Anti-PDGF-B Antibodies and Methods of Use for Treating Pulmonary Arterial Hypertension (PAH)

Abstract

The present disclosure provides anti-platelet-derived growth factor subunit B (PDGF-B) antibodies and antigen-binding fragments thereof, and methods of using such antibodies or antigen-binding fragments thereof to treat subjects with pulmonary arterial hypertension (PAH).

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 141,030, filed January 25, 2021, the entire contents of which are expressly incorporated by reference herein in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on January 20, 2022, is named 118003-00720_SL.txt and is 33,624 bytes in size.

[0003] The present invention relates to human antibodies and antigen-binding fragments thereof that specifically bind to platelet-derived growth factor subunit B (PDGF-B), and to therapeutic and diagnostic methods using such antibodies and fragments. [Background technology]

[0004] Platelet-derived growth factor (PDGF) is a potent mitogen that exists as five different dimeric forms consisting of four different isoform subunits: A, B, C, and D. The five dimeric forms of PDGF are AA, BB, AB, CC, and DD, which are formed by disulfide bonds of corresponding individual PDGF monomers. PDGF ligands exert their biological effects through interaction with PDGF receptors (PDGFRs). PDGFRs are single-pass, transmembrane, tyrosine kinase receptors consisting of heterodimeric or homodimeric associations of alpha (a) receptor chains (PDGFR-alpha) and / or beta (β) receptor chains (PDGF-B). Thus, active PDGFRs can consist of αα, ββ, or αβ receptor chain pairs. PDGFRs share a common domain structure that includes five extracellular immunoglobulin (Ig) loops, a transmembrane domain, and a split intracellular tyrosine kinase (TK) domain. The interaction between dimeric PDGF ligands and PDGFRs results in receptor chain dimerization, receptor autophosphorylation, and intracellular signal transduction. It has been demonstrated in vitro that ββ receptors are activated by PDGF-BB and -DD, whereas αβ receptors are activated by PDGF-BB, -CC, -DD, and -AB, and αα receptors are activated by PDGF-AA, -BB, -CC, and -AB (see Andrae et al. (2008) Genes Dev 22(10):1276-1312).

[0005] PDGF signaling is involved in a variety of human diseases, including pulmonary arterial hypertension (PAH), a progressive disorder characterized by a persistent increase in pulmonary arterial pressure that damages both large and small pulmonary arteries. PAH is hemodynamically defined as a systolic pulmonary arterial pressure of more than 30 mmHg, or a mean pulmonary arterial pressure of more than 25 mmHg with pulmonary capillaries, or an assessment of a left atrial pressure of less than 15 mmHg. See, for example, Zaiman et al., Am. J. Respir. Cell MoI. Biol. 33:425-31 (2005). The persistent vasoconstriction in PAH leads to structural remodeling in which pulmonary vascular smooth muscle cells and endothelial cells undergo a phenotypic switch from a contractile normal phenotype to a synthetic phenotype, resulting in cell proliferation and matrix deposition. As the walls of the smallest blood vessels thicken, they are no longer able to transport oxygen and carbon dioxide normally between the blood and the lungs, and eventually pulmonary hypertension results in thickening of the pulmonary arteries and narrowing of the passageways through which blood flows. Eventually, proliferation of vascular smooth muscle and endothelial cells leads to remodeling of the blood vessels with obstruction of the lumen of the pulmonary vasculature. Histological examination of tissue samples from patients with pulmonary hypertension shows intimal thickening, and smooth muscle cell hypertrophy, especially for blood vessels less than 100 μm in diameter. This causes a progressive increase in pulmonary pressure as blood is pumped through a reduced luminal area. As a result, the right side of the heart works harder to compensate, and the increased effort causes the right ventricle to hypertrophy and thicken. With an enlarged right ventricle, there is a risk of pulmonary embolism, as blood tends to pool in the ventricle and legs. If clots form in the pooled blood, they can eventually dislodge and lodge in the lungs. Ultimately, the additional workload placed on the right ventricle causes cardiac failure in these patients, leading to early death.

[0006] Standard therapies for the treatment of subjects with PAH are primarily hemodynamic and affect vascular tone, including, for example, prostacyclin analogues, endothelin receptor antagonists, phosphodiesterase inhibitors, and soluble guanylate cyclase activators / stimulators, which provide symptomatic relief and improve prognosis. However, these therapies are insufficient and do not re-establish the structural and functional integrity of the pulmonary vasculature to provide patients with PAH with unimpaired long-term survival. Despite all the advances in the treatment of PAH, there remains no cure for this deadly disease and the majority of patients continue to progress to right ventricular failure. Thus, there is a need in the art for new, highly specific and potent inhibitors of PDGF signaling. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Andrae et al. (2008) Genes Dev 22(10):1276-1312 [Non-Patent Document 2] Zaiman et al.,Am.J.Respir.Cell MoI.Biol.33:425-31(2005) Summary of the Invention [Means for solving the problem]

[0008] The present disclosure provides fully human monoclonal antibodies (mAbs) and antigen-binding fragments thereof that specifically bind to platelet-derived growth factor subunit B (PDGF-B). Such antibodies may be useful in treating subjects with pulmonary arterial hypertension (PAH).

[0009] Circulating and pulmonary expression of PDGF-B, a potent mitogen for pulmonary smooth muscle cells and fibroblasts, is increased in both patients with idiopathic PAH and in hypoxic / sugen PAH mouse models. PDGF-B interactions with PDGFRββ, PDGFRαβ, and PDGFRαα are involved in promoting pathological angiogenesis and pulmonary vascular remodeling. Thus, targeting PDGF-B may provide enhanced efficacy over PDGFRβ by targeting signaling through PDGFRαα, PDGFRαβ, and PDGFRββ, which cooperate to promote abnormal vascular remodeling.

[0010] Accordingly, in one aspect, the disclosure provides an isolated human monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to human platelet-derived growth factor subunit B (PDGF-B).

[0011] In some embodiments, the antibody or antigen-binding fragment has (a) a binding dissociation equilibrium constant (K) of less than about 1.84 pM as measured by surface plasmon resonance. D ) to human PDGF-subunit B homodimer (PDGF-BB) at 37°C; (b) a K of less than about 1.36 pM as measured by surface plasmon resonance. D (c) binds to human PDGF-BB at 37° C. with a t of about 1155 minutes or more as measured by surface plasmon resonance; and (d) has a K of less than about 2.79 pM as measured by surface plasmon resonance. D (e) binds to human PDGF-BB at 25° C. with a t of about 1155 minutes or more as measured by surface plasmon resonance; and (f) has an IC of less than about 1.9 nM as measured by a competitive ELISA assay at 25° C. 50 (g) inhibiting PDGF-B activation against human PDGF-BB with an IC of less than about 8.8 nM as measured by competitive ELISA assay at 25°C. 50and (h) inhibiting PDGF-B activation against human PDGF-subunit A and subunit B heterodimer (PDGF-AB); and (b) blocking the interaction between human PDGF-BB and one or more of human PDGFR-αα, PDGFR-αβ, and PDGFR-ββ.

[0012] In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 2 and 22, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 10 and 30.

[0013] In some embodiments, the isolated human antibody or antigen-binding fragment thereof comprises a HCVR comprising an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:2 or SEQ ID NO:22.

[0014] In some embodiments, the antibody or antigen-binding fragment comprises a LCVR comprising an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:10 or SEQ ID NO:30.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises (a) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22, and (b) an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30.

[0016] In some embodiments, the antibody or antigen-binding fragment comprises (a) an HCDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 24; (b) an HCDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 26; (c) an HCDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 28; (d) an LCDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 32; (e) an LCDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 34; and / or (f) an LCDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 36.

[0017] In some embodiments, the antibody or antigen-binding fragment comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 22 / 30.

[0018] In some embodiments, the antibody or antigen-binding fragment comprises (i) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:20 and a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:18, and / or (ii) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:40 and a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:38.

[0019] In some embodiments, the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single chain Fv (scFv) molecule, or a dAb fragment.

[0020] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to the same epitope on human PDGF-B as an antibody or antigen-binding fragment comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 2 and 22, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 10 and 30.

[0021] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that competes for binding to human PDGF-B with an antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 2 and 22, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 10 and 30.

[0022] In another aspect, the invention provides nucleic acid molecules encoding anti-PDGF antibodies or fragments thereof. Recombinant expression vectors carrying the nucleic acids of the invention, and host cells into which such vectors have been introduced, are also included in the invention, as are methods of producing the antibodies by culturing the host cells under conditions that allow for the production of the antibodies, and recovering the antibodies produced.

[0023] In another aspect, the disclosure provides a method for making an antibody or antigen-binding fragment disclosed herein, the method comprising: (i) introducing into a host cell one or more polynucleotides encoding a light immunoglobulin chain of the antibody or fragment and a heavy immunoglobulin chain of the antibody or fragment; (ii) culturing the host cell in a growth medium under conditions favorable for expression of the polynucleotides; and (iii) optionally isolating the antibody or fragment from the host cell and / or the medium in which the host cell is growing.

[0024] In another aspect, the disclosure provides an antibody or antigen-binding fragment produced by a method comprising: (i) introducing into a host cell one or more polynucleotides encoding a light immunoglobulin chain of the antibody or fragment and a heavy immunoglobulin chain of the antibody or fragment; (ii) culturing the host cell in a growth medium under conditions favorable for expression of the polynucleotides; and (iii) optionally isolating the antibody or fragment from the host cell and / or the medium in which the host cell is growing.

[0025] In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 2 and 22, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 10 and 30.

[0026] In another aspect, the present disclosure provides an injection device or container comprising an antibody or antigen-binding fragment disclosed herein.

[0027] In another aspect, the disclosure provides a pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof that binds to human PDGF-B as described above or herein, a pharma- ceutically acceptable carrier or diluent, and, optionally, one or more additional therapeutic agents.

[0028] In some embodiments, the one or more additional therapeutic agents comprises an iron supplement.

[0029] In another aspect, the disclosure provides a method for preventing or treating pulmonary arterial hypertension (PAH) in a patient in need thereof, comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof that binds human PDGF-B as disclosed herein, or a pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof that binds human PDGF-B as disclosed herein.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, orally, or intramuscularly.

[0031] In some embodiments, the PAH results in a condition selected from the group consisting of thickening of the subject's pulmonary artery, reduced stroke volume in the subject, reduced right ventricular cardiac output in the subject, and reduced survival time in the subject, and administration of the antibody or antigen-binding fragment treats the condition or reduces the severity of one or more symptoms of the condition.

[0032] In another aspect, the disclosure provides an antibody, or antigen-binding fragment thereof, that binds human PDGF-B, as disclosed herein, for use in treating a patient having PAH.

[0033] In another aspect, the disclosure provides compositions comprising one or more antibodies or antigen-binding fragments that bind human PDGF-B disclosed herein for use in treating PAH.

[0034] In another aspect, the disclosure provides the use of an isolated antibody, or antigen-binding fragment thereof, that binds human PDGF-B, as disclosed herein, in the manufacture of a medicament for treating a patient having PAH.

[0035] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can neutralize PDGF-BB, PDGF-AB, and / or PDGF-AB / BB and prevent PDGF-driven calcium flux in cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can neutralize PDGF-BB and prevent PDGF-driven calcium flux in cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can neutralize PDGF-AB and prevent PDGF-driven calcium flux in cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can neutralize PDGF-AB / BB and prevent PDGF-driven calcium flux in cells.

[0036] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can neutralize PDGF-BB, PDGF-AB, and / or PDGF-AB / BB and prevent PDGF-driven cell proliferation. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can neutralize PDGF-BB and prevent PDGF-driven cell proliferation. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can neutralize PDGF-AB and prevent PDGF-driven cell proliferation. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can neutralize PDGF-AB / BB and prevent PDGF-driven cell proliferation.

[0037] In some embodiments, the cells are human cells, hi some embodiments, the cells are selected from the group consisting of pulmonary artery smooth muscle cells and primary human lung fibroblasts.

[0038] In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the outer edge of the beta sheet region of PDGF-BB. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the interchain loops of PDGF-BB. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the outer edge of the beta sheet region and to the interchain loops of PDGF-BB. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to one or more residues of PDGF-BB selected from the group consisting of W40, R73, K80, K81, P82, F84, K86, and R56. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the W40 residue of PDGF-BB. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the R73 residue of PDGF-BB. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the K80 residue of PDGF-BB. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the K81 residue of PDGF-BB. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the P82 residue of PDGF-BB. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the F84 residue of PDGF-BB. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the K86 residue of PDGF-BB. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the R56 residue of PDGF-BB.

[0039] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are internalized into the cell in a ligand-dependent manner via the PDGF receptor. In some embodiments, the cell is a human cell. In some embodiments, the cell is selected from the group consisting of pulmonary artery smooth muscle cells and primary human lung fibroblasts.

[0040] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce right ventricular systolic pressure in a subject in need thereof. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce right ventricular hypertrophy in a subject in need thereof. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce right ventricular systolic pressure and right ventricular hypertrophy in a subject in need thereof.

[0041] Other embodiments will be apparent from review of the detailed description that follows. [Brief description of the drawings]

[0042] [Figure 1] 1 is a graph showing the results of screening monoclonal anti-PDGF antibodies derived from VI-3 mice. [Diagram 2] FIG. 1 is a schematic diagram showing a study performed to determine the binding kinetics of 17 anti-PDGF-B antibodies at 25° C. and 37° C. [Figure 3A] FIG. 1 is a schematic showing cross-competition studies between purified anti-PDGF-BB monoclonal antibodies. [Figure 3B] 1 is a matrix showing the results of an antibody cross-competition assay in which a first anti-PDGF-B antibody (mAb-1) was applied to a sensor chip coated with human PDGF-BB, followed by treatment with a second anti-PDGF-B antibody (mAb-2). The binding response for each antibody combination tested is shown. [Figure 4A]Five potent anti-PDGF-B antibodies were identified with >70% blocking, three anti-PDGF-B antibodies blocked both PDGF-BB and PDGF-AB with PDGF-BB IC50 values ​​ranging from 0.23 to 1.8 nM, two anti-PDGF-B antibodies blocked only PDGF-BB with IC50 values ​​ranging from 0.55 to 0.64 nM, six moderate anti-PDGF-B antibodies blocked >45%, two anti-PDGF-B antibodies blocked both PDGF-BB and PDGF-AB with PDGF-BB IC50 values ​​ranging from 2.8 to 13 nM, four anti-PDGF-B antibodies blocked only PDGF-BB with IC50 values ​​ranging from 0.64 to 2.8 nM, and six anti-PDGF-B antibodies were non-blockers. [Figure 4B] Five potent anti-PDGF-B antibodies were identified with >70% blocking, three anti-PDGF-B antibodies blocked both PDGF-BB and PDGF-AB with PDGF-BB IC50 values ​​ranging from 0.23 to 1.8 nM, two anti-PDGF-B antibodies blocked only PDGF-BB with IC50 values ​​ranging from 0.55 to 0.64 nM, six moderate anti-PDGF-B antibodies blocked >45%, two anti-PDGF-B antibodies blocked both PDGF-BB and PDGF-AB with PDGF-BB IC50 values ​​ranging from 2.8 to 13 nM, four anti-PDGF-B antibodies blocked only PDGF-BB with IC50 values ​​ranging from 0.64 to 2.8 nM, and six anti-PDGF-B antibodies were non-blockers. [Figure 4C] 1 is a table summarizing the characteristics of anti-PDGF-B antibodies in a blocking ELISA using soluble PDGF-BB and PDGF-AB and plate-captured PDGFR-beta. [Figure 5A] FIG. 5A is a graph showing that 13 of 17 anti-hPDGF-B antibodies inhibited 500 pM hPDGF BB with IC50 values ​​ranging from 88 pM to 7.2 nM and maximum inhibition rates ranging from 42% to 99%. [Figure 5B] Additionally, as shown in FIG. 5B, a graph showing that the two anti-PDGF-B antibodies were activated by human PDGF-BB. [Figure 5C]Figure 5C is a graph showing that 8 of 17 anti-hPDGF-B antibodies inhibited 5 nM human PDGF-AB with IC50 values ​​ranging from 99 pM to 50 nM and maximal inhibition ranging from 32 to 99%. H4H13132P and H4H13145P blocked to baseline with IC50 values ​​of 8.8 and 2.6 nM. [Figure 5D] Finally, Figure 5D is a graph showing that 10 of the 17 anti-hPDGF-B antibodies inhibited 600 pM mouse PDGF-BB with IC50 values ​​ranging from 450 pM to 6.4 nM and maximum inhibition rates ranging from 39 to 98%. [Figure 6A] FIG. 13 is a graph showing chromatograms from samples forming the most distinct 2:2 mAb:human PDGF-BB species with no detectable higher order complexes observed. [Figure 6B] FIG. 13 is a graph showing chromatograms from samples forming the most distinct 2:2 mAb:human PDGF-BB species with no detectable higher order complexes observed. [Figure 6C] FIG. 13 is a graph showing chromatograms from samples forming the most distinct 2:2 mAb:human PDGF-BB species with no detectable higher order complexes observed. [Figure 7] 1 is a graph showing that H4H13145P is more potent than H4H13132P against both human and cynomolgus PDGF-BB proteins. [Figure 8] 1 is a table showing that commercial monkey PDGF-BB demonstrated specific binding to anti-PDGF-B mAbs H4H13132P and H4H13145P. [Figure 9] Western blot analysis of antibody binding epitopes shows that H4H13145P was confirmed to bind to a linear epitope on human rPDGF-BB. [Figure 10] FIG. 1 is a panel of graphs showing that anti-PDGF-B provided superior efficacy in two clinically relevant endpoints in PAH (right ventricular pressure and hypertrophy) when compared directly to anti-PDGFR-β. [Figure 11]FIG. 1 is a panel of graphs showing that prophylactic treatment with anti-PDGF-B reduced both hemodynamic endpoints (RVSP) and right ventricular hypertrophy in this rat model of PAH. [Figure 12] 1 is a panel of graphs showing that anti-PDGF-B robustly improved survival time in a severe MCT rat model of PAH. [Figure 13A] FIG. 1 is a panel of graphs showing that anti-PDGF-B at a dose as low as 1 mg / kg sc provided efficacy in a clinically relevant endpoint (right ventricular pressure) in PAH. [Figure 13B] 1 is a panel of graphs showing circulating PDGF-B levels and anti-PDGF-B concentrations in Hy / Su mice 42 days after administration of anti-PDGF-B antibody. [Figure 14] Panel of pericyte images showing that systemic delivery of anti-PDGF-Rβ antibody (2C5, REGN764) effectively depleted pericytes, whereas anti-PDGF-BB antibody had no apparent effect in the retinal vessel formation (RVD) model at 3 mg / kg. [Figure 15] FIG. 13 is a panel of pericyte images showing that a higher dose (25 mg / kg) of H4H13145P has a moderate effect on pericyte depletion in the RVD model. [Figure 16] 1 is a panel of graphs and tables showing the pharmacokinetic profile of anti-PDFG-B antibody in C57BL / 6 wild type mice at three single subcutaneous doses of 0.1, 1, or 10 mg / kg. [Figure 17] FIG. 1 is a schematic showing the study design to evaluate the effects of chronic treatment of healthy C57BL adult mice with high doses of anti-PDGF-B antibody on gastrointestinal (GI), pulmonary, or cerebrovascular permeability. [Figure 18] 1 is a panel of graphs showing that administration of anti-PDGF-B and anti-PDGFRβ antibodies did not significantly interfere with weight gain in animals. [Figure 19]1 is a graph showing that there was no significant change in gastrointestinal (GI) water retention / edema in the small intestine following treatment with isotype control IgG, anti-PDGF-B, and anti-PDGFRβ antibodies in healthy mice. [Figure 20] 1 is a panel of graphs and images showing that treatment of mice with anti-PDGF-B and anti-PDGFRβ antibodies did not significantly alter vascular permeability in the small intestine. [Figure 21] 13 is a graph showing that there was no significant change in intragastric GI water retention / edema by anti-PDGF-B and anti-PDGFRβ antibody treatment in healthy and captopril-treated mice. [Figure 22] 1 is a panel of graphs and images showing that treatment of mice with anti-PDGF-B and anti-PDGFRβ antibodies did not significantly alter vascular permeability in the stomach. [Figure 23] 13 is a graph showing that anti-PDGF-B and anti-PDGFRβ antibody treatment in healthy and captopril-treated mice did not significantly alter GI fluid retention / edema in the lungs. [Figure 24] 1 is a panel of graphs and images showing that anti-PDGF-B and anti-PDGFRβ antibody treatment in healthy and captopril-treated mice did not significantly alter vascular permeability in the lungs. [Diagram 25] 13 is a graph showing that there was no significant change in cerebral GI water retention / edema by anti-PDGF-B and anti-PDGFRβ antibody treatment in healthy and captopril-treated mice. [Figure 26] 1 is a panel of graphs and images showing that anti-PDGF-B and anti-PDGFRβ antibody treatment in healthy and captopril-treated mice did not significantly alter vascular permeability in the brain. [Figure 27] 1 is a panel of graphs showing inhibition of PDGF-induced calcium flux in human pulmonary artery smooth muscle cells (HPASMC) by anti-PDGF-B antibodies. [Figure 28]1 is a panel of graphs showing inhibition of PDGF-induced cell proliferation in human pulmonary artery smooth muscle cells (HPASMC) by anti-PDGF-B antibodies. [Figure 29] FIG. 1 is a panel of graphs showing a fluorescent imaging plate reader (FLIPR) receptor internalization assay in pulmonary artery smooth muscle cells (PASMC) pretreated with anti-PDGF-B antibody-ligand complex. [Diagram 30] 1 is a graph showing a study in monocrotaline treated rats for catheter assessment of cardiac right ventricular pressure demonstrating that anti-PDGF-B antibody treatment reduced right ventricular systolic pressure compared to isotype control treated rats. [Diagram 31] 1 is a graph showing that prophylactic treatment with anti-PDGF-B antibody reduced right ventricular hypertrophy compared to isotype control treated rats. [Diagram 32] 1 is a graph showing that the combination of the endothelin receptor antagonist macitentan with anti-PDGF-B treatment did not provide additional survival benefit compared to monotherapy with an anti-PDGF-B antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Before describing the methods of the present invention, it is to be understood that the invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, examples of preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

[0045] definition The terms "PDGF-B", "PDGFB", and "platelet-derived growth factor B" synonymously refer to the human PDGF-B protein having the amino acid sequence of SEQ ID NO: 41 (see also UniProt Accession No. P01127). All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless specifically identified as being from a non-human species (e.g., "mouse PDGF-B", "monkey PDGF-B", etc.). Circulating and pulmonary expression of PDGF-B, a potent mitogen for pulmonary smooth muscle cells and fibroblasts, is increased in both patients exhibiting idiopathic PAH and in hypoxic / sugen PAH mouse models. Interaction of PDGF-B with PDGFRββ, PDGFRαβ, and PDGFRαα is involved in promoting pathological angiogenesis and pulmonary vascular remodeling. Mechanistically, targeting PDGF-B may provide enhanced efficacy over PDGFRβ by targeting signaling through PDGFRαα, PDGFRαβ, and PDGFRββ, which cooperate to promote aberrant vascular remodeling.

[0046] The term "antibody", as used herein, is intended to refer to an immunoglobulin molecule (i.e., a "complete antibody molecule") consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain consists of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (consisting of domains CH1, CH2, and CH3). 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 more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the aligned analysis of two or more CDRs.

[0047] Substitution of one or more CDR residues or elimination of one or more CDRs is also possible. In the scientific literature, antibodies are described that can remove one or two CDRs for binding. Padlan et al. (FASEB J. 1995, 9:133-139) analyzed the contact areas between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0048] CDR residues that do not contact the antigen can be identified based on previous studies, by molecular modeling and / or empirically. If a CDR or its residues are missing, they are usually replaced with an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of such sequences. The positions for substitution within the CDR and the amino acids to be substituted can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[0049] The fully human PDGF-B monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LAll of the framework and / or CDR residues within the domain are mutated back to the 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, e.g., only the mutated residues are found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues are found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present disclosure.

[0050] The present disclosure also includes fully human anti-PDGF-B monoclonal antibodies that include 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 disclosure includes anti-PDGF-B antibodies that have HCVR, LCVR, and / or CDR amino acid sequences that have, for example, 10 or less, 8 or less, 6 or less, or 4 or less conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0051] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the present disclosure may include, for example, amino acid residues in the CDRs, particularly in CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences.

[0052] The term "pulmonary hypertension" ("PH") is the term used to describe high blood pressure in the lungs from any cause, while the terms "hypertension" or "high blood pressure" refer to high blood pressure in the arteries of the entire body.

[0053] The term "pulmonary arterial hypertension" ("PAH") refers to a progressive lung disease characterized by persistently elevated pulmonary artery pressure. Patients with PAH typically have a pulmonary artery pressure of 25 mmHg or greater and a pulmonary capillary or left atrial pressure of 15 mmHg or less. These pressures are typically measured in subjects at rest using right heart catheterization. If untreated, PAH is fatal (on average) within 2.8 years after diagnosis.

[0054] The World Health Organization (WHO) provides a clinical classification of five groups of PAH (described in Simonneau, et al. J Am Coll Cardiol. 2013;62(25_S), the entire contents of which are incorporated herein by reference): 1. Pulmonary Arterial Hypertension (PAH) Idiopathic 1.2.Heritage BMPR2 1.2.2.ALK1, ENG, SMAD9, CAV1, KCNK3 Unknown 1.3. Drug and toxin induction 1.4. Associated with: 1.4.1. Connective tissue diseases 1.4.2.HIV infection Portal hypertension 1.4.4. Congenital heart disease 1.4.5.Schistosomiasis 1'. Pulmonary veno-occlusive disease (PVOD) and / or pulmonary capillary hemangiomatosis (PCH) 1. Persistent Pulmonary Hypertension of the Newborn (PPHN) 2. Pulmonary hypertension due to left heart disease 2.1. Left ventricular systolic dysfunction 2.2. Left ventricular diastolic dysfunction 2.3. Valvular disease 2.4.Congenital / acquired left heart inflow / outflow tract obstruction and congenital cardiomyopathy 3. Pulmonary hypertension due to lung disease and / or hypoxia 3.1. Chronic obstructive pulmonary disease Interstitial lung disease 3.3. Other pulmonary diseases with a mixed restrictive and obstructive pattern 3.4. Sleep-disordered breathing 3.5. Alveolar hypoventilation disorder 3.6. Chronic exposure to high altitude 3.7. Developmental abnormalities 4. Chronic Thromboembolic Pulmonary Hypertension (CTEPH) 5. Multifactorial pulmonary hypertension with unknown mechanisms 5.1. Blood disorders: chronic hemolytic anemia, myeloproliferative disorders, splenectomy 5.2. Systemic diseases: sarcoidosis, pulmonary histiocytosis, lymphangioleiomyomatosis 5.3. Metabolic disorders: glycogen storage disease, Gaucher disease, thyroid disorders 5.4.Others: Tumor obstruction, fibrosing mediastinitis, chronic renal failure due to dialysis, segmental PH.

[0055] In one embodiment, subjects who may benefit from the methods of the present disclosure are those with Group I (WHO) PAH.

[0056] PAH at baseline (e.g., when diagnosed) can be mild, moderate, or severe, as measured, for example, by the WHO functional class, which is a measure of disease severity in patients with PAH. The WHO functional classification is an adaptation of the New York Heart Association (NYHA) system, which is routinely used, for example, to qualitatively assess activity tolerance in monitoring disease progression and response to treatment (Rubin (2004) Chest 126:7-10). There are four functional classes recognized in the WHO system: Class I: pulmonary hypertension not limiting physical activity; Ordinary physical activity does not cause undue shortness of breath or fatigue, chest pain, or dizziness; Class II: pulmonary hypertension resulting in minor limitations in physical activity; The patient is comfortable at rest; ordinary physical activity causes undue dyspnea or fatigue, chest pain, or dizziness; Class III: Pulmonary hypertension resulting in significant limitation of physical activity; patient is comfortable at rest; less than usual activity causes excessive dyspnea or fatigue, chest pain or dizziness; and Class IV: Pulmonary hypertension in which any physical activity cannot be performed without symptoms; the patient shows signs of right heart failure; dyspnea and / or fatigue may be present even at rest; discomfort is increased by any physical activity.

[0057] In one embodiment, subjects who may benefit from the methods of the present disclosure are those with WHO class I PAH, e.g., Group I (WHO) PAH, at baseline. In another embodiment, subjects who may benefit from the methods of the present disclosure are those with WHO class II PAH, e.g., Group I (WHO) PAH, at baseline. In another embodiment, subjects who may benefit from the methods of the present disclosure are those with WHO class III PAH, e.g., Group I (WHO) PAH, at baseline.

[0058] As used herein, a "subject" is an animal, such as a primate (human, non-human primate, such as monkeys and chimpanzees), non-primate (such as cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales), mammal, etc.

[0059] In one embodiment, the subject is a human, such as a human being treated or evaluated for PAH, e.g., Group I (WHO) PAH; a human at risk for PAH, e.g., Group I (WHO) PAH; a human having PAH, e.g., Group I (WHO) PAH; and / or a human being treated for PAH, e.g., Group I (WHO) PAH), as described herein.

[0060] As used herein, the term "treat" or "treatment" refers to a beneficial or desired result, including, but not limited to, alleviation or amelioration of one or more symptoms associated with PAH, e.g., Group I (WHO) PAH. "Treatment" can also mean slowing the course of a disease or reducing the onset of symptoms of a disease, reducing the severity of late-onset disease, or prolonging survival compared to expected survival in the absence of treatment. For example, a reduction in the onset of symptoms associated with such a disease, disorder, or condition (e.g., at least about 10% on a clinically accepted scale for the disease or disorder), or a delayed onset of symptoms (e.g., by days, weeks, months, or years) is considered effective treatment.

[0061] Terms such as "specifically binds" or "binds specifically to" mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1×10 -6 M or less (e.g., a smaller K Dindicates stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies that specifically bind to PDGF-B have been identified by surface plasmon resonance, e.g., BIACORE™. Furthermore, as used herein, multispecific antibodies that bind to one domain of PDGF-B and one or more additional antigens, or bispecific antibodies that bind to two different regions of PDGF-B, are nevertheless considered to be "specifically binding" antibodies.

[0062] The term "high affinity" antibody refers to an antibody that has an affinity of at least 10 as measured by surface plasmon resonance, e.g., BIACORE™ or solution affinity ELISA. -7 M, preferably 10 -8 M, more preferably 10 -9 M, and even more preferably 10 -10 M, and even more preferably 10 -11 K of M D The terms refer to those mAbs that have binding affinity for PDGF-B, expressed as .

[0063] The term "slow off rate", "Koff" or "kd" refers to a slow off rate of 1×10 as determined by surface plasmon resonance, e.g., BIACORE™. -3 s -1 Less than or equal to 1×10 -4 s -1 It is meant to describe an antibody that dissociates from PDGF-B with the following rate constant:

[0064] An "antigen-binding portion" of an antibody, an "antigen-binding fragment" of an antibody, and similar terms, as used herein, include naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind an antigen to form a complex. The term "antigen-binding fragment" of an antibody or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to bind to PDGF-B.

[0065] In certain embodiments, the antibodies or antibody fragments of the disclosure may be conjugated to a therapeutic moiety (an "immunoconjugate") such as an antibiotic, a second anti-PDGF-B antibody, or an antibody against a cytokine such as IL-1, IL-6, or TGF-β, or any other therapeutic moiety useful in the treatment of pulmonary arterial hypertension.

[0066] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody or fragment thereof that specifically binds PDGF-B is substantially free of Abs that specifically bind to antigens other than PDGF-B).

[0067] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows analysis of real-time biomolecular interactions through detection of changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0068] "K D The term "as used herein" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0069] 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. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen to which an antibody binds. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes, and have residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural and / or specific charge characteristics.

[0070] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicates that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, 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.

[0071] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT, using predefined gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions are not expected to substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acids substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45, incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0072] Polypeptide sequence similarity is typically measured using sequence analysis software. Protein analysis software matches similar sequences using a measure of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the region of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, e.g., 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.

[0073] In certain embodiments, antibodies or antibody fragments for use in the methods of the present disclosure may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen binding domains specific for epitopes of more than one target polypeptide.

[0074] The phrase "therapeutically effective amount," as used herein, is intended to include an amount of an anti-PDGF-B antibody or antigen-binding fragment thereof that, when administered to a subject with PAH, e.g., Group I (WHO) PAH, is sufficient to effect treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease) or to manage the disease. A "therapeutically effective amount" may vary depending on the anti-PDGF-B antibody or antigen-binding fragment thereof, the method of administration of the anti-PDGF-B antibody or antigen-binding fragment thereof, the disease and its severity, as well as the medical history, age, weight, family history, genetic makeup, stage of PAH, type of prior or concomitant treatment (if any), and other individual characteristics of the patient being treated.

[0075] A "therapeutically effective amount" is also intended to include an amount of an anti-PDGF-B antibody or antigen-binding fragment thereof that, when administered to a subject, is sufficient to ameliorate a disease or one or more symptoms of a disease. Ameliorating a disease includes slowing the progression of a disease or reducing the severity of later onset disease.

[0076] A "therapeutically effective amount" also includes that amount of an anti-PDGF-B antibody or antigen-binding fragment thereof that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The anti-PDGF-B antibody or antigen-binding fragment thereof used in the methods of the present disclosure may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0077] overview Platelet-derived growth factor (PDGF) is a potent mitogen that exists as five different dimeric forms consisting of four different isoform subunits: A, B, C, and D. The five dimeric forms of PDGF are AA, BB, AB, CC, and DD, which are formed by disulfide bonds of corresponding individual PDGF monomers. PDGF ligands exert their biological effects through interaction with PDGF receptors (PDGFRs). PDGFRs are single-pass, transmembrane, tyrosine kinase receptors consisting of heterodimeric or homodimeric associations of alpha (a) receptor chains (PDGFR-alpha) and / or beta (β) receptor chains (PDGF-B). Thus, active PDGFRs can consist of αα, ββ, or αβ receptor chain pairs. PDGFRs share a common domain structure that includes five extracellular immunoglobulin (Ig) loops, a transmembrane domain, and a split intracellular tyrosine kinase (TK) domain. The interaction between dimeric PDGF ligands and PDGFRs results in receptor chain dimerization, receptor autophosphorylation, and intracellular signal transduction. It has been demonstrated in vitro that ββ receptors are activated by PDGF-BB and -DD, whereas αβ receptors are activated by PDGF-BB, -CC, -DD, and -AB, and αα receptors are activated by PDGF-AA, -BB, -CC, and -AB (see Andrae et al. (2008) Genes Dev 22(10):1276-1312).

[0078] The antibodies described herein exhibit specific binding to PDGF-B and, in some embodiments, may be useful in treating patients suffering from pulmonary arterial hypertension. They may be used alone or as adjunctive therapy with other treatment sites or modalities known in the art for treating pulmonary arterial hypertension, such as, but not limited to, iron supplementation with iron supplements, dietary modifications to promote serum iron, and / or intravenous delivery of iron, blood transfusions, and iron-promoting drugs. They may be used in conjunction with additional antibodies specific for antigens other than PDGF-B, or in conjunction with other types of treatment.

[0079] In some embodiments, the antibodies described herein may be useful in the prevention, treatment, or management of pulmonary arterial hypertension.

[0080] In certain embodiments, antibodies of the present disclosure are obtained from mice immunized with a primary immunogen, such as native full-length human PDGF-B (SEQ ID NO: 41) or a PDGF-B fragment, followed by immunization with a secondary immunogen or an immunogenically active fragment of PDGF-B.

[0081] The immunogen can be an immunogenic fragment of PDGF-B or DNA encoding the fragment. The immunogen can be PDGF-B linked to a histidine tag and / or a fragment of the Fc region of an antibody.

[0082] The amino acid sequence of full-length human PDGF-B is shown as SEQ ID NO:41.

[0083] In certain embodiments, antibodies that specifically bind to PDGF-B may be prepared using fragments of the above regions or peptides extending beyond the designated regions by about 5 to about 20 amino acid residues from either or both of the N-terminus or C-terminus of the regions described herein. In certain embodiments, any combination of the above regions or fragments thereof may be used in the preparation of PDGF-B-specific antibodies. In certain embodiments, any one or more of the above regions of PDGF-B, or fragments thereof, may be used in the preparation of monospecific, bispecific, or multispecific antibodies.

[0084] Antigen-binding fragment of an antibody Unless otherwise indicated, the term "antibody" as used herein should be understood to encompass an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "complete antibody molecule"), as well as antigen-binding fragments thereof. An "antigen-binding portion" of an antibody, an "antigen-binding fragment" of an antibody, and similar terms, as used herein, include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind an antigen to form a complex. The term "antigen-binding fragment" of an antibody or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to PDGF-B. An antibody fragment may include a Fab fragment, a F(ab')2 fragment, a Fv fragment, a dAb fragment, a fragment comprising a CDR, or an isolated CDR. An antigen-binding fragment of an antibody may be derived from a complete antibody molecule using any suitable standard technique, such as, for example, proteolytic or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated by using chemical or molecular biology techniques, for example, to arrange one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids.

[0085] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constraining FR3-CDR3-FR4 peptide. Domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the expression "antigen-binding fragment" as used herein.

[0086] An antigen-binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and will generally contain at least one CDR adjacent to or in frame with one or more framework sequences. L Domain and associated V H For antigen-binding fragments containing domains, V H and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer and has a V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V H Or V L It may contain domains.

[0087] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary arrangements of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H -CH 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -C L , (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C L In any arrangement of the variable and constant domains, including any of the exemplary arrangements listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain arrangements listed above in non-covalent association with the domains (e.g., via disulfide bonds).

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

[0089] The disclosure includes anti-PDGF-B antibodies and antigen-binding fragments having immunoglobulin chains comprising the amino acid sequences described herein, as well as variants having cellular and / or in vitro post-translational modifications. For example, the disclosure includes antibodies and antigen-binding fragments thereof that specifically bind to PDGF-B comprising the heavy and / or light chain amino acid sequences (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3) described herein, as well as antibodies and fragments in which one or more amino acid residues are glycosylated, one or more Asn residues are deamidated, one or more residues (e.g., Met, Trp, and / or His) are oxidized, the N-terminal Gln is pyroglutamate (pyroE), and / or the C-terminal lysine is missing.

[0090] The disclosure includes recombinant methods for making an anti-PDGF-B antibody or antigen-binding fragment thereof, or an immunoglobulin chain thereof, e.g., a polynucleotide is in a vector and / or operably linked to a promoter; (i) the antibody or antigen-binding fragment (e.g., a heavy chain or a V chain thereof); H or an immunoglobulin comprising HCDR1, HCDR2, and HCDR3 thereof, and / or a light chain or V thereof Lor an immunoglobulin comprising its LCDR1, LCDR2 and LCDR3; (ii) culturing a host cell (e.g., a Chinese hamster ovary (CHO) cell or a Pichia cell or a Pichia pastoris cell) under conditions favorable for expression of the polynucleotides; and (iii) optionally isolating the antibody or fragment or chain from the host cell and / or the medium in which the host cell is growing. When making an antibody or antigen-binding fragment comprising more than one immunoglobulin chain, e.g., an antibody comprising two immunoglobulin heavy chains and two immunoglobulin light chains, co-expressing the chains in a single host cell results in association of the chains inside or on the cell surface or outside the cell, e.g., when such chains are secreted to form an antibody or antigen-binding fragment molecule. Methods include those in which only an immunoglobulin heavy chain or only an immunoglobulin light chain (e.g., any of those discussed herein including mature fragments and / or variable domains thereof) is expressed. Such chains are useful, for example, as intermediates in the expression of antibodies or antigen-binding fragments that contain such chains. The present disclosure also provides products of such expression methods (e.g., antibodies, antigen-binding fragments, V H , or V L ).

[0091] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method may be used in the context of the present disclosure to generate human antibodies that specifically bind to PDGF-B.

[0092] Using VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, a high affinity chimeric antibody against PDGF-B is first isolated, having a human variable region and a mouse constant region. VELOCIMMUNE® technology involves the generation of a transgenic mouse with a genome that includes human heavy and light chain variable regions operably linked to an endogenous mouse constant region locus, such that the mouse generates antibodies that include the human variable region and the mouse constant region in response to antigenic stimulation. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

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

[0094] First, a high affinity chimeric antibody having a human variable region and a mouse constant region is isolated. The antibody is characterized and selected for desired characteristics including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to generate a fully human antibody of the present disclosure, e.g., a wild-type or modified IgG1 or IgG4. While the constant region selected may vary depending on the particular application, the characteristics of high affinity antigen binding and target specificity reside in the variable region.

[0095] In general, the antibodies of the present disclosure have very high affinities, as measured by binding to antigen immobilized in either a solid or solution phase, typically on the order of about 10 -12 ~about 10 -7 K of M D The mouse constant regions are replaced with the desired human constant regions to generate the fully human antibodies of the present disclosure. While the constant region selected may vary depending on the particular application, the characteristics of high affinity antigen binding and target specificity reside in the variable regions.

[0096] bioequivalence The anti-PDGF-B antibodies and antibody fragments of the present disclosure include proteins having amino acid sequences that differ from those of the described antibodies but that retain the ability to bind PDGF-B. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Similarly, DNA sequences encoding the antibodies of the present disclosure include sequences that encode antibodies or antibody fragments that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but are essentially biologically equivalent to the antibodies or antibody fragments of the present disclosure.

[0097] Two antigen-binding proteins or antibodies are considered to be bioequivalents if, for example, they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in the rate and extent of absorption when administered under similar experimental conditions, at the same molar dose, either in single or multiple doses. Some antibodies are considered to be equivalents or pharmaceutical substitutes if their extent of absorption is comparable, but their rates of absorption are not, and furthermore, such differences in rates of absorption may be considered to be bioequivalents because they are intentional, are reflected in the label, are not essential to achieving effective body drug concentrations in, for example, chronic use, and are not considered medically significant for the particular drug product studied.

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

[0099] In one embodiment, two antigen binding proteins are bioequivalent if a patient can make one or more switches between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or decreased efficacy, compared to continuous therapy without such switches.

[0100] In one embodiment, two antigen binding proteins are biologically equivalent if they both act by a common mechanism or mode of action for a condition or condition of use, to the extent that such mechanism is known.

[0101] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurement methods include, for example, (a) in vivo studies in humans or other mammals in which concentrations of the antibody or its metabolites are measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effects of the antibody (or its target) are measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0102] Biologically equivalent variants of the antibodies of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent antibodies can include antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0103] Anti-PDGF-B Antibodies, Including Fc Variants According to certain embodiments of the present disclosure, anti-PDGF-B antibodies are provided that include an Fc domain that includes one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides an Fc domain that includes one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2nd Area or C H Anti-PDGF-B antibodies include those containing mutations in region 3 that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0) and can result in increased serum half-life of the antibody when administered to an animal.

[0104] All possible combinations of the aforementioned Fc domain mutations, as well as other mutations in the antibody variable domains disclosed herein, are contemplated within the scope of this disclosure.

[0105] The present disclosure also provides a chimeric heavy chain constant (C H ) region, and H The region is a C region of more than one immunoglobulin isotype. H For example, the antibodies of the present disclosure may include segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H C derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule in combination with some or all of the three domains. H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibodies of the present disclosure may comprise a chimeric C region having a chimeric hinge region. H For example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216-227 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues at positions 228-236 according to EU numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric C described herein may be H Antibodies comprising the region may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Provisional Patent Application No. 61 / 759,578, filed February 1, 2013, the disclosure of which is incorporated herein by reference in its entirety).

[0106] Biological properties of antibodies In general, the antibodies of the disclosure may function by binding to PDGF-B. In some embodiments, the antibodies of the disclosure may bind to another antigen (a cross-reactive antibody).

[0107] In certain embodiments, the antibody of the present disclosure may be a bispecific antibody. The bispecific antibody of the present disclosure may bind to one epitope in one domain and also bind to one epitope in a second domain of PDGF-B. In certain embodiments, the bispecific antibody of the present disclosure may bind to two different epitopes in the same domain.

[0108] In one embodiment, the disclosure provides a fully human monoclonal antibody or antigen-binding fragment thereof that binds to PDGF-B, the antibody or fragment thereof exhibiting one or more of the following characteristics: (i) comprising an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) comprising an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and (iii) comprising an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 28, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 36, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. (iv) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 24, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 26, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 32, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 34, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; -7 The following K D Binds to PDGF-B.

[0109] Certain anti-PDGF-B antibodies of the present disclosure are capable of binding to and neutralizing the activity of PDGF-B as determined by in vitro or in vivo assays. The ability of an antibody to bind to and neutralize the activity of PDGF-B can be measured using any standard method known to those of skill in the art, including the binding or activity assays described herein.

[0110] The peptides can be modified to include the addition or substitution of certain residues for tagging or for the purpose of conjugation to a carrier molecule such as KLH. For example, a cysteine ​​can be added to either the N- or C-terminus of the peptide, or a linker sequence can be added to prepare the peptide for conjugation to KLH, for example, for immunization.

[0111] Antibodies specific for PDGF-B may contain no additional labels or moieties, or they may contain N-terminal or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the location of the label (if any) may determine the orientation of the peptide relative to the surface to which it binds. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal to the surface. In one embodiment, the label may be a radionuclide, a fluorescent dye, or an MRI-detectable label. In certain embodiments, such labeled antibodies may be used in diagnostic assays, including imaging assays.

[0112] Epitope mapping and related techniques The present disclosure includes anti-PDGF-B antibodies that interact with one or more amino acids found within one or more regions of PDGF-B. The epitope to which the antibody binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids located within any of the above-mentioned regions of the PDGF-B molecule (e.g., a linear epitope in a domain). Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located in either or both of the aforementioned regions of the PDGF-B molecule (e.g., a conformational epitope).

[0113] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" in a polypeptide or protein. Exemplary techniques include 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 truncation analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Protein Science 9:487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, hydrogen / deuterium exchange involves deuterium labeling of the protein of interest and then binding of the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo deuterium to hydrogen back-exchange at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface can retain deuterium and therefore exhibit a relatively high mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing peptides containing deuterium-labeled residues that contain specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259, Engen and Smith (2001) Anal.Chem.73:256A-265A.

[0114] The term "epitope" refers to a site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed both from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least 3, more usually at least 5, or 8-10 amino acids in a unique spatial conformation.

[0115] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) against the same antigen according to the similarity of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see US2004 / 0101920, specifically incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is distinct or partially overlapping with the epitopes represented by another category. This technique 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 with desired characteristics. MAP can be used to sort the antibodies of the present disclosure into groups of antibodies that bind to different epitopes.

[0116] In certain embodiments, the anti-PDGF-B antibody or antigen-binding fragment thereof binds to an epitope within any one or more of the regions exemplified in human PDGF-B, as exemplified by SEQ ID NO:41, or a fragment thereof.

[0117] The present disclosure includes human anti-PDGF-B antibodies that bind to the same epitope or portion of an epitope as any of the specific exemplary antibodies described herein, or antibodies that have the CDR sequences of any of the exemplary antibodies described herein. Similarly, the present disclosure also includes anti-PDGF-B antibodies that compete with any of the specific exemplary antibodies described herein for binding to PDGF-B or a PDGF-B fragment, or antibodies that have the CDR sequences of any of the exemplary antibodies described herein.

[0118] Using routine methods known in the art, one can easily determine whether an antibody binds to the same epitope as a reference anti-PDGF-B antibody or competes for binding with it. For example, to determine whether a test antibody binds to the same epitope as a reference anti-PDGF-B antibody of the present disclosure, the reference antibody can be bound to a PDGF-B protein or peptide under saturating conditions. The ability of the test antibody to bind to a PDGF-B molecule is then evaluated. If the test antibody is able to bind to PDGF-B after saturation binding with the reference anti-PDGF-B antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-PDGF-B antibody. On the other hand, if the test antibody is unable to bind to the PDGF-B chain after saturation binding with the reference anti-PDGF-B antibody, the test antibody may bind to the same epitope as the reference anti-PDGF-B antibody of the present disclosure binds.

[0119] To determine whether an antibody competes for binding with a reference anti-PDGF-B antibody, the binding method described above is carried out from two orientations. In the first orientation, the reference antibody is allowed to bind to PDGF-B protein under saturating conditions, and then the binding of the test antibody to the PDGF-B molecule is evaluated. In the second orientation, the test antibody is allowed to bind to the PDGF-B molecule under saturating conditions, and then the binding of the reference antibody to the PDGF-B molecule is evaluated. In both orientations, if only the first (saturating) antibody can bind to the PDGF-B molecule, it is concluded that the test antibody and the reference antibody compete for binding to PDGF-B. As will be recognized by those skilled in the art, an antibody that competes for binding with a reference antibody does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0120] Two antibodies bind to the same or overlapping epitopes if each of them competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0121] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0122] Immunoconjugates The present disclosure encompasses human anti-PDGF-B monoclonal antibodies conjugated to a therapeutic moiety ("immunoconjugate"), such as an agent capable of reducing the severity of pulmonary arterial hypertension or an agent capable of ameliorating at least one symptom associated with pulmonary arterial 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 moiety, enzyme, toxin, or therapeutic agent. The antibody may be linked to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, toxin, or therapeutic agent at any position along the molecule, so long as it is capable of binding to its target. An example of an immunoconjugate is an antibody drug conjugate. In some embodiments, the agent may be a second, different antibody against PDGF-B, or a cytokine such as IL-1, IL-6, or a chemokine such as TGF-β. The type of therapeutic moiety that may be conjugated to the anti-PDGF-B antibody takes into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art (see, e.g., WO05 / 103081). The preparation of immunoconjugates and immunotoxins is generally well known in the art (see, e.g., U.S. Pat. No. 4,340,535). Immunoconjugates are described in detail, for example, in US7250492, US7420040, and US7411046, each of which is incorporated herein in its entirety.

[0123] multispecific antibodies The antibodies of the present disclosure may be monospecific, bispecific, or multispecific. A multispecific antibody may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The antibodies of the present disclosure may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment thereof may be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent association, etc.) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity. For example, the present disclosure includes bispecific antibodies in which one arm of the immunoglobulin is specific for the N-terminal region of PDGF-B or a fragment thereof, and the other arm of the immunoglobulin is specific for the C-terminal region of PDGF-B or a second therapeutic target, or is conjugated to a therapeutic moiety. An exemplary bispecific antibody format that can be used in the context of the present disclosure is H3 Domain and second Ig C H3 The first and second Ig C domains are H3 The domains differ from one another by at least one amino acid, which at least one amino acid difference reduces binding of the bispecific antibody to PDGF-B compared to a bispecific antibody lacking that amino acid difference. Variations on the above-described bispecific antibody formats are contemplated within the scope of this disclosure.

[0124] Other exemplary bispecific formats that can be used in the context of the present disclosure include, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zipper, duobodies, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 Bispecific formats include, but are not limited to, bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6,1-11, and references cited therein, for a discussion of the above formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimers with defined composition, valency, and shape. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub:Dec.4,2012]).

[0125] Therapeutic Administration and Formulations The present disclosure provides therapeutic compositions comprising the anti-PDGF-B antibodies or antigen-binding fragments thereof discussed herein. The therapeutic compositions according to the present disclosure may be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerability, and the like. Numerous suitable formulations may be found in a formulary known to any pharmacist: 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 (such as LIPOFECTIN®), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0126] The dose of the antibody may vary depending on the age and size of the subject to be administered, the target disease, the pathology, the route of administration, etc. When the antibody of the present disclosure is used to prevent or treat pulmonary arterial hypertension, it is advantageous to administer the antibody of the present disclosure intravenously, usually at a single dose of about 0.1 to about 100 mg per kg of body weight, more preferably at a single dose of about 5 to about 100, about 10 to about 90, or about 20 to about 70 mg per kg of body weight. The frequency and duration of treatment can be adjusted depending on the severity of the pathology. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, about 10 to about 100 mg, or about 10 to about 50 mg. In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be about the same as or less than the initial dose, where the subsequent doses are separated by at least 1 to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0127] Various delivery systems are known and may be used to administer the pharmaceutical compositions of the present disclosure, 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). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (such as oral, rectal, and intestinal mucosa), and may be administered together with other biologically active agents. Administration may be systemic or local. The pharmaceutical compositions may also be delivered in vesicles, particularly liposomes (see, for example, Langer (1990) Science 249:1527-1533).

[0128] The use of nanoparticles to deliver the antibodies of the present disclosure is also contemplated herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are detailed 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), which are incorporated herein by reference. Nanoparticles for drug delivery are also described, for example, in US8277812, US8258256, US8257740, US8246995, US8236330, each of which is incorporated herein in its entirety.

[0129] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose.

[0130] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium that is conventionally used for injections. The aqueous medium for injection may be, for example, physiological saline, a glucose-containing isotonic solution, or other auxiliary agent, which may be used in combination with a suitable solubilizing agent such as alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), or a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. The oily medium may be, for example, sesame oil, soybean oil, or the like, which may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol. The injection thus prepared is preferably filled into a suitable ampoule.

[0131] The pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device easily finds use in delivering the pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0132] Numerous reusable pen and autoinjector delivery devices have application in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include the AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG® pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN® I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD pen (Becton Dickinson, Franklin Examples of disposable pen delivery devices that have application in the subcutaneous delivery of pharmaceutical compositions of the present disclosure include, but are in no way limited to, the SOLOSTAR pen (Sanofi-aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (HUMALOG), SURECLICK Autoinjector (PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Mylan and HUMIRA pens (Abbott Labs, Abbott Park, Ill.), and the like.

[0133] Advantageously, the pharmaceutical compositions for oral or parenteral use are prepared in a suitable unit dose dosage form to suit the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose, and particularly in the form of injection, the antibody is preferably contained in about 5 to about 100 mg, and for other dosage forms, it is preferably contained in about 10 to about 250 mg. The present disclosure includes an injection device (e.g., a pre-filled syringe or a pre-filled autoinjector) or a vial (e.g., a glass or plastic vial) containing the antibody or antigen-binding fragment of the present disclosure or a pharmaceutical composition thereof, including a pharma- ceutically acceptable carrier.

[0134] Therapeutic Uses of Antibodies In certain embodiments of the present disclosure, the antibody is useful for treating pulmonary arterial hypertension or at least one symptom associated with pulmonary arterial hypertension. The antibody of the present disclosure is also contemplated for prophylactic use in patients at risk of developing pulmonary arterial hypertension. These patients include elderly or patients with immunodeficiency due to disease or treatment with immunosuppressive therapy. It is contemplated that the antibody of the present disclosure can be used alone or in combination with a second or third agent to treat pulmonary arterial hypertension or to alleviate at least one symptom or complication associated with pulmonary arterial hypertension. The second or third agent can be delivered simultaneously with the antibody of the present disclosure, or they can be administered separately, either before or after the antibody of the present disclosure. Patients who may receive the antibody or antigen-binding fragment of the present disclosure or pharmaceutical composition thereof include animals such as, for example, humans (e.g., elderly, e.g., 65 years or older), rabbits, mice, rats, cattle, pigs, dogs, primates, horses, or sheep, and other mammals.

[0135] In a further embodiment of the disclosure, the antibody is used for the preparation of a pharmaceutical composition for treating a patient suffering from pulmonary arterial hypertension.

[0136] Combination therapy The present disclosure includes compositions and therapeutic formulations comprising any of the anti-PDGF-B antibodies described herein in combination with one or more additional therapeutically active ingredients, as well as methods of treatment comprising administering such combinations to a subject in need thereof.

[0137] The anti-PDGF-B antibodies of the disclosure may be formulated and / or administered in combination with, for example, a VEGF antagonist, e.g., a "VEGF trap" such as aflibercept or other VEGF inhibitory fusion proteins described in US 7,087,411, an anti-VEGF antibody or antigen-binding fragment thereof (e.g., bevacizumab, ranibizumab), a small molecule kinase inhibitor of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib), or an anti-VEGF receptor antibody. Anti-PDGF-B antibodies may also be combined with PDGF ligand antagonists (e.g., anti-PDGF-BB antibodies, anti-PDGF-DD antibodies, anti-PDGF-CC antibodies, anti-PDGF-AB antibodies, or other PDGF ligand antagonists such as aptamers (e.g., anti-PDGF-B aptamers such as Fovista™, Ophthotech Corp., Princeton, NJ), antisense molecules, ribozymes, siRNAs, peptibodies, nanobodies or antibody fragments directed against a PDGF ligand). In other embodiments, the anti-PDGF-B antibodies of the disclosure may be used in combination with other agents, such as an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or a small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member, such as Her2 / ErbB2, ErbB3, or ErbB4 (e.g., an anti-ErbB2, anti-ErbB3, or anti-ErbB4 antibody, or a small molecule inhibitor of ErbB2, ErbB3, or ErbB4 activity), an antagonist specific for EGFRvlll (e.g., an antibody that specifically binds EGFRvlll), a cMET antagonist (e.g., an anti-cMET antibody), an IGF1 R antagonist (e.g., an anti-IGF1R antibody), an IGF1R ... R antibody), or B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720).In some cases, the anti-PDGF-B antibodies of the present disclosure are combined, co-formulated, and / or administered in combination with a PDGFR-alpha inhibitor (e.g., an anti-PDGFR-alpha antibody), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in US 2009 / 0142354, such as REGN421), an Ang2 antagonist (e.g., an anti-Ang2 antibody disclosed in US 2011 / 0027286, such as H1 H685P), etc. Other agents that may be beneficially administered in combination with the anti-PDGF-B antibodies of the present disclosure include cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-, IL-12, IL-13, IL-17, IL-18, etc., or their respective receptors.

[0138] The anti-PDGF-B antibodies of the disclosure may also be administered and / or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, metal chelators, IFN-gamma, and / or NSAIDs. The anti-PDGF-B antibodies of the disclosure may also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy (e.g., in the context of a method of treating cancer or inhibiting tumor growth).

[0139] Any of the aforementioned additional therapeutic active ingredients may be administered in combination with any of the anti-PDGF-B antibodies of the present disclosure for the treatment of any disease or disorder for which administration of an anti-PDGFR-beta antibody is beneficial, including, for example, any of the ocular diseases, fibrotic diseases, vascular diseases, and / or cancers mentioned herein.For example, in the context of treating ocular diseases (e.g., wet AMD, diabetic retinopathy, CRVO, or any of the other ocular diseases described herein), the anti-PDGF-B antibodies of the present disclosure may be formulated and / or administered in combination with a VEGF antagonist, such as aflibercept or other VEGF-inhibiting fusion proteins described in US 7,087,411, or an anti-VEGF antibody or antigen-binding fragment thereof (e.g., bevacizumab, or ranibizumab).

[0140] In exemplary embodiments in which an anti-PDGF-B antibody of the present disclosure is administered in combination with a VEGF antagonist (e.g., a VEGF trap such as aflibercept), including administration of a co-formulation comprising an anti-PDGF-B antibody and a VEGF antagonist, the individual components may be administered to a subject and / or co-formulated using various dosage combinations. For example, an anti-PDGF-B antibody may be administered to a subject and / or contained in a co-formulation in an amount selected from the group consisting of 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, and 5.5 mg, and a VEGF antagonist (e.g., a VEGF trap such as aflibercept) may be administered to a subject and / or contained in a co-formulation in an amount selected from the group consisting of 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, and 5.5 mg. A VEGF trap such as aflibercept) may be administered to a subject and / or included in a co-formulation in an amount selected from the group consisting of 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, and 3.0 mg. Exemplary anti-PDGF-B antibody / aflibercept dosage combinations of the present disclosure include, for example, (i) 0.2 mg of anti-PDGF-B antibody + 2 mg of aflibercept, (ii) 0.5 mg of anti-PDGF-B antibody + 2 mg of aflibercept, (iii) 1 mg of anti-PDGF-B antibody + 2 mg of aflibercept, (iv) 3 mg of anti-PDGF-B antibody + 2 mg of aflibercept, and (v) 4 mg of anti-PDGFR-beta antibody + 2 mg of aflibercept. This combination / co-formulation can be administered to a subject according to any of the dosing regimens disclosed elsewhere herein, including, for example, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, etc.

[0141] The additional therapeutically active ingredient may be administered to the subject prior to administration of the anti-PDGF-B antibody of the present disclosure. For example, if the first ingredient is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration of the second ingredient, the first ingredient may be considered to be administered "before" the second ingredient. In other embodiments, the additional therapeutically active ingredient may be administered to the subject after administration of the anti-PDGF-B antibody of the present disclosure. For example, a first component may be considered to be administered "after" the second component if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours after administration of the second component. In yet other embodiments, an additional therapeutically active component may be administered to a subject simultaneously with administration of an anti-PDGF-B antibody.

[0142] For purposes of this disclosure, "concurrent" administration includes, for example, administration of an anti-PDGF-B antibody and an additional therapeutically active ingredient to a subject in a single dosage form (e.g., co-formulated) or in separate dosage forms administered to the subject within about 30 minutes of each other. When administered in separate dosage forms, each dosage form can be administered by the same route (e.g., both the anti-PDGFR-beta antibody and the additional therapeutically active ingredient can be administered intravitreally, subcutaneously, etc.), or alternatively, each dosage form can be administered by a different route (e.g., the anti-PDGF-B antibody can be administered intravitreally and the additional therapeutically active ingredient can be administered systemically). In any event, administration of the ingredients in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered "concurrent administration" for purposes of this disclosure. For purposes of this disclosure, administration of an anti-PDGF-B antibody "before," "concurrent with," or "after" administration of an additional therapeutically active ingredient will be considered administration of an anti-PDGF-B antibody "in combination with" the additional therapeutically active ingredient.

[0143] The present disclosure includes pharmaceutical compositions in which an anti-PDGFR-beta antibody of the present disclosure is co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein.

[0144] The present disclosure also includes additional therapeutic compositions that include a combination of a PDGF antagonist and a VEGF antagonist. PDGF antagonists according to this aspect of the disclosure include PDGF receptor antagonists as well as PDGF ligand antagonists. Similarly, VEGF antagonists according to this aspect of the disclosure include VEGF receptor antagonists as well as VEGF ligand antagonists.

[0145] The additional therapeutically active ingredients may be administered prior to, concurrently with, or following administration of an anti-PDGF-B antibody of the present disclosure, and for purposes of this disclosure, such administration regimens will be considered administration of an anti-PDGF-B antibody "in combination" with one or more additional therapeutically active ingredients.

[0146] Diagnostic Uses of Antibodies The anti-PDGF-B antibodies of the present disclosure may also be used to detect and / or measure PDGF-B in a sample, e.g., for diagnostic purposes. An exemplary diagnostic assay for PDGF-B may include, for example, contacting a sample obtained from a patient with an anti-PDGF-B antibody of the present disclosure, which is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate PDGF-B from the patient sample. Alternatively, an unlabeled anti-PDGF-B antibody may be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be 3 H, 14 C. 32 P, 35 S, or 125The immunoassay may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Certain exemplary assays that can be used to detect or measure PDGF-B in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

[0147] Samples that can be used in the PDGF-B diagnostic assays according to the present disclosure include any tissue or fluid sample obtainable from a patient that contains a detectable amount of PDGF-B or a fragment thereof under normal or pathological conditions. In general, the level of PDGF-B in a particular sample obtained from a healthy patient (e.g., a patient not suffering from pulmonary arterial hypertension) is measured to first establish a baseline or standard level of PDGF-B. This baseline level of PDGF-B can then be compared to the level of PDGF-B measured in a sample obtained from an individual suspected of having a condition associated with pulmonary arterial hypertension or a symptom associated with such a condition.

[0148] Antibodies specific for PDGF-B may contain no additional labels or moieties, or they may contain N-terminal or C-terminal labels or moieties. In one embodiment, the label or moiety is biotin. In binding assays, the location of the label (if any) may determine the orientation of the peptide relative to the surface to which it binds. For example, if the surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal to the surface. In some embodiments, the label may be a detectable label, such as a radionuclide, a fluorescent dye, or an MRI detectable label. A detectable label may be linked to the antibody such that such antibodies may be used in imaging assays.

[0149] Treatment method The present disclosure provides methods for treating a subject with pulmonary arterial hypertension. The methods generally involve administering to the subject a therapeutically effective amount of an anti-PDGF-B antibody, or antigen-binding fragment thereof.

[0150] In some embodiments, administration of an anti-PDGF-B antibody or antigen-binding fragment thereof inhibits pulmonary artery thickening in a subject, e.g., inhibits further thickening of the pulmonary artery in a subject from baseline, e.g., at the time of diagnosis. Pulmonary artery thickening can be determined, for example, by chest CT (e.g., unenhanced axial 10 mm CT slices, etc.) and used to calculate the main pulmonary artery diameter (mPA). The main pulmonary artery diameter in a normal subject is about 2.4 cm to about 3.0 cm. The main pulmonary artery diameter in a subject with pulmonary arterial hypertension is about 3.1 cm to about 3.8 cm or greater. See, e.g., Edwards, et al. (1998) Br J Radiol 71(850):1018-20.

[0151] In other aspects, administration of an anti-PDGF-B antibody or antigen-binding fragment thereof increases stroke volume and / or stroke volume to end-systolic volume ratio ("SV / ESV") in a subject. "Stroke volume" ("SV") is the volume of blood pumped from the right or left ventricle per beat. Stroke volume can be calculated by measuring ventricular volume from an echocardiogram and subtracting the volume of blood in the ventricle just before a beat (called the "end-diastolic volume," "ESV") from the volume of blood in the ventricle at the end of the beat (called the "end-systolic volume," "EDV"). Stroke volume can also be calculated as the cardiac output measured by thermodilution during right heart catheterization divided by the heart rate, or as EDV minus ESV, indexed by body surface area. The term stroke volume can apply to each of the two ventricles of the heart. The stroke volume of each ventricle is generally equal, both being about 70 mL in healthy subjects. The SV / ESV of healthy subjects is about 0.9 to about 2.2, and the SV / ESV of subjects with PAH is about 0.2 to about 0.9. See, e.g., Brewis, et al. (2016) Int J Cardiol 218:206-211.

[0152] In yet other aspects, administration of an anti-PDGF-B antibody or antigen-binding fragment thereof increases right ventricular cardiac output and / or cardiac index (CI) in a subject. "Cardiac output" ("CO") is defined as the volume of blood pumped by the ventricle per unit time. "Cardiac index" ("CI") is a hemodynamic parameter relating cardiac output (CO) from the left ventricle per minute to "body surface area" ("BSA"), and thus relating cardiac performance to the size of an individual. Echocardiography and radionuclide imaging techniques can be used to estimate real-time changes in ventricular dimensions, thereby calculating stroke volume, which when multiplied by heart rate gives cardiac output, and BSA can be calculated using any one of the formulas known to those skilled in the art, including, for example, the Du Bois formula (Verbraecken, J, et al. (2006) Metabolism-Clin Exper 55(4):515-24) or the Mosteller formula (Mosteller (1987) N Engl J Med 317:1098). Subjects without PAH have a cardiac output in the range of about 4.0 to 8.0 L / min and a cardiac index of about 2.6 to about 4.2 L / min per square meter. Subjects with PAH have a cardiac index of about 1.9 to about 2.3 L / min per square meter (Ryan and Archer (2016) Circ Res 115:176-188).

[0153] Administration of an anti-PDGF-B antibody or antigen-binding fragment thereof to a subject with PAH in the methods of the present disclosure may improve other hemodynamic measurements in a subject with PAH, such as, for example, right atrial pressure, pulmonary artery pressure, end expiratory pressure, systemic arterial pressure, heart rate, pulmonary vascular resistance, and / or pulmonary capillary wedge pressure in the presence of systemic vascular resistance. Methods and devices for measuring right atrial pressure, pulmonary artery pressure, end expiratory pressure, systemic arterial pressure, heart rate, pulmonary vascular resistance, and / or pulmonary capillary wedge pressure in the presence of systemic vascular resistance are known to those of skill in the art.

[0154] Subjects without PAH have right atrial pressures of about 1 mmHg to about 5 mmHg, and subjects with PAH have right atrial pressures of about 11 mmHg to about 13 mmHg.

[0155] Subjects without PAH have a pulmonary artery pressure of about 9 mmHg to about 20 mmHg, and subjects with PAH have a pulmonary artery pressure of about 57 mmHg to about 61 mmHg.

[0156] Subjects without PAH have a pulmonary capillary wedge pressure in the presence of an end-expiratory pressure of about 4 mmHg to about 12 mmHg, and subjects with PAH have a pulmonary capillary wedge pressure in the presence of an end-expiratory pressure of about 9 mmHg to about 11 mmHg.

[0157] Subjects without PAH have a systemic arterial pressure of about 90 mmHg to about 96 mmHg, and subjects with PAH have a systemic arterial pressure of about 87 mmHg to about 91 mmHg.

[0158] Subjects without PAH have a heart rate of about 60 beats per minute (bpm) to about 90 bpm, and subjects with PAH have a systemic arterial pressure of about 84 bpm to about 88 bpm.

[0159] Subjects without PAHs had a mean of approximately 20 dyne sec / cm 5 ~About 130 dyne sec / cm 5 (or about 0.25 to about 1.625 wood units), and subjects with PAH have a pulmonary vascular resistance of about 1200 dyne seconds / cm 5 ~ approx. 1360 dyne sec / cm 5 (or about 15 to about 17 wood units) of pulmonary vascular resistance.

[0160] Subjects without PAHs have a mean of approximately 700 dyne sec / cm 5 ~ approx. 1600 dyne sec / cm 5 (or about 9 to about 20 dyne units), and subjects with PAH have a systemic vascular resistance of about 1840 dyne seconds / cm 5 ~About 2000 dyne sec / cm 5 (or about 23 to about 25 wood units) of systemic vascular resistance.

[0161] The disclosed methods may also improve other clinical parameters, such as pulmonary function, in treated subjects. For example, during or after a treatment period, a subject may have increased exercise capacity or activity, or a reduced Borg Dyspnea Index (BDI), as measured, for example, by the 6-minute walking distance (6MWD) test or activity scale.

[0162] The methods of the present disclosure may also improve one or more quality of life parameters, e.g., an increase in score on at least one of the SF-36® Health Survey functional scales, compared to baseline, an improvement in the severity of the condition, e.g., by moving to a lower WHO functional class, and / or an extension of life span, compared to baseline.

[0163] Any suitable measure of exercise capacity can be used to determine whether a subject has increased exercise capacity or activity. One suitable measure is the 6-minute walk test (6MWT), which measures how far a subject can walk in 6 minutes, i.e., 6-minute walk distance (6MWD). Another suitable measure is the Borg dyspnea index (BDI), which is a numerical scale for assessing perceived dyspnea (respiratory discomfort). The degree of shortness of breath after completion of the 6-minute walk test (6MWT) is measured, with a BDI of 0 indicating no shortness of breath and 10 indicating maximum shortness of breath. In one embodiment, the method of the present disclosure provides the subject with an increase in 6MWD from baseline for at least about 10 minutes, e.g., about 10 minutes, 15 minutes, 20 minutes, or about 30 minutes. In another embodiment, following the 6MWT, the method of the present disclosure provides the subject with a decrease in 6MWD from baseline of at least about 0.5 to about 1.0 index points.

[0164] Any suitable measure of quality of life may be used. For example, the SF-36® Health Survey provides self-reported multi-item scales measuring eight health parameters: physical functioning, role limitations due to physical health problems, bodily pain, general health, vitality (energy and fatigue), social functioning, role limitations due to emotional problems, and mental health (psychological distress and psychological well-being). The survey also provides a physical component summary and a mental component summary. In one embodiment, the disclosed method provides the subject with an improvement over baseline in at least one of the SF-36 physical health-related parameters (physical health, role-physical, bodily pain, and / or general health) and / or at least one of the SF-36 mental health-related parameters (vitality, social functioning, role-emotional, and / or mental health). Such improvement may take the form of an increase of at least 1, e.g., at least 2, or at least 3 points on the scale for any one or more parameters.

[0165] The disclosed methods may also improve the prognosis of the treated subject. For example, the disclosed methods may provide a subject with a reduced probability of clinically adverse events during treatment and / or a reduction in serum brain natriuretic peptide (BNP) or NT pro-BNP or its N-terminal prohormone, NT-pro-BNP concentration from a baseline where the time from initial diagnosis of the condition in the subject is about 2 years or less.

[0166] In various embodiments, the time from initial diagnosis can be, for example, about 1.5 years or less, about 1 year or less, about 0.75 years or less, or about 0.5 years or less. Clinically worsening events (CWE) include death, lung transplant, hospitalization for PAH, atrial septal resection, initiation of additional pulmonary hypertension therapy, or combinations thereof. Time to clinical worsening of PAH is defined as the time from initiation of treatment to the first occurrence of CWE.

[0167] In one embodiment, the methods of the disclosure provide a decrease in BNP or NT-pro-BNP concentration from baseline of at least about 15%, such as at least about 25%, at least about 50%, or at least about 75%.

[0168] In one embodiment, the methods of the present disclosure provide a reduction of at least about 25%, e.g., at least about 50%, at least about 75%, or at least about 80%, in the probability of death, lung transplant, hospitalization for pulmonary arterial hypertension, atrial septal resection, and / or initiation of additional pulmonary hypertension therapy during the treatment period.

[0169] The methods of the present disclosure may also extend the life span (prolonged survival time) of a subject with PAH, for example, by at least about 30 days from the time treatment is initiated.

[0170] A therapeutically effective amount of an anti-PDGF-B antibody or antigen-binding fragment thereof for use in the methods of the disclosure can be from about 0.05 mg to about 600 mg, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, or about 160 mg of the antibody. About 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 3 00mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440m g, about 450mg, about 460mg, about 470mg, about 480mg, about 490mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, About 590mg, about 600mg, about 610mg, about 620mg, about 630mg, about 640mg, about 650mg, about 660mg, about 670mg, about 680mg, about 690mg, about 700mg, about 710mg, about 720mg, about 73 The amount of the active ingredient may be about 0 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg, about 900 mg, about 910 mg, about 920 mg, about 930 mg, about 940 mg, about 950 mg, about 960 mg, about 970 mg, about 980 mg, about 990 mg, or about 1000 mg.

[0171] The amount of anti-PDGF-B antibody or antigen-binding fragment thereof contained in an individual dose can be expressed in milligrams of antibody per kilogram of patient body weight (i.e., mg / kg). For example, the anti-PDGF-B antibody or antigen-binding fragment thereof can be administered in an amount of about 0.0001 to about 50 mg / kg of patient body weight (e.g., 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg). The composition may be administered to the patient at a dose of 10.0 mg / kg, 10.5 mg / kg, 11.0 mg / kg, 11.5 mg / kg, 12.0 mg / kg, 12.5 mg / kg, 13.0 mg / kg, 13.5 mg / kg, 14.0 mg / kg, 14.5 mg / kg, 15.0 mg / kg, 15.5 mg / kg, 16.0 mg / kg, 16.5 mg / kg, 17.0 mg / kg, 17.5 mg / kg, 18.0 mg / kg, 18.5 mg / kg, 19.0 mg / kg, 19.5 mg / kg, 20.0 mg / kg, etc.

[0172] Multiple doses of an anti-PDGF-B antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising an anti-PDGF-B antibody or antigen-binding fragment thereof, may be administered to a subject over a defined time course. The method according to this aspect of the disclosure includes sequentially administering multiple doses of an active ingredient of the disclosure to a subject. As used herein, "sequentially administering" means that each dose of an active ingredient is administered to a subject at different times, e.g., different days, separated by a predetermined interval (e.g., hours, days, weeks, or months). The disclosure includes methods that include sequentially administering to a patient a single initial dose of an active ingredient, followed by one or more secondary doses of the active ingredient, optionally followed by one or more tertiary doses of the active ingredient.

[0173] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the time sequence for administration of an anti-PDGF-B antibody or antigen-binding fragment thereof or combination therapy of the present disclosure. Thus, an "initial dose" is the dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is the dose administered after the initial dose, and a "tertiary dose" is the dose administered after the secondary dose. The initial dose, secondary dose, and tertiary dose may all contain the same amount of anti-PDGF-B antibody or antigen-binding fragment thereof, but may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of anti-PDGF-B antibody or antigen-binding fragment thereof contained in the initial dose, secondary dose, and / or tertiary dose differs from each other during the course of treatment (e.g., adjusted up or down as necessary). In certain embodiments, two or more doses (e.g., two, three, four, or five) are administered as a "loading dose" at the beginning of a treatment regimen, with subsequent doses administered on a less frequent basis (e.g., "maintenance doses").

[0174] In certain exemplary embodiments of the present disclosure, each secondary dose and / or tertiary dose is administered within 1 to 26 (e.g., 1, 1 and 1 / 2, 2, 2 and 1 / 2, 3, 3 and 1 / 2, 4, 4 and 1 / 2, 5, 5 and 1 / 2, 6, 6 and 1 / 2, 7, 7 and 1 / 2, 8, 8 and 1 / 2, 9, 9 and 1 / 2, 10, 10 and 1 / 2, 11, 11 and 1 / 2, 12, 12 and 1 / 2, 1 3, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks later. The phrase "immediately preceding administration" as used herein refers to a dose of an anti-PDGF-B antibody or antigen-binding fragment thereof in a series of multiple doses that is administered to a patient prior to administration of the immediately following dose with no intervening doses.

[0175] Methods according to this aspect of the disclosure may include administering any number of secondary and / or tertiary doses to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) tertiary doses are administered to the patient.

[0176] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding administration. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding administration. In certain embodiments of the present disclosure, the frequency at which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical testing.

[0177] In some embodiments, the anti-PDGF-B antibody or antigen-binding fragment thereof may be administered as a monotherapy (i.e., as the only therapeutic agent), hi other embodiments, the anti-PDGF-B antibody or antigen-binding fragment thereof may be administered in combination with one or more additional therapeutic agents.

[0178] In combination methods including administering to a subject an anti-PDGF-B antibody or antigen-binding fragment thereof and at least one additional therapeutic agent, the antibody and additional therapeutic agent may be administered to the subject simultaneously or substantially simultaneously, e.g., in a single therapeutic dose, or simultaneously with each other, or in two separate doses administered within less than about 5 minutes of each other. Alternatively, the antibody and additional therapeutic agent may be administered to the subject sequentially, e.g., in separate therapeutic doses separated in time from each other by more than about 5 minutes.

[0179] Thus, in one embodiment, the method further comprises administering a therapeutically effective amount of at least one therapeutic agent selected from the group consisting of anticoagulants, diuretics, cardiac glycosides, calcium channel blockers, vasodilators, prostacyclin analogs, endothelial antagonists, phosphodiesterase inhibitors, endopeptidase inhibitors, lipid lowering agents, and thromboxane inhibitors. In one embodiment, the method of the present disclosure further comprises administering a therapeutically effective amount of at least one or more additional therapeutic antibodies or antibodies, or antigen-binding fragments or fragments thereof. In one embodiment, the one or more additional antibodies or antibodies are selected from the group consisting of anti-Grem 1 antibody or antibodies, anti-PDGFRβ antibody or antibodies, anti-TLR4 antibody or antibodies, anti-TLR2 antibody or antibodies, anti-EDN1 antibody or antibodies, and anti-ASIC1 antibody or antibodies.

[0180] Examples of suitable anticoagulants include, but are not limited to, warfarin, which is useful, for example, in treating patients with pulmonary hypertension who are at increased risk of thrombosis and thromboembolism.

[0181] Examples of suitable calcium channel blockers include, but are not limited to, diltiazem, felodipine, amlodipine, and nifedipine.

[0182] Suitable vasodilators include, but are not limited to, for example, prostacyclin, epoprostenol, treprostinil, and nitric oxide (NO).

[0183] Suitable exemplary phosphodiesterase inhibitors include, but are not limited to, phosphodiesterase V inhibitors such as, for example, tadalafil, sildenafil, and vardenafil, among others.

[0184] Examples of suitable endothelin antagonists include, but are not limited to, bosentan and sitaxsentan.

[0185] Suitable prostacyclin analogs include, but are not limited to, for example, ilomedin, treprostinil, and epoprostenol.

[0186] Suitable lipid-lowering agents include, but are not limited to, HMG CoA reductase inhibitors, such as simvastatin, pravastatin, atorvastatin, lovastatin, itavastatin, fluvastatin, pitavastatin, rosuvastatin, ZD-4522, and cerivastatin.

[0187] Diuretics suitable for use in the combination therapy of the present disclosure include, but are not limited to, for example, chlorothalidone, indapamide, bendro-flumethiazide, metolazone, cyclopenthiazide, polythiazide, mefruside, quimapide, chlorothiazide, and hydrochlorothiazide.

[0188] Examples of other therapeutic agents include, but are not limited to, ACE inhibitors such as enalapril, ramipril, captopril, cilazapril, trandolapril, fosinopril, quinapril, moxipril, lisinopril, and perindopril, or ATII inhibitors such as losartan, candesartan, irbesartan, embusartan, valsartan, and telmisartan, or iloprost, betaprost, L-arginine, omapatrilat, oxygen, and / or digoxin.

[0189] The methods may also include the use of combination kinase inhibitors (e.g., BMS-354825, canertinib, erlotinib, gefitinib, imatinib, lapatinib, lestautinib, lonafarnib, pegaptanib, pelitinib, semaxanib, tandutinib, tipifarnib, vatalanib, lonidamine, fasudil, leflunomide, bortezomib, imatinib, erlotinib, and Gleevec) and / or elastase inhibitors.

[0190] The additional therapeutically active ingredient may be administered to the subject prior to administration of the anti-PDGF-B antibody. For example, a first component may be considered to be administered "before" the second component if the first component is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less than 1 minute before administration of the second component. In other embodiments, the additional therapeutically active ingredient may be administered to the subject after administration of the anti-PDGF-B antibody or antigen-binding fragment thereof. For example, a first component can be considered to be administered "after" the second component if the first component is administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after administration of the second component.

[0191] In yet other embodiments, the additional therapeutically active ingredient may be administered to the subject simultaneously with administration of the anti-PDGF-B antibody or antigen-binding fragment thereof of the present disclosure. For purposes of this disclosure, "concurrent" administration includes, for example, administration of the anti-PDGF-B antibody and the additional therapeutically active ingredient to the subject in a single dosage form or in separate dosage forms administered to the subject within about 30 minutes of each other. When administered in separate dosage forms, each dosage form can be administered by the same route (e.g., both the anti-PDGF-B antibody and the additional therapeutically active ingredient can be administered intravenously, subcutaneously, intravitreally, etc.), or alternatively, each dosage form can be administered by a different route (e.g., the anti-PDGF-B antibody can be administered locally (e.g., intravitreally) and the additional therapeutically active ingredient can be administered systemically). In any event, administration of the ingredients in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes, are all considered "concurrent administration" for purposes of this disclosure. For purposes of this disclosure, administration of an anti-PDGF-B antibody "before," "concurrent with," or "after" administration of an additional therapeutically active ingredient will be considered administration of the anti-PDGF-B antibody, or antigen-binding fragment thereof, "in combination with" the additional therapeutically active ingredient. EXAMPLES

[0192] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, but are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is in ° C., and pressure is at or near atmospheric.

[0193] The terms REGN13335 and H4H13145P are used interchangeably herein. Additionally, the terms REGN15171 and H4H13132P are used interchangeably herein.

[0194] Example 1. Screening and in vitro characterization of anti-PDGF antibodies For isolation and primary screening of anti-PDGF-B antibodies, Adam6 / VI-3, ULC1633, and ULC1635 mice were utilized. Considerations included the need to cross with monkeys and with mice, and the need to block PDGF-BB and PDGF-AB signaling. Cross-reactivity to DD was deemed unlikely, and cross-reactivity to PDGF AA was not expected. AF-220-NA goat a-human PDGFBB polyclonal neutralizing antibody was used as a control / comparison antibody.

[0195] PES sorted B cells were isolated from 12 mice. Adam6 / VI-3, ULC1633, and ULC1635 mice were used to sort the antigen biotin-hPDGFBB or mPDGFBB and 6387 B cells were collected. 11 plates of B cells were processed (3614 B cells) and VH was amplified only for ULC mice. A total of 1056 PCR pairs were cloned into hIgG1 BST plasmid. Primary screening of Ag+ samples was performed by ELISA. A total of 854 Ag+ (81%) samples were identified (hPDFGBB (Peprotech) from MFI >1000). Secondary screening was performed for Ag+ samples using blocking ELISA, blocking Luminex, Biacore, and Bioassay. Mice matings were analyzed using mouse Biacore and Bioassay. The results of screening monoclonal antibodies from VI-3 mice are summarized in FIG. 1 and Table 1 below. [Table 1]

[0196] The equilibrium dissociation constants (K DValues) were determined using a real-time surface plasmon resonance (SPR) biosensor instrument, MASS-1. All binding studies were performed at 25° C. and 37° C. in a running buffer of 10 mM HEPES, 300 mM NaCl, 3 mM EDTA, 1 μg / mL heparin, and 0.05% v / v surfactant Tween®-20, pH 7.4 (HBS-T) ( FIG. 2 ). The HCA sensor surface was first derivatized with amines to couple a monoclonal mouse anti-human Fc antibody (GE, #BR100839) and separately capture an anti-PDGF-B monoclonal antibody. Different concentrations of human PDGF-B (hPDGF-B; 50 nM, 12.5 nM, 3.125 nM) or mouse PDGF-B (mPDGF-B; 50 nM, 12.5 nM, 3.125 nM) prepared in HBS-EHT running buffer were injected over the captured anti-PDGF-B monoclonal antibody at a flow rate of 50 μL / min for 4 min, and dissociation of the PDGF-B reagent bound to the captured anti-PDGF-B monoclonal antibody was monitored for 10 min in HBS-T running buffer. Kinetic binding (k a ) and dissociation (k d The binding and dissociation equilibrium constants (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows:

number

[0197] The binding kinetic parameters of hPDGF-B or mPDGF-B binding to different anti-PDGF-B monoclonal antibodies of the present disclosure at 25° C. and 37° C. are shown in Tables 2-4. [Table 2] [Table 3] [Table 4]

[0198] The binding kinetic parameters of hPDGF-B or mPDGF-B binding to exemplary anti-PDGF-B monoclonal antibodies of the disclosure at 37° C. are shown in Tables 5-6. [Table 5] [Table 6]

[0199] These binding data demonstrate that anti-PDGF-B-mAbs (eg, H4H13132P and H4H13145P) can specifically bind human, cynomolgus monkey, rat, and mouse PDGF-BB at pM concentrations.

[0200] Example 2. Amino acid sequences of heavy and light chain variable regions Table 7 lists the amino acid sequence pairs of the heavy and light chain variable regions of selected antibodies specific for PDGF-B and their corresponding antibody identifiers. Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H"), followed by a numerical identifier (e.g., "3132" as shown in Table 7), followed by a "P" suffix. Thus, according to this nomenclature, an antibody may be referred to as, for example, "H4H13132P". The H4H prefix in the antibody name used herein indicates the particular Fc region of the antibody. For example, an "H4H" antibody has a human IgG4 Fc. [Table 7]

[0201] Example 3. Cross-competition between purified anti-PDGF-BB monoclonal antibodies To determine cross-competition between purified anti-PDGF-BB monoclonal antibodies (mAbs), approximately 1.2-1.6 nM of anti-human PDGF-BB mAb is captured by immersing an α-hFc coated Octet biosensor in a well containing 50 μg / mL of anti-human PDGF-BB mAb for 3 min (Figure 3A). H4H hFc (isotype control) was used as a negative control. Unoccupied α-hFc Octet sensors were saturated by immersion for 4 min in a well containing blocking mAb solution (200 μg / mL of H4H human Fc (isotype control) 1) and 100 nM of human PDGF-BB (R&D) pre-incubated with 1 uM of anti-PDGF-BB mAb for at least 2 h. Blocking mAb saturated Octet biosensors were immersed for 4 min in a well containing a pre-mixture of anti-PDGF-BB mAb and human PDGF-BB. At the end of each cycle, the α-hFc Octet sensor was regenerated in 10 mM HCl. During analysis, the self-background binding signal caused by mAb binding to the capture surface was subtracted from the entire column. The binding responses of mAb-1 were compared, and competing and non-competing mAbs were binned based on their respective mAb-1 binding responses.

[0202] Cross-competition studies were performed on an Octet HTX instrument at 25°C using a running buffer of HBST + 0.1 mg / mL BSA. The sensor type was anti-His, the capture flow rate / time was 1000 rpm for 3 min, and the sample injection flow rate / time was 1000 rpm for various times. Figure 3B shows a matrix showing the binding response results of the antibody cross-competition assay. The affinities of the purified antibodies range from 0.002 nM to 2 nM.

[0203] Example 4. Characterization of anti-PDGF-B antibodies by use of ELISA Anti-PDGF-B antibodies were characterized in two blocking ELISA formats: blocking PDGF-BB and PDGF-AB binding to plate-captured PDGFR-B. To perform the study, 1 ug / ml of human PDGFR-beta-mmH (REGN979 lot number 01-100326) was coated onto plates overnight at 4°C.

[0204] Pre-binding: 12-point 3-fold serial dilutions of Abs from 100nM + 100pM final concentration PDGF-AB (R&D, 222-AB) or 60pM final concentration PDGF-BB (R&D, 220-BB). 1 hour at room temperature. Detection: 1:10,000 SA-HRP.

[0205] As shown in Figure 4A-C, five potent anti-PDGF-B antibodies were identified that blocked >70%, and three anti-PDGF-B antibodies had IC of 0.23-1.8 nM PDGF-BB. 50 The two anti-PDGF-B antibodies blocked both PDGF-BB and PDGF-AB with IC values ​​ranging from 0.55 to 0.64 nM. 50 Only 16 anti-PDGF-B antibodies blocked PDGF-BB with IC values ​​ranging from 2.8 to 13 nM. Six moderate anti-PDGF-B antibodies blocked more than 45% and two anti-PDGF-B antibodies had IC values ​​ranging from 2.8 to 13 nM. 50 The four anti-PDGF-B antibodies blocked both PDGF-BB and PDGF-AB with IC values ​​ranging from 0.64 to 2.8 nM. 50 Only PDGF-BB with a range of values ​​was blocked, and six anti-PDGF-B antibodies were non-blockers.

[0206] Example 5. Anti-PDGF-B antibodies inhibit human PDGF-BB To determine the activity of anti-PDGF-B antibodies, a bioassay was performed using PDGF-BB. To perform the bioassay, 20,000 HEK293 / SRE-Luc / hPDGFRβ single cell sorted cells / well were seeded overnight in 0.1% FCS Optimem. Dose response was determined for human PDGF BB (Peprotech, Cat# 100-14B, Lot# 111004, from E. coli) in a 1:3 serial dilution starting at 100 nM. Inhibition was determined by adding anti-PDGF-B, anti-PDGFRb (REGN2176, 08-R120731), and purified antibodies (1:3 serial dilutions starting at 100 nM) to cells with 500 pM human PDGF-BB. Plates were incubated at 37° C. for 5.5 hours and luminescence was measured using One-Glo (Promega).

[0207] As shown in Figure 5A, 13 of the 17 anti-hPDGF-B antibodies inhibited 500 pM hPDGF BB with IC 50 The IC values ​​ranged from 88 pM to 7.2 nM, with maximum inhibition rates ranging from 42 to 99%. Furthermore, as shown in Figure 5B, two anti-PDGF-B antibodies were activated by human PDGF-BB. Figure 5C shows that eight of the 17 anti-hPDGF-B antibodies inhibited human PDGF-AB at 5 nM with IC 50 The IC values ​​ranged from 99 pM to 50 nM, and the maximum inhibition rates ranged from 32 to 99%. H4H13132P and H4H13145P had IC 50 Finally, Figure 5D shows that 10 of the 17 anti-hPDGF-B antibodies blocked mouse PDGF-BB at 600 pM with IC 50 These results show that the IC values ​​ranged from 450 pM to 6.4 nM, with maximum inhibition rates ranging from 39 to 98%. 50The results demonstrate that H4H13145P shows complete inhibition of human PDGF-BB with values ​​ranging from 310 pM to 2 nM. Specifically, H4H13145P inhibits all three ligands, hPDGF BB, hPDGF AB, and mPDGF BB, to baseline. Inhibition of PDGF-B activation by use of exemplary anti-hPDGF-B antibodies in HEK293 / SRE-luc / hPDGFRβ cells is summarized in Tables 8-9 below. [Table 8]

[0208] H4H13132P showed IC expression against hPDGF-BB, hPDGF-AB, mPDGF-BB, and cynoPDGF-BB. 50 Values ​​between 1.9 and 9.0 nM indicate over 87% inhibition. [Table 9]

[0209] H4H13145P showed IC expression against hPDGF-BB, hPDGF-AB, mPDGF-BB, and cynoPDGF-BB. 50 Values ​​of 0.5 to 3 nM indicate complete inhibition.

[0210] Example 6. Analysis of the complex formed between recombinant human PDGF-BB and anti-PDGF-B monoclonal antibody (mAb) Size-exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) was used to assess the relative size distribution of complexes formed between recombinant human PDGF-BB (Peprotech) and several anti-PDGF lead mAbs. 5 mM PDGF-BB + 5 mM mAb (equimolar ratio) was prepared in 1x PBS, pH 7.4 for each anti-PDGF-B mAb tested and incubated for 3 h at room temperature prior to fractionation of total protein by SEC-MALLS. Under SEC-MALLS conditions, 100 mg (total protein) of each sample was injected in duplicate onto a GE Healthcare Superose 6 10 / 300 GL column in 1x PBS, pH 7.4 (mobile phase) with a run time of 90 min per injection.

[0211] H4H13145P (peak 1) largely formed a distinct complex with human PDGF-BB (peak 2) consistent with a 2:2 mAb:human PDGF-BB species (Figure 6A). In addition, H4H13145P (peak 1) largely formed a distinct complex with human PDGF-BB (peak 2) consistent with a 2:2 mAb:human PDGF-BB species (Figure 6B). Figure 6C shows overlaid chromatograms from samples that form the most distinct 2:2 mAb:human PDGF-BB species, with no detectable higher order complexes observed.

[0212] In general, most anti-PDGF mAbs appeared to form significantly different complexes with human PDGF-BB, consistent with a 2:2 mAb:hPDGF-BB species, with few or no higher order complexes ("paper dolls") observed. Small amounts of higher order complexes larger than the 2:2 mAb:hPDGF-BB species were observed with samples H4H13132P, H4H13145P, and H4H13162P, but it is unclear whether these complexes formed as a result of interaction of hPDGF-BB with the monomeric form of the antibody or antibody multimers (HMW species), which were present in varying amounts in each mAb sample. Alternatively, the higher order complexes in these samples could be due to interaction of the monomeric antibody with the small amount of multimeric hPDGF-BB that was detected. Samples H4H13148P and H4H13169P appeared to form the most distinct 2:2 mAb:hPDGF-BB species, with no detectable higher order complexes. Interestingly, the complexes formed by these two samples had similar calculated molar masses, but the complex formed by H4H13148P eluted significantly later than the equivalent H4H13169P complex, suggesting that the molecular shape (hydrodynamic radius) of the complex formed by H4H13148P is essentially more compact. A broad distribution of complexes of the estimated molar mass of the 2:2 mAb:hPDGF-BB complex was observed in the H4H13155P sample, which may indicate that the complex formed by hPDGF-BB in solution had dissociated during the fractionation process or that the sample did not reach equilibrium under the experimental conditions tested.

[0213] Example 7. Validation of anti-PDGF-B monoclonal antibodies H4H13132P and H4H13145P in a cynomolgus monkey PDGF-BB protein bioassay Cynomolgus monkey PDGF-BB was obtained as an Fc fusion from KingFisher and Sino Biological. HEK293 / SRE-Luc / mfPDGFBB-ecto / hPDGFRb-cyto cells p2 were seeded in 96-well plates, and the cells were cultured at 2.5 × 10 5Cells / ml (20,000 cells / well) were added at 80ul / well overnight at 37°C, 5% CO2. PDGF-BB dose response was determined at 1-3 dilutions starting at 20nM. To detrmening inhibition, 1:3 serially diluted Abs were analyzed starting at 500nM with 500pM PDGF-BB as a constant. Plates were incubated at 37°C, 5% CO2 for 5.5 hours. Plates were removed from the incubator and equilibrated at room temperature (RT) for approximately 30 minutes. 100ul of One-glo substrate (equilibrated to RT) was added to all wells of the plate and mixed for 10 minutes at RT on a plate shaker. Luminescence was measured using a SpectraMaxi3X (PerkinElmer™). As shown in FIG. 7, H4H13145P was identified as being more potent than H4H13132P against both human and cynomolgus PDGF-BB proteins in this assay system.

[0214] The following assay was used to determine the potency of anti-PDGF-B antibodies to block cynomolgus PDGF-BB-induced signaling in cells with expression of the extracellular domain of monkey PDGFRβ.

[0215] Luciferase bioassay An engineered cell line, HEK293 / SRE-Luc / mfPDGFRβ-ecto / hPDGFRb-cyto cells (ACL7804), was used to assay the ability of H4H13145P and H4H13132P to neutralize luciferase expression driven by cynomolgus PDGF-BB. Cells were seeded in 96-well plates using assay medium (0.5% bovine serum albumin, 1% penicillin-streptomycin-glutamine in DMEM medium) and serum starved overnight (37° C., 5.0% CO2).

[0216] Generation of dose-response curves Dose-response curves for cynomolgus PDGF-BB were determined by adding ligand diluted in assay medium to HEK293 / SRE-Luc / mfPDGFRβ-ecto / hPDGFRb-cyto cells at concentrations ranging from 3 pM to 20 nM. Each concentration was tested in duplicate, and wells without added ligand served as negative controls. After addition of cynomolgus PDGF-BB, cells were incubated at 37°C, 5.0% CO2 for 5.5 hours and then equilibrated to room temperature for 30 minutes. An equal volume of ONE-Glo luciferase substrate was added to each well, and the plate was incubated at room temperature for an additional 5 minutes. Relative light units (RLU) were measured on a SpectraMaxi3X plate reader, and values ​​were analyzed by a 4-parameter logistic equation on a 10-point dose-response curve (GraphPad Prism).

[0217] Generation of inhibition curves H4H13145P and H4H13132P were tested for inhibition of cynomolgus PDGF-BB-mediated mfPDGFRβ signaling in the HEK293 / SRE-Luc / mfPDGFRβ-ecto / hPDGFRb-cyto cell line. H4H13145P, H4H13132P, or a negative control antibody (REGN1945) was added in duplicate to cells at concentrations ranging from 228 pM to 500 nM, followed by addition of cynomolgus PDGF-BB at 500 pM. Plates were incubated at 37°C, 5.0% CO2 for 5.5 hours and equilibrated to room temperature for 30 minutes. An equal volume of ONE-Glo luciferase substrate was added to each well and plates were incubated at room temperature for an additional 5 minutes. Relative light units (RLU) were measured on a SpectraMaxi3X plate reader and values ​​were analyzed by a four-parameter logistic equation on a 10-point dose-response curve (GraphPad Prism).

[0218] result Blockade of ligand-mediated cynomolgus PDGF-BB-mediated mfPDGFRβ signaling 50% of the maximum activity level (EC50 The concentration of Sino Biologics Cynomolgus PDGF-BB Fc Tag required to stimulate mfPDGFRβ signaling to 50% of maximal activity (IC) was 167 pM (Figure 7). 50 The concentration of antibody required to reduce PDGF-BB signaling to IC was determined for H4H13132P, H4h13145P, and the isotype control antibody REGN1945 (an antibody raised against feline Fel d 1 with no binding to mfPDGFRβ). As shown in FIG. 7, H4H13132P and H4H13145P each had an IC 50 Values ​​of 781 pM and 16 pM effectively blocked cynomolgus PDGF-BB signaling induced by a fixed concentration of 500 pM cynomolgus PDGF-BB. In contrast, the IgG4 isotype control antibody REGN1945 was ineffective in blocking cynomolgus PDGF-BB signaling induced by cynomolgus PDGF-BB.

[0219] Example 8. Validation of Biacore binding of commercial monkey PDGF-BB reagents to anti-PDGF-B monoclonal antibodies H4H13132P and H4H13145P To determine the binding kinetics of commercial monkey PDGF-BB (Kingfisher, Sino biological) to anti-PDGF-B mAbs H4H13132P and H4H13145P, approximately 250-350 RU of anti-PDGF-B mAb was captured with a negative control (REGN1945) on an anti-hFc HCA chip in MASS-2 at 25°C. 90 nM human and monkey PDGF-BB were prepared and serially diluted 3-fold. Samples were injected at 25 uL / min for 3 min and dissociation was monitored for 10 min. Kinetic parameters were evaluated by fitting the real-time data using a 1:1 binding model with mass transport limitation. As shown in Figure 8, commercial monkey PDGF-BB showed specific binding to anti-PDGF-B mAbs H4H13132P and H4H13145P.

[0220] Example 9. Western blot detection of mAb binding epitopes on PDGF-BB The key to understanding the different performances of antibodies is to determine the binding sites or epitopes they recognize. Epitopes are generally divided into two categories: linear epitopes, where a continuous stretch of amino acids is sufficient for binding, and conformational epitopes, where key amino acid residues are brought together by protein folding. Linear epitopes may be preferred for applications where the protein target is fully or partially denatured during sample preparation prior to immunoassays such as Western Blot (WB). Thus, Western Blot analysis of PDGF-BB with H4H13145P and H4H13132P was performed to determine whether these mAbs bind to linear epitopes.

[0221] Human recombinant PDGF-BB dimeric protein (CF) from R&D systems 1 μg was resolved on SDS PAGE 5-20% gradient gels under reducing (R) and non-reducing (NR) conditions. The following steps were used to detect PDGF-BB bands under both R and NR conditions with PDGF-BB mAb H4H13145P and commercial anti-PDGF-BB ab (R&D anti-human PDGF-BB ab: human PDGF-BB antibody AF-220-NA). For non-specific blocking: PVDF membrane was blocked in 5% BSA for 1 hour at room temperature. For primary Ab blotting: PVDF membrane was blocked with (1) PDGF-BB mAb H4H13145P or (2) R&D anti-PDGF-BB mAb at 1.0ug / ml diluted at 4C in 5% BSA TBST overnight. Membrane was washed in TBST wash buffer for 10 minutes (3 times) at room temperature. Secondary antibody incubation included incubating the membrane in HRP-conjugated anti-mouse secondary Ab in 5% BSA TBST for 1 hour at room temperature. Membrane washing included using TBST buffer for 10 minutes (3 times) at room temperature. Signal was developed using Pierce™ Western Blot Signal Enhancer.

[0222] Recombinant human PDGF-BB, 1 μg / lane, was resolved by SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by silver staining to show single bands at 13 kDa and 28 kDa, respectively. Figure 9 shows that Western blot analysis of the antibody binding epitope confirmed that H4H13145P binds to a linear epitope on human rPDGF-BB.

[0223] Example 10. Preclinical PAH Efficacy Evaluation of H4H13145P Lead mAb-mediated PDGF-B blockade (H4H13145P) provided robust therapeutic benefit in multiple models of PAH (mouse + rat). These effects were more impressive than any previous treatment or benchmark, including SOC. These results demonstrate that anti-PDGF-B antibodies provide an effective non-vasodilator PAH therapy.

[0224] To evaluate the effect of the anti-PDGF-B antibody H4H13145P in pulmonary arterial hypertension, separate studies were performed using a chronic hypoxia-induced pulmonary arterial hypertension mouse model and a monocrotaline rat model.

[0225] The PAH model is summarized in Table 10 below. [Table 10]

[0226] In these studies, the following materials and methods were used.

[0227] Materials and Methods mouse Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling leading to a progressive increase in vascular resistance. Elevated pulmonary arterial pressure induces compensatory right ventricular hypertrophy and eventual failure. Platelet-derived growth factor-B (PDGF-B) is a potent mitogen that is a member of the platelet-derived growth factor (PDGF) family. PDGF-B / PDGFRβ signaling has been shown to be a central signaling pathway regulating pathological pulmonary arterial vascular remodeling. This study was to compare the efficacy of anti-PDGF-B and anti-PDGFRβ antibodies in hypoxic / sugen PAH mice when administered therapeutically.

[0228] In the first study, 14- to 16-week-old male humanized PDGFRβ mice were hu-hu Mice (MAID#1639) were used. Mice were divided into treatment groups by weight so that starting weights were similar between different groups. Cages were either kept at approximately 21% O2 (normobaric normoxia) or placed in a 10% O2 (normobaric hypoxia) chamber (modified 6' semi-rigid isolator unit, Charles River) where low O2 levels were maintained by adjusting the N2 flow rate to a steady inhalation of room air. Mice in the hypoxic chamber were subcutaneously dosed with 20mg / kg of the VEGF receptor inhibitor Sugen5146 once a week for 6 weeks. Mice were dosed with antibodies starting on day 21 for 3 weeks as outlined in Table 11. By the end of the 6th week, right ventricular systolic pressure (RVSP) was measured by right heart catheterization and RV hypertrophy was calculated by Fulton's index as the weight ratio of RV to (LV+septum).

[0229] In the second study (Study 2), 12-14 week old male C57 / BL mice (Taconic) were used for the study. Mice were divided into treatment groups by weight so that starting weights were similar between the different groups. Cages were either kept at approximately 21% O2 (normobaric normoxia) or placed in a 10% O2 (normobaric hypoxia) chamber (modified 6' semi-rigid isolator unit, Charles River) where low O2 levels were maintained by adjusting the N2 flow rate to a steady inhalation of room air. Mice in the hypoxic chamber were administered 20 mg / kg of the VEGF receptor inhibitor Sugen5146 subcutaneously once a week for 6 weeks. Mice were administered antibodies starting on day 21 for 3 weeks as outlined in Table 12. By the end of the 6th week, right ventricular systolic pressure (RVSP) was measured by right heart catheterization and RV hypertrophy was calculated by the Fulton index as the weight ratio of RV to (LV + septum). Mouse serum was collected on day 42 for human antibody IgG assessment.

[0230] Right heart catheterization and right ventricular systolic pressure Mice were anesthetized with isoflurane and maintained at approximately 37°C using a heating platform (Heated Hard Pad 1, Braintree Scientific) and a circulating heated water pump (T / Pump Classic, Gaymar Industries). The neck region of each mouse was prepared for surgery by depilating the right common carotid artery and right jugular vein. An incision was made and the right jugular vein was isolated, taking care not to damage the carotid artery and / or vagus nerve. A piece of 5-0 silk suture was placed under the isolated jugular vein to retract the vessel cephalad, after which the jugular vein was punctured with a 30-gauge needle. A pressure catheter (Microtip Catheter Transducer SPR-1000, Millar Instruments, Inc.) was inserted into the opening of the jugular vein and advanced through the right atrium into the right ventricle. The catheter was connected to a pressure / volume instrument (MPVS-300, Millar Instruments, Inc.) that measured both heart rate and diastolic and systolic right ventricular pressures. These parameters were digitally acquired using a data acquisition system (PowerLab 4 / 35, AD Instruments). Right ventricular pressure was analyzed using LabChart Pro 7.0 software (AD Instruments). Measurements were quantified from 60-second intervals of pressure tracings (after 2 minutes of recording to allow for pressure stabilization). Parameters analyzed were right ventricular systolic pressure (RVSP), heart rate (HR), and right ventricular pressure rise rate (dP / dt max).

[0231] Right heart hypertrophy assessment After in vivo hemodynamic measurements, animals were euthanized by exsanguination under anesthesia, and then RV free wall, left ventricle (LV) and septal tissues were harvested and weighted. RV hypertrophy was calculated by Fulton's index as the weight ratio of RV to (LV + septum). RV tissues were then frozen and stored (-80°C) for biochemical analysis. Hematocrit was obtained for each mouse immediately after euthanasia.

[0232] Serum human IgG evaluation Serum PDGF-B antibody human IgG is captured on a Gyrolab Bioaffy™ 200 CD by biotinylated mouse anti-human IgG1 / IgG4 monoclonal antibody (REGN2567) diluted in glycerol and detected by Alexa Fluor 647-conjugated mouse anti-human kappa antibody diluted in glycerol. Serum samples are processed as described below: 1) Dilute serum samples 1:50 with dilution buffer and dilute standards to a starting concentration of 2 μg / mL using dilution buffer for a total of 8 standards. 2) During the wash, dilute biotinylated mouse anti-human IgG1 / IgG4 to 100 ug / mL. 3) Dilute Alexa Fluor 647-conjugated mouse anti-human kappa antibody to 10 ug / mL in wash buffer. 4) Centrifuge capture and detection antibodies at 15,000 RCF for 5 minutes. 5) Further dilute the detection antibody in detection buffer to a final concentration of 0.5ug / mL (use only the top layer of centrifuged detection antibody). 6) Using the GYROS generated plate map, load samples, standards, wash buffers, capture and detect antibodies on a 96-well PCR plate.

[0233] statistical analysis Quantitative data are expressed as mean ± standard deviation. Statistical analysis performed between two groups was analyzed by t-test. Comparison of time courses was analyzed by two-way analysis of variance (ANOVA). Comparison of treatment effects of multiple drug treatment groups was analyzed by one-way ANOVA. PRISM software was used for all statistical analyses. A p-value <0.05 was considered statistically significant.

[0234] The dosing schedule for Study 1 is shown in Table 11. [Table 11]

[0235] The dosing schedule for Study 2 is shown in Table 12. [Table 12]

[0236] Ultrasound Evaluation and Analysis On the final day of each study, the size of the pulmonary artery and the function and dimensions of the right ventricle of each mouse were evaluated using a high-frequency ultrasound system (Vevo 2100, VisualSonics). For the evaluation, mice were anesthetized (1.5% isoflurane at a rate of 1.0 cc / mL of medical-grade air) and their temperature was monitored with a rectal temperature probe and maintained at approximately 37°C using a heating platform (MouseMonitorS, Indus Instruments) and a warming lamp. Both brightness mode (B-mode) and motion mode (M-mode) imaging were used. B-mode imaging of mouse hearts in cross-section was used to determine the pulmonary artery cross-sectional area (PA CSA) at the level of the pulmonary valve. M-mode imaging was used to determine the pulse wave velocity time integral (VTI), which is derived from the area under the curve of a representative Doppler tracing of blood flow through the pulmonary artery. The right ventricular stroke volume (RV SV) was calculated from the product of PA CSA and VTI. Right ventricular cardiac output (RV CO) was calculated from the product of SV and heart rate (HR). M-mode imaging was used to determine right ventricular free wall (RVFW) thickness during diastole and systole. Prior to right ventricular pressure assessment, animals were returned to their home cages.

[0237] Right ventricular pressure assessment Right ventricular pressure was then assessed for all treatment groups. Mice were anesthetized with isoflurane and maintained at approximately 37°C using a heating platform (Heated Hard Pad 1, Braintree Scientific) and a circulating heated water pump (T / Pump Classic, Gaymar Industries). The neck region of each mouse was prepared for surgery by depilating the right common carotid artery and right jugular vein. An incision was made and the right jugular vein was isolated, taking care not to damage the carotid artery and / or vagus nerve. A piece of 5-0 silk suture was placed under the isolated jugular vein to retract the vessel cephalad, after which the jugular vein was punctured with a 30-gauge needle. A pressure catheter (Microtip Catheter Transducer SPR-1000, Millar Instruments, Inc.) was inserted into the opening of the jugular vein and advanced through the right atrium into the right ventricle. The catheter was connected to a pressure / volume instrument (MPVS-300, Millar Instruments, Inc.) that measured heart rate and both diastolic and systolic right ventricular pressure. These parameters were digitally acquired using a data acquisition system (PowerLab 4 / 35, ADInstruments). Right ventricular pressure was analyzed using LabChart Pro 7.0 software (ADInstruments). Measurements were quantified from 60-s intervals of pressure tracings (after 2 min of recording to allow for pressure stabilization). Parameters analyzed were right ventricular systolic pressure (RVSP), heart rate (HR), and rate of right ventricular pressure rise (dP / dt max). Serum / tissue collection and assessment of right ventricular hypertrophy.

[0238] After completion of right ventricular pressure measurements, the catheter was removed and each animal was sacrificed. The abdomen was opened and blood was collected from the vena cava for hematocrit assessment and serum collection. The thoracic cavity was then opened and the middle lobe of the right lung was excised by ligation with 5-0 silk suture, and later placed in RNA (Sigma-Aldrich, Cat. No. R0901) and frozen at -80°C after 24 hours. The heart was excised from each animal and the right ventricle (RV) was carefully dissected from the left ventricle and septum (LV+S). Both cardiac tissues were weighed separately on a microbalance (AJ000, Mettler) and the RV hypertrophy index [RV / (LV+S); Fulton index] was calculated.

[0239] Half of the animals in each treatment group had their lungs perfused with phosphate buffered saline (PBS, pH 7.4) at 20-25 mmHg and then fixed in 10% neutral buffered formalin (NBF). Lungs were immersed in 10% NBF for 24 hours, followed by immersion in 70% ethanol for at least 48 hours prior to tissue processing and paraffin embedding. For animals that did not undergo lung perfusion fixation, the right lower lobe was ligated with 5-0 silk suture, then excised, weighed, and frozen in liquid N2.

[0240] Rat Male Sprague Dawley rats aged 6-7 weeks were used. Rats were divided into treatment groups so that body weights were similar between the different groups. In study 1, rats in the antibody treatment group received either 25 mg / kg anti-PDGF-B antibody or isotype control IgG subcutaneously twice a week for 28 days, 1 day before monocrotaline injection. The standard treatment group received 30 mg / kg macitentan orally daily for 28 days. On day 1, rats received either 40 mg / kg monocrotaline or 5 mL / kg saline subcutaneously. By the end of day 28, right ventricular systolic pressure (RVSP) was measured by right heart catheterization, and RV hypertrophy was calculated by the Fulton index as the weight ratio of RV to (LV + septum). In study 2, rats received either 60 mg / kg monocrotaline or 5 mL / kg saline subcutaneously. Starting on day 14, rats in the antibody treatment groups were administered 25 mg / kg of either anti-PDGF-B antibody or isotype control IgG subcutaneously twice weekly for 21 days. Body weight changes from day 0 to day 35 were used for general toxicity assessment and animal mortality was calculated up to day 35.

[0241] The dosing schedule for Study 1 is shown in Table 13. [Table 13]

[0242] The dosing schedule for Study 2 is shown in Table 14. [Table 14]

[0243] Right heart catheterization and right ventricular systolic pressure Rats were anesthetized with isoflurane and maintained at approximately 37°C using a heating platform (Heated Hard Pad 1, Braintree Scientific) and a circulating heated water pump (T / Pump Classic, Gaymar Industries). The neck region of each rat was prepared for surgery by depilating the right common carotid artery and right jugular vein. An incision was made and the right jugular vein was isolated, taking care not to damage the carotid artery and / or vagus nerve. A piece of 5-0 silk suture was placed under the isolated jugular vein to retract the vessel cephalad before puncturing the jugular vein with a 23-gauge needle. A pressure catheter (Microtip Catheter Transducer SPR-1000, Millar Instruments, Inc.) was inserted into the opening of the jugular vein and advanced through the right atrium into the right ventricle. The catheter was connected to a pressure / volume instrument (MPVS-300, Millar Instruments, Inc.) that measured both heart rate and diastolic and systolic right ventricular pressures. These parameters were digitally acquired using a data acquisition system (PowerLab 4 / 35, AD Instruments). Right ventricular pressure was analyzed using LabChart Pro 7.0 software (AD Instruments). Measurements were quantified from 60-second intervals of pressure tracings (after 2 minutes of recording to allow for pressure stabilization). Parameters analyzed were right ventricular systolic pressure (RVSP), heart rate (HR), and right ventricular pressure rise rate (dP / dt max).

[0244] Right heart hypertrophy assessment After in vivo hemodynamic measurements, animals were euthanized by exsanguination under anesthesia, and then RV free wall, left ventricle (LV) and septal tissues were harvested and weighted. RV hypertrophy was calculated by Fulton's index as the weight ratio of RV to (LV + septum). RV tissues were then frozen and stored (-80°C) for biochemical analysis.

[0245] statistical analysis Quantitative data are expressed as mean ± standard deviation. Statistical analysis performed between two groups was analyzed by t-test. Comparison of time courses was analyzed by two-way analysis of variance (ANOVA). Comparison of treatment effects of multiple drug treatment groups was analyzed by one-way ANOVA. PRISM software was used for all statistical analyses. A p-value <0.05 was considered statistically significant.

[0246] result Anti-PDGF-B provided superior efficacy in two clinically relevant endpoints in PAH when compared head-to-head with anti-PDGFRβ Body weight changes in hypoxic / sugen mice Mice exposed to hypoxia showed slower weight gain at 6 weeks compared to normoxia animals. Anti-PDGF-B antibody treatment significantly attenuated the weight loss compared to the isotype control hIgG4 treatment group. Anti-PDGFRβ antibody treatment did not show any effect on the weight change caused by hypoxia exposure (Figure 10). Significant differences were only observed between Hy / Su+isotype control IgG and Hy / Su+anti-PDGF-B at day 42 (**p<0.01).

[0247] Increase in right ventricular pressure induced in hypoxia / sugen mice Catheter-based assessment of cardiac right ventricular pressure revealed a significant increase in right ventricular systolic pressure in the isotype antibody-treated group of hypoxic / sugen mice at 6 weeks. Anti-PDGF-B antibody treatment significantly reduced the increase in right ventricular systolic pressure by 9 mmHg, whereas anti-PDGFRβ antibody also reduced right ventricular systolic pressure to a lesser extent (Figure 10). Significant differences were observed between Hy / Su+isotype control IgG and Hy / Su+anti-PDGF-B (****p<0.0001) and Hy / Su+anti-PDGFRβ (****p<0.0001). Significant differences were also observed between Hy / Su+PDGFRβ and Hy / Su+anti-PDGF-B (*p<0.05).

[0248] Right ventricular hypertrophy induced in hypoxia / sugen mice Postmortem analysis of mouse right heart weight showed significant changes in hypoxia / sugen exposed mice (Figure 10). The ratio of right ventricular weight to left ventricular + septum weight provides an index of right ventricular hypertrophy (i.e., Fulton index), and the 6 week group in hypoxia / sugen showed a greater ratio compared to normoxic animals, indicating the presence of right ventricular hypertrophy. Therapeutic treatment with anti-PDGF-B antibody reduced right ventricular hypertrophy by approximately 35% compared to hypoxia / sugen isotype control treated mice. Use of anti-PDGFRβ antibody failed to reduce right ventricular hypertrophy in hypoxia / sugen exposed animals. Significant differences were observed between Hy / Su+isotype control IgG and Hy / Su+anti-PDGF-B (****p<0.0001) and Hy / Su+anti-PDGFRβ (*p<0.05). There was also a significant difference between Hy / Su+PDGFRβ and Hy / Su+anti-PDGF-B (*p<0.05).

[0249] In summary, anti-PDGF-B antibodies provided superior efficacy in two clinically relevant endpoints of PAH (right ventricular pressure and hypertrophy) when compared directly with anti-PDGFRβ in the hypoxic / sugen mouse mode.

[0250] Prophylactic treatment with anti-PDGF-B strongly protects animals in MCT rats, and therapeutic treatment with anti-PDGF-B improves survival in high-dose MCT rats. Increase in right ventricular pressure induced by monocrotaline in rats. In Study 1, catheter-based assessment of cardiac right ventricular pressure revealed a significant increase in right ventricular systolic pressure in the isotype antibody-treated group of monocrotaline rats at week 4. Both the vasodilator macitentan and anti-PDGF-B antibody treatment significantly reduced the increase in right ventricular systolic pressure. Significant differences were observed between monocrotaline + isotype control IgG and monocrotaline + macitentan (***p<0.001) and monocrotaline + anti-PDGF-B (*p<0.05) (Figure 11).

[0251] Right ventricular hypertrophy induced in monocrotaline rats. Postmortem analysis of the right heart weight of the rats showed a significant change in monocrotaline rats receiving isotype control IgG treatment. The ratio of right ventricular weight to left ventricular + septum weight provides an index of right ventricular hypertrophy (i.e., Fulton index), and the 4-week group in monocrotaline-treated rats showed a greater ratio compared to saline-treated animals, indicating the presence of right ventricular hypertrophy. (Prophylactic treatment with anti-PDGF-B antibody reduced right ventricular hypertrophy by approximately 40% compared to monocrotaline with isotype control-treated rats (Figure 11). Use of the vasodilator macitentan failed to reduce right ventricular hypertrophy in monocrotaline rats. Significant differences were observed between monocrotaline + isotype control IgG and monocrotaline + anti-PDGF-B (*p<0.05).

[0252] Animal survival in monocrotaline rats In Study 2, rats injected with 60 mg / kg monocrotaline developed severe pulmonary hypertension symptoms and showed high mortality. Notably, 90% of animals in the monocrotaline with isotype control IgG treatment group died by day 35. Anti-PDGF-B antibody treatment was started 14 days after monocrotaline injection and significantly reduced the weight loss of animals. Significant differences were observed between monocrotaline + isotype control IgG and monocrotaline + anti-PDGF-B at days 28 (*p<0.05) and 35 (***p<0.001). PDGF-B antibody treatment also rescued 58% of rats from death by day 35. Significant differences were observed between monocrotaline + isotype control IgG and monocrotaline + anti-PDGF-B (*p<0.05), rescued 58% of rats from death by day 35 (Figure 12).

[0253] In summary, prophylactic treatment with anti-PDGF-B antibody reduced both hemodynamic endpoints (right ventricular systolic pressure) and right ventricular hypertrophy in the monocrotaline model. Therapeutic treatment with anti-PDGF-B antibody significantly improved animal survival in severe PAH in monocrotaline rats.

[0254] Anti-PDGF-B demonstrated efficacy in low-dose therapeutic treatment Increase in right ventricular pressure induced in hypoxia / sugen mice Catheter-based assessment of cardiac right ventricular pressure revealed a significant increase in right ventricular systolic pressure in isotype antibody-treated groups of hypoxic / sugen mice by week 6. Anti-PDGF-B antibody treatment significantly reduced the increase in right ventricular systolic pressure at doses of 1, 3, 10, and 25 mg / kg. Significant differences were observed between the Hy / Su+isotype control IgG and Hy / Su+anti-PDGF-B groups at 1 mg / kg (*p<0.05), 3 mg / kg (*<0.01), 10 mg / kg (****p<0.0001), and 25 mg / kg (****p<0.0001). There were no statistically significant differences between different doses of PDGF-B treatment (Figure 13A).

[0255] Right ventricular hypertrophy induced in hypoxia / sugen mice Postmortem analysis of mouse right heart weight showed significant changes in hypoxia / sugen exposed mice (Figure 13A). The ratio of right ventricular weight to left ventricular plus septum weight provides an index of right ventricular hypertrophy (i.e., Fulton index), and the 6 week group in hypoxia / sugen showed a greater ratio compared to normoxic animals, indicating the presence of right ventricular hypertrophy. Therapeutic treatment with anti-PDGF-B antibody did not alter right ventricular hypertrophy compared to hypoxia / sugen isotype control treated mice. Significant differences were observed between the normoxia group and all Hy / Su groups ( #### p<0.0001).

[0256] Serum PDGF-B protein and antibody levels in hypoxic / Sugen mice By week 6, serum was collected for circulating PDGF-B protein and human antibody analysis. Circulating levels of PDGF-B were significantly elevated in the hypoxic / sugen mice group (Figure 13B). Measurement of human IgG in mouse serum confirmed that antibody levels in the 1-25 mg / kg anti-PDGF-B treatment groups increased in a dose-dependent manner. Repeated subcutaneous administration of 1-25 mg / kg achieved nM-level serum antibody levels, several thousand times higher than circulating PDGF-B (Figure 13B).

[0257] In summary, circulating PDGF-BB was elevated in Hy / Su PAH mice. Anti-PDGF-B antibodies showed efficacy in improving pulmonary hemodynamics (right ventricular pressure) up to a dose of 1 mg / kg in the hypoxic / sugen mouse model.

[0258] The in vivo efficacy for targeting PDGF-B signaling is summarized in Table 15 below. [Table 15]

[0259] Targeting PDGF-B via the lead mAb (H4H13145P) demonstrated consistent and robust efficacy across clinically relevant PAH endpoints (right ventricular systolic pressure - RVSP; right ventricular hypertrophy - RV) in rodent models. These effects were more impressive than any previous treatment or benchmark, including standard of care, comparative clinical trial agents, and in-house PDGFRb blockade. PDGF-B blockers may provide an effective non-vasodilator PAH therapy.

[0260] Example 11. Safety evaluation of PDGFR-β blockade PDGF-B / PDGFRb signaling is involved in pericyte proliferation, migration, survival and attachment. Sprouting endothelial cells secrete PDGF-B, which binds to the pericyte-specific receptor PDGFRb, resulting in pericyte recruitment and attachment. Impaired PDGF-B / PDGFRb signaling leads to failure of pericyte recruitment and reduced microvascular pericyte coverage, ultimately resulting in endothelial hyperplasia, abnormal vascular morphogenesis, and formation of microaneurysms. Mice ablated for endothelium-derived PDGF-B exhibited less than 52% normal pericyte density leading to variable capillary and venous diameters and retracted capillary branches. Specific overexpression of PDGF-B in photoreceptor cells led to increased proliferation of pericytes, but also increased astrocytes and endothelial cells. Long-term treatment with PDGF-B vs. PDGF-Rb antagonists could potentially cause pericyte loss / vascular damage and subsequent edema and hemorrhage in humans. Furthermore, systemic toxicity of anti-PDGFRβ pegylated di-Fab (Celltech / Zymogenetics) has been reported in humans (peripheral edema in cancer patients) and the safety of long-term anti-PDGF-B therapy is unknown.

[0261] We hypothesized that specific PDGF-B ligand blockade would allow continued receptor signaling through remaining PDGF ligands with less detrimental effects on pericyte homeostasis. It was found that systemic administration of anti-PDGF-B ab at 3 mg / kg sc did not cause loss of retinal vascular pericytes in neonatal mice, whereas anti-PDGFRb ab completely depleted retinal vascular pericytes. Adult rodents were observed to tolerate high doses of anti-PDGF-B and anti-PDGFRb treatment. Furthermore, high doses of anti-PDGFRb treatment in adult mice for 8 months did not induce any vascular damage, hemorrhage, or obvious edema.

[0262] When analyzing the effect of anti-PDGF-B antibodies on the retinal vasculature of P2 wild-type mice, no effect was observed on pericyte coverage in anti-PDGF BB-treated retinas at a dose of 3 mg / kg. Although systemic delivery of anti-PDGF-Rβ antibodies (2C5, REGN764) effectively depleted pericytes, anti-PDGF-BB antibodies (H4H13132P and H4H13145P) had no apparent effect in the retinal angiogenesis (RVD) model at 3 mg / kg (Figure 14).

[0263] To determine the effect of anti-PDGF-B antibody on the retinal vasculature of P2 WT mice, anti-PDGF-B antibody was injected at P2 and tissues were collected at P5. All retinas were incubated with anti-NG2 antibody to detect vascular pericytes. It was observed that a higher dose of anti-PDGF-B antibody (H4H13145P) showed a moderate effect on pericyte depletion in the RVD model (Figure 15).

[0264] Thus, this study demonstrates that the use of anti-PDGF-B antibodies offers a better safety profile compared with the use of anti-PDGFR-β antibodies and that specific PDGF-B ligand blockade may allow continued receptor signaling through remaining PDGF ligands to maintain pericyte function.

[0265] Example 12. Pharmacokinetic profile of anti-PDGF-B antibodies The pharmacokinetic (PK) profile of anti-PDGF-B antibodies was determined at three different doses in C57BL / 6 WT mice. Both anti-PDGF-B monoclonal antibodies used in this study cross-react with mouse PDGF-BB with sub-nanomolar affinity at 37°C. 26-week-old female mice (total = 36 mice; 100% WT on a C57BL / 6 background) were administered a single subcutaneous dose of 0.1, 1, or 10 mg / kg and analyzed at 6 h, 1, 2, 3, 4, 7, 10, 15, 22, and 30 days of bleeding. Total human IgG was determined by Gyros: Mouse anti-human kappa light chain constant (REGN654*biotin) mAb capture / mouse anti-human IgG1 / IgG4 (REGN2567*Alexa647) mAb Detect. Functional binding was determined by Gyros: Mouse PDGF-BB*Biotin Capture / Mouse anti-human IgG1 / IgG4 (REGN2567*Alexa647) mAb Detect.

[0266] The doses of anti-PDGF-B antibodies and hIgG4 isotype controls used in the pharmacokinetic studies are listed in Table 16. [Table 16]

[0267] Overall, differences were observed between H4H13145P and H4H13132P (Figure 16). H4H13145P shows faster clearance than the isotype control at 1 mg / kg and 0.1 mg / kg, and has a similar PK profile to the isotype control at 10 mg / kg. H4H13132P shows a similar PK profile to the isotype control at all doses. The greatest differences in drug exposure (AUClast) are observed at 1 mg / kg and 0.1 mg / kg when compared to H4H13145P. All monoclonal antibodies tested reached similar Cmax at each dose. However, a lower than expected Cmax was observed for all three monoclonal antibodies administered at 0.1 mg / kg.

[0268] The clearance observed with H4H13145P at 1 mg / kg and 0.1 mg / kg suggests target-mediated clearance that may be driven by sub-picomolar affinity for mPDGF-BB. It is important to note that although the affinity of each molecule for hPDGF-BB is very similar, there are differences in the affinities between the molecules for mPDGF-BB.

[0269] A functional (mouse PDGF-BB) binding assay can be performed to examine the unbound antibody concentration relative to the total hIgG concentration.

[0270] Similar studies are performed in cynomolgus monkeys to evaluate the PK profile of anti-PDGF antibodies. Cynomolgus monkeys (total=27) are administered a single subcutaneous dose of 0.1, 1, or 10 mg / kg and analyzed at 6 hours, 1, 2, 3, 4, 7, 10, 15, 22, and 30 bleed time points. Plasma is collected for PK analysis to determine endpoints of antibody target levels (total bound and unbound PDGF-B), hFc levels (total), and free target levels (free PDGF-B).

[0271] Example 13. Vascular permeability study of anti-PDGF-B antibodies The pathobiological mechanism of angioedema with angiotensin-converting enzyme (ACE) inhibitor therapy (captopril) is thought to be related to the kallikrein-kinin plasma effector system (see Craig et al., 2014 Int Arch Allergy Immunol. 2014;165(2):119-27, which is incorporated herein by reference in its entirety). Angioedema may result from insufficient degradation of bradykinin (BK). BK has a very short half-life of 17 seconds because it is rapidly metabolized by various metalloproteinases, such as ACE. Captopril, an ACE inhibitor, increases bradykinin levels by blocking the degradation of bradykinin, resulting in vasodilation and increased vascular permeability.

[0272] Studies were conducted to evaluate whether chronic treatment of healthy C57 adult mice with high doses of PDGF-BB antibodies had any additive effects on gastrointestinal (GI), pulmonary, or cerebrovascular permeability. Taconic C57BL male mice, aged 11-16 weeks, were administered subcutaneous doses of 25 mg / kg of control IgG, anti-PDGF-B, and anti-PDGFRβ antibodies twice weekly for 4 weeks, as described in Figure 17. The Evans Blue (EB) assay was used to evaluate the effects of 4-week treatment of anti-PDGF-B and PDGFRβ antibodies on baseline and ACE II inhibitor-induced GI vascular permeability. Evans Blue (EB) dye reversibly binds to serum albumin with moderate affinity and has a long blood half-life. Binding of EB to albumin has been utilized to quantify protein leakage as an index of increased vascular permeability. Under physiological conditions, the endothelium is impermeable to albumin, so Evans Blue-bound albumin remains restricted within the blood vessels. Under pathological conditions that promote increased vascular permeability, endothelial cells partially lose close contact and the endothelium becomes permeable to small proteins such as albumin. Organs with increased permeability will show significantly increased blue coloration compared to organs with an intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue.

[0273] Figure 18 shows that administration of anti-PDGF-B and anti-PDGFRβ antibodies did not significantly interfere with the weight gain of the animals. Furthermore, no changes in the behavior of the animals were observed.

[0274] Furthermore, as shown in FIG. 19, there was no significant change in GI water retention / edema in the small intestine after treatment with isotype control IgG, anti-PDGF-B, and anti-PDGFRβ antibodies in healthy mice. Captopril caused a mild and statistically insignificant increase in wet weight in the small intestine. Treatment with isotype control IgG, anti-PDGF-B, and anti-PDGFRβ antibodies did not significantly change the tissue edema in the small intestine caused by captopril. Similarly, FIG. 20 shows that there was no significant change in vascular permeability in the small intestine by treatment with anti-PDGF-B and anti-PDGFRβ antibodies in mice. Treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not exacerbate captopril, which caused acute vascular permeability increase in the small intestine.

[0275] Furthermore, as shown in Figure 21, there was no significant change in GI water retention / edema in the stomach by treatment with anti-PDGF-B and anti-PDGFRβ antibodies in healthy mice and captopril-treated mice. Captopril did not cause a statistically significant increase in wet weight in the stomach. Similarly, Figure 22 shows that there was no significant change in vascular permeability in the stomach by treatment with anti-PDGF-B and anti-PDGFRβ antibodies in mice. Treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not exacerbate captopril, which caused an acute increase in vascular permeability in the stomach.

[0276] Figure 23 shows results showing that there was no significant change in GI water retention / edema in the lungs by treatment with anti-PDGF-B and anti-PDGFRβ antibodies in healthy and captopril-treated mice. Captopril did not cause significant weight gain in the lungs. Similarly, as shown by Figure 24, there was no significant change in vascular permeability in the lungs by treatment with anti-PDGF-B and anti-PDGFRβ antibodies in healthy and captopril-treated mice. Captopril acute treatment did not cause vascular hyperpermeability in the lungs.

[0277] Furthermore, Figure 25 shows results showing that there was no significant change in GI water retention / edema in the brain by treatment with anti-PDGF-B and anti-PDGFRβ antibodies in healthy and captopril-treated mice. Captopril did not cause significant weight gain in the brain. Similarly, as shown by Figure 26, there was no significant change in vascular permeability in the brain by treatment with anti-PDGF-B and anti-PDGFRβ antibodies in healthy and captopril-treated mice. Acute captopril treatment did not cause vascular permeability in the brain.

[0278] Overall, anti-PDGF-B and anti-PDGFRβ antibody treatment did not cause any significant tissue edema or changes in GI vascular permeability in mice. Thus, there is no negative effect of PDGF-B and PDGFRβ neutralizing antibodies on GI vascular permeability in adult mice. Furthermore, this study suggests that PDGF-B signaling may be unnecessary in maintaining the integrity of mouse vasculature pericytes.

[0279] Example 14. Biacore binding kinetics of PDGF-BB monoclonal antibodies to human PDGF-BB, monkey PDGF-BB, and rat PDGF-BB at 25° C. The equilibrium dissociation constants (K D) was determined using a real-time surface plasmon resonance biosensor using a Biacore T200 or MASS-2 instrument. All binding studies were performed at 25° C. in a running buffer of 10 mM HEPES, 300 mM NaCl, and 0.05% v / v surfactant Tween®-20, pH 7.4 (HBS-P). The surface of the sensor chip was first derivatized by amine coupling with a human Fc-specific mouse mAb (REGN2567) to capture different PDGF-BB mAbs. Different concentrations (90–1.11 nM, 3-fold serial dilutions) of human PDGF-BB, monkey (Macaca fascicularis) PDGF-BB, or rat PDGF-BB prepared in HBS-P running buffer were injected over the anti-PDGF-BB mAb capture surface at flow rates of 25–50 μL / min for 1–3 min, and their dissociation in HBS-P running buffer was monitored for 5–10 min. At the end of each cycle, the PDGF-BB mAb capture surface was regenerated using a 10–12 s injection of 20 mM phosphate.

[0280] Association rates (k) were determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve fitting software. a ) and dissociation rate (k d The binding / dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rates as follows:

number

[0281] The binding kinetic parameters of PDGF-BB binding to different PDGF-BB mAbs of the invention at 25° C. are listed in Tables 17-19. result [Table 17] [Table 18] [Table 19]

[0282] Example 15. Antibody inhibition studies of human PDGF-BB, PDGF-AB, or combined PDGF-BB / AB-induced calcium flux using FLIPR with primary human pulmonary artery smooth muscle cells and primary human lung fibroblasts. For calcium flux assays, primary human pulmonary smooth muscle cells (Lonza, Cat. No. CC-2581) and human pulmonary fibroblasts (Lonza, Cat. No. 2512) were seeded onto 96-well assay plates at a cell density of 10k / well. Smooth muscle cells were cultured in the supplier's recommended smooth muscle medium (Lonza, CC-3182) and fibroblasts were cultured in DMEM-10% FBS-1x penicillin / streptomycin / L-glutamine in a humidified incubator at 37°C with 5% CO2.

[0283] Upon reaching 100% confluency in the wells, PDGF-driven calcium flux was assessed using the FLIPR® Calcium 5 Assay Kit (Molecular Devices, Cat. No. R8185) on the FLIPR Tetra system (Molecular Devices) according to the manufacturer's protocol. Briefly, complete medium was replaced with 50 μL of assay medium (DMEM-0.1% BSA-1× penicillin / streptomycin / L-glutamine). Calcium 5 dye was then reconstituted in the supplied buffer in the presence of probenecid (Thermofisher, Cat. No. P36400). 50 μL of reconstituted calcium 5 dye was then added to the cells and incubated at 37° C. in 5% CO2 for 1 hour before being placed in the FLIPR Tetra system. Individual dosing plates were generated using human PDGF-BB (R&D Systems, Cat. No. 220-GMP), human PDGF-AB (R&D Systems, Cat. No. 222-AB), and PDGF-AB / PDGF-BB in a 3:1 ratio. To evaluate antibody inhibition, 1 nM concentration of PDGF-AB, PDGF-BB, and combination treatment PDGF-AB / BB were chosen as constants. Antibodies were tested in the range of 500 nM to 0.06 nM (1:6 serial dilutions) in the presence of 1 nM PDGF protein H4H13145P or H4H13132P. Antibodies were added to the cells via the FLIPR tetra system after preincubation with PDGF protein for 30 min. Calcium flux traces were evaluated over both 60 s and 5 min full time courses. These calcium traces were analyzed for their maximum and minimum values ​​using SoftMax Pro Software (Molecular Devices).

[0284] As shown in Table 20, both antibodies, H4H13145P and H4H13132P, demonstrated complete inhibition of 1 nM PDGF-BB, PDGF-AB, and PDGF-AB / BB (3:1) when calcium flux was recorded for 60 seconds in human pulmonary smooth muscle cells. Both antibodies showed IC values ​​ranging from 0.18 nM to 4.5 nM.50 H4H13145P demonstrated inhibition of PDGF treatment with IC values ​​of 0.18 nM and 0.64 nM, respectively. 50 demonstrated greater potency against PDGF-BB than H4H13132P. These antibodies are more potent inhibitors of PDGF-BB alone than PDGF-AB and the combination of PDGF-AB / BB (3:1).

[0285] When calcium flux was recorded over a full 5 min time course, H4H13145P demonstrated a higher ability to neutralize PDGF-BB and prevent PDGF-driven calcium flux compared to H4H13132P, with IC values ​​of 0.36 nM for inhibition of PDGF-BB, 16 nM for inhibition of PDGF-AB, and 5.7 nM for the combination of PDGF-AB / BB (3:1). 50 The potent inhibition of PDGF-mediated calcium flux was maintained at 100 mM MgCl2 and 100 mM NaCl2. Isotype control antibodies demonstrated no inhibition of PDGF-mediated calcium flux. [Table 20]

[0286] When tested in human lung fibroblasts, H4H13145P and H4H13132P demonstrated complete inhibition of all PDGF treatments when calcium flux was recorded over 60 seconds. Both antibodies had IC values ​​ranging from 0.59 nM to 1.6 nM. 50 H4H13145P demonstrated inhibition of PDGF treatment with IC values ​​of 0.59 nM and 0.99 nM, respectively. 50 H4H13145P also demonstrated slightly higher potency in neutralizing PDGF-BB compared to H4H13132P, which has IC 50These antibodies were slightly more potent in neutralizing the PDGF-AB / BB treatment combination than H4H13132P, which has a PDGF-AB and PDGF-AB / BB treatment combination value. These antibodies are more potent inhibitors of PDGF-BB alone than of the PDGF-AB and PDGF-AB / BB treatment combination.

[0287] When calcium flux was recorded over a full 5-minute time course, H4H13145P demonstrated a significantly higher ability to neutralize PDGF-BB, PDGF-AB, and the combined treatment PDGF-AB / BB compared to H4H13132P. H4H13132P demonstrated a lower ability to maximally inhibit the 1 nM constant of all PDGF treatments. H4H13145P had ICs of 0.63 nM and 3.8 nM, respectively. 50 H4H13132P showed greater potency in inhibiting PDGF-BB-driven calcium flux compared to H4H13132P, which has an isotype control antibody that did not demonstrate inhibition of PDGF-mediated calcium flux in human lung fibroblasts. [Table 21]

[0288] Example 16: Structural analysis of antibody-PDGF-BB complexes by cryo-electron microscopy (Cryo-EM) method Preparation of Fab fragments H4H13145P, H4H13132P, and H4H13127P IgG were purified by F(ab') purification using Fabricator enzyme (Genovis) according to the manufacturer's standard protocol. 2 and Fc fragments. F(ab') 2was reduced to Fab using 2-mercaptoethylamine (2-MEA, ThermoFisher) followed by removal of the Fc fragment using CaptureSelect IgG-Fc(ms) affinity resin (ThermoFisher). The Fab fragment was further purified by injection onto a size-exclusion chromatography (SEC) column (Superdex 200 Increase 15 / 300 GL, GE Healthcare) connected to an AKTA Avant 25 chromatography system (GE Healthcare) with a running buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl. Peak fractions were pooled and concentrated into a 30 kDa cutoff centrifugal filter (Millipore Sigma) and then used for conjugate preparation.

[0289] Complex preparation Recombinant human PDGF-BB protein (R&D) was mixed with H4H13145P Fab or H4H13132P Fab at a molar ratio of 1:2.2. Excess non-competitive H4H13127P Fab was further added to two samples to increase the size of the two complexes, making them more suitable targets for EM characterization. This additional Fab can also improve the orientation distribution of the complex particles on the EM grid. Samples were incubated at 4°C for 30 min and then concentrated in 30 kDa cut-off centrifugal filters (Millipore Sigma) to a concentration of 2.5 mg / mL, measured using a Nanodrop instrument (ThermoFisher).

[0290] Cryo-EM sample preparation and data collection Freshly made PDGFBB-H4H13145P Fab-H4H13127 Fab complexes or PDGFBB-H4H13132P Fab-H4H13127 Fab complexes were mixed with approximately 0.15% Amphipol PMAL-C8 (Anatrace) immediately prior to pipetting 3.5 μL of the mixture onto an UltrAufoil R1.2 / 1.3, 300 mesh grid (Quantifoil). Excess liquid was blotted using filter paper and flash frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (ThermoFisher) operated at 4°C and 100% humidity. Grids were then loaded onto a Titan Krios G3i microscope (ThermoFisher) equipped with a K3 camera (Gatan). Approximately 7,000 movies were collected for both complexes using EPU software (ThermoFisher) in counting mode at a nominal magnification of 105,000× (0.86 Å pixel size). Each movie contained 46 dose fractions over a 2 s exposure and spanned Å. 2 The total dose obtained per unit time was approximately 40 electrons.

[0291] Cryo-EM data processing and map generation Cryo-EM data were processed using Cryosparc v2.14.2. Movies were motion corrected with patch motion correction and CTF parameters were estimated with patch CTF estimation. Particles were first picked using Blob Picker to generate 2D class averages that were used as templates for subsequent template picking. After multiple rounds of 2D classification to remove junk particles, 249,832 and 429,948 particles remained for the PDGFBB-H4H13145P Fab-H4H13127 Fab complex and the PDGFBB-H4H13132P Fab-H4H13127 Fab complex, respectively. Ab initial reconstruction, homogeneous purification, and heterogeneous purification identified 110,261 particles corresponding to the PDGFBB-H4H13145P Fab-H4H13127 Fab complex and 159,372 particles corresponding to the PDGFBB-H4H13132P Fab-H4H13127 Fab complex. Using heterogeneous purification, these particles were further refined to reconstructions at 3.4 Å and 3.5 Å resolution for the two complexes, respectively.

[0292] Model construction and refinement Manual model building was performed using Coot version 0.8.9, and real-space refinement was performed in Phenix version 1.17. The crystal structure of human PDGF-BB (PDB:1PDG) was docked into the cryo-EM density maps of the PDGFBB-H4H13145P Fab-H4H13127 Fab complex and the PDGFBB-H4H13132P Fab-H4H13127 Fab complex using fit-in maps from UCSF Chimera. Homology models of the Fab fragments of H4H13145P, H4H13132P, and H4H13127P were generated from the previously determined REGN Fab structure. Unambiguous docking of the Fab models into their respective densities was aided by the clearly interpretable side chain densities corresponding to their distinct CDR sequences. After docking, the models were manually refined in Coot, followed by real-space refinement of the entire PDGFBB-H4H13145P Fab-H4H13127 Fab complex or the PDGFBB-H4H13132P Fab-H4H13127 Fab complex in Phenix.

[0293] result The PDGFBB ligands are homodimers that contain three interchain loops (L1 formed by residues 25-38, L2 formed by residues 53-58, and L3 formed by residues 78-81) followed by two clamp regions formed by the C-terminal segment.

[0294] According to cryo-EM studies, each PDGFBB homodimer binds two H4H13145P or H4H13132P Fab molecules and two H4H13127P Fab molecules. The two complexes both have two-fold symmetry with two H4H13127P Fab molecules binding to the outer edge of the β-sheet region. Both H4H13145P Fab and H4H13132P Fab bind to three interchain loops that also engage PDGFRβ in the PDGFBB-PDGFRβ signaling complex (PDB:3MJG), which has a similar binding site. The data suggest that the H4H13145P and H4H13132P antibodies bind at the ends of the PDGFBB dimer and that a single Fab contacts both monomers in the dimer, i.e., the antibodies bind across the dimer interface.

[0295] H4H13145P: Both the heavy and light chains of H4H13145P interact with the PDGFBB dimer at three interchain loops.

[0296] Antibody to dimer interactions: CDRs H1, H2, and H3 (residues A31, Y32, W50, Y54, N57, W100) in the heavy chain are involved in interactions with both chains of the PDGFBB homodimer. CDR L3 (residues Y91, Y92, N93, L94, and F96) in the light chain is involved in interactions with chain 1 of the PDGFBB dimer.

[0297] Dimer-to-antibody interaction: Chain 1 of PDGFBB (residues F37, W40, R73, K80, K81, P82, F84, and K86) interacts with both chains of H4H13145P. Residues I13 and R56 of chain 2 of PDGFBB are involved in the interaction with the heavy chain of H4H13145P. Loop 1 and Loop 3 of chain 1 play a major role in the interaction.

[0298] H4H13132P: Both the heavy and light chains of H4H13132P interact with the PDGFBB dimer at three interchain loops.

[0299] Antibody to dimer interactions: CDRs H1, H2, and H3 (residues S31, A33, I52, I54, F55, D103, Y104, Y105) in the heavy chain are involved in interactions with both chains of the PDGFBB dimer. CDRs L1 and L3 (residues Y31, T97, and W99) in the light chain are involved in interactions with chain 1 of the PDGFBB dimer.

[0300] Dimer-to-antibody interaction: Chain 1 of PDGFBB (residues L38, W40, R73, I75, K80, K81, P82, I83, F84, and K86) interacts with both chains of H4H13132P. Residue R56 of chain 2 of PDGFBB is involved in the interaction with the heavy chain of H4H13132P. Loop 1 and loop 3 of chain 1 play a major role in the interaction.

[0301] Consideration Cryo-EM data show that the two antibodies, H4H13145P and H4H13132P, have nearly identical binding sites on the PDGFBB dimer. Multiple residues within chain 1 of PDGFBB interact with both antibodies (W40, R73, K80, K81, P82, F84, and K86). There are fewer interactions between chain 2 of PDGFBB and the antibodies (two in the case of H4H13145P and one in the case of H4H13132P), but both interactions share the common R56 residue.

[0302] Example 17: Testing antibody or small molecule inhibition of calcium flux induced by human PDGF-BB, PDGF-AA, or PDGF-DD using FLIPR or cell proliferation in primary human pulmonary artery smooth muscle cells. reagent Reagents used to test antibody or small molecule inhibition of calcium flux induced by human PDGF-BB, PDGF-AA, or PDGF-DD using FLIPR or cell proliferation in primary human pulmonary artery smooth muscle cells include: Human Pulmonary Artery Smooth Muscle Cells (PASMC), Lonza Catalog No. CC-2581 Lot No. 0000559495; Smooth Muscle Cell Medium, Lonza, CC-3182; DMEM Medium, Thermofisher, Catalog No. 11965092; Bovine Serum Albumin Solution, Millipore-Sigma, Catalog No. A9576; PDGF-BB, R&D Systems, Catalog No. 220-GMP; PDGF-AA, R&D Systems, Catalog No. 221-AA; PDGF-DD, R&D Systems, Catalog No. 1159-SB; Calcium 5 Assay Kit, Molecular Devices, catalog number R8185; probenecid, Thermofisher, catalog number P36400; seraltinib (GB002), MedChemExpress, catalog number HY-109190; and imatinib mesylate, MedChemExpress, catalog number HY-50946.

[0303] Experimental procedure For calcium flux and proliferation assays, primary human pulmonary smooth muscle cells were seeded onto 96-well assay plates at cell densities of 10,000 / well and 2,000 / well, respectively. Smooth muscle cells were cultured in the supplier's recommended smooth muscle medium in a humidified incubator at 37°C with 5% CO2.

[0304] Upon reaching 100% confluency in each well, PDGF-driven calcium flux was assessed using the FLIPR® Calcium 5 Assay Kit on a FLIPR Tetra system (Molecular Devices) according to the manufacturer's protocol. Briefly, complete medium was replaced with 50 μL of assay medium (DMEM-0.1% BSA-1× penicillin / streptomycin / L-glutamine). Calcium 5 dye was then reconstituted in the supplied buffer in the presence of probenecid. 50 μL of reconstituted calcium 5 dye was then added to the cells and incubated for 1 hour at 37° C. in 5% CO2 before being placed in the FLIPR Tetra system. Serial dilutions and inhibition constants of human PDGF-AA, PDGF-BB, and PDGF-DD were generated on separate dosing plates. PDGF-BB at 0.5 nM, PDGF-DD at 1 nM, and PDGF-AA at 3 nM were chosen as constants for evaluating antibody and small molecule inhibition. Serial dilutions of H4H13145P (anti-PDGFb) and H4H1238N (isotype control) (250 nM to 0.2 nM, 1:6 dilution) were tested in the presence of PDGF constants. Antibodies were preincubated with PDGF protein for 30 min before being added to cells via the FLIPR tetra system. Small molecules seraltinib and imatinib were preincubated with cells and calcium dye in assay medium at concentrations ranging from 10,000 nM to 8 nM (1:6 serial dilution) for 30 min. These calcium traces were analyzed for their maximum and minimum values ​​using SoftMax Pro Software (Molecular Devices).

[0305] Proliferation was assessed using an IncuCyte live cell imaging system (Essen BioScience). After overnight seeding, the complete medium was replaced with serum-free smooth muscle medium supplemented with 0.1% BSA. Antibodies were pre-incubated with PDGF constant for 30 min, while cells were pre-treated with small molecule compounds imatinib and seraltinib prior to the addition of PDGF. Plates were imaged every 3 h for 7 days, and proliferation was determined by measuring cell confluence in wells on day 6 using IncuCyte-based analysis software (Sartorius).

[0306] result The PDGF growth factor family is a potent series of mitogens of interest in pulmonary arterial hypertension. Multiple therapies target the PDGF / PDGFR signaling pathway, including small molecule receptor kinase inhibitors called seraltinib and imatinib. In this study, we directly compared the anti-PDGF-BB molecule H4H13145P to these small molecule PDGFR inhibitors in two in vitro cellular assays.

[0307] Both seraltinib and imatinib completely inhibited PDGF-AA, PDGF-DD, and PDGF-BB driven calcium flux and proliferation. PDGF-AA signals through PDGFRa, PDGF-DD signals through PDGFRb, and PDGF-BB signals through both dimeric receptors. As shown in Table 22, seraltinib had ICs ranging from 6 to 27 nM. 50 It is more potent than imatinib in this cell-based assay with IC 50 H4H13145P was able to completely inhibit PDGF signaling with IC values ​​ranging from 110 to 650 nM. 50 H4H13145P was able to completely inhibit PDGF-BB-driven calcium flux and proliferation at 0.11 nM and 0.14 nM, respectively. H4H13145P is more potent against PDGF-BB compared to the small molecule PDGFR inhibitors, seraltinib and imatinib (Table 22 and Figures 27-28). [Table 22]

[0308] Example 18: Examination of receptor-mediated antibody internalization in primary human pulmonary artery smooth muscle cells and primary human lung fibroblasts. reagent Reagents used to test receptor-mediated antibody internalization in primary human pulmonary artery smooth muscle cells and primary human lung fibroblasts include: human pulmonary artery smooth muscle cells (PASMC), Lonza catalog number CC-2581 lot number 0000559495; normal human lung fibroblasts (NHLF) Lonza catalog number CC-2512 lot number 0000494609; DMEM medium, Thermofisher, catalog number 11965092; bovine serum albumin solution, Millipore-Sigma, catalog number A9576; smooth muscle cell medium, Lonza, CC-3182; pHrodo™ Deep Red Antibody Labeling Kit catalog number P35355 Thermo Fisher; CellTrkr™ Violet Cell Proliferation Kit catalog number C34571 Thermo Fisher; PDGF-BB-R&D Systems, catalog number 220-GMP; PDGF-AB-R&D Systems, Catalog No. 222-AB; Calcium 5 Assay Kit, Molecular Devices, Catalog No. R8185; probenecid, Thermofisher, Catalog No. P36400; PDGFRa-APC, Abcam, Catalog No. AB119838; PDGFRb-APC, R&D Systems, Catalog No. FAB1263A; isotype antibody-APC, Abcam, Catalog No. AB37391; DAPI viability dye, Thermofisher, Catalog No. 62248; and TrypLE™ Express Enzyme, Thermofisher, Catalog No. 12604013.

[0309] Experimental procedure Antibody labeling: pHrodo Deep Red Antibody labeling was completed according to the user guide provided by the Invitrogen pHrodo™ Deep Red Antibody Labeling Kit (Cat. Nos. P35355 and P35356). Antibody internalization assays were performed using the following procedure:

[0310] Antibody internalization assay 1: (1) 20,000 PASMCs were seeded per well of a 96-well plate, and the cells were incubated overnight at 37° C. in 5% CO2 in complete medium, (2) the complete medium was removed and washed once with PBS, then the cells were labeled with CellTrkr Violet dye for 15 min, (3) the CellTrkr solution was removed, (4) anti-PDGF-B mAb labeled with PDGF-BB or PDGF-AB (pHrodo) was pre-bound for 30 min at room temperature before cells were treated, (5) antibody ligand solution was added to the cells without pre-binding as a control, (6) live cell imaging was acquired every hour for up to 21 h on an Opera Phenix Imaging System, and (7) the data was analyzed by Harmony 4.9 software.

[0311] Antibody internalization assay 2 (with receptor blocking): (1) 20,000 PASMCs were seeded per well of a 96-well plate and cells were incubated overnight at 37° C., 5% CO2 in complete medium; (2) complete medium was removed and washed once with PBS, then cells were labeled with CellTrkr Violet dye for 15 min; (3) CellTrkr solution was removed; (4) anti-PDGFRα (REGN1574), anti-PDGFRβ (REGN764) were added at 500 nM each or in combination with cells for 30 min; (5) labeled (pHrodo) anti-PDGF-B mAb was pre-bound with PDGF-BB or PDGF-AB, respectively, at room temperature for 30 min before treating cells; (6) antibody ligand solution was added to cells; (7) live cell imaging was acquired every hour for up to 72 h on an Opera Phenix Imaging System; (8) data was analyzed with Harmony 4.9 software.

[0312] FLIPR (Fluorescence Imaging Plate Reader) antibody internalization assay: (1) 10,000 PASMCs were seeded per well of a 96-well plate and the cells were incubated overnight in complete medium at 37°C with 5% CO2, (2) a mixture of antibodies against PDGF-BB was pre-incubated at a ratio of 10-1 nM in assay medium (DMEM-0.1% BSA-1x penicillin / streptomycin / L-glutamine) for 10 min, (3) the complete medium was replaced with a ratio of 10-1 nM of REGN1945 and H4H13145P against PDGF-BB, (4) the cells were treated with the antibody-ligand mixture for 3, 24, and 48 h, (5) the cell monolayer was washed 3 times with PBS, (6) 50 uL of assay medium and 50 uL of calcium 5 dye were added, and (7) the cells were incubated for 3 h with FLIPR. Plates are incubated for 1 hour after dye addition before reading the plates using a Tetra imager; (8) the treatment plate is assembled and inserted into the FLIPR system to treat the cells with serial dilutions of PDGF-BB (25-0.04 nM, 1:6 dilution); (9) the calcium traces are analyzed for max-min values ​​using SoftMax Pro software (Molecular Devices).

[0313] Flow cytometry antibody internalization assay: (1) seed 300,000 PASMC per well of a 6-well plate and incubate the cells overnight in complete medium at 37° C. with 5% CO2, (2) preincubate a mixture of antibodies against PDGF-BB at a ratio of 10-1 nM in assay medium (DMEM-0.1% BSA-1x penicillin / streptomycin / L-glutamine) for 10 min, (3) replace the complete medium with a ratio of 10-1 nM of antibodies against PDGF-BB, (4) treat the cells with the antibody-ligand mixture for 24 h, (5) wash the cell monolayer three times with PBS, (6) dissociate the cells from the 6-well plate using TrypLE™ Express Enzyme, (7) precipitate and stain with cell staining buffer (10% DMEM, 5% (8) stain the cells for 1 hour on ice with APC-conjugated antibodies against PDGFRa and PDGFRb according to the manufacturer's protocol, (9) spin the cells and wash three times, (10) add DAPI viability dye to the cells at a final concentration of 1 μg / mL before inserting into the CytoFlex flow cytometer, (11) record 10,000 cells on the CytoFlex flow cytometer with gating for vital single cells, and (12) analyze the flow cytometer data in FlowJo software (BD Biosciences).

[0314] result Across multiple separate assays, it was shown that H4H13145P could be internalized into a variety of cell lines in a ligand-dependent manner via the PDGF receptor.

[0315] Antibody internalization: Labeling of anti-PDGF-B antibodies with pHrodo Deep Red enabled the assessment of antibody internalization in human primary PASMC assays. pHrodo Deep Red emits light at pH levels triggered in late endosomes or lysosomes. At normal pH, no light is emitted. This allows the assessment of time-dependent internalization using confocal imaging. As shown in Tables 23-24, co-treatment of labeled H4H13145P-PDGFBB had an increase of 108-127 fold change in fluorescent signal compared to labeled isotype control REGN1945 in both PASMCs and NHLFs. A similar trend is observed for PDGF-AB, but at a lower magnitude. This increase in fluorescent signal, which is only emitted when the labeled antibody is internalized into the cells, is reduced when the cells are pre-treated with two antibodies that inhibit both PDGFRa and PDGFRb receptors. This data suggests that H4H13145P is internalized into cells in a ligand-dependent manner via the PDGF receptor.

[0316] FLIPR Data: In this assay, PDGF-BB binds to the PDGF receptor, causing rapid receptor internalization and desensitization of cells to subsequent PDGF-BB treatment. As shown in Table 25 and Figure 29, PASMCs pretreated with REGN1945-PDGFBB (10:1 nM) were desensitized to subsequent PDGF-BB-induced calcium flux. H4H13145P was able to neutralize PDGF-BB and limit the observed loss of responsiveness to PDGF-BB. Compared to the medium control, the H4H13145P group was unable to completely prevent PDGF-BB desensitization. H4H13145P-PDGFBB treatment had a slight rightward shift in potency and a slight decrease in the maximum-minimum calcium flux response at 5 nM of PDGF-BB treatment. This may be explained by the antibody-ligand complex binding to the PDGFR receptor and causing receptor internalization, albeit at a significantly slower rate and magnitude than unbound PDGF-BB.

[0317] Flow cytometry: PASMC express both PDGFRa and PDGFRb. In the REGN1945 ligand-treated group, no fluorescent signal was detected for both PDGFRa and PDGFRb. This finding confirms the rapid ligand-dependent internalization of the receptor in response to PDGF-BB. H4H13145P prevented the observed loss in PDGFRb fluorescent signal. In the H4H13145P-treated group, there was a slight shift and loss of PDGFR cell surface expression compared to the medium control indicating receptor internalization, albeit at a reduced rate and magnitude compared to unbound PDGF-BB. [Table 23] [Table 24] [Table 25]

[0318] Example 19: Evaluation of the effects of two inhibitory anti-PDGF-B antibodies in the rat monocrotaline model of pulmonary arterial hypertension. reagent Reagents used for evaluation of the effect of two inhibitory anti-PDGF-B antibodies in the rat monocrotaline model of pulmonary arterial hypertension include: Monocrotaline (crotaline) Sigma Catalog No. PHL89251 - CAS No.: 315-22-0, MDL: MFCD00084656, Formula: C 16 H 23 NO6, Formula weight: 325.36 g / mol, Storage temperature: 2-8°C, Purity: 98%, Macitentan MedChemExpress Catalog No. HY-14184 - Orally active endothelin receptor antagonist, CAS No.: 441798-33-0, Formula: C 19 H 20 Br2N6O4S, formula weight: 588.27 g / mol, lot number: 10673, storage temperature: -20°C for 3 years, purity: 98%; and PEG-400 (50:50 v / v, Affymetrix Inc., #19957).

[0319] Experimental procedure To evaluate the effects of two inhibitory anti-PDGF-B antibodies in the rat monocrotaline model of pulmonary arterial hypertension, two studies were performed: Study No. 1 was performed in monocrotaline PAH rats for 4 weeks with preventive drug treatment, and Study No. 2 was performed in monocrotaline severe PAH rats survival for 5 weeks with therapeutic drug treatment (Tables 26-27).

[0320] Male Sprague Dawley rats aged 6-7 weeks were used. Rats were divided into treatment groups so that body weights were similar between the different groups. In study number 1, rats in the antibody-treated group were subcutaneously administered either anti-PDGF-B antibody or isotype control IgG at 10 mg / kg 1 day before monocrotaline injection, and administration continued twice a week for 28 days. On day 1, rats were subcutaneously administered either monocrotaline at 40 mg / kg or saline at 5 mL / kg as a control. On day 28, right ventricular systolic pressure (RVSP) was measured by right heart catheterization, and RV hypertrophy was calculated by the Fulton index as the weight ratio of RV to (LV + septum). In study number 2, rats were subcutaneously administered either monocrotaline at 60 mg / kg or saline at 5 mL / kg. Beginning on day 14, rats in the antibody treatment groups were administered either anti-PDGF-B antibody or isotype control IgG subcutaneously twice weekly at 25 mg / kg. The small molecule group received macitentan orally at 30 mg / kg daily. Body weight changes from days 0 to 35 were used for general toxicity assessment, and animal mortality and median survival were calculated through day 35. [Table 26] [Table 27]

[0321] Right heart catheterization and right ventricular systolic pressure: Rats were anesthetized with isoflurane and maintained at approximately 37°C using a heating platform (Heated Hard Pad 1, Braintree Scientific) and a circulating heated water pump (T / Pump Classic, Gaymar Industries). The neck region of each rat was prepared for surgery by depilating the right common carotid artery and right jugular vein. An incision was made and the right jugular vein was isolated, taking care not to damage the carotid artery and / or vagus nerve. A piece of 5-0 silk suture was placed under the isolated jugular vein to retract the vessel cephalad, after which the jugular vein was punctured with a 23-gauge needle. A pressure catheter (Microtip Catheter Transducer SPR-1000, Millar Instruments, Inc.) was inserted into the opening of the jugular vein and advanced through the right atrium into the right ventricle. The catheter was connected to a pressure / volume instrument (MPVS-300, Millar Instruments, Inc.) that measured heart rate and both diastolic and systolic right ventricular pressure. These parameters were digitally acquired using a data acquisition system (PowerLab 4 / 35, AD Instruments). Right ventricular pressure was analyzed using LabChart Pro 7.0 software (AD Instruments). Measurements were quantified from 60-second intervals of pressure tracings (after 2 minutes of recording to allow for pressure stabilization). Parameters analyzed were right ventricular systolic pressure (RVSP) and heart rate (HR).

[0322] Right heart hypertrophy assessment: After in vivo hemodynamic measurements, animals were euthanized by exsanguination under anesthesia, and then RV free wall, left ventricle (LV) and septal tissues were harvested and weighted. RV hypertrophy was calculated by Fulton's index as the weight ratio of RV to (LV + septum).

[0323] result Induced increase in right ventricular pressure in monocrotaline rats: In study number 1 in monocrotaline-treated rats, catheter-based assessment of cardiac right ventricular pressure revealed a significant increase in right ventricular systolic pressure in the isotype antibody-treated group at week 4. The two anti-PDGF-B antibody treatments significantly reduced right ventricular systolic pressure (Figure 30 and Table 28).

[0324] Right ventricular hypertrophy induced in monocrotaline rats: An increase in right ventricular heart weight was observed in monocrotaline rats in the isotype control IgG treatment group. The ratio of right ventricular weight to left ventricular + septum weight provides an index of right ventricular hypertrophy (i.e., Fulton index). Increased futon index was observed in monocrotaline-treated rats compared to saline-treated controls, thereby indicating the presence of right ventricular hypertrophy. Prophylactic treatment with anti-PDGF-B antibodies H4H13145P and H4H13132P at 10 mg / kg reduced right ventricular hypertrophy by 36% and 30%, respectively, compared to rats treated with isotype control (Figure 31 and Table 28).

[0325] Animal survival in monocrotaline rats: In study #2, rats injected with 60 mg / kg monocrotaline developed severe pulmonary hypertension symptoms and showed high mortality. Notably, 15 of 16 rats in the monocrotaline with isotype control IgG treatment group died by day 35. Anti-PDGF-B antibody H4H13145P, started 14 days after monocrotaline injection, significantly extended survival time and improved mortality. Standard of care endothelin receptor antagonist macitentan failed to extend survival. Combination of macitentan with anti-PDGF-B treatment showed no additional survival benefit compared to monotherapy with anti-PDGF-B antibody (Figure 32 and Table 29).

[0326] In summary, prophylactic treatment with Regeneron anti-PDGF-B antibody reduced both hemodynamic endpoints (right ventricular systolic pressure) and right ventricular hypertrophy in the monocrotaline rat model of PAH. Therapeutic treatment with Regeneron anti-PDGF-B antibody demonstrated a survival benefit superior to the vasodilator endothelin receptor antagonist macitentan in the severe monocrotaline rat model of PAH. Anti-PDGF-B antibody H4H13145P significantly rescued animal mortality and extended survival time. [Table 28] [Table 29]

[0327] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein which equivalents are intended to be encompassed by the following claims.

[0328] Unofficial sequence listing H4H13145P SEQ ID NO:1; HCVR CAGGTTCAGCTGGTGCAGTCTGGAACTGAGGTGAAGAAGCCTGGGGCCTCAGTAAAGGTCTCCTGCAAGGCCTCTGGTTATACTTATGGTGCCTATGCAATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACA AACTATGCACAGAAATTCCAGGACAGAGTCACCATGACCACAGACACATCCACGAACACAGCCTACATGGAACTGAGGGGCCTAAAATCTGACGACACGGCCGTGTATTTCTGTGCGAGGGCCTGGAACTCCTTTGACTACTGGGGCCAGGGCACCCTGGTCACTGTCTCCTCA SEQ ID NO:2; HCVR QVQLVQSGTEVKKPGASVKVSCKASGYTYGAYAISWVRQAPGQGLEWMGWISAYNGNTNYAQKFQDRVTMTTDTSTNTAYMELRGLKSDDTAVYFCARAWNSFDYWGQGTLVTVSS SEQ ID NO:3; HCDR1 GGT TAT ACT TAT GGT GCC TAT GCA SEQ ID NO:4; HCDR1 GYTYGAYA SEQ ID NO:5; HCDR2 ATC AGC GCT TAC AAT GGT AAC ACA SEQ ID NO:6; HCDR2 ISAYNGNT SEQ ID NO:7; HCDR3 GCG AGG GCC TGG AAC TCC TTT GAC TAC SEQ ID NO:8; HCDR3 ARAWNSFDY SEQ ID NO: 9; LCVR GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCGTCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGGAAAAATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCCGATGCATCCACTT TAGAAACAGGGGTCCCATCAAGATTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAAAATATTACTGTCAACAATATTATAATCTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA SEQ ID NO:10; LCVR DIQMTQSPSSLSASVGDRVTITCQASQDIRKNLNWYQQKPGKAPKLLISDASTLETGVPSRFSGSGSGTDFTFTISSLQPEDIAKYYCQQYYNLPFTFGPGTKVDIK SEQ ID NO:11; LCDR1 CAG GAC ATT AGG AAA AAT SEQ ID NO:12; LCDR1 QDIRKN SEQ ID NO:13; LCDR2 GAT GCA TCC SEQ ID NO:14; LCDR2 DAS SEQ ID NO:15; LCDR3 CAA CAA TAT TAT AAT CTC CCA TTC ACT SEQ ID NO:16; LCDR3 QQYYNLPFT SEQ ID NO:17;HC SEQ ID NO:18;HC QVQLVQSGTEVKKPGASVKVSCKASGYTYGAYAISWVRQAPGQGLEWMGWISAYNGNTNYAQKFQDRVTMTTDTSTNTAYMELRGLKSDDTAVYFCARAWNSFDYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* SEQ ID NO:19; LC GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCGTCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGGAAAAATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCCGATGCATCCACTTTAGAAACAGGGGTCCCATCAAGATTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAAAATATTACTGTCAACAATATTATAATCTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG Sequence number 20; LC DIQMTQSPSSLSASVGDRVTITCQASQDIRKNLNWYQQKPGKAPKLLISDASTLETGVPSRFSGSGSGTDFTFTISSLQPEDIAKYYCQQYYNLPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* H4H13132P Sequence number 21; HCVR CAGGTGCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACG CACAGAAGTTCCAGGGCAGAGTCACGATTACCACGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTATATTACTGTGCGAGAGAGGGCTACGGTGACTACTACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCCA Highway 22;HCVR QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITTDESTSTAYMELSSLRSEDTAVYYCAREGYGDYYFGMDVWGQGTTVTVSS Highway 23;HCDR1 GGA GGC ACC TTC AGC AGC TAT GCT Hotline 24;HCDR1 GGTFSSYA Honeycomb 25;HCDR2 ATC ATC CCT ATC TTT GGT ACA GCA Honeycomb 26;HCDR2 IIPIFGTA Honeycomb 27;HCDR3 GCG AGA GAG GGC TAC GGT GAC TAC TAC TTC GGT ATG GAC GTC Honeycomb 28;HCDR3 AREGYGDYYFGMDV Municipality 29;LCVR GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACAGTGATGGAAACACCTACTTGAATTGGTTTCAGCAGAGGCCAGACCAATCTCCAAGGCGCCTAATTTATAAGATT TCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCTCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO:30; LCVR DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPDQSPRRLIYKISNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIK SEQ ID NO:31; LCDR1 CAA AGC CTC GTA TAC AGT GAT GGA AAC ACC TAC SEQ ID NO:32; LCDR1 QSLVYSDGNTY SEQ ID NO:33; LCDR2 AAG ATT TCT SEQ ID NO:34; LCDR2 KIS SEQ ID NO:35; LCDR3 ATG CAA GGT ACA CAC TGG CCT CCC ACT SEQ ID NO:36; LCDR3 MQGTHWPPT SEQ ID NO:37;HC SEQ ID NO:38;HC QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITTDESTSTAYMELSSLRSEDTAVYYCAREGYGDYYFGMDVWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* SEQ ID NO:39; LC GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACAGTGATGGAAACACCTACTTGAATTGGTTTCAGCAGAGGCCAGACCAATCTCCAAGGCGCCTAATTTATAAGATTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCTCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG Accession number 40; LC DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPDQSPRRLIYKISNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* hPDGF-B SEQ ID NO:41; P01127 | PDGFB_human platelet-derived growth factor subunit BOS=Homo sapiens OX=9606 GN=PDGFB PE=1 SV=1 MNRCWALFLSLCCYLRLVSAEGDPIPEELYEMLSDHSIRSFDDLQRLLHGDPGEEDGAELDLNMTRSHSGGELESLARGRRSLGSLTIAEPAMIAECKTRTEVFEISRRLIDRTNANFLV WPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVTRSPGGSQEQRAKTPQTRVTIRTVRVRRPPKGKHRKFKHTHDKTALKETLGA

Claims

1. 1. An isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human platelet-derived growth factor subunit B (PDGF-B), comprising: (a) a binding-dissociation equilibrium constant (K) of less than about 1.84 pM as measured by surface plasmon resonance; Ｄ ) to human PDGF-subunit B homodimer (PDGF-BB) at 37° C.; (b) a K of less than about 1.36 pM as measured by surface plasmon resonance Ｄ binds to human PDGF-BB at 37° C. (c) binds to human PDGF-BB at 37° C. with a t1 / 2 of about 1155 minutes or greater, as measured by surface plasmon resonance; (d) a K of less than about 2.79 pM as measured by surface plasmon resonance Ｄ binds to human PDGF-BB at 25° C. (e) binds to human PDGF-BB at 25° C. with a t1 / 2 of about 1155 minutes or greater, as measured by surface plasmon resonance; (f) an IC of less than about 1.9 nM as measured in a competitive ELISA assay at 25° C. ５０ and inhibiting PDGF-B activation against human PDGF-BB; (g) an IC of less than about 8.8 nM as measured by competitive ELISA assay at 25° C. ５０ and inhibiting PDGF-B activation against human PDGF-subunit A and subunit B heterodimer (PDGF-AB); and (h) an isolated human monoclonal antibody or antigen-binding fragment thereof, which exhibits one or more properties selected from the group consisting of: blocking the interaction between human PDGF-BB and one or more of human PDGFR-αα, PDGFR-αβ, and PDGFR-ββ.

2. An isolated human antibody or antigen-binding fragment thereof that specifically binds to human PDGF-B, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within any one of the heavy chain variable region (HCVR) sequences selected from the group consisting of SEQ ID NOs: 2 and 22, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within any one of the light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 10 and 30.

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

22.

4. The isolated human antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises an LCVR comprising an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 10 or SEQ ID NO:

30.

5. The isolated human antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment comprises: (a) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22; and (b) an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30.

6. the antibody or antigen-binding fragment being (a) a HCDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 24; (b) a HCDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 26; (c) a HCDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 28; (d) an LCDR1 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 32; (e) an LCDR2 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 34; and / or (f) an LCDR3 domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 36;

7. 7. The isolated human antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 22 / 30.

8. the antibody or antigen-binding fragment being (i) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 20; and A heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 18, and / or (ii) a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 40; and 8. The isolated human antibody or antigen-binding fragment of any one of claims 1 to 7, comprising a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO:

38.

9. The antigen-binding fragment may be a Fab fragment, F(ab') ２ 9. The antigen-binding fragment of any one of claims 1 to 8, which is a fragment, an Fd fragment, an Fv fragment, a single chain Fv (scFv) molecule, or a dAb fragment.

10. An isolated antibody or antigen-binding fragment thereof that binds to the same epitope on human PDGF-B as the antibody or antigen-binding fragment of any one of claims 1 to 9.

11. An isolated antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of any one of claims 1 to 9 for binding to human PDGF-B.

12. A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 11, comprising the steps of: (i) introducing into a host cell one or more polynucleotides encoding a light immunoglobulin chain of the antibody or fragment and a heavy immunoglobulin chain of the antibody or fragment; (ii) culturing the host cell in a growth medium under conditions favorable for expression of the polynucleotide; (iii) optionally isolating said antibody or fragment from said host cell and / or the medium in which said host cell is growing.

13. 13. An antibody or antigen-binding fragment produced by the method of claim 12.

14. An injection device or container comprising an antibody or antigen-binding fragment according to any one of claims 1 to 11 and 13.

15. A pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof that binds to human PDGF-B according to any one of claims 1 to 11 and 13, a pharma- ceutically acceptable carrier or diluent, and, optionally, one or more additional therapeutic agents.

16. 16. The pharmaceutical composition of claim 15, wherein the one or more additional therapeutic agents comprises an iron supplement.

17. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 and 13, or the pharmaceutical composition of any one of claims 15 to 16, for preventing or treating pulmonary arterial hypertension (PAH) in a patient in need thereof.

18. 20. The composition of claim 17, wherein the composition is administered subcutaneously, intravenously, intradermally, orally, or intramuscularly.

19. wherein the PAH results in a condition selected from the group consisting of thickening of the pulmonary artery in the subject, reduced stroke volume in the subject, reduced right ventricular cardiac output in the subject, and reduced survival time in the subject; 19. The composition of claim 17 or 18, wherein administration of the composition treats the condition or reduces the severity of one or more symptoms of the condition.

20. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and 13 for use in treating a patient with PAH.

21. A composition comprising one or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 11 and 13 for use in the treatment of PAH.

22. 14. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 11 and 13 in the manufacture of a medicament for treating a patient with PAH.

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