutes or less. When administered in separate dosage forms, each dosage form may be administered by the same route (eg, both the anti-No.P.1 antibody and the additional therapeutically active component may be administered intravenously, etc.); alternatively, each dosage form can be administered by different routes (eg, anti-NPR! antibody can be administered intravenously, and the additional therapeutically active component can be administered orally). In any case, simultaneous administration for the purposes of the present invention refers to the administration of components in the same dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes. For purposes of the present invention, administration of an hhtu-NPR1 antibody before, simultaneously with, or after (as those terms are defined hereinabove) administration of an additional therapeutically active component should be considered as administration of an hhtu-NPR1 antibody in combination with an additional therapeutically active component.

The present invention includes pharmaceutical compositions in which the anti-No.P.1 antibody of the present invention is formulated in one formulation with one or more additional components.

- 26 046104 peutically active components, as described elsewhere in this document.

Diagnostic use of antibodies.

Antibodies of the present invention can be used to detect and/or measure NPR1 in a sample, for example, for diagnostic purposes. Some embodiments provide for the use of one or more antibodies of the present invention in assays for detecting an NPR1-associated disease or disorder. Exemplary diagnostic assays for NPR1 may include, for example, contacting a sample obtained from a patient with an anti-NoK.1 antibody of the invention, wherein the anti-NoK.1 antibody is labeled with a detectable label or reporter molecule or used as a capture ligand for selective isolating NPR1 from patient samples. Alternatively, for diagnostic purposes, an unlabeled anti-NPR1 antibody can be used in combination with a secondary antibody that is itself a detectable label. The detectable label or reporter molecule may be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure NPR1 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in the NPR1 diagnostic assays of the present invention include any tissue or fluid sample obtained from a patient that contains detectable amounts of NPR1 protein or fragments thereof in a normal or pathological state. Typically, to initially determine a baseline or standard NPR1 level, NPR1 protein levels are measured in a specific sample obtained from a healthy patient (eg, a patient not suffering from an NPR1-associated disease). This baseline NPR1 level can then be compared to NPR1 levels measured in samples obtained from individuals suspected of having an NPR1-associated condition or symptoms associated with such a condition.

Antibodies specific for the NPR1 protein may contain no additional tags or fragments, or may contain an N-terminal or C-terminal tag or fragment. In one embodiment, the tag or moiety is biotin. In a binding assay, the location of the label (if present) can determine the orientation of the peptide relative to the surface on which the peptide is bound. For example, if the surface is coated with avidin, the peptide containing N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal to the surface.

Examples

The following examples are provided for the purpose of providing those skilled in the art with complete disclosure and description of how to develop and apply the methods and compositions of the present invention, and are not intended to limit the scope of the present invention. Although every effort has been made to ensure the accuracy of the numbers used (e.g. quantity, temperature, etc.), some experimental errors and deviations should be taken into account. Unless otherwise stated, 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 pressure.

Example 1: Generation of human antibodies to natriuretic peptide receptor 1 (NPR1).

Human antibodies to the NPR1 protein were generated in VELOCIMMUNE® mice containing DNA encoding the human immunoglobulin heavy and kappa light chain variable regions. Mice were immunized with human NPR1 and mouse ANP DNA by DNA hydrodynamic delivery and stimulated with the extracellular domain of the human NPR1 protein complexed with mouse ANP.

The antibody response was monitored using an NPR1-specific immunoassay. When the required immune response was achieved, splenocytes were collected and fused with murine myeloma cells to maintain their viability and generate hybridoma cell lines. Hybridoma cell lines were screened and cell lines that produced NPR1-specific antibodies were selected for identification. Cell lines were used to generate several chimeric anti-NPR1 antibodies (ie, antibodies containing human variable domains and mouse constant domains).

Anti-NPRI antibodies were also isolated directly from mouse antigen-positive B cells without fusion with myeloma cells, as described in US Pat. No. 7,582,298, which is specifically incorporated herein by reference in its entirety. Using this method, several fully human anti-K.1 antibodies (ie, antibodies having human variable domains and human constant domains) were produced.

Exemplary antibodies prepared by the method described above are designated mAb22033, mAb22035, mAb22805, mAb22809, mAb22810, mAb25479, mAb25491, mAb25497, mAb25498, mAb25502, mAb25508, and mAb25545 .

- 27 046104

The biological properties of exemplary antibodies produced in accordance with the methods of this example are described in detail in the examples set forth below.

Example 2. Amino acid and nucleotide sequences of the variable regions of the heavy and light chains.

In table 1 shows identifiers of the amino acid sequences of the variable regions of the heavy and light chains and CDRs of selected anti-NPR1 antibodies according to the invention.

Table 1

Amino acid sequence identifiers

SEQ ID NO: Antibody designation HCV R HCDR 1 HCDR 2 HCD R3 LCVR LCDR1 LCDR2 LCDR 3 shаb22033 2 4 6 8 10 12 14 16 shаb22035 18 20 22 24 26 28 thirty 32 shаb22805 34 36 38 40 42 44 46 48 tAL22809 50 52 54 56 58 60 62 64 shаb22810 66 68 70 72 74 76 78 80 shаb25479 82 84 86 88 90 92 94 96 wAL25491 98 100 102 104 106 108 110 112 shАb25497 114 116 118 120 122 124 126 128 shаb25498 130 132 134 136 138 140 142 144 wAL25502 146 148 150 152 154 156 158 160 shаb25508 162 164 166 168 170 172 174 176 shаb25545 178 180 182 184 186 188 190 192

Identifiers of the corresponding nucleic acid sequences are presented in table. 2.

table 2

Nucleic acid sequence identifiers

SEQ ID NO: Antibody designation HCVR HCDR 1 HCDR 2 HCDR 3 LCV R LCD R.I. LCD R2 LCD R3 shаb22033 1 3 5 7 9 eleven 13 15 shаb22035 17 19 21 23 25 27 29 31 shаb22805 33 35 37 39 41 43 45 47 shаb22809 49 51 53 55 57 59 61 63 shаb22810 65 67 69 71 73 75 77 79 shАb25479 81 83 85 87 89 91 93 95 wAL25491 97 99 101 103 105 107 109 111 shАb25497 113 115 117 119 121 123 125 127 shаb25498 129 131 133 135 137 139 141 143 wAL25502 145 147 149 151 153 155 157 159 shаb25508 161 163 165 167 169 171 173 175 shаb25545 177 179 181 183 185 187 189 191

Antibodies provided herein generally have fully human variable regions, but may have human or murine constant regions. As one skilled in the art will appreciate, an antibody having a particular Fc domain isotype can be converted to an antibody with a different Fc domain isotype (e.g., a mouse IgG1 Fc domain antibody can be converted to a human IgG4 antibody, etc. .), but in any case the variable domains (including CDRs), which are designated by the numeric identifiers shown in table. 2 remain the same and the antigen binding properties are expected to be identical or substantially identical regardless of the nature of the Fc domain. In some embodiments, the selected mouse IgG1 Fc domain antibodies are converted to human IgG4 Fc domain antibodies. In one embodiment, the IgG4 Fc domain contains two or more amino acid substitutions, as described in US 2010/0331527. In one embodiment, the Fc domain of human IgG4 contains a mutation

- 28 046104 serine to proline in the hinge region (S108P), which helps stabilize the dimer. Unless otherwise noted, all antibodies used in the examples below contain the human IgG4 isotype.

Control structures used in the examples below.

For purposes of comparison, the following control construct (anti-NPR1 antibody) is included in the experiments reported herein: Comparator 1, an anti-human NPR1 monoclonal antibody having the VH/VL sequences of mAb5591 according to US Pat. No. 2012/0114659 (Morphosys).

Example 3. Antibody binding to NPR1 determined by surface plasmon resonance.

Experimental procedure.

The equilibrium dissociation constant (KD) for various NPR1 reagents that bind to purified monoclonal anti-NPR1 antibodies (mAbs) was determined using a Biacore 4000 biosensor by real-time surface plasmon resonance. All binding experiments were performed in a running buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v. surfactant Tween-20, pH 7.4 (HBS-ET) at 25 and 37°C. The surface of the Biacore CM5 sensor chip was first derivatized by amino immobilization of either mouse anti-human Fc antibody (GE Healthcare, #BR100839) or goat polyclonal anti-human Fcy antibody (Jackson ImmunoResearch Laboratories, #BR-1008-39) to capture the anti-NPR1 mAb . Binding experiments were performed using the extracellular domain of human NPR1 expressed with a C-terminal myc-myc-hexahistidine (hNPR1-MMH) (SEQ ID NO: 194), the extracellular domain of monkey NPR1 expressed with a C-terminal myc-myc-hexahistidine ( mfNPR1-MMH) (SEQ ID NO: 195), extracellular domain of mouse NPR1 expressed with a C-terminal myc-myc-hexahistidine (mNPR1-MMH) (SEQ ID NO: 196), extracellular domain of human NPR1 expressed with a C-terminal mouse IgG2a (hNPR1-mFc) (SEQ ID NO: 197), extracellular domain of monkey NPR1 expressed with the C-terminal mouse IgG2a (mfNPR1-mFc) (SEQ ID NO: 198), extracellular domain of mouse NPR1 expressed with the 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, mNPR1-mFc+mBNP . Different concentrations of hNPR1-MMH, mfNPR1-MMH (100-3.7 nM, 3-fold serial dilution; or 100-6.25 nM, 4-fold serial dilution); hNPR1-mFc, mfNPR1.mFc (100-1.56 nM, 4-fold serial dilution; or 100-3.7 nM, 3-fold serial dilution); mNPR1.mmh (100 nM), hNPR1-mFc or mfNPR1-mFc complexed with hANP or hBNP at 10-fold concentration (100, 25, 6.25 or 100-3.7 nM, 3-fold serial dilution); mNPR1.mFc complexed with mANP or mBNP at 10-fold concentration (100, 25, or 100-3.7 nM, 3-fold serial dilution) or hNPR1-hFc or hNPR1-hFc complexed with hANP at 10-fold concentration ( 100-6.25 nM, 4-fold serial dilution), prepared in HBS-ET running buffer, were injected over 4 min at a flow rate of 30 μl/min, while the dissociation of mAb bound to various NPR1 reagents was monitored over 10 min in HBS-ET running buffer. At the end of each cycle, the NPR1 mAb capture surface was regenerated using either a 10-second injection of 20 mM phosphoric acid for mouse anti-human Fc-specific mAbs or a 40-second injection of 10 mM glycine, HCl, pH 1.5, for goat anti- human Fcy-specific polyclonal antibodies, or two injections of 10 mM Gly, pH 1.5, for 1 min. The rate of association ( _ka ) and rate of dissociation (kd) were determined by fitting real-time binding sensorgrams to a mass transfer-limited 1:1 binding model using Scrubber 2.0c curve fitting software. The equilibrium dissociation constant for binding ( _KD ) and the half-life due to dissociation (t1/2) were calculated from the kinetic rates as follows:

K _D (M) = (kd/k _a ) and t'/2 (min) = 1p(2)/(60*ka)

Results.

The binding kinetics parameters for various NPR1 proteins that bind to selected anti-NPR1 antibodies of the invention at 25 and 37°C are shown in Table 1. 3-26.

- 29 046104

Table 3 Binding kinetics parameters for the binding of hNPRl-MMH to anti-NPRl monoclonal antibodies at 25°C________

Captured mAbs mAb Capture Level (RU) 100 nM bound Ag (RU) k _a (1/Ms) kd(l/c) KD (M) t ^X /2 (min) shаb22033 377 + 0.9 thirty 8.55E+03 4.06E-04 4.75E-08 28.4 shаb22035 368 + 0.6 210 2.03E+05 1.50E-04 7.38E-10 77.3 shаb22805 356 + 0.7 0 NB* NB* NB* NB* shаb22809 359+ 1.1 0 NB* NB* NB* NB* shаb22810 386 + 3.8 1 NB* NB* NB* NB* shаb25479 338+ 1.2 16 1.40E+04 6.73E-03 4.82E-07 1.7 wAL25491 275 ± 0.9 37 2.62E+04 9.83E-04 3.76E-08 11.7 shАb25497 314 + 0.6 22 ZD7E+04 1.67E-03 5.27E-08 6.9 shаb25498 325 + 0.8 68 2.25E+04 1.82E-03 8D0E-08 6.3 wAL25502 349 + 2.3 2 NB* NB* NB* NB* shаb25508 296+ 1 192 4.32E+05 1.34E-04 3.09E-10 86.5 shаb25545 360 + 0.7 2 NB* NB* NB* NB* IgG4 isotype control 315 + 0.7 1 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions. Indicates that the observed binding data is not informative.

Table 4

Binding kinetics parameters for the binding of hNPR1-MMN ________ to anti-NPR1 monoclonal antibodies at 37°C ______

Captured mAbs mAb Capture Level (RU) 100 nM bound oAg(RU) k _a (1/Ms) kd(l/c) KD(M) 1½ (min) shаb22033 473 + 2.1 52 6.03E+03 8D9E-04 l,36E-07 14.1 shаb22035 492 ± 2.3 284 2.40E+05 ZD2E-04 l,30E-09 37.1 shаb22805 451 + 2 1 NB* NB ^3, NB ^3, NB ^3, shаb22809 466 + 2 -1 NB* NB* NB* NB* shаb22810 428+ 1.2 1 NB* NB* NB* NB* shаb25479 443 ± 4.5 16 2.28E+04 l,57E-02 6.89E-07 0.7 wAL25491 357+ 1.8 51 3.56E+04 4.53E-03 l,27E-07 2.5 shАb25497 402 + 3 31 1.86E+04 2.58E-03 l,39E-07 4.5 shаb25498 418 + 2.8 97 2.25E+04 4D5E-03 l,84E-07 2.8 wAL25502 452 + 3.3 4 NB* NB* NB* NB* shаb25508 397 + 2.1 252 4.53E+05 2.22E-04 4.90E-10 52.1 shаb25545 463 ± 1.7 7 IC* IC* IC* IC* IgG4 isotype control 417 + 3.1 3 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions. Indicates that the observed binding data is not informative.

- 30 046104

Table 5 Binding kinetics parameters for the binding of mfNPRl-MMN ____with anti-NPR1 monoclonal antibodies at 25°C________

Captured Shab Capture level shab (RU) 100 nM bound oAg (RU) ka (1/Ms) kd (1/s) KD (M) 1½ (min) shаb22033 376 ±0.8 79 1.79E+04 2.91E-04 1.62E-08 39.6 shаb22035 368 ±0.8 245 4.26E+05 1.43E-04 3.36E-10 80.8 shаb22805 356± 1.1 0 NB* NB* NB* NB ^S shаb22809 359 ±0.9 0 NB* NB* NB* NB ^S shаb22810 381 ±0.8 1 NB* NB* NB* NB ^S shаb25479 338 ±0.5 41 2.77E+04 6.82E-03 2.46E-07 1.7 wAL25491 275 ± 0.4 81 4.20E+04 1.28E-03 3.05E-08 9.0 shАb25497 315 ±0.6 49 3.78E+04 2.26E-03 5.98E-O8 5.1 shаb25498 327 ± 0.5 114 5.79E+04 1.58E-O3 2.72E-08 7.3 wAL25502 350 ±0.9 3 NB ^S NB ^S NB ^S NB ^S shаb25508 298 ± 1.1 213 7.06E+05 1.32E-04 1.87E-10 87.5 tAL25545 360 ± 0.5 4 IC* IC* IC* IC* IgG4 isotype 315 ±0.6 0 NB ^$ NB ^$ NB ^$ NB ^$ control

$ Indicates that no binding was observed under the current experimental conditions. Indicates that the observed binding data is not informative.

Table 6

Binding kinetics parameters for mfNPR1-MMN binding to anti-NPR1 monoclonal antibodies at 37°C

Captured Shab mAb Capture Level (RU) 100 nM bound oAg (RU) ka (1/Ms) kd (1/s) KD(Μ) 1½ (min) shаb22033 470 ± 1.4 131 2.51E+04 6.97E-04 2.78E-08 16.6 shаb22035 490 ± 1.3 319 5.32E+05 3.74E-04 7.02E-10 30.9 shаb22805 449 ± 1.3 3 NB* NB* ΝΒ* NB* shаb22809 465 ± 1 1 NB* NB* ΝΒ* NB* tAL22810 426 ± 1.1 0 NB ^S NB ^4, ΝΒ ^4, NB ^4, shаb25479 440 ± 1.8 43 3.96E+04 1.42E-02 3.60E-07 0.8 wAL25491 353 ± 1.2 106 5.83E+04 4.57E-03 7.84E-08 2.5 wAL25497 397 ± 1.4 69 2.77E+04 3.84E-03 1.39E-07 3.0 shаb25498 414 ± 1.7 151 7.07E+04 3.69E-03 5.21E-08 3.1 wAL25502 449 ± 1.7 6 NB* NB* ΝΒ* NB* shаb25508 394 ± 1.1 275 7.82E+05 2.73E-04 3.49E-10 42.3 shаb25545 461 ± 1 10 IC* IC* IC* IC* IgG4 isotype control 413 ± 1.3 3 NB ^$ NB ^$ ΝΒ ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions. Indicates that the observed binding data is not informative.

- 31 046104

Table 7

Binding kinetic parameters for mNPRl-MMH binding to anti-NPRl monoclonal antibodies at 25°C

Captured Shab Capture level shab (RU) 100 nM bound Ag (RU) k _a (1/Ms) ka (1/s) KD(M) 1½ (min) shаb22033 376 ± 0.7 0 NB* NB* NB* NB* shаb22035 368 ± 0.2 0 NB* NB* NB* NB* shаb22805 358 ± 1.9 0 NB* NB* NB* NB* shаb22809 358 ±0.3 0 NB* NB* NB* NB* shаb22810 378 ± 0.3 -1 NB* NB* NB* NB* shаb25479 338 ± 1.2 0 NB* NB* NB* NB* wAL25491 276 ± 0.3 0 NB* NB* NB* NB* shАb25497 315 ± 0.1 0 NB* NB* NB* NB* shаb25498 326 ± 0.6 0 NB* NB* NB* NB* wAL25502 349 ± 0.7 0 NB* NB* NB* NB* shаb25508 296 ± 1.2 0 NB* NB* NB* NB* shаb25545 359 ±0.1 0 NB* NB* NB* NB* IgG4 isotype control 315 ± 0.1 0 NB ^$ NB ^$ NB ^$ NB ^$

Means that no binding was observed under the current experimental conditions. Indicates that the observed binding data is not informative.

# Indicates that the conditions have not been tested.

Table 8

Binding kinetics parameters for mNPR1-MMN binding to anti-NPR1 monoclonal antibodies at 37°C

Captured niAbs mAb Capture Level (RU) 100 nM bound Ag (RU) ka (1/Mc) kd(l/c) KD(M) 1½ (min) mAb22033 468 ± 0.6 1 NB* NB* NB* NB* mAb22035 491 ± 0.9 1 NB* NB* NB* NB* mAb22805 450 ± 0.2 1 NB* NB* NB* NB* mAb22809 466 ±0.1 0 NB* NB* NB* NB* mAb22810 420 ±0.1 -2 NB* NB* NB* NB* mAb25479 438 ± 0.2 1 NB* NB* NB* NB* mAb25491 353 ± 1.8 0 NB* NB* NB* NB* mAb25497 395 ± 0.7 0 NB* NB* NB* NB* mAb25498 413 ±0.2 0 NB* NB* NB* NB* mAb25502 447 ± 0.9 0 NB* NB* NB* NB* mAb25508 392 ± 0.3 0 NB* NB* NB* NB* mAb25545 460 ± 0.4 1 NB* NB* NB* NB* IgG4 isotype control 412 ±0.4 1 NB ^$ NB ^$ NB ^$ NB ^$

$

Means that no binding was observed under the current experimental conditions.

Indicates that the observed binding data is not informative.

# Indicates that the conditions have not been tested.

- 32 046104

Table 9

Binding kinetics parameters for the binding of hNPR1-mFc or hNPR1-hFc to anti-NPRl monoclonal antibodies at 25°C

Captured mAbs mAb Capture Level (RU) 100 nM bound oAg (RU) ka (1/Ms) kd (1/s) KD (M) 1½ (min) shаb22033 351 ± 1.3 56 6D8E+04 6.38E-05 1.03E-09 181.0 shаb22035 347 ± 1.2 198 2.25E+05 8.20E-05 3.65E-10 140.9 shаb22805 331 ±0.8 2 NB* NB* NB* NB* shаb22809 327 ±0.7 -2 NB* NB* NB* NB* shаb22810 380 ±0.4 4 IC* IC* IC* IC* shаb25479 327 ±2.1 26 3.80E+04 8.52E-04 2.24E-08 13.6 wAL25491 249 ± 2.8 36 1.01E+05 1.24E-04 1.24E-09 92.8 shАb25497 302 ±25.8 49 2.03E+05 4.83E-05 2.38E-10 239.3 shаb25498 312 ± 0.8 94 6.98E+04 2.55E-04 3.65E-09 45.3 wAL25502 321 ± 1.6 17 8.79E+04 8.74E-05 9.94E-10 132.1 shаb25508 276 ± 1.5 211 7.89E+05 6.85E-05 8.68E-11 168.6 shаb25545 340 ± 1.1 7 IC* IC* IC* IC* IgG4 isotype control 298 ± 1.6 -14 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions. Indicates that the observed binding data is not informative.

Table 10

Binding kinetics parameters for binding of hNPR1-mFc or hNPR1-hFc to anti-NPR1 monoclonal antibodies at 37°C

Captured mAbs mAb Capture Level (RU) 100 nM bound oAg (RU) k _a (1/Ms) kd(l/c) KD(Μ) 1½ (min) shаb22033 423 ±3.1 75 3.68E+04 1.31E-04 3.56E-09 88.2 shаb22035 454 ±3.6 257 6.36E+05 1.27E-04 2.00E-10 90.8 shаb22805 406 ± 1.8 8 NB ^3, NB ^3, NB ^3, NB ^3, shаb22809 413 ± 1.6 -1 NB* NB* NB* NB* shаb22810 423 ± 0.8 9 IC* IC* IC* IC* shаb25479 414 ±5.6 47 3.37E+04 1.39E-03 4D2E-08 8.3 wAL25491 298 ± 1.6 70 4D9E+04 3.63E-04 8.66E-09 31.8 shАb25497 344 ± 1.7 43 4.23E+04 ZD0E-04 7.31E-09 37.3 shаb25498 381 ±2 129 8.86E+04 5.68E-04 6D1E-09 20.3 wAL25502 396 ±2.8 9 NB* NB* NB* NB* shаb25508 353 ± 1.3 242 8.38E+05 1.01E-04 l,21E-10 113.9 shаb25545 415 ±2.8 15 IC* IC* IC* IC* IgG4 isotype control 366 ±3.9 -19 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions.

* Indicates that the observed binding data is not informative.

- 33 046104

Table 11

Binding kinetics parameters for binding of hNPR1-mFc:hANP or hNPR1-hFc:hANP complex to anti-NPR1 monoclonal antibodies at 25°C

Captured whores SHL grip level (RU) 100 nM bound oAg(RU) k _a (1/Ms) ka (1/s) KD (M) 1½ (min) shаb22033 350 ±0.4 84 6.03E+04 8.60E-05 1DZE-09 134.4 shаb22035 347 ± 0.3 186 1J9E+05 7.78E-05 4.34E-10 148.4 shаb22805 331 ± 1.8 84 4.31E+04 1D1E-04 3.28E-09 81.7 shаb22809 327 ± 1.5 53 3.92E+04 1.93E-04 4.94E-09 59.7 shаb22810 378 ± 0.6 14 8.31E+O3 3.44E-04 4D4E-08 34 shаb25479 327 ± 2.4 49 ZD0E+04 1.09E-04 3.53E-09 105.8 wAL25491 250 ± 1.7 71 5.22E+04 4D5E-05 7.94E-10 278.5 shАb25497 292 ±2.1 59 1.63E+05 4.56E-05 2.79E-10 253.1 shаb25498 313 ±0.6 52 4.58E+04 ZD7E-04 6.93E-09 36.4 wAL25502 321 ±4.3 81 6.06E+04 2.53E-04 4D7E-09 45.7 shаb25508 278 ± 4.2 199 2.64E+05 8.97E-05 ZD0E-10 128.8 shаb25545 341 ± 1.8 28 2.34E+04 4.63E-05 1.98E-09 249.7 IgG4 isotype control 297 ± 1.5 -12 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions.

* Indicates that the observed binding data is not informative.

Table 12

Binding kinetics parameters for the binding of hNPR1-mFc:hANP or hNPR1-hFc:hANP complex to anti-NPR1 monoclonal antibodies at 37°C

Captured whores Capture level shab (RU) 100 nM bound oAg(RU) ka (1/Ms) kd(l/c) KD (M) 1½ (min) shаb22033 412 ± 1.2 108 5.79E+04 9.80E-05 1.69E-09 117.9 shаb22035 444 ±2 237 2.25E+05 1D4E-04 5.08E-10 101.0 shаb22805 399 ± 1.9 113 6.01E+04 2.95E-04 4.91E-09 39.2 shаb22809 406 ±0.8 68 5.04E+04 1.66E-04 3.29E-09 69.5 shаb22810 421 ±0.8 26 1.37E+04 1.04E-03 7.55E-08 eleven shаb25479 408 ± 7.6 84 4.97E+04 1.34E-04 2.70E-09 86.1 wAL25491 291 ± 1.3 96 5.69E+04 1D1E-04 1.95E-09 104.1 shАb25497 336 ± 1.6 60 4.85E+04 1.02E-04 2.09E-09 113.7 shаb25498 375 ± 1.9 74 5.30E+04 7.20E-04 1.36E-08 16.0 wAL25502 388 ±5.1 105 7.20E+04 4.67E-04 6D9E-09 24.7 shаb25508 348 ±0.8 230 2.93E+05 1D8E-04 5.06E-10 77.9 shаb25545 409 ± 2.7 58 3.67E+04 8.61E-05 2.35E-09 134.2 IgG4 isotype control 358 ±4.9 -18 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions.

* Indicates that the observed binding data is not informative.

- 34 046104

Table 13

Binding kinetic parameters for the binding of the hNPR1-mFc:hBNP complex to anti-NPR1 monoclonal antibodies at 25°C

Captured aphids Capture level shab (RU) 100 nM bound oAg(RU) k _a (1/Ms) kd(l/c) KD (M) t7 ₂ (min) tAL22033 350 ±0.4 85 5DZE+04 6.67E-05 1.23E-09 173.2 tAL22035 348 ±2.9 194 1J1E+05 8.70E-05 5.08E-10 132.7 tAL22805 330 ±0.4 84 4D4E+04 1.52E-04 3.68E-09 75.8 tAL22809 327 ±0.4 51 3.28E+04 2.21E-04 6.72E-09 52.4 tAL22810 NT ^# NT ^# NT ^# NT ^# NT ^# NT ^# tAL25479 326 ±0.5 50 2.92E+04 1.23E-04 4.21E-09 94.1 tAL25491 250 ±0.3 72 5D6E+04 4.96E-05 9.08E-10 232.9 tAL25497 292 ± 0.4 59 1.53E+05 5.49E-05 3.58E-10 210.5 tAL25498 313 ± 0.4 55 4.85E+04 2.56E-04 5.28E-09 45.1 tAL25502 317 ± 1.5 78 6.94E+04 2.36E-04 3.40E-09 49.0 tAL25508 275 ± 0.6 200 2.50E+05 8.57E-05 ZDZE-10 134.7 tAL25545 340 ±0.2 27 2.30E+04 6.26E-05 2.72E-09 184.6 IgG4 isotype control 296 ± 0.5 -eleven NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions.

* Indicates that the observed binding data is not informative.

# Indicates that the conditions have not been tested.

Table 14

Binding kinetic parameters for the binding of the hNPR1-mFc:hBNP complex to anti-NPR1 monoclonal antibodies at 37°C

Captured niAb mAb Capture Level (RU) 100 nM bound oAg(RU) ka (1/Ms) kd(l/c) KD (M) 1½ (min) shаb22033 409 ± 1.4 108 5.37E±04 9.60E-05 1.79E-09 120.3 shаb22035 440 ± 0.8 243 2D2E+05 1.28E-04 5.53E-10 90.0 shаb22805 398 ± 1.4 112 5.20E+04 3.1 ZE-04 6.02E-09 36.9 shаb22809 405 ± 0.5 63 4.65E+04 1.66E-04 3.56E-09 69.8 shаb22810 shаb25479 wAL25491 shАb25497 shаb25498 wAL25502 NT ^# 402 ±0.4 289 ±1.5 333 ±1.1 372 ±0.9 391 ±0.7 NT ^# 85 96 59 76 102 NT ^# 4.27E+04 5D7E+04 4.92E+04 5.29E+04 6.03E+04 NT ^# 1.63E-04 1.34E-04 1D6E-04 9D8E-04 4.72E-04 NT ^# 3.83E-09 2D5E-09 2.35E-09 1.79E-08 7.82E-09 NT ^# 70.7 86.2 99.7 12.2 24.5 shаb25508 347 ± 0.9 236 3.52E+05 1.64E-04 4.66E-10 70.3 shаb25545 405 ± 1.2 56 ZD6E+04 9.84E-05 2.85E-09 117.3 IgG4 isotype control 354 ±0.4 -17 NB ^$ NB ^$ NB ^$ NB ^$

Means that no binding was observed under the current experimental conditions.

* Indicates that the observed binding data is not informative.

# Indicates that the conditions have not been tested.

- 35 046104

Table 15

Binding kinetic parameters for mfNPR1-mFc binding to anti-NPR1 monoclonal antibodies at 25°C

Captured whores Capture level shab (RU) 100 nM bound oAg(RU) ka (1/Ms) kd(l/c) KD(M) 1½ (min) shаb22033 351 ±0.7 96 5D7E+04 4.85E-05 9.38E-10 238.0 shаb22035 346 ± 0.6 231 2.44E+05 5.57E-05 2.29E-10 207.2 shаb22805 331 ± 1.6 10 IC* IC* IC* IC* shаb22809 328 ± 0.7 6 NB ^S NB ^S NB ^S NB ^S shаb22810 NT* NT* NT* NT* NT* NT* shаb25479 325 ± 0.7 54 ZD2E+04 5.07E-04 l,62E-08 22.8 wAL25491 251 ± 1.4 81 5D0E+04 7.54E-05 1D0E-09 153.1 shАb25497 292 ± 1 76 1J2E+05 5.79E-05 3.37E-10 199.4 shаb25498 313 ±0.6 128 7.20E+04 1J7E-04 2D5E-09 65.4 wAL25502 321 ± 1.9 22 8.83E+04 1D1E-04 l,60E-09 82.0 shаb25508 276 ± 0.9 230 5D5E+05 5.98E-05 1D6E-10 193.3 shаb25545 340 ± 0.7 13 IC* IC* IC* IC* IgG4 isotype control 297 ± 0.6 5 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that ' no binding was observed under the current experimental conditions. Indicates that the observed binding data is not informative.

# Indicates that the conditions have not been tested.

Table 16

Binding kinetics parameters for mfNPR1-mFc binding to anti-NPR1 monoclonal antibodies at 37°C

Captured niAbs mAb Capture Level (RU) 100 nM bound oAg (RU) k _a (1/Mc) ka (1/c) KD(M) 1½ (min) mAb22033 415 ± 1.3 126 4.82E+04 9.90E-05 2.06E-09 116.7 mAb22035 447 ± 1.5 291 4.59E+05 8D4E-05 l,84E-10 136.9 mAb22805 402 ± 1.7 16 IC* IC* IC* IC* mAb22809 409 ± 1.6 8 NB ^S NB ^S NB ^S NB* mAb22810 NT* NT* NT* NT* NT* NT* mAb25479 408 ± 1.3 89 3.82E+04 l,08E-03 2.81E-08 10.7 mAb25491 294 ± 1.6 114 4.70E+04 l,64E-04 ZD9E-09 70.4 mAb25497 340 ± 1.1 79 4.25E+04 2.21E-04 5.20E-09 52.3 mAb25498 377 ± 1.4 170 9D6E+04 3.72E-04 4.06E-09 31.1 mAb25502 393 ± 1.7 17 IC* IC* IC* IC* mAb25508 350± 1.1 268 6.34E+05 8D6E-05 l,32E-10 138.2 mAb25545 410± 1 26 5.51E+04 1D4E-04 2.62E-09 80.0 IgG4 isotype control 360 ± 1.4 8 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions. Indicates that the observed binding data is not informative.

# Indicates that the conditions have not been tested.

- 36 046104

Table 17

Binding kinetic parameters for the binding of the mfNPR1-mFc:hANP complex to anti-NPRl monoclonal antibodies at 25°C

Captured whores mAb Capture Level (RU) 100 nM bound oAg (RU) ka (1/Mc) kd(1/c) KD(M) 1½ (min) shаb22033 349 ±0.5 133 6.58E+04 5.36E-05 8D5E-10 215.5 shаb22035 346 ±0.7 227 1.89E+05 4.66E-05 2.46E-10 247.9 shаb22805 330 ±0.5 116 4.84E+04 9.51E-05 l,96E-09 121.4 tAL22809 327 ±0.5 86 4.47E±04 l,73E-04 3.88E-09 66.6 tAL22810 NT ^# NT ^# NT ^# NT ^# NT ^# NT ^# tAL25479 326 ±0.3 86 4.20E±04 l,03E-04 2.44E-09 112.7 tAL25491 250 ±0.8 111 6.01E±04 4.55E-05 7.57E-10 253.8 tAL25497 292 ± 0.6 95 l.56E+05 3.75E-05 2.40E-10 307.7 tAL25498 313 ±0.4 81 4.19E±04 1D2E-04 2.68E-09 102.9 tAL25502 316 ±2.7 113 7.40E±04 2.64E-04 3.56E-09 43.8 tAL25508 273 ± 0.5 224 3.14E±05 7.32E-05 2.33E-10 157.7 tAL25545 339 ±0.4 45 2.28E±04 5.37E-05 2.35E-09 215.1 IgG4 isotype 296 ± 0.5 7 NB ^$ NB ^$ NB ^$ NB ^$ th control

$ Indicates that no binding was observed under the current experimental conditions.

* Indicates that the observed binding data is not informative.

# Indicates that the conditions have not been tested.

Table 18

Binding kinetic parameters for binding of the mfNPR1-mFc:hANP complex to anti-NPR1 monoclonal antibodies at 37°C

Captured mAbs mAb Capture Level (RU) 100 hM bound ⁰ Ag (RU) k _a (1/Mc) ka(l/c) KD(M) 1½ (min) mAb22033 407 ± 1.1 169 6.80E±04 8.90E-05 l,31E-09 129.8 mAb22035 438 ±0.6 283 2.55E±05 7.70E-05 3.02E-10 150.1 mAb22805 395 ±0.3 153 6.33E±04 2.09E-04 3.3OE-O9 55.3 mAb22809 403 ± 0.4 106 5.55E±04 2.05E-04 3.70E-09 56.2 mAb22810 NT ^# NT ^# NT ^# NT ^# NT ^# NT ^# mAb25479 401 ± 1.6 138 5.47E±04 2D9E-04 4.00E-09 52.8 mAb25491 288 ± 1 148 7.10E±04 l,06E-04 l,49E-09 109.0 mAb25497 332 ±0.6 102 5.84E±04 l,52E-04 2.60E-09 75.9 mAb25498 370 ± 1.5 114 5.01E+04 4.45E-04 8.89E-09 25.9 mAb25502 390 ±0.7 143 9D0E+04 5.24E-04 5.76E-09 22.0 mAb25508 346 ±0.3 262 4D1E+05 l,46E-04 3.55E-10 79.3 mAb25545 404 ± 1.2 86 4.01E±04 8D8E-O5 2.04E-09 141.2 IgG4 isotype 353 ±0.8 9 NB ^$ NB ^$ NB ^$ NB ^$ th control

$ Indicates that no binding was observed under the current experimental conditions. Indicates that the observed binding data is uninformative, # Indicates that the conditions were not tested.

- 37 046104

Table 19

Binding kinetic parameters for the binding of the mfNPR1-mFc:hBNP complex to anti-NPR1 monoclonal antibodies at 25°C

Captured whores mAb Capture Level (RU) 100 nM bound oAg(RU) ka (1/Ms) ka(1/s) KD (M) 1½ (min) shаb22033 349 ±0.7 132 6.59E±04 5D5E-05 7.81E-10 224.2 shаb22035 346 ±0.3 231 2.05E±05 6DZE-05 3.00E-10 188.3 shаb22805 330 ± 1.5 113 4.53E±04 9.60E-05 2D2E-09 120.4 shаb22809 327 ±0.6 80 4D4E+04 1.60E-04 3.85E-09 72.4 shаb22810 NT ^# NT ^# NT ^# NT ^# NT ^# NT ^# shаb25479 325 ±0.1 86 3.81E±04 1.1 ZE-04 2.95E-09 102.6 wAL25491 249 ± 0.7 BY 5.86E±04 3.71E-05 6.32E-10 311.7 shАb25497 291 ±0.1 93 1.40E+05 4.47E-05 3.20E-10 258.4 shаb25498 312 ±0.6 81 4.32E±04 1.09E-04 2.53E-09 106.0 wAL25502 314 ± 3.4 BY 6.90E±04 2.82E-04 4.08E-09 41.0 shаb25508 273 ± 0.8 229 3.08E±05 8.91E-05 2.90E-10 129.6 shаb25545 339 ±0.1 42 2.39E±04 4.97E-05 2.08E-09 232.5 IgG4 isotype control 296 ± 1.1 7 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions.

*#

Indicates that the observed binding data is not informative;

Indicates that the conditions have not been tested.

Table 20

Binding kinetic parameters for binding of the mfNPR1-mFc:hBNP complex to anti-NPR1 monoclonal antibodies at 37°C

Captured mAbs mAb Capture Level (RU) 100 nM bound oAg(RU) ka (1/Ms) kd(l/c) KD (M) 1½ (min) shаb22033 406 ± 1.4 167 6.75E±04 8.8OE-O5 1,ZOE-09 131.3 shаb22035 436 ± 1.1 286 2.58E±05 9D8E-05 3.68E-10 121.9 shаb22805 394 ± 1 150 5.96E+04 2.02E-04 3.39E-09 57.2 shаb22809 402 ± 0.9 99 5.34E+04 2.29E-04 4.29E-09 50.5 shаb22810 NT ^# NT ^# NT ^# NT ^# NT ^# NT ^# shаb25479 399 ± 0.7 135 5.30E+04 2.93E-04 5.53E-09 39.4 wAL25491 286 ± 0.7 146 6.89E+04 1D0E-04 1.60E-09 105.0 shАb25497 330 ±0.5 100 5.74E+04 1.66E-04 2.90E-09 69.5 shаb25498 369 ±0 113 5.21E+04 4.64E-04 8.91E-09 24.9 wAL25502 388 ± 1.5 139 8.52E+04 4.81E-04 5.65E-09 24.0 shаb25508 344 ± 0.3 266 4.23E+05 1.50E-04 3.54E-10 77.1 shаb25545 402 ± 0.7 82 3.71E+04 7.77E-05 2.09E-09 148.6 IgG4 isotypic 351 ±0.5 9 NB ^$ NB ^$ NB ^$ NB ^$ control

Means that no binding was observed under the current experimental conditions.

Indicates that the observed binding data is not informative.

Indicates that the conditions have not been tested.

T *

#

- 38 046104

Table 21

Binding kinetics parameters for mNPR1-mFc binding to anti-NPR1 monoclonal antibodies at 25°C

Captured mAbs mAb Capture Level (RU) 100 nM bound Ag (RU) k _a (1/Ms) kd(l/c) KD(M) ^tl /z (min) shаb22033 350 + 0.5 0 NB* NB* NB* NB* shаb22035 346 ± 0.8 -1 NB* NB* NB* NB* tAL22805 330 + 0.5 0 NB* NB* NB* NB* tAL22809 328 ± 0.7 0 NB* NB* NB* NB* tAL22810 NT* NT* NT* NT* NT* NT* tAL25479 325 ± 0.3 -1 NB* NB* NB* NB* tAL25491 250+ 1.1 1 NB* NB* NB* NB* tAL25497 292 ± 0.7 1 NB* NB* NB* NB* tAL25498 313 + 1 0 NB* NB* NB* NB* tAL25502 321 ± 1.1 1 NB* NB* NB* NB* tAL25508 275 ± 0.7 -1 NB* NB* NB* NB* tAL25545 340 ± 0.2 0 NB* NB* NB* NB* IgG4 296 ± 0.6 0 NB* NB* NB* NB* isotypic control

$ Indicates that no binding was observed under the current experimental conditions. Indicates that the observed binding data is not informative.

# Indicates that the conditions have not been tested.

Table 22

Binding kinetic parameters for mNPR1-mFc binding to anti-NPR1 monoclonal antibodies at 37°C

Captured mAbs mAb Capture Level (RU) 100 nM bound Ag (RU) ka (1/Mc) ka (1/c) KD(M) 1½ (min) mAb22033 413 + 0.4 1 NB* NB* NB* NB* mAb22035 444 ± 0.5 0 NB* NB* NB* NB* mAb22805 400 ± 0.2 0 NB* NB* NB* NB* mAb22809 407+ 1 0 NB* NB* NB* NB* mAb22810 NT* NT* NT* NT* NT* NT* mAb25479 406 ± 0.3 2 NB* NB* NB* NB* mAb25491 291 + 0.9 1 NB* NB* NB* NB* mAb25497 336 + 0.7 1 NB* NB* NB* NB* mAb25498 375 +1 0 NB ³¹ NB* NB* NB* mAb25502 391 + 0.6 1 NB* NB* NB* NB* mAb25508 347 ± 0.4 0 NB* NB* NB* NB* mAb25545 407 ± 0.7 1 NB* NB* NB* NB* IgG4 isotype control 359 + 0.6 2 NB ^$ NB ^$ NB ^$ NB ^$

Means that no binding was observed under the current experimental conditions. # Indicates that the conditions have not been tested.

- 39 046104

Table 23

Binding kinetics parameters for the binding of the mNPR1-mFc:hANP complex to anti-NPR1 monoclonal antibodies at 25°C

Captured whores mAb Capture Level (RU) 100 nM bound oAg(RU) k _a (1/Ms) kd(l/c) KD(M) 1½ (min) shаb22033 348 ± 0.4 1 NB* NB* NB* NB* shаb22035 345 ± 0.4 0 NB* NB* NB* NB* shаb22805 330+ 1.1 18 1.98E+05 8.39E-02 4.24E-07 0.14 shаb22809 326 ± 0.4 0 NB* NB* NB* NB* shаb22810 NT* NT* NT* NT* NT* NT* shаb25479 324 ± 0.8 1 NB* NB* NB* NB* wAL25491 249 ± 0.9 1 NB* NB* NB* NB* shАb25497 290 ± 0.5 1 NB* NB* NB* NB* shаb25498 311 + 0.9 0 NB* NB* NB* NB* wAL25502 317 + 2.4 25 2.37E+05 7.99E-02 3.37E-07 0.14 shаb25508 274 ± 0.8 0 NB* NB* NB* NB* shаb25545 339 + 0.3 1 NB* NB* NB* NB* IgG4 isotype control 295 ± 0.8 1 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions. # Indicates that the conditions have not been tested.

Table 24

Binding kinetic parameters for binding of the mNPR1-mFc:hANP complex to anti-NPR1 monoclonal antibodies at 37°C

Captured mAbs mAb22033 mAb22035 mAb capture rate (RU) 402+ 1.8 435 + 0.8 100 nM bound Ag (RU) 2 1 k _a (1/Ms) NB ³³ NB* ka (1/c) NB* NB* KD(M) NB* NB* 1½ (min) NB* NB* mAb22805 392+1 15 IC* IC* IC* IC* mAb22809 400 ± 0.7 1 NB* NB* NB* NB* mAb22810 NT* NT* NT* NT* NT* NT* mAb25479 397+ 1.2 2 NB* NB* NB* NB* wAL25491 284 ± 0.7 1 NB ^3, NB* NB* NB* mAb25497 328 + 0.8 1 NB ^3, NB* NB* NB* shаb25498 367 + 0.4 1 NB ^3, NB* NB* NB* wAL25502 386 + 2.1 35 2.64E+05 8.83E-02 3.34E-07 0.13 shаb25508 343 + 1.1 0 NB ^3, NB* NB* NB* shаb25545 401 + 0.8 3 NB* NB* NB* NB* IgG4 isotype 349 + 0.5 2 NB ^$ NB ^$ NB ^$ NB ^$ control

$ Indicates that no binding was observed under the current experimental conditions.

Indicates that the observed binding data is not informative.

Indicates that the conditions have not been tested.

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Table 25

Binding kinetics parameters for the binding of the mNPR1-mFc:hBNP complex to anti-NPR1 monoclonal antibodies at 25°C

Captured mAbs mAb Capture Level (RU) 100 nM bound oAg(RU) ka (1/Ms) kd(1/c) KD(M) 1½ (min) shаb22033 347 ± 1.1 5 NB* NB* NB* NB* shаb22035 345 ± 0.8 0 NB* NB* NB* NB* shаb22805 329 ± 0.9 21 1.53E+05 6.82E-02 4.45E-07 0.17 shаb22809 326 ± 1.1 1 NB* NB* NB* NB* shаb22810 NT* NT* NT* NT* NT* NT* shаb25479 325 ± 0.2 1 NB* NB* NB* NB* wAL25491 249 ± 0.4 5 NB* NB* NB* NB* shАb25497 291 ± 0.2 2 NB* NB* NB* NB* shаb25498 312 ±0.8 1 NB* NB* NB* NB* wAL25502 318 ±0.3 25 2.34E+05 5.03E-02 2D5E-07 0.23 shаb25508 274 ± 0.6 1 NB* NB* NB* NB* shаb25545 339 ±0.4 3 NB* NB* NB* NB* IgG4 isotype control 296 ± 0.6 2 NB ^$ NB ^$ NB ^$ NB ^$

$ Indicates that no binding was observed under the current experimental conditions. # Indicates that the conditions have not been tested.

Table 26

Binding kinetic parameters for the binding of the mNPR1-mFc:hBNP complex to anti-NPR1 monoclonal antibodies at 37°C

Captured mAbs mAb22033 mAb22035 mAb capture rate (RU) 401 ± 0.2 433 ± 0.5 100 nM bound oAg (RU) 4 1 k _a (1/Mc) NB* NB* kd(l/c) NB* NB* KD(M) NB* NB* 1½ (min) NB* NB* mAb22805 389 ±0.8 17 IC* IC* IC* IC* mAb22809 397 ± 1.3 1 NB* NB* NB* NB* mAb22810 NT* NT* NT* NT* NT* NT* mAb25479 397 ±0.9 2 NB* NB* NB* NB* mAb25491 282 ±0.9 4 NB* NB* NB* NB* mAb25497 327 ±0.5 2 NB* NB* NB* NB* mAb25498 366 ±0.6 1 NB* NB* NB* NB* mAb25502 384 ± 1.3 34 2.91E+05 9.98E-02 3.42E-07 0.12 mAb25508 342 ±0.9 1 NB* NB* NB* NB* mAb25545 400 ± 0.4 4 NB* NB* NB* NB* IgG4 isotype 348 ±0.7 3 NB ^$ NB ^$ NB ^$ NB ^$ control

$

Means that no binding was observed under the current experimental conditions.

Indicates that the observed binding data is not informative.

# Indicates that the conditions have not been tested.

As shown in table. 3-6, selected anti-NPR1 antibodies bind to monomeric human and monkey NPR1.

As shown in table. 9-20, antibodies bind to dimeric human and monkey NPR1 in both the presence and absence of ANP or BNP.

The antibodies do not bind to mouse NPR1, with the exception of mAb25502, which binds to mNPR1 in the presence of ANP/BNP (Tables 21-26).

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Example 4. Cross-competition between selected monoclonal antibodies to NPR1 agonists.

Experimental procedure.

Binding competition within a panel of anti-NPR1 mAbs was determined using a real-time label-free biolayer interferometry assay on the Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25°C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v. surfactant Tween-20, 1 mg/ml BSA, buffer pH 7.4 (HBS-EBT), while shaking the plate at 1000 rpm. To assess whether the 2 antibodies were able to compete with each other for binding to the respective epitopes, 100 nM recombinant human NPR1 expressed with C-terminal mouse IgG2a (hNPR1-mFc; SEQ ID NO: 453) was first incubated with 2 μM human ANP in for at least 2 h. The recombinant hNPR1-mFc/hANP complex, approximately 0.3-0.5 nm in size, was first captured by dipping Octet biosensor tips coated with anti-mouse Fc fragment antibodies (Fortebio Inc, #18-5090). for 45 s into wells containing the hNPR1-mFc/hANP complex. The antigen-captured biosensor tips were then saturated with the first anti-NPR1 monoclonal antibody (termed mAb-1) by dipping into wells containing 50 μg/mL mAb-1 solution for 5 min. The biosensor tips were then immersed in wells containing a 50 μg/mL solution of a second anti-NPR1 monoclonal antibody (called mAb-2) for 3 min. Between each experimental step, the biosensor tips were washed with HBS-EBT buffer. The binding reaction was monitored in real time throughout the experiment, and the binding reaction was recorded at the end of each step. The binding reaction of mAb-2 with hNPR1-mFc/hANP complexed with mAb-1 was compared, and the competitive/noncompetitive behavior of various anti-NPR1 monoclonal antibodies was determined.

Results.

Table 27

Cross-competition between anti-NPR1

- 42 046104 shаb25502

tAL25545 tAL22805 tAL25479 tAL22810 shАb25497 wAL25502 shаb25545 shаb22805 tAL22809 tAL22033 tAL25491 tAL25502 tAL25545 tAL22805 tAL25479 tAL22810 tAL22809 tAL22033 tAL25491 tAL25497 tAL25545 tAL22805 tAL25479 tAL22810 tAL22809 tAL22033 tAL25491 tAL25497 tAL25502 tAL22805 tAL25479 tAL22810 tAL22809 tAL22033 tAL25491 tAL25497 tAL25502 tAL25545 tAL22810 shаb25479 tAL22809 tAL22033 tAL25491 tAL25497 tAL25502 tAL25545 tAL22810 tAL22810 tAL22809 tAL25445

In table 27 shows cross-competition between selected anti-NPRl antibodies.

Example 5: Antibody binding to cells expressing NPR1.

Experimental procedure.

The ability of monoclonal antibodies to human (h)NPR1 to bind to cells expressing human NPR1 (hNPR1), with or without one of the ligands human ANP (hANP) was determined by electrochemiluminescence (ECL) detection.

Briefly, cells expressing HEK293/hNPR1.myc.DKK were constructed by transfecting neomycin-resistant human embryonic kidney (HEK) 293 cells with the expression plasmid pLVX.hNPR1.myc.DDK encoding human NPR1 (amino acids M1-G1061, UniProtKB - P16066). Untransfected HEK293 cells, which lack NPR1 expression detected by fluorescence-activated cell sorting (FACS) with commercial a-hNRP1 antibodies, were included as a control for nonspecific binding.

Experiments were performed according to the following procedure. Cells from the lines described above were washed once in 1x PBS buffer without Ca ²⁺ /Mg ²⁺ and incubated for 10 min at 37°C with an enzyme-free cell dissociation solution to separate the cells from the tube. All

- 43 046104 cells were washed once with 1x PBS with Ca ²⁺ /Mg ²⁺ and counted using a Ceilometer™ Auto T4 cell counter (Nexcelom Bioscience). Approximately 2.0 x ^{10 4} HEK293/hNPR1.myc.DDK or HEK293 cells were individually seeded into 96-well carbon electrode plates (MULTI-ARRAY High Binding Plate, Meso Scale Discovery (MSD, Rockville, MD)) and incubated for 1 h at 37°C. Nonspecific binding sites were blocked with 2% BSA (w/v) in 1x PBS with Ca ²⁺ /Md ²⁺ for 1 h at room temperature (RT). HEK293/hNPR1.myc.DDK cells were incubated for 0.5 h at room temperature in sample dilution buffer with or without 10 nM human ANP (Tocris, Minneapolis, MN) and HEK293 cells were incubated in sample dilution buffer only. same conditions. Without washing, serial dilutions of anti-NPR1 antibodies, COMP1 or isotype control ranging from 1.7 pM to 100 nM, or buffer containing no antibodies, were added to plate-bound cells for 1 h at room temperature. The plates were then washed to remove unbound antibodies and/or hANP using an AquaMax2000 plate washer with cell wash head (MDS Analytical Technologies, Sunnyvale, CA). Plate-bound antibodies were detected using SULFO-TAG-conjugated goat polyclonal anti-human IgG heavy- and light-chain-specific antibodies (Jackson Immunoresearch, West Grove, PA) for 1 h at RT.

After washing, plates were developed using reading buffer (MSD, Rockville, MD) according to the manufacturer's recommended procedure, and luminescent signals were recorded using a SECTOR Imager 600 (MSD, Rockville, MD). Luminescence intensity, measured in relative light units (RLU), was recorded to determine the binding intensity of each antibody over a range of concentrations. The level of signal detected upon binding of 3.7 nM antibody to cells engineered to express NPR1, with or without 10 nM hANP, relative to the same concentration of antibody binding to parental cells without the addition of hANP, was recorded as an indicator of the specificity of NPR1 binding. Antibodies with a binding coefficient greater than or equal to 3 were classified as specific binders, and antibodies with a binding coefficient less than 3 were classified as non-binding agents.

In addition, direct binding (RLU) signals were analyzed as a function of antibody concentration, and data were fitted using a sigmoidal (four-parameter logistic) dose-response model using the R statistical package (open source). The EC ₅₀ value, defined as the antibody concentration at which 50% of the maximum binding signal is detected, was determined to indicate binding ability to cells engineered to express NPR1 with or without 10 nM hANP. In table 28 presents EC ₅₀ values for specific binders only and is marked (-) for antibodies with levels below 3.

Results.

In table 28 shows the binding of selected anti-NaD1 antibodies to cells engineered to express human NPR1 or the NPR1-ANP complex.

- 44 046104

Table 28

Binding of anti-NPRl antibodies to cells engineered to express the human NPR1 or NPR1-ANP complex on the cell surface

PID AB Cell binding signal level (RLU) HEK293/hNPRl.myc.DKK at an antibody concentration of 3.7 nM relative to parental HEK293 Binding ability to HEK293/hNPRl.myc.DKK cells, ec ₅₀ (M) + 10 nM hANP OhANP + 10 nM hANP OhANP Peptide-dependent NPR1 binding shаb22809 166 2 4.8E-10 - shаb22805 222 1 6.5E-10 - shаb25545 79 1 1.9E-09 - shаb22810 24 1 3.7E-09 - wAL25502 14 1 INC - Comparator! 103 2 1.5E-10 - Peptide-independent NPR1 binders shаb22033 236 207 8.2E-10 4.9E-10 shАb25497 87 50 8.7E-10 E5E-09 shаb22035 220 151 8.9E-10 2,ZE-09 wAL25491 78 58 1.0E-09 1.6E-09 shАb25479 51 thirty 1.2E-09 1.7E-09 shаb25508 35 18 1,ZE-09 INC tAL25498 49 41 1.8E-09 4.6E-09 h!gG4 isotype control 1 1 - -

(INC) = not informative, no upper plateau in the four-parameter sigmoid curve fitted to calculate the EC50 value, but the antibody bound specifically to cells engineered to express NPR1 with or without 10 nM hANP at a level equal to or greater than 3.

According to the results in table. 28 all anti-NPR1 antibodies specifically bound to cells engineered to express hNPR1 in the presence of 10 nM hANP with a ratio of >2. The effectiveness of these antibodies against HEK293/hNPR1 myc.DDK cells with 10 nM hANP ranged from EC ₅₀ values of 0.15 to 3.7 nM. Five antibodies and Comparator 1 specifically bound to cells engineered to express NPR1 only in the presence of 10 nM hANP and were classified as peptide-dependent NPR1 binders. The remaining seven antibodies bound to cells engineered to express hNPR1 with and without 10 nM hANP, and these antibodies were classified as peptide-independent NPR1 binders. The effectiveness of these antibodies against HEK293/hNPR1.myc.DDK cells without the addition of hANP was within the EC ₅₀ range of 0.49 to 4.6 nM.

Example 6. Activation of NPR1 by NPR1 agonist monoclonal antibodies.

Experimental procedure.

To assess transcriptional activation of human natriuretic peptide receptor 1 (hNPR1), a stable cell line stably expressing cyclic nucleotide-gated channel alpha 2 (CNGA2) and hNPR1 in HEK293 was generated. CNGA2 is a calcium channel that can be activated by cGMP, so it can be used as a sensor for cGMP generation (Wunder et al. 2005, PMID:15766716). Because NPR1 is activated by ligand and produces cGMP (Zois et al., 2014, PMID:24820868), CNGA2 is activated and calcium influx can be measured using a fluorescent Ca indicator. The cell line was sorted for high expression of hNPR1, HEK293/hNPR1.MycDDK/CNGA2.Myc HS or HEK293/CNGA2/hNPR1 for short, and maintained in DMEM containing 10% FBS, NEAA, penicillin/streptavidin/glutamate, 100 μg/ml hygromycin and 500 μg/ml G418 sulfate.

A bioassay was performed to measure the effect of anti-hNPR1 antibodies on human NPR1 signaling in the absence of ANP. For bioassay, HEK293/CNGA2/hNPR1 cells were seeded in black transparent bottom PDL plates at 30,000 cells/well in DMEM containing 10% FBS, NEAA, fine

- 45 046104 cillin/streptavidin/glutamate, and incubated at 37°C and 5% CO2 overnight. The next morning, the medium was removed and 80 μl of FLUO4-NW assay buffer with probenecid (Thermo Scientific) was added to the cells for 30 min at 37°C. Purified anti-hNPR1 antibody or isotype control antibody was serially diluted 1:3 from ~0.4 nM to 1 μM (8 spots) or ANP 1:3 from ~0.3 pM to 2 nM (10 spots), transferred to an assay plate using FLIPR TETRA® (Molecular Devices). Baseline and response images were obtained. The dose-response curve was determined based on Max-Min or area under the curve (shown in the description) and the results were analyzed by non-linear regression (4-parameter logistic curve) using Prism 6 software (GraphPad) to obtain EC ₅₀ values. % antibody activation was calculated with the maximum RFU (relative fluorescence units) range achieved by the antibody relative to the maximum RFU range achieved by the ANP.

Results.

In table 29 shows activation of human NPR1 by anti-NPR1 antibodies.

Table 29

Activation of human NPR1 by anti-NPR1 antibodies

Experiment 1 Experiment II Cells 293/CNGA2/hNPRl 293/CNGA2/hNPRl Mode Activation Activation Ligand ANP ANP EC50 (pM) 60.5-98.8 78.8 Constant No No Antibody EC ₅₀ (nM) % activation (vs. ANP max.) EC ₅₀ (nM) % activation (vs. ANP max.) shаb22033 83.7 129.54 41.6 129.20% shаb22810 Not a tester. Not a tester. 50.8 125.50% shаb25545 187.9 123.14% 74.1 122.70% wAL25502 161.4 92.90% >200 109.80% shаb22805 239 91.61% 112.3 118.90% shаb22809 270 86.72% Not a tester. Not a tester. wAL25491 101.8 74.64% 43.4 103.70% shаb22035 383.9 73.07% >200 56.80% shАb25497 72.62 71.09% 56.4 103.40% shаb25498 335.3 67.74% Not a tester. Not a tester. shаb25479 174.3 66.73% 101.9 82.80% shаb25508 325.7 56.08% Not a tester. Not a tester. Comparator 1 >2000 53.45% Activations No 13.80% Isotypic control Activations No -6.03% Activations No 1.90%

Activation of hNPR1 by selected anti-NPR1 antibodies of the invention was tested by measuring calcium flux activity in HEK293/CNGA2/hNPR1 in two experiments (Experiments I and II) (Table 29). As shown in table. 29 (in experiment I), 11 purified anti-NPR1 antibodies showed activation in a Ca ²⁺ current with maximum activation ranging from 55 to 130% with an EC50 in the range of 83.7383.9 nM. 19 antibodies showed weak activation with maximum activation of less than 31% (data not shown). In experiment II, 9 anti-NPR1 antibodies showed activation in a Ca ²⁺ current with maximum activation ranging from 57-129% with an EC50 of 41.6 - >200 nM (Table 29). ANP is activated with an EC ₅₀ of 60.5-98.8 pM. Activation was not observed with the isotype control antibody hIgG4.

Example 7 Effect of a Single Dose of NPR1 Agonist Monoclonal Antibody on Systemic Blood Pressure in Normotensive NPR1 ^hu/hu Mice

Experimental procedure.

The purpose of this study was to evaluate the effects of selected NPR1 agonist antibodies on baseline systemic blood pressure in telemetrically monitored normotensive NPR1 ^hu/hu mice. Male NPR1 ^hu/hu mice (n=50) ~10-20 weeks old were implanted with PA-C10 telemetry sensors (DSI, St. Paul, MN) and allowed to recover for 7 days before distribution.

- 46 046104 by group (groups 1-12) (Table 30).

Table 30

Summary of Doses and Dose Groups

No. groups Test or control product Dose level (mg/mg/dose) Dose volume (ml/kg) quantity animals males 1 PBS control 25 5 9 3 shаb22035 5 4 shаb22033 4 5 shАb25497 3 6 shаb22805 4 7 shаb25545 3 8 shаb22809 5 9 shаb25479 4 10 wAL25491 4 eleven wAL25502 5 12 shаb22810 4

Animals were housed singly under standard conditions (temperature, 64 to 84°F (18 to 29°C); relative humidity, 30 to 70%) and maintained on a 12-hour day/12-hour night cycle. Food (Research Diets Standard pelleted food) and water were freely available (ad libitum).

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

During testing, systolic pressure, diastolic pressure, mean arterial pressure, and heart rate were recorded for 10 s every minute. Efficacy assessments were performed using data collected from days 3 to 7 of the study. Long-term effects of NPR1 agonist antibodies were assessed using data recorded daily for 24 hours and averaged over 28 days. All data are presented as mean ± standard error of the mean (SEM).

Results.

Primary screening in vivo antibodies NPR1 demonstrated that compared to the control animals introduced by PBS 3-7 days after the administration of the dose of 9 antibodies (MAB22033, MAB25479, MAB25497, MAB22805, MAB25545, MAB22809, MAB25491, MAA, MA B25502 and MAB22810 ), decreased significantly, and one antibody (mAb22035) had no effect on systemic blood pressure (Fig. 1). The magnitude of blood pressure reduction, as assessed by the mean change (3 to 7 days post-dose) in systolic blood pressure from baseline, ranged from -3.2±0.2 (mAb25497N) to -11.5±0.8 (mAb22810 ) mmHg Art. (mmHg)

Long-term effects of NPR1 agonist antibodies were assessed over 28 days (Table 31).

Table 31

28-day mean arterial pressure and heart rate

Dosing group systolic (mmHg) diastolic (mmHg) Mean arterial (mmHg) Heart rate (BRM) PBS control 0.08+0.09 1.09+0.25 1.19+0.27 11.79+3.17 shаb25545 -4.34+0.18* 0.13+0.38* -2.0+0.46* 5.35+3.13* shаb22805 -3.90+0.17* -1.51+0.30* -3.40+0.40* 18.83+2.73* shАb25497 -3.04+0.20* 2.37+0.36* 1.04+0.44 12.07+2.64* shаb22035 3.80+0.32* 2.05+0.22* 2.95+0.26* 2.86+2.33* shаb22033 -11.07+0.59* -4.55+0.38* -8.01+0.48* -2.14+2.83* shаb22810 -7.88+0.57* -2.24+0.31* -5.21+0.42* -6.10+2.96* wAL25502 -3.63+0.49* -0.41+0.40* -2.13+0.44* -14.27+2.87* shаb22809 -2.01+0.58* 2.95+0.70* 0.30+0.62 3.95+2.33* wAL25491 -4.90+0.35* -1.22+0.26* -3.19+0.30* -12.9+3.22* shаb25479 -3.44+0.50* 0.51+0.42* -1.60+0.44* -4.01+3.03*

- 47 046104

Telemetry-controlled normotensive NPR1 ^hu/hu mice were divided into groups depending on body weight. Animals received a single subcutaneous injection of 25 mg/kg NPR1 agonist mAb or control PBS as described in Table 1. 1. All values represent mean ± SEM, n=3-9 per group. Statistics - two-way ANOVA with Dunnett's test; *p<0.05 compared to PBS control

After one subcutaneous injection on day 0, one antibody (mAb22035) significantly increased blood pressure by ∼4–7 mmHg, while the other antibodies decreased systemic blood pressure by 2–11 mmHg. in normotensive NPR1 ^hu/hu mice, heart rate responses to both elevated and decreased systemic blood pressure varied, with increases in some groups and decreases in other groups.

Example 8. Effect of dose of NPR1 agonist monoclonal antibody on systemic blood pressure in normotensive NPR1 ^hu/hu mice.

Experimental procedure.

The purpose of this study was to evaluate the dose effect of the NPR1 agonist antibody mAb22033 on systemic blood pressure in telemetrically monitored normotensive NPR1 ^hu/hu mice. Male NPR1 ^hu/hu mice (n=30) ~20 weeks old were implanted with PA-C10 telemetry sensors (DSI, St. Paul, MN) and allowed to recover for 7 days before being allocated to groups (groups 1-5). . (Table 32).

Table 32

Summary of Doses and Dose Groups

No. groups Test or control product Dose level (mg/mg/dose) Ob I eat doses (ml/kg) quantities about animals males 1 IgG4 isotype control 25 5 6 2 tAL22033 1 5 7 3 shаb22033 5 5 6 4 shаb22033 25 5 7 5 shаb22033 50 5 7

Animals were housed singly under standard conditions (temperature, 64 to 84°F (18 to 29°C); relative humidity, 30 to 70%) and maintained on a 12-hour day/12-hour night cycle. Food (Research Diets Standard pelleted food) and water were provided ad libitum.

Test proteins were administered to the respective animals as a single subcutaneous injection on day 0. The dose volume for each animal was determined based on the most recent body weight measurement. To assess urine and serum biomarkers, urine was collected on day 28, and blood samples were collected on study day 14 and at the end of the experiment. Echocardiography was performed on day 28 before urine output.

During testing, systolic pressure, diastolic pressure, mean arterial pressure, and heart rate were recorded for 10 s every minute. Graphically displayed telemetry data was obtained from animals with clear signals in the live mouse portion of the study.

Results.

Blood pressure (Fig. 2, 3 and 5) decreased by 10-15 mm Hg. Art. for up to 4 weeks after administration of a single dose of mAb22033 to normotensive NPR1 ^hu/hu mice. The peak blood pressure reduction was similar for all doses, with a duration of effect on blood pressure of approximately 10 days with 1 mg/kg and over 28 days with 50 mg/kg.

Table 33

28-day mean arterial pressure and heart rate

Dosing group systolic (mmHg) diastolic (mmHg) Mean arterial Heart rate (BRM)

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(mmHg) Isotype control IgG4 (25 mg/kg) 126+.3 97.0+0.3 112.2+0.3 524.9+3.2 mAL22033 (1 mg/kg) 124.4+0.5 95.1+0.4 110.4+0.4 530.6+4.0 mAL22033 (5 mg/kg) 120.0+0.4**** 93.8+0.3** 107.6+0.3**** 543.4+4.0** ** tAL22033 (25 mg/kg) 119.7+0.5**** 93.7+0.4* 107.1+0.4**** 524.5+3,,4 tAL22033 (50 mg/kg) 118.5+0.5**** 94.2+0.3* 106.8+0.3**** 553.6+3.5** **

Telemetry-controlled normotensive NPR1 ^hu/hu mice were randomly divided into five equal body weight groups and given a single subcutaneous injection of mAb22033 at the doses listed in Table 1. 1. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=6-7 per group. Statistics - two-way ANOVA with Dunnett's test; *p<0.05 compared to IgG4 isotype control, **p<0.01 compared to IgG4 isotype control; ***p<0.001 compared with IgG4 isotype control; ****p<0.0001 compared to IgG4 isotype control

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

Table 34

Urine and serum markers

Dosing group: Urine volume (ml/day) cGMP in urine (mmol/day) Serum NTproANP (nmol/L) day 14 Serum NTproANP (nmol/L) day 29 Isotype control IgG4 (25 mg/kg) 1.4+0.2 5.0+1.3 0.21+.06 0.49+0.17 mAL22033 (1 mg/kg) 1.2+0.2 5.2+1.3 0.35+0.11 0.52+0.12 mAL22033 (5 mg/kg) 0.9+0.1 3.7+0.8 0.20+0.08 0.20+0.05 wAL22033 (25 mg/kg) 1.2+0.2 5.9+0.8 0.14+0.06 0.24+0.06 wAL22033 (50 mg/kg) 1.4+0.2 11.9+2.1** 0.26+0.08 0.56+0.20

Telemetry-controlled normotensive NPR1 ^hu/hu mice were randomly divided into five groups of equal body weight and given a single subcutaneous injection of mAb22033 at the doses listed in Table 1. 1. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=6-7 per group. Statistics - one-way ANOVA with Dunnett's test; **p<0.01 compared with IgG4 isotype control.

Relative heart weight (Table 35) was lower in the 50 mg/kg group with a downward trend in the 1, 5 and 25 mg/kg groups. There was no significant effect on body weight (Table 35), absolute heart weight (Table 35), standard serum chemistry (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).

Table 35

Final body and organ masses

Dosing group: Ultimate body mass (G) heart weight (mg) Heart mass: length of tibia Heart weight: body weight Isotype control IgG4 (25 mg/kg) 33.1+1.7 0.143+0.003 0.0079+0.0002 0.0043+0.0001 mAL22033 (1 mg/kg) 34.5+2.3 0.140+0.005 0.0078+0.0002 0.0041+0.0001 mAL22033 (5 mg/kg) 34.3+1.4 0.131+0.006 0.0075+0.0004 0.0039+0.0003 wAL22033 (25 mg/kg) 32.4+1.4 0.126+0.006 0.0071+0.0003 0.0039+0.0002 mAb22033 (50 mg/kg 34.3+2.1 0.124+0.004 0.0069+0.0002 0.0037+0.0002*

Telemetry-controlled normotensive NPR1 ^hu/hu mice were randomly divided into five groups of equal body weight and given a single subcutaneous injection of mAb22033 at the doses listed in Table 1. 1. IgG4 antibody was used as isotype control. All values represent mean ± SEM, n=6-7 per group. Statistics - one-way ANOVA with Dunnett's test; *p<0.05 compared with IgG4 isotype control

Myocardial function (Figs. 6 and 7A) improved following administration of mAb22033, with a statistically significant reduction in end-systolic volume observed in the 25 mg/kg dose group (Fig. 7A) and a trend toward increases in both shortening fraction (Fig. 7C) and release (Fig. 7B), most evident in the 25 and 50 mg/kg groups.

Key findings are that the NPR1 agonist mAb mAb22033 caused significant reductions in systolic and mean arterial blood pressure that lasted up to 28 days. A compensatory increase in heart rate was initially observed in all groups, with a persistent slight increase in the mAb22033 50 mg/kg group relative to the isotype control mAb.

Example 9. Effect of a single dose of an NPR1 agonist monoclonal antibody on systemic blood pressure in hypertensive NPR1 ^hu/hu mice.

Experimental procedure.

The purpose of this study was to evaluate the effects of a single dose of NPR1 agonist antibody (mAb22033 or mAb22810) on systemic blood pressure in NPR1 ^hu/hu mice with angiotensin II-induced (Ang II) hypertension under telemetry monitoring. Male NPR1 ^hu/hu mice (n=36), ~13 weeks old, were implanted with PA-C10 telemetry sensors (DSI, St. Paul, MN) and allowed to recover for 7 days before being allocated to groups (groups 1–6) ( table 36).

Table 36

Summary of Doses and Dose Groups

No. groups Test or control product Dose level (mg/mg) Dose volume (ml/kg) quantity animals males 1 IgG4 isotype control 25 5 5 2 tAL22033 1 5 4 3 tAL22033 5 5 5 4 tAL22033 25 5 6 5 tAL22810 5 5 5 6 tAL22810 19 5 6

The animals were then implanted with osmotic mini-pumps (Alzet micro-osmotic pump; model 1004; lot 10335-14). Minipumps were filled with angiotensin II acetate (Bachem; lot # 1066804) and set to an average pumping rate of 0.11 μL/h to deliver 1.5 mg/kg/day Ang II. Minipumps were implanted subcutaneously in the scapular region 3 days before dosing. Animals were housed singly under standard conditions (temperature, 64 to 84°F (18 to 29°C); relative humidity, 30 to 70%) and maintained on a 12-hour day/12-hour night cycle. Food (Research Diets Standard pelleted food) and water were provided ad libitum.

The test proteins were administered subcutaneously to the respective animals once on day 3 by subcutaneous injection. Dose volumes for each animal were determined based on the most recent body weight measurement.

During testing, systolic pressure, diastolic pressure, mean arterial pressure, and heart rate were recorded for 10 s every minute. Urine was collected on days 14 and 20.

Results.

A single dose of NPR1 agonist mAb (mAb22033 or mAb22810) significantly reduced systemic blood pressure (Figs. 8, 9 and 11) when administered to NPR1 ^hu/hu mice with angiotensin-II-induced hypertension. All 23-day mean arterial pressure values (Table 37) were significantly lower in animals that received either mAb22033 or mAb22810 compared to the IgG4 isotype control.

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Table 37

23-day mean arterial pressure and heart rate

Dosage group: Systolic (mmHg) Diastolic (mmHg) Mean arterial (mmHg) heart rate (VRM) Isotype control IgG4 (25 mg/kg) 176±2 147±2 155±2 507±4 mAL22033 (1 mg/kg) 163±2**** I34-I-2**** 147±2 541±4**** mAL22033 (5 mg/kg) 153±2**** 120±1**** 132±4**** 547±7**** wAL22033 (25 mg/kg) 149-1-2**** 121±1**** -|_/| I am < I < 544±7**** mAL22810 (5 mg/kg) 162±2**** 128±2**** 145±2* 532±5** mAb22810 (19 mg/kg) 159±2**** 127±2**** 139±3*** 532±4**

All values represent mean ± SEM, n=3-6 per group. Statistics - two-way ANOVA with Dunnett's test; *p<0.05 compared with isotype control IgG4 **p<0.01 compared with isotype control IgG4; ***p<0.001 compared with IgG4 isotype control; ****p<0.0001 compared with IgG4 isotype control.

Table 38

Urine and serum biomarkers

Volume Volume NTproANP urine cGMP in urine urine cGMP in urine in plasma Group (ml/day) (mmol/day (ml/day) (mmol/day (nmol/l) - dosing: - Day 14 ) - day 14 - Day 20 ) - day 20 day 20 Isotypically th control IgG4 (25 2.2±0.7 8.7±2.5 5.2±0.9 13.9±3.1 0.7±0.1 mg/kg) wAL22033 (1 mg/kg) 3.0±0.7 13.8±2.3 6.6±0.6 23.4±2.8 0.8±0.2 mAL22033 (5 mg/kg) 1.3±0.5 11.0±3.2 3.9±0.3 20.8±0.8 1.0±0.3 wAL22033 (25 mg/kg) 2.1±0.3 23.5±5.3* 5.6±0.8 44.9±7.8*** 1.5±0.2 wAL22810(5 1.5±0.4 8.3±2.0 4.1±0.8 19.2±5.3 1.3±0.4 mg/kg) wAL22810(19 mg/kg) 2.3±0.3 13.6±1.8 4.9±0.7 18.8±1.9 1.0±0.2

All values represent mean ± SEM, n=3-6 per group. Statistics are one-way ANOVA with Dunnett's test. *p<0.05 compared with IgG4 isotype control; ***p<0.001 compared with IgG4 isotype control.

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Table 39

Final body and organ masses

Dosage group: Final body weight (g) Heart weight (mg) Heart mass: length of tibia Heart weight: body weight Isotype control IgG4 (25 mg/kg) 29±1 0.166±0.011 0.0093±0.0006 0.0057±0.0003 mAL22033 (1 mg/kg) 32±1 0.168±0.005 0.0094±0.0004 0.0053±0.0003 mAL22033 (5 mg/kg) 29±2 0.149±0.017 0.0085±0.0009 0.0051±0.0005 giAB22033 (25 mg/kg) 32±1 0.162±0.008 0.0090±0.0004 0.0051±0.0002 mAL22810 (5 mg/kg) 30±1 0.150±0.004 0.0084±0.0002 0.0050±0.0001 mAb22810 (19 mg/kg) 30±1 0.146±0.015 0.0082±0.0008 0.0049±0.0003

All values represent mean ± SEM, n=3-6 per group. Statistics are two-way ANOVA with Dunnett's test.

Heart rate effects (Fig. 10) increased sharply with more moderate chronic increases. The mean 23-day heart rate (Table 37) was significantly higher in all groups treated with the test drug compared to isotype control animals. Urinary cGMP levels tended to increase in most groups administered either mAb22033 or mAb22810, with animals receiving mAb22033 at 25 mg/kg having significantly higher urinary cGMP levels on days 14 and 20 compared to isotype animals. IgG control (Table 38). No effects on body weight or absolute or relative organ weight were observed (Table 39).

Example 10. Effect of repeated doses of an NPR1 agonist monoclonal antibody on systemic blood pressure in hypertensive NPR1 ^hu/hu mice.

Experimental procedure.

The purpose of this study was to evaluate the effects of repeated dosing of the NPR1 agonist antibody mAb22033 on systemic blood pressure in NPR1 ^hu/hu mice with angiotensin II (Ang II)-induced hypertension under telemetry monitoring. Male NPR1 ^hu/hu mice (n=30), ~26 weeks old, were implanted with PA-C10 telemetry sensors (DSI, St. Paul, MN) and allowed to recover for 7 days before being allocated to groups (groups 1-5) ( table 40).

Table 40

Summary of Doses and Dose Groups

No. groups Test or control product Dose level (mg/mg/dose) Ob I eat doses (ml/kg) quantities about animals males 1 IgG4 isotype control 25 5 6 2 tAL22033 1 5 6 3 tAL22033 5 5 6 4 tAL22033 25 5 6 5 tAL22033 50* 5 6

*One dose.

The animals were then implanted with osmotic mini-pumps (Alzet micro-osmotic pump; model 1004; lot 10335-14). Minipumps were filled with angiotensin II acetate (Bachem; lot # 1066804) and set to an average pumping rate of 0.11 μL/h to deliver 1.5 mg/kg/day Ang II. Minipumps were implanted subcutaneously in the scapular region 7 days before dosing. Animals were housed singly under standard conditions (temperature, 64 to 84°F (18 to 29°C); relative humidity, 30 to 70%) and maintained on a 12-hour day/12-hour night cycle. Food (Research Diets Standard pelleted food) and water were provided ad libitum.

Animals were divided into groups based on systolic blood pressure. Test proteins were administered subcutaneously to the respective animals either once on day 0 (group 5) or twice a week for three weeks (groups 1-4) starting on day 6 by subcutaneous injection. Dose volumes for each animal were determined based on the most recent body weight measurement.

During testing, systolic pressure, diastolic pressure, mean arterial pressure, and heart rate were recorded for 10 s every minute.

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Results.

In NPR1 ^hu/hu mice with angiotensin II-induced hypertension, single or repeated doses of the NPR1 agonist mAb mAb22033 reduced systemic blood pressure (Figs. 12, 13 and 15) to near normal levels. Repeated doses of 1, 5, or 25 mg/kg mAb22033 reduced systolic blood pressure in a dose-dependent manner, with peak reductions from baseline in each group of 11, 19, and 39 mmHg. Art. respectively, after 3 weeks of administration twice a week. One dose of 50 mg/kg reduced systolic blood pressure by 31 mmHg. Art. by day 7 with a gradual return to more hypertensive levels. Mean 21-day blood pressure (Table 41) was significantly lower in animals receiving single or repeated doses of mAb22033 compared to the IgG4 isotype control.

Table 41

21-day mean arterial pressure and heart rate

Dosing group: Systolic (mmHg) Diastolic (mmHg) Mean arterial e (mmHg) Heart rate (BRM) Isotype control IgG4 (25 mg/kg) 173.4±1.3 138.6±0.7 154.9±1.2 538.2±1.8 mAL22033 (1 mg/kg) 163.0±1.2** 132.9±1.0 146.8±1.0** 532.5±3.2 mAL22033 (5 mg/kg) 151.9±0.9**** 124.7±0.7**** 137.8±0.8**** 524.8±3.5** wAL22033 (25 mg/kg) 149 1±2 2**** 127.0±2.0*** 137.1±2.0**** 511.3±2.9**** wAL22033 (50 mg/kg)*oflHa dose* 156.2±1.4**** 135.0±1.7 144 9±1 5**** 552.4±3.1**

All values represent mean ± SEM, n=3-6 per group. Statistics - two-way ANOVA with Dunnett's test; **p<0.01 compared with IgG4 isotype control; ***p<0.001 compared with IgG4 isotype control; ****p<0.0001 compared with IgG4 isotype control.

Table 42

Final body and organ masses

Dosage group: Final body weight (g) Heart weight (mg) Heart mass: length of tibia Heart weight: body weight Isotype control IgG4 (25 mg/kg) 30.9±1.5 0.165±0.006 0.0091±0.00032 0.0054±0.0002 mAL22033 (1 mg/kg) 31.1±1.1 0.151±0.007 0.0084±0.00043 0.0049±0.0002 mAL22033 (5 mg/kg) 29.6±1.0 0.149±0.004 0.0082±0.00020 0.0050±0.0001 wAL22033 (25 mg/kg) 32.4±1.9 0.165±0.012 0.009±0.00067 0.0051±0.0003 wAL22033 (50 mg/kg) *one dose* 31.0±1.3 0.153±0.006 0.0085±0.00034 0.0050±0.0003

All values represent mean ± SEM, n=3-6 per group. Statistics are one-way ANOVA with Dunnett's test.

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Table 43

Urine and serum markers

Dosage group: Urine volume (ml/day) cGMP in urine (mmol/day) Serum NTproANP (nmol/L) Day 20 Serum NTproANP (nmol/L) Day 29 Isotype control IgG4 (25 mg/kg) 2.74±0.74 30.5±6.3 0.58±0.05 0.57±0.09 mAL22033 (1 mg/kg) 3.08±0.51 61.6±20.4 0.75±0.03 0.51±0.05 mAL22033 (5 mg/kg) 2.48±0.68 36.6±10.4 0.45±.013 0.36±0.08 wAL22033 (25 mg/kg) 3.2±0.80 *106.3±25.4* 0.59±0.13 0.63±0.14 wAL22033 (50 mg/kg) *one dose* 3.27±0.89 52.8±21.5 0.56±0.18 1.27±0.43

All values represent mean ± SEM, n=3-6 per group. Statistics are one-way ANOVA with Dunnett's test. *p<0.05 compared with IgG4 isotype control.

The acute effects of mAb22033 were analyzed with a reduction in blood pressure of 10-20 mmHg. within 24 hours after the first dose. Heart rate effects (FIG. 14) were variable, with higher and lower values observed over the 21-day dosing 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 increase in the 50 mg/kg single dose group. Urinary cGMP levels were significantly increased in the 25 mg/kg booster group, with all other groups showing a trend of non-statistically significant increases compared to animals administered 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 Figs. 16, 17).

Example 11: Effects of NPR1 agonist antibodies on body weight, metabolic rate, and glucose homeostasis in diet-induced obese (DIO) mice.

Experiment 1.

This experiment tested the effects of the NPR1 agonist mAb mAb22810 on body weight, metabolic rate, and glucose homeostasis in diet-induced obesity (DIO) mice.

Male NPR1 ^hu/hu mice were placed on a 60% high-fat diet for 10 weeks and then randomized into three groups (n=10 per group): isotype control antibody (human IgG4), NPR1 agonist antibody mAb22810 or hFc.FGF21.FGF21, which 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, PMID: 22798348). The latter drug was used in this study as a positive control for the endpoints. Treatment was performed using saline by subcutaneous injection (SC) either weekly (for control antibody and mAb22810) or twice weekly (for hFc.FGF21). In table 44 lists the groups, number of animals, and doses used in the study.

Table 44

Summary of treatment groups

Group Treatment Dose Dosing schedule Mice 1 control shаb 25 mg/kg lx/week, S.C. 10 2 shаb22810 25 mg/kg 1x/week, S.C. 10 3 hFc.FGF21 3.2 mg/kg 2x/week, S.C. 10

The body weight of each mouse was recorded before dosing and twice weekly thereafter. During the second week of the study, mice were placed in metabolic cages to assess energy expenditure. Oral glucose tolerance was assessed during the third week of the study and the animal's constitution was determined after six weeks of treatment using EchoMRI.

In fig. 18A shows changes in body weight following administration of the NPR1 agonist mAb mAb22810, hFc.FGF21, or isotype control mAb. In fig. 18B and 18C show total fat and total muscle mass, respectively, after six weeks of treatment, measured using EchoMRI. The main results are that although the hFc.FGF21 molecule caused a significant reduction

- 54 046104 body weight and reduced obesity during the treatment period, this did not happen with the introduction of the NPR1 agonist antibody mAb22810.

In fig. 19A-19C show changes in VO ₂ (A), VCO ₂ (B), or energy expenditure (C) by mean of each day/night cycle after one week of treatment with either the NPR1 agonist mAb mAb22810, hFc.FGF21, or Isotype control mAb. The main results are that while the hFc.FGF21 molecule resulted in significant increases in VO ₂ , VCO 2 and energy expenditure during the treatment period, this did not occur with the NPR1 agonist antibody mAb22810.

In fig. 20A shows changes in glucose tolerance measured by an oral glucose tolerance test (2 g/kg glucose) after two weeks of treatment with either the NPR1 agonist mAb mAb22810, mAb hFc.FGF21, or isotype control mAb. In fig. 20B shows the overnight fasting glucose levels recorded at the start of the study in A. The main results are that both mAb22810 and hFc.FGF21 molecules caused a significant improvement in glucose tolerance after two weeks. In addition, the improvement in glucose tolerance provided by mAb22810 was not dependent on changes in body weight or energy expenditure (as shown in Figs. 18 and 19).

Experiment 2.

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

male NPR1 ^hu/hu mice were placed on a 60% high-fat diet for 10 weeks, then randomized into three groups (n=10 per group): isotype control antibody (human IgG4), NPR1 agonist antibody mAb22033, or hFc .FGF21 as a positive control. Treatment was performed using saline by subcutaneous injection (SC) either weekly (for control antibody and mAb22033) or twice weekly (for hFc.FGF21). In table 45 lists the groups, number of animals, and doses in the study.

Table 45

Summary of treatment groups

Group Treatment Dose Dosing schedule Mice 1 control mAb 25 mg/kg lx/week, S.C. 10 2 shаb22033 25 mg/kg 1x/week, S.C. 10 3 hFc.FGF21 3.2 mg/kg 2x/week, S.C. 10

The body weight of each mouse was recorded before dosing and twice weekly thereafter. During the second week of the study, mice were placed in metabolic cages to assess energy expenditure. Oral glucose tolerance was assessed during the third week of the study, and the animal's constitution was determined after six weeks of treatment using EchoMRI.

In fig. 21A shows changes in body weight following administration of the NPR1 agonist mAb mAb22033, hFc.FGF21, or isotype control mAb. In fig. 21B and 21C show total fat and total muscle mass, respectively, after six weeks of treatment, measured using EchoMRI. The main results are that while the hFc.FGF21 molecule caused significant weight loss and reduced obesity during the treatment period, this did not occur when administered with the NPR1 agonist antibody mAb22033.

In fig. 22A-22C show changes in VO ₂ (A), VCO ₂ (B), or energy expenditure (C) by average of each day/night cycle after one week of treatment with any of: the NPR1 agonist mAb mAb22033, hFc.FGF21, or Isotype control mAb. The main results are that while the hFc.FGF21 molecule resulted in significant increases in VO ₂ , VCO 2 and energy expenditure during the treatment period, this did not occur with the NPR1 agonist antibody mAb22033

In fig. 23A shows changes in glucose tolerance measured by an oral glucose tolerance test (2 g/kg glucose) after two weeks of treatment with either the NPR1 agonist mAb mAb22033, mAb hFc.FGF21, or isotype control mAb. In fig. Figure 23B shows overnight fasting glucose levels recorded at the start of the study in A. The main results are that both mAb22033 and hFc.FGF21 molecules caused a significant improvement in glucose tolerance after two weeks. In addition, the improvement in glucose tolerance provided by mAb22033 was not dependent on changes in body weight or energy expenditure (as shown in Figs. 21 and 22).

Example 12. HDX epitope mapping.

To determine the epitopes of human NPR1 recognized by anti-NPR1 antibodies, mAb22033 and mAb22810 were respectively studied by hydrogen-deuterium exchange (HDX). Previous experiments performed in the laboratory showed that binding of NPR1 to mAb22810 requires the presence of the ANP peptide. Therefore, in addition to the standard HDX experiment using the NPR1/mAb22033 complex, HDX experiments were also performed on the complexes

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NPR1/ANP/mAb22033 and NPR1/ANP/mAb22810.

For this study, anti-NPRl antibodies (mAb22033 and mAb22810) were covalently coupled to N-hydroxysuccinimide (NHS)-agarose beads (GE Lifescience, catalog no. 17-0906-01) according to the manufacturer's protocol. The human recombinant NPR1 protein expressed by CHO cells contained the ecto-domain of the human NPR1 protein (Uniprot, accession number P16066) with a C-terminal myc-myc-hexahistidine tag (SEQ ID NO: 194). ANP peptide was purchased from TOCRIS (cat. no. 1906).

The deuteration buffer was prepared in D ₂ O containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na ₂ HPO ₄ and 2 mM KH ₂ PO ₄ (pD = 7.4). For antigen-on or complex-on format experiments, 30 μl of antibody-bound bead suspension (equivalent to 15 μl of beads) was mixed with hNPR1.mmh or hNPR1.mmh/ANP complex. The mixture was incubated at room temperature, swirling gently. The deuteration process was stopped along with the elution process of hNPR1.mmh from the antibody-bound beads using 0.075% ice-cold TFA. After stopping the reaction, the sample was immediately introduced into the Waters HDX Manager for online pepsin digestion (Waters Enzymate BEN pepsin column, 2.1x30 mm). Digested peptides were captured on an ACQUITY UPLC VEN C18 1.7 μm, 2.1 x 5 mm VanGuard precolumn at 0°C and eluted on an ACQUITY UPLC VEN C18 1.7 μm, 2.1 x 50 mm column using an 8-minute 1-30 gradient separation % 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 taper voltage of 37 V, a scan time of 0.5 s, and a mass/charge ratio range of 50–1700 Th.

To map the hNPR.mmh binding epitope recognized by mAb22033, two sets of H/D exchange experiments were performed. The first experiment used the antigen format (HDX of antigen only followed by binding to antibody-bound beads). For the antigen format experiment, hNPR1.mmh was deuterated for 3 and 8 min (in two separate sub-experiments) in phosphate buffer prepared in D2O (PBS-D, pD=7.4) at room temperature. Deuterated hNPR1.mmh was then added to mAb22033 beads washed with PBS-D for 2 min incubation at room temperature, resulting in a total deuteration time of 5 and 10 min, respectively. Bound hNPR.mmh was then eluted from the beads using an aqueous 0.075% glacial trifluoroacetic acid (TFA) solution. The eluted hNPR.mmh was immediately introduced into the Waters HDX control system for pepsin digestion in flow mode, followed by measurement of peptide mass.

The second experiment is called the complex format (HDX beads with antigen/antibody complex). For this experiment, hNPR.mmh was first coupled to mAb22033 beads in regular PBS (pH 7.4) for 2 min. The complex was then incubated in PBS-D (pD = 7.4) for 5 or 10 min (in separate sub-experiments) for deuteration. The next steps (elution, injection, pepsin digestion, and MS analysis) were performed as described in the previous antigen procedure.

To identify peptides and perform deuterium uptake measurements, LC-MSE data obtained for nondeuterated hNPR1.mmh were first processed and database searches including hNPR1.mmh were performed using Waters ProteinLynx Global Server (PLGS) software. The identified peptides were imported into DynamX software and filtered using two criteria: 1) minimum number of products per amino acid: 0.3 and 2) replication file threshold: 2. DynamX software was then able to determine the deuterium uptake of each peptide used in the experiments in antigen and complex formats, based on each peptide's retention time and mass accuracy (<30 ppm) at two time points. All identified peptides were verified and screened manually to minimize false positive hits.

Centroid values or the average mass-to-charge ratio (m/z) of all detected peptides were calculated and compared between antigen and complex experiments at the two time points. Peptides that exhibit increased mass after deuteration in the antigen format compared to deuteration in the complex format include amino acids that are protected from deuterium exchange by antibody binding and therefore reveal epitope binding sites.

In the HDX experiment with NPR1/mAb22033, a total of 101 peptides were identified from hNPR1.mmh, representing 74% sequence coverage. Among these peptides, ten peptides spanning amino acids 29-50 and 328-347 had significantly increased mass after deuteration in the antigen format compared to deuteration in the complex format, as shown in Table 1. 46.

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Table 46

Effect on H/D exchange of mAb22033 binding to hNPRl.mmh measured using MH ⁺ peptide values after pepsin digestion

NPR1- P1P1N residues 5 min deuteration 10 min deuteration With antigen With a complex Δ With antigen With a complex Δ 29-50 2426.346 2424.541 1.80 2426.649 2424.925 1.72 32-50 2128.200 2126.776 1.42 2128.488 2127.088 1.40 46-54 950.481 950.333 0.15 950.549 950.527 0.02 328-335 925.039 925.027 0.01 925.065 925,030 0.03 328-347 2084.447 2081.863 2.58 2084.306 2081.925 2.38 329-347 1921.240 1918.335 2.91 1921.305 1918.614 2.69 331-347 1679.173 1677.107 2.07 1679.106 1677.176 1.93 332-347 1607.797 1605.754 2.04 1607.799 1605.842 1.96 334-347 1407.379 1405.529 1.85 1407.335 1405.612 1.72 335-347 1278.196 1276.411 1.78 1278.225 1276.463 1.76 336-347 1176.174 1174.913 1.26 1176.181 1174.926 1.26 337-347 1063.079 1061.760 1.32 1063.014 1061.745 1.27

Because two peptides, amino acids 46-54 and 328-335, showed no difference in deuterium uptake between the antigen and complex treatments, the regions protected from deuterium exchange of peptides 29-50 and 328-347 are reduced to residues 29-45 and 336-347. Therefore, two segments, including amino acids 29-45 and 336-347, were identified as the binding epitope of mAb22033 to the hNPR1.mmh protein.

For the HDX experiment with NPR1/ANP/mAb22033, a total of 95 peptides were identified from hNPR.mmh, representing 68% sequence coverage. Among these peptides, nine peptides spanning amino acids 29-50 and 331-347 had significantly increased mass after deuteration in the antigen format compared to deuteration in the complex format, as shown in Table 1. 47.

Table 47

Effect on H/D exchange of mAb22033 binding to hNPR1.mmh/ANP measured using MH ⁺ peptide values after pepsin digestion

NPR1- P1P1N 5 min deuteration 10 min deuteration WITH WITH Δ WITH WITH Δ leftovers antigen complex antigen complex 29-50 2425.746 2424.277 1.47 2426.686 2425.079 1.61 32-50 2127.514 2126.429 1.08 2128.333 2127.126 1.21 35-50 1828.391 1827.331 1.06 1829.026 1827.974 1.05 46-54 950.149 950,080 0.07 950.312 950.323 -0.01 331-347 1679.030 1677.150 1.88 1679.016 1677.216 1.80 332-347 1607,700 1605.825 1.88 1607.738 1605.943 1.79 334-347 1407.282 1405.558 1.72 1407.271 1405.715 1.56 335-347 1278.028 1276.463 1.56 1278.087 1276.658 1.43 336-347 1176.211 1175.009 1.20 1176.186 1175.009 1.18 337-347 1063.036 1061.743 1.29 1063.019 1061.807 1.21

Because the other peptide, amino acids 46-54, showed no difference in deuterium uptake between the antigen and complex treatments, the region protected from deuterium exchange in peptide 29-50 is reduced to residues 29-45. Thus, two segments, including amino acids 29-45 and 331-347, were identified as the epitope for binding of the mAb22033 antibody to the hNPR1.mmh/ANP protein complex.

For the HDX experiment with NPR1/ANP/mAb22810, a total of 93 peptides were identified from hNPR.mmh, representing 70% sequence coverage. Among these peptides, ten peptides covering amino acids 29-50, 70-81 and 331-347 had significantly increased mass after treatment.

- 57 046104 deuteration in the antigen format compared to deuteration in the complex format, as shown in table. 48.

Table 48

Effect on H/D exchange of mAb22810 binding to hNPR1.mmh/ANP measured using MH ⁺ peptide values after pepsin digestion

NPRl-mmH leftovers 5 min deuteration 10 min deuteration With antigen With a complex Δ With antigen With a complex Δ 29-50 2426.295 2425.324 0.97 2426.927 2426.077 0.85 32-50 2128.086 2127.361 0.72 2128.584 2128.016 0.57 33-50 2056.507 2056.083 0.42 2056.723 2056.405 0.32 35-50 1828.973 1828.597 0.38 1829.278 1828.952 0.33 46-54 950.063 950,090 -0.03 950.267 950,087 0.18 70-81 1456.036 1455.661 0.37 1456.127 1455.699 0.43 72-81 1241.898 1241.496 0.40 1241.969 1241.536 0.43 331-347 1678.712 1678.316 0.40 1678.888 1678.589 0.30 332-347 1607.457 1607,060 0.40 1607.617 1607.323 0.29 334-347 1407.018 1406.698 0.32 1407.217 1406.970 0.25 335-347 1277.816 1277.550 0.27 1278.075 1277.824 0.25 336-347 1175.926 1175.894 0.03 1176.094 1176.051 0.04 337-347 1062.703 1062.646 0.06 1062.844 1062.767 0.08

Since three peptides, amino acids 46-54, 336-347, 337-347, showed no difference in deuterium uptake between the antigen and complex treatments, the regions protected from deuterium exchange in peptides 29-50 and 331-347 are reduced to residues 29-45 and 331-335. Thus, three segments, including amino acids 29-45, 70-81 and 331-335, were identified as the epitope for binding of the mAb22810 antibody to the hNPR1.mmh/ANP protein complex.

Example 13: Intravitreal injection of anti-NPR1 antibody into humanized NPR1 mice reduces intraocular pressure.

This example describes the effect of intravitreal injection (IVT) of an exemplary anti-human NPR1 antibody mAb22033 on intraocular pressure (IOP) in mice expressing humanized NPR1.

Methods: NPR1-humanized mice (NPR1 ^hu/hu ) were generated using VelociGene technology (Regeneron). A single IVT injection of 40 μg mAb22033 or control Ab was administered to NPR1 humanized or wild-type (WT) mice. IOP was measured daily for four days after injection. In the second experiment, to study dose response, 40, 12.6, or 4 μg of mAb22033 or 40 μg of control Ab were administered intravitreally and IOP was monitored daily. In another experiment, to test the effect of long-term delivery Abs, AAV2 vectors expressing anti-NPR1 or eGFP were administered, and IOP was observed for 7 weeks.

Results: IVT 40 μg mAb22033 to NPR1 ^hu/hu mice significantly reduced IOP from days 1 to 3 compared to control antibody. The mean change in IOP was 5 mmHg. Art. However, no IOP-lowering effect was observed in WT mice. A dose-response study showed that intravenous doses of 40 or 12.6 μg of mAb22033 had a similar IOP-lowering effect, with the effect of 40 μg of mAb22033 lasting longer than 12.6 μg. IVT 4 μg of NPR1 antibody did not reduce IOP. Following IVT of AAV2-GFP or AAV2-NPR1 antibodies, no IOP effect was observed at all time points tested. This could be due to low expression of the NPR1 antibody, i.e. only 10 ng was detected in the total lysate obtained from the eye.

Conclusion: IVT administration of human anti-NPR1 antibody (mAb22033)

NPR1-humanized mice significantly reduced IOP, thereby demonstrating the potential of NPR1 agonist antibodies to reduce IOP in glaucoma.

Example 14 Structural analysis of the antibody-NPR1 complex by electron microscopy.

Methods.

Titration using size exclusion chromatography with a multi-angle light scattering (SEC-MALS) detector.

Several series of titrations of the extracellular domain of human NPR1 with a C-terminal myc-myc-6xHis tag (hNPR1-mmh; SEQ ID NO: 194) complexed with various antibodies in various molar ratios were performed. The following antibodies were tested: mAb22033, REGN5308 (Fab fragment of mAb22033), mAb22810 and REGN5314 (Fab fragment of mAb22810). All titration series were performed as with

- 58 046104 using atrial natriuretic factor (ANP, Tocris), and without it at a 2-fold molar excess compared to hNPR1-mmh. After overnight incubation in PBS at 4°C, the complexes were injected into the SEC-MALS system, which consisted of a Superdex 200 Increase 10/300 G1 column on an ACT microsystem (GE Healthcare Life Sciences), and then into a miniDAWN Treos and Optilab T-system. rEX (Wyatt Technology Corporation). Because phosphate buffers are incompatible with negative staining for electron microscopy, the SEC column was equilibrated in 50 mM Tris pH 7.5, 150 mM NaCl running buffer, and all larger scale complexes obtained below were contained 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 Technology).

Preparation of samples for negative spot electron microscopy.

hNPRl complexes were prepared on a larger scale for use in negative spot electron microscopy. Five samples were prepared as follows: sample 1 = hNPR1-mmh only (SEQ ID NO: 194); sample 2 = hNPR1-Fc only (SEQ ID NO: 197); sample 3 = hNPR1-mmh+ANP, molar ratio 1:2; sample 4 = hNPR1-mmh+REGN5308, molar ratio 1:1.5; sample 5=hNPR1-mmh+ANP+REGN5308, molar ratio 1:2:1.5. Samples 1, 3, 4, and 5 were purified by size exclusion chromatography in the same manner as in 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, the buffer was replaced with 50 mM Tris, pH 7.5 + 150 mM NaCl, diluted to 1.5 mg/ml, frozen at -80°C and shipped with other samples.

Collection and processing of negative spot electron microscopy data.

Negative spot EM grids were prepared for five protein samples using a standard method using uranyl formate (NanoImaging Services). The grids contained a thin continuous layer of carbon placed on top of a carbon grid with C-shaped holes. TEM images were acquired at room temperature on a Tecnai T12 electron microscope (FEI/Thermo Fisher) operating at 120 keV with an FEI Eagle 4k x 4k CCD camera. Images were collected at various nominal magnifications, mainly 67,000x and 110,000x. The collected images were further processed in-house.

NanoImaging micrographs were inspected by eye, and images that had poor spot contrast were removed, approximately 1/5 of the total images. The remaining images were separated using magnification; the 110,000x images were not as useful for further analysis because there were fewer particles in each image. Consequently, all subsequent processing steps were performed using 67000x images. All images were CTF corrected using CTFFIND4.

Detection of EM particles and 2D class averaging.

Particle distribution for all five negative spot samples was quite good, with uniform particle size, very little clumping, and good particle density. In samples 4 and 5, particles were detected using Relion with autorun patterns derived from an initial round of manual collection and 2D class averaging. For sample 4 (hNPR1+REGN5308), 19,184 good particles were identified from a total of 75 micrographs. For sample 5 (hNPR1+ANP+REGN5308), 20,318 good particles were initially identified from a total of 88 micrographs. Initial 2D class averaging with Relion revealed significant heterogeneity in both data sets, with a significant number of classes showing only Fab REGN5308 or only NPR1.

The 2D class average for sample 5 was further refined by removing particles corresponding to Fab only or NPR1 only. The remaining 9219 particles were used to calculate new 2D class averages, which showed a better distribution of images for the NPR1+REGN5308 complex and showed that a minority of complexes contained only one Fab bound to the NPR1 dimer, while the majority of complexes contained two Fabs. Removal of the oneFab complexes further reduced the particle set to 6728 particles.

Three-dimensional image reconstruction from negative spot EM data.

An initial 3D model of the NPR1-ANP-REGN5308 complex was built in Relion using a stochastic gradient descent procedure for the initial 3D model with a resolution limited to 40 A. This model was then low-pass filtered to 60 A and used as a reference for 3D classification in Relion for The 6728 particles mentioned above, with the wait step resolution limited to 25 A. The best 3D class was then further refined in Relion until it converged to a final resolution of 22 A, measured by the FSC gold standard. No 2-fold symmetry was applied during 3D classification or refinement. The density map obtained from the 3D reconstruction clearly showed that two Fab fragments associated with one side of the square particle corresponded to the crystal structure of ANP-bound NPR1 (PDB code 1T34).

- 59 046104

Sample preparation and data collection for cryoelectron microscopy.

A sample of the NPR1-ANP-REGN5308 complex was prepared for cryoelectron microscopy (CryoEM) in the same manner as the negative-spot EM sample described above. The final concentration of the complex was 0.8 mg/ml in 50 mM Tris, pH 7.5, 150 mM NaCl. CryoEM samples were prepared by standard methods using UltrAuFoil (Quantifoil Micro Tools GmbH) and Vitrobot (FEI/Thermo Fisher) grids. Data acquisition was performed on a Titan Krios electron microscope operating at 300 kV (FEI/Thermo Fisher) using a K2 direct electron detector in counting mode and a GIF energy filter (Gatan, Inc.). 1409 films were collected at 130,000x magnification (1.04 A/pixel) with a defocus range of -0.5 to -1.5 µm and a total dose of 45.44 e ^- /A2. Data collection was controlled using Leginon software.

CryoEM data processing and structure determination.

All CryoEM movies were motion corrected, dose weighted, and CTF corrected using the cisTEM package. The images were then manually checked, removing those with thick ice, poor CTF parameters (fitting resolution worse than 6 A), missing particles, contamination, etc. This filtering left 1172 images, which were then used to collect template-free particles in cisTEM, resulting in 872915 particle positions. After 2D classification with bad particle removal, 686,709 particles were included for 3D automatic refinement using cisTEM, and an initial 3D reference volume was generated ab initio. The three-dimensional refinement converged to a single solution with a resolution of 2.8 A, estimated from the Fourier-shell correlation curve.

This 3D map was then used to refine the structure, starting with the model embedded in the EM negative spot 3D map mentioned above. The N- and C-terminal domains of both NPR1 molecules were refined in real space as rigid bodies in the EM map and then manually rearranged in the few places where the model did not fit the EM density. The REGN5308 homology model was manually placed into the EM density; careful inspection of the CDR regions allowed the orientation of the heavy and light chains to be determined. The CDR regions of the model required extensive restructuring to accommodate EM density. Finally, positional refinement in real space using Phenix allowed us to obtain the current structural model of the complex.

Results/discussion.

Titration using size exclusion chromatography with a multi-angle light scattering (SEC-MALS) detector.

When hNPR1-mmh interacts with mAb22033, hNPR1-mmh itself behaves as a dimer with a molecular mass of approximately 110 kDa in the presence and absence of ANP, with a slight increase in molecular mass upon binding of ANP to NPR1. No free monomer peak was observed for hNPR1-mmh itself. Titration of mAb22033 in the absence of ANP revealed two main species of the complex: a species with a molecular mass equal to one IgG bound to one NPR1 dimer, and a species with a molecular mass equal to one IgG bound to two NPR1 dimers. However, NPR1 plus mAb22033 in the presence of ANP produced higher molecular weight particles that could represent NPR1 and paper doll-type IgG polymers.

The system was subsequently simplified by considering REGN5308, a Fab fragment of mAb22033. The hNPR1-mmh and REGN5308 complexes show a completely different SEC profile with bound ANP compared to the same complexes without ANP. The NPR1-REGN5308 complex has a molecular mass of approximately 155 kDa, corresponding to one bound Fab per NPR1 dimer. In the presence of ANP, the molecular weight of the NPR1-ANP-REGN5308 complex increases by ∼50 kDa, corresponding to two bound Fabs per NPR1 dimer. It has been hypothesized that the previously described conformational change of NPR1 upon binding to ANP (Ogawa, H. et al., 2004) allows the binding of a second Fab and that the 2 Fab+2 NPR1+ANP complex is required for the growth of paper doll polymers observed with full-length IgG mAb22033 (see below for further discussion).

Titrations were also performed with hNPR1-mmh and mAb22810. SEC-MALS analysis revealed that a small fraction of the mAb22810 sample was dimeric with a molecular mass of approximately 315 kDa compared to the standard IgG molecular mass of 150 kDa. SEC-MALS titrations with the NPR1-mAb22810-ANP complex showed a heterogeneous mixture of species in the range of 430–700 kDa; this profile was too complex to be reliably interpreted, possibly due to contamination of the IgG dimer in the mAb22810 protein. SEC-MALS titration with REGN5314, a Fab fragment of mAb22810, showed that binding of REGN5314 to NPR1 in the absence of ANP is too weak or transient to produce complex species that can be isolated by SEC. When ANP is present, a single NPR1-REGN5314-ANP complex is formed with a molecular mass of approximately 170 kDa, corresponding to one bound Fab per NPR1 dimer.

Average 2D class values of negative spots.

The hNPR1+REGN5308 and hNPR1+ANP+REGN5308 complexes were further analyzed by negative-spot electron microscopy. 2D classification and averaging of complex particles revealed significant heterogeneity in the samples. Most of the particles on the EM grids can be classified as hNPR1 only or REGN5308 Fab only. Since protein samples

- 60 046104 submitted for imaging were purified homogeneous complexes, it appears that these complexes dissociate into their components during the preparation of the negative spot grid. However, a significant part of the complexes remained intact and can be used for analysis.

The negative spot 2D class averages for hNPR1+REGN5308 show a single-arm complex in which only one Fab fragment can be seen binding to the hNPR1 dimer, consistent with the SEC-MALS results. In contrast, the negative spot 2D class averages for hNPR1+ANP+REGN5308 show a two-armed complex, with two Fabs associated with the dimer. Different average values represent different projections of the real 3D complex. In a single class average, the hNPR1 dimer is visible in a side view with two density lobes: one corresponding to the N-terminal domains of the two monomers superimposed on each other, and the other lobe corresponding to the two C-terminal domains superimposed on each other. In this orientation, the two bound Fabs appear as rabbit ears positioned on top of the NPR1 density. On the other hand, all four hNPR1 domains can be viewed as four dense patches forming a square, with the two REGN5308 Fabs above the square intersecting each other to form an inverted V. The hNPR1+ANP+REGN5308 class averages also showed single-armed complexes, but these are likely , are associated with the same dissociation of the complex that produces free Fab and free NPR1.

Average 2D class values calculated from CryoEM data for hNPR1+ANP+REGN5308 show different complex species compared to the negative spot data; in particular, there is no bunny ears orientation. However, other 2D class means of the CryoEM data can be accurately compared to the corresponding means in the negative spot data. There was not sufficient evidence of complex dissociation in the CryoEM data, likely because the original sample was more homogeneous and also because cryofreezing conditions better preserve the native conformation of the complex in solution.

3D image reconstruction.

A 3D CryoEM density map for hNPR1+ANP+REGN5308 was constructed using the 2D class averages as a starting point. This reconstruction procedure does not require any prior information about the expected shape or size of the complex other than a rough estimate of the diameter of the complex, so the resulting CryoEM map is independent of the researchers' expectations of how the antibody-target complex should form. The known crystal structure of hNPR1+ANP was then placed and refined into this EM density map along with REGN5308 Fab models generated by homology to known Fab structures. The NPR1 and Fab structures were placed in their approximate locations manually and then refined to solids in the correct locations using Phenix. The resolution of CryoEM maps is sufficient to manually rearrange residues at the NPR1:antibody interface, especially residues in the REGN5308 complementarity determining regions (CDRs) that cannot be accurately modeled by homology. The current structural model contained all antibody CDR residues as well as NPR1 residues in contact with them. More distant regions of the model (NPR1 C-terminal domains and antibody constant Fab domains) were modeled using a combination of the current CryoEM map and previously determined X-ray crystal structure information for NPR1 (PDB code 1T34) and the structure of the isolated antibodies .

The mAb22033 epitope on NPR1: inspection of the hNPR1+ANP+REGN5308 structure reveals which NPR1 residues Fab REGN5308 (and by extension the parent IgG mAb22033) binds to. This epitope consists of four distinct stretches of amino acids in NPR1 that combine to form a continuous surface in three dimensions: residues 2-4, 41-45, 47, 73-79, 332, 336-344 and 347 (numbered according to SEQ ID NO: 194). As a result of earlier hydrogen/deuterium exchange (HDX) mass spectrometry experiments, some of these residues were identified as significant (see Example 12), but the CryoEM structure provides finer epitope detail.

Structural mechanism of action of mAb22033.

The two Fabs in this model of the NPRl-antibody complex are within ∼10 A of each other at a point near the bend between the Fab variable domains and the Fab constant domains. The C-termini of these two Fab fragments are much further apart, about 100 A, and therefore cannot be two arms of the same IgG molecule. The Fab fragments do not come close enough to the simulated ANP peptide (distance of approximately 30 Å at the closest approximation), and there does not appear to be any direct interaction between Fab and ANP.

Based on the assumption of a fixed position and relative orientation of the Fab relative to its binding site in the N-terminal domain of NPR1, an explanation for the ANP-dependent binding of Fab REGN5308 arises. NPR1 has been shown to undergo a conformational change upon binding to ANP, in which one NPR1 monomer rotates relative to the other while remaining dimerized. Applying this rotation to one half of the NPR1+2 Fab complex 2 yields a model in which the NPR1 dimer resembles the crystal structure of NPR1 without the ANP. However, one of the REGN5308 Fabs is now rotated to a position where it sterically conflicts with the other Fab, a physically impossible situation. This steric hindrance is the reason why only one Fab

-

Claims (9)

REGN5308 can bind to the NPR1 dimer in the absence of ANP: both antibody binding sites on the two monomers are equally accessible, but binding of the first Fab blocks binding of the second Fab. Viewing this effect another way, binding of two Fabs to an ANP-containing NPR1 dimer will prevent this complex from relaxing back to an ANP-free conformation as long as both Fabs are bound. At equilibrium, this could result in a large proportion of NPR1 molecules being in an active state, capable of signaling downstream - assuming that the effects observed in this case are retained at the cell surface. Another possible effect of antibody binding is the formation of oligomeric clusters of the antibody with NPR1+ANP, as described above. This effect is only possible if each NPR1 dimer can bind two Fab arms of two separate IgG molecules; in the absence of ANP, only one Fab arm can bind to each NPR1 dimer, and complex formation ceases to produce much smaller compounds containing at most one IgG with an NPR1 dimer bound to each Fab arm. Both of these effects, receptor clustering and prolongation of receptor active state, may explain the activating effect of mAb22033 on the NPR1 receptor. The present invention is not limited to the scope of the specific embodiments disclosed in the present description. Indeed, various modifications of the invention in addition to those disclosed herein will become apparent to those skilled in the art from the above description and the accompanying drawings. It should be understood that such modifications are within the scope of the appended claims. CLAIM 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 contains three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained in the variable a heavy chain region (HCVR) containing the amino acid sequence of SEQ ID NO: 2; three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2 and LCDR3) contained in a light chain variable region (LCVR) containing the amino acid sequence of SEQ ID NO: 10. 2. The antibody or antigen-binding fragment thereof according to claim 1, containing HCVR with the amino acid sequence SEQ ID NO: 2. 3. The antibody or antigen-binding fragment thereof according to claim 1, containing LCVR with the amino acid sequence SEQ ID NO: 10. 4. Antibody or antigen-binding fragment thereof according to claim 1, containing: (a) HCDR1 with amino acid sequence SEQ ID NO: 4; (b) HCDR2 with amino acid sequence SEQ ID NO: 6; (c) HCDR3 with amino acid sequence SEQ ID NO: 8; (d) LCDR1 with amino acid sequence SEQ ID NO: 12; (e) LCDR2 having the amino acid sequence SEQ ID NO: 14 and (f) LCDR3 having the amino acid sequence SEQ ID NO: 16. 5. An antibody or an antigen-binding fragment thereof according to claim 4, containing a pair of amino acid sequences HCVR/LCVR SEQ ID NO: 2/10. 6. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof reacts with one or more amino acids contained in the extracellular domain of NPR1 (amino acids 29-347 SEQ ID NO: 194) 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) activates NPR1. 7. The isolated antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof reacts with an amino acid sequence selected from the group consisting of: (a) amino acids 29-45 SEQ ID NO: 194 and (b) amino acids 336-347 SEQ ID NO: 194. 8. The isolated antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody or antigen-binding fragment thereof reacts with an amino acid sequence selected from the group consisting of: (a) amino acids 29-45 SEQ ID NO: 194 and (b) amino acids 331-347 SEQ ID NO: 194, in the presence of ANP. 9. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is a fully human monoclonal antibody. -