Method of use for anti-PDGF-B antibodies and 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 on 25 January 2021, the entirety of which is expressly incorporated herein by reference.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The 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 a human antibody that specifically binds to platelet-derived growth factor subunit B (PDGF-B), its antigen-binding fragment, and therapeutic and diagnostic methods using such antibody and fragment. [Background technology]

[0004] Platelet-derived growth factor (PDGF) is a potent mitogen that exists as five distinct 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 between the corresponding individual PDGF monomers. PDGF ligands exert their biological effects through interaction with the PDGF receptor (PDGFR). PDGFR is a single-pass, transmembrane, tyrosine kinase receptor consisting of a heterodimer or homodimer association of an alpha (a) receptor chain (PDGFR-alpha) and / or a beta (β) receptor chain (PDGF-B). Therefore, active PDGFR can consist of αα, ββ, or αβ receptor chain pairs. PDGFR shares a common domain structure containing five extracellular immunoglobulin (Ig) loops, a transmembrane domain, and a split intracellular tyrosine kinase (TK) domain. The interaction between dimeric PDGF ligands and PDGFR leads to receptor chain dimerization, receptor autophosphorylation, and intracellular signaling. It has been demonstrated in vitro that ββ receptors are activated by PDGF-BB and -DD, while 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 various human diseases, including pulmonary arterial hypertension (PAH). PAH is a progressive disorder characterized by a persistent increase in pulmonary artery pressure that damages both the large and small pulmonary arteries. Hemodynamically, PAH is defined as a systolic pulmonary artery pressure greater than 30 mmHg, or a mean pulmonary artery pressure greater than 25 mmHg with pulmonary capillaries, or a left atrial pressure of 15 mmHg or less. See, for example, Zaiman et al., Am.J.Respir.Cell MoI.Biol.33:425-31 (2005). Persistent vasoconstriction in PAH leads to structural remodeling of pulmonary vascular smooth muscle cells and endothelial cells, resulting in a phenotypic shift from a contractile normal phenotype to a synthetic phenotype, leading to cell proliferation and matrix deposition. When the walls of the smallest blood vessels thicken, they become unable to properly transport oxygen and carbon dioxide between the blood and the lungs, eventually leading to pulmonary hypertension, which results in thickening of the pulmonary arteries and narrowing of the blood vessels. Ultimately, proliferation of vascular smooth muscle and endothelial cells leads to vascular remodeling, accompanied by obstruction of the lumen of the pulmonary vascular system. Histological examination of tissue samples from patients with pulmonary hypertension shows intimal thickening and smooth muscle cell hypertrophy, especially in vessels less than 100 μm in diameter. This causes a progressive increase in pulmonary pressure as blood is pumped through the reduced lumen area. As a result, the right side of the heart works harder to compensate, and this increased effort causes the right ventricle to enlarge and thicken. Because blood tends to pool in the ventricles and legs, right ventricular hypertrophy carries a risk of pulmonary embolism. If a thrombus forms in the pooled blood, it can eventually travel and remain in the lungs. Ultimately, the additional workload placed on the right ventricle leads to cardiac dysfunction in these patients, resulting in premature death.

[0006] Standard therapies for the treatment of patients with PAH are primarily hemodynamic, affecting vascular tone and including, for example, prostacyclin analogs, endothelin receptor antagonists, phosphodiesterase inhibitors, and soluble guanylate cyclase activators / stimulants, 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 vascular system to provide handicap-free long-term survival for patients with PAH. Despite all the advances in the treatment of PAH, there is still no prospect of a cure for this fatal disease, and the vast majority of patients continue to progress to right ventricular failure. Therefore, there is a need in this field for novel, 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) [Overview of the Initiative] [Means for solving the problem]

[0008] This disclosure provides a fully human monoclonal antibody (mAb) and its antigen-binding fragment that specifically bind to platelet-derived growth factor subunit B (PDGF-B). Such an antibody may be useful in treating subjects with pulmonary arterial hypertension (PAH).

[0009] The circulation and pulmonary expression of PDGF-B, a potent mitogen for pulmonary smooth muscle cells and fibroblasts, are increased in both patients exhibiting idiopathic PAH and in hypoxia / sugen PAH mouse models. The interaction between PDGF-B and PDGFRββ, PDGFRαβ, and PDGFRαα is involved in promoting pathological angiogenesis and pulmonary vascular remodeling. Therefore, targeting PDGF-B may offer enhanced efficacy over PDGFRβ by targeting PDGFRαα, PDGFRαβ, and PDGFRββ-mediated signaling that coordinates to promote abnormal vascular remodeling.

[0010] Accordingly, in one embodiment, the present disclosure provides an isolated human monoclonal antibody or an 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 binding-dissociation equilibrium constant (K) less than approximately 1.84 pM when measured by surface plasmon resonance. D (b) It binds to human PDGF-subunit B homodimer (PDGF-BB) at 37°C, and (b) when measured by surface plasmon resonance, it has a K content of less than approximately 1.36 pM. D (c) Binds to human PDGF-BB at 37°C, with a t1 / 2 of approximately 1155 minutes or more as measured by surface plasmon resonance, and (d) has a K content of less than approximately 2.79 pM as measured by surface plasmon resonance. D (e) Binds to human PDGF-BB at 25°C, with a t1 / 2 of approximately 1155 minutes or more as measured by surface plasmon resonance, and (f) IC20 is less than approximately 1.9 nM as measured by competitive ELISA assay at 25°C. 50 Therefore, it inhibits PDGF-B activation against human PDGF-BB, and (g) when measured by a competitive ELISA assay at 25°C, the IC50 is less than approximately 8.8 nM. 50The substance exhibits one or more properties selected from the group consisting of (h) inhibiting PDGF-B activation in relation to human PDGF-subunit A and subunit B heterodimer (PDGF-AB), and (h) 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 includes 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 its antigen-binding fragment contains an HCVR having an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 2 or SEQ ID NO: 22.

[0014] In some embodiments, the antibody or antigen-binding fragment includes an LCVR having an amino acid sequence that is at least 90% identical 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 includes (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 an 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 described in SEQ ID NO: 20 and a heavy chain immunoglobulin comprising the amino acid sequence described in SEQ ID NO: 18, and / or (ii) a light chain immunoglobulin comprising the amino acid sequence described in SEQ ID NO: 40 and a heavy chain immunoglobulin comprising the amino acid sequence described in SEQ ID NO: 38.

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

[0020] In another embodiment, the present disclosure provides an isolated antibody or antigen-binding fragment that binds to the same human PDGF-B epitope as the 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 embodiment, the present disclosure provides an isolated antibody or antigen-binding fragment that competes for binding to human PDGF-B with 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.

[0022] In another embodiment, the present invention provides nucleic acid molecules encoding an anti-PDGF antibody or a fragment thereof. Recombinant expression vectors containing the nucleic acid of the present invention, and host cells into which such vectors have been introduced, are also included in the present invention, as are methods for producing antibodies by culturing host cells under conditions that enable antibody production, and by recovering the produced antibodies.

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

[0025] In some embodiments, the antibody or antigen-binding fragment includes 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 Disclosure provides an injection device or container containing an antibody or antigen-binding fragment disclosed herein.

[0027] In another embodiment, the present 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 disclosed herein, a pharmaceutically acceptable carrier or diluent, and optionally one or more additional therapeutic agents.

[0028] In some embodiments, one or more additional therapeutic agents include iron supplements.

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

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

[0031] In some embodiments, PAH causes a condition selected from the group consisting of thickening of the pulmonary artery in the subject, decreased stroke volume in the subject, decreased right ventricular output in the subject, and decreased survival time in the subject, and administration of an 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 to human PDGF-B as disclosed herein, for use in the treatment of patients with PAH.

[0033] In another embodiment, the Disclosure provides a composition for use in the treatment of PAH, comprising one or more antibodies or antigen-binding fragments that bind to human PDGF-B as disclosed herein.

[0034] In another aspect, the disclosure provides the use of isolated antibodies or antigen-binding fragments thereof that bind to human PDGF-B as disclosed herein in the manufacture of a pharmaceutical product for treating patients with PAH.

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

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

[0037] In some embodiments, the cells are human cells. In 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 antibody or antigen-binding fragment described herein binds to the outer edge of the β-sheet region of PDGF-BB. In some embodiments, the antibody or antigen-binding fragment described herein binds to the interchain loop of PDGF-BB. In some embodiments, the antibody or antigen-binding fragment described herein binds to the outer edge of the β-sheet region and the interchain loop of PDGF-BB. In some embodiments, the antibody or antigen-binding fragment described herein binds 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 antibody or antigen-binding fragment described herein binds to the W40 residue of PDGF-BB. In some embodiments, the antibody or antigen-binding fragment described herein binds to the R73 residue of PDGF-BB. In some embodiments, the antibody or antigen-binding fragment described herein binds to the K80 residue of PDGF-BB. In some embodiments, the antibody or antigen-binding fragment described herein binds to the K81 residue of PDGF-BB. In some embodiments, the antibody or antigen-binding fragment described herein binds to the P82 residue of PDGF-BB. In some embodiments, the antibody or antigen-binding fragment described herein binds to the F84 residue of PDGF-BB. In some embodiments, the antibody or antigen-binding fragment described herein binds to the K86 residue of PDGF-BB. In some embodiments, the antibody or antigen-binding fragment described herein binds to the R56 residue of PDGF-BB.

[0039] In some embodiments, the antibodies or antigen-binding fragments described herein are internalized in cells in a ligand-dependent manner via PDGF receptors. In some embodiments, the cells are human cells. In some embodiments, the cells are 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 the right ventricular systolic pressure in a subject that needs it. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce right ventricular hypertrophy in a subject that needs it. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can reduce both the right ventricular systolic pressure and right ventricular hypertrophy in a subject that needs it.

[0041] Other embodiments will become apparent from a review of the detailed description of the invention below. The present invention provides, for example, the following items: (Item 1) An isolated human monoclonal antibody or its antigen-binding fragment that specifically binds to human platelet-derived growth factor subunit B (PDGF-B), wherein the antibody or antigen-binding fragment is (a) When measured by surface plasmon resonance, the coupling dissociation equilibrium constant (K) is less than approximately 1.84 pM. D ) and bind to human PDGF-subunit B homodimer (PDGF-BB) at 37°C. (b) When measured by surface plasmon resonance, K is less than approximately 1.36 pM. D And it binds to human PDGF-BB at 37°C. (c) When measured by surface plasmon resonance, it binds to human PDGF-BB at 37°C with a t1 / 2 of approximately 1155 minutes or more. (d) When measured by surface plasmon resonance, K is less than approximately 2.79 pM. D And it binds to human PDGF-BB at 25°C. (e) When measured by surface plasmon resonance, it binds to human PDGF-BB at 25°C with a t1 / 2 of approximately 1155 minutes or more. (f) IC < approximately 1.9 nM when measured by competitive ELISA assay at 25°C 50 Therefore, inhibiting PDGF-B activation in human PDGF-BB, (g) IC < approximately 8.8 nM when measured by competitive ELISA assay at 25°C 50 <00(00319> (h) An isolated human monoclonal antibody or its antigen-binding fragment exhibiting 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. (Item 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. (Item 3) The isolated human antibody or antigen-binding fragment according to item 1 or 2, wherein the antibody or antigen-binding fragment comprises an HCVR having an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 22. (Item 4) An isolated human antibody or antigen-binding fragment according to any one of items 1 to 3, wherein the antibody or antigen-binding fragment comprises an LCVR having an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 10 or SEQ ID NO: 30. (Item 5) The isolated human antibody or antigen-binding fragment according to any one of items 1 to 4, wherein the antibody or antigen-binding fragment comprises (a) an HCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22, and (b) an LCVR containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30. (Item 6) The antibody or antigen-binding fragment is (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) 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 isolated human antibody or antigen-binding fragment thereof according to any one of items 1 to 5, comprising an LCDR3 domain containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 36. (Item 7) The isolated human antibody or antigen-binding fragment described in any one of items 1 to 6, comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 22 / 30. (Item 8) The antibody or antigen-binding fragment is (i) Light chain immunoglobulin containing the amino acid sequence described in Sequence ID No. 20, and Heavy chain immunoglobulins containing the amino acid sequence described in SEQ ID NO: 18, and / or (ii) Light chain immunoglobulin containing the amino acid sequence described in SEQ ID NO: 40, and An isolated human antibody or antigen-binding fragment according to any one of items 1 to 7, comprising a heavy chain immunoglobulin containing the amino acid sequence described in SEQ ID NO: 38. (Item 9) The antigen-binding fragment is a Fab fragment, F(ab') 2 An antigen-binding fragment as described in any one of items 1 to 8, which is a fragment, Fd fragment, Fv fragment, single-chain Fv(scFv) molecule, or dAb fragment. (Item 10) An isolated antibody or antigen-binding fragment thereof that binds to the same human PDGF-B epitope as the antibody or antigen-binding fragment described in any one of items 1 to 9. (Item 11) An isolated antibody or antigen-binding fragment that competes for binding to human PDGF-B with any one of the antibodies or antigen-binding fragments described in item 1 to 9. (Item 12) A method for producing an antibody or antigen-binding fragment as described in any one of items 1 to 11, (i) Introducing one or more polynucleotides encoding the light immunoglobulin chain of the antibody or fragment and the heavy immunoglobulin chain of the antibody or fragment into a host cell, (ii) Culturing the host cells in a growth medium under conditions favorable for the expression of the polynucleotide, (iii) A method comprising optionally isolating the antibody or fragment from the host cells and / or the culture medium in which the host cells are growing. (Item 13) An antibody or antigen-binding fragment produced by the method described in item 12. (Item 14) An injection device or container containing an antibody or antigen-binding fragment as described in any one of items 1-11 and 13. (Item 15) A pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof that binds to human PDGF-B as described in any one of items 1 to 11 and 13, a pharmaceutically acceptable carrier or diluent, and optionally one or more additional therapeutic agents. (Item 16) The pharmaceutical composition according to item 15, wherein the one or more additional therapeutic agent comprises an iron supplement. (Item 17) A method for preventing or treating pulmonary arterial hypertension (PAH) in a patient requiring prevention or treatment of PAH, comprising administering to the patient an effective amount of an antibody or antigen-binding fragment thereof described in any one of items 1 to 11 and 13, or a pharmaceutical composition described in item 15 or 16. (Item 18) The method according to item 17, wherein the antibody or its antigen-binding fragment is administered subcutaneously, intravenously, intradermally, orally, or intramuscularly. (Item 19) The aforementioned PAH causes a condition selected from the group consisting of thickening of the pulmonary artery of the subject, a decrease in the stroke volume of the subject, a decrease in the right ventricular output of the subject, and a decrease in the survival time of the subject. The method according to item 17 or 18, wherein the administration of the antibody or antigen-binding fragment treats the condition or reduces the severity of one or more symptoms of the condition. (Item 20) An antibody or antigen-binding fragment thereof, as described in any one of items 1-11 and 13, for use in the treatment of patients with PAH. (Item 21) A composition comprising one or more antibodies or antigen-binding fragments thereof as described in any one of items 1 to 11 and 13, for use in the treatment of PAH. (Item 22) Use of an isolated antibody or its antigen-binding fragment as described in any one of items 1-11 and 13 in the manufacture of a pharmaceutical product for the treatment of a patient with PAH. [Brief explanation of the drawing]

[0042] [Figure 1] This graph shows the screening results for monoclonal anti-PDGF antibodies derived from VI-3 mice. [Figure 2] This is a schematic diagram showing studies conducted to determine the binding reaction rates of 17 anti-PDGF-B antibodies at 25°C and 37°C. [Figure 3A] This is a schematic diagram illustrating a cross-competition study among purified anti-PDGF-BB monoclonal antibodies. [Figure 3B] This matrix shows 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 reactions for each antibody combination tested are shown. [Figure 4A] Five potent anti-PDGF-B antibodies were identified that blocked more than 70% of PDGF-BB. Three of these antibodies blocked both PDGF-BB and PDGF-AB with an IC50 value range of 0.23–1.8 nM, two blocked only PDGF-BB with an IC50 value range of 0.55–0.64 nM, six moderate anti-PDGF-B antibodies blocked more than 45% of PDGF-BB. Two of these antibodies blocked both PDGF-BB and PDGF-AB with an IC50 value range of 2.8–13 nM, four blocked only PDGF-BB with an IC50 value range of 0.64–2.8 nM, and six of these antibodies were nonblockers. [Figure 4B]Five potent anti-PDGF-B antibodies were identified that blocked more than 70% of PDGF-BB. Three of these antibodies blocked both PDGF-BB and PDGF-AB with an IC50 value range of 0.23–1.8 nM, two blocked only PDGF-BB with an IC50 value range of 0.55–0.64 nM, six moderate anti-PDGF-B antibodies blocked more than 45% of PDGF-BB. Two of these antibodies blocked both PDGF-BB and PDGF-AB with an IC50 value range of 2.8–13 nM, four blocked only PDGF-BB with an IC50 value range of 0.64–2.8 nM, and six of these antibodies were nonblockers. [Figure 4C] This table summarizes the characteristics of anti-PDGF-B antibodies in blocking ELISA using soluble PDGF-BB and PDGF-AB, as well as plate-captured PDGFR-beta. [Figure 5A] Figure 5A is a graph showing that 13 out 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] Furthermore, as shown in Figure 5B, this graph shows that two anti-PDGF-B antibodies were activated by human PDGF-BB. [Figure 5C] Figure 5C is a graph showing that 8 out of 17 anti-hPDGF-B antibodies inhibited 5 nM human PDGF-AB with IC50 values ​​ranging from 99 pM to 50 nM and maximum inhibition rates ranging from 32% to 99%. H4H13132P and H4H13145P blocked up to baseline with IC50 values ​​of 8.8 and 2.6 nM, respectively. [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 with maximum inhibition rates ranging from 39% to 98%. [Figure 6A] This graph shows chromatograms from samples forming the most distinct 2:2 mAb:human PDGF-BB species in which no detectable higher-order complexes were observed. [Figure 6B] This graph shows chromatograms from samples forming the most distinct 2:2 mAb:human PDGF-BB species in which no detectable higher-order complexes were observed. [Figure 6C] This graph shows chromatograms from samples forming the most distinct 2:2 mAb:human PDGF-BB species in which no detectable higher-order complexes were observed. [Figure 7] This graph shows that H4H13145P is even more potent than H4H13132P against both human and cynomolgus monkey PDGF-BB protein. [Figure 8] This table shows that commercially available monkey PDGF-BB exhibited specific binding to anti-PDGF-B mAbs H4H13132P and H4H13145P. [Figure 9] Western blot analysis of the antibody-conjugated epitope H4H13145P confirms its binding to the linear epitope of human rPDGF-BB. [Figure 10] This is a panel of graphs showing that anti-PDGF-B provided superior efficacy in two clinically relevant endpoints of PAH (right ventricular pressure and hypertrophy) when directly compared with anti-PDGFR-β. [Figure 11] This panel of graphs shows that in this rat model of PAH, prophylactic treatment with anti-PDGF-B reduced both the hemodynamic endpoint (RVSP) and right ventricular hypertrophy. [Figure 12] This is a panel of graphs showing that anti-PDGF-B robustly improved survival time in a severe MCT rat model of PAH. [Figure 13A] This is a panel of graphs showing that low doses of 1 mg / kg of sc (anti-PDGF-B) provided efficacy in clinically relevant endpoints of PAH (right ventricular pressure). [Figure 13B] This 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]This panel of pericyte images shows that systemic delivery of anti-PDGF-Rβ antibody (2C5, REGN764) effectively depleted pericytes, while anti-PDGF-BB antibody at 3 mg / kg did not have a clear effect in a retinal angiogenesis (RVD) model. [Figure 15] This panel of pericytes shows that a higher dose (25 mg / kg) of H4H13145P has a moderate effect on pericyte depletion in the RVD model. [Figure 16] This panel contains graphs and tables showing the pharmacokinetic profiles of anti-PDFG-B antibodies in C57BL / 6 wild-type mice at three single subcutaneous doses of 0.1, 1, or 10 mg / kg. [Figure 17] This is a schematic diagram illustrating a study design to evaluate the effects of chronic treatment of healthy adult C57BL mice with high doses of anti-PDGF-B antibodies on gastrointestinal (GI), pulmonary, or cerebral vascular permeability. [Figure 18] This panel of graphs shows that administration of anti-PDGF-B and anti-PDGFRβ antibodies did not significantly interfere with weight gain in animals. [Figure 19] This graph shows that there were no significant changes in gastrointestinal (GI) fluid retention / edema in healthy mice after treatment with isotype control IgG, anti-PDGF-B, and anti-PDGFRβ antibodies. [Figure 20] This panel of graphs and images shows that treatment with anti-PDGF-B and anti-PDGFRβ antibodies in mice did not result in significant changes in vascular permeability in the small intestine. [Figure 21] This graph shows that there was no significant change in gastric GI fluid retention / edema in healthy mice and captopril-treated mice after treatment with anti-PDGF-B antibodies and anti-PDGFRβ antibodies. [Figure 22] This panel of graphs and images shows that treatment with anti-PDGF-B and anti-PDGFRβ antibodies in mice did not result in significant changes in gastric vascular permeability. [Figure 23]This graph shows that there was no significant change in pulmonary GI fluid retention / edema in healthy mice and captopril-treated mice after treatment with anti-PDGF-B antibodies and anti-PDGFRβ antibodies. [Figure 24] This panel of graphs and images shows that treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not result in significant changes in pulmonary vascular permeability in healthy and captopril-treated mice. [Figure 25] This graph shows that treatment with anti-PDGF-B antibodies and anti-PDGFRβ antibodies did not result in significant changes in glycemic acid (GI) fluid retention / edema in the brain of healthy mice and captopril-treated mice. [Figure 26] This panel of graphs and images shows that treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not result in significant changes in cerebral vascular permeability in healthy and captopril-treated mice. [Figure 27] This is a panel of graphs showing the inhibition of PDGF-induced calcium flux in human pulmonary artery smooth muscle cells (HPASMCs) by anti-PDGF-B antibodies. [Figure 28] This panel displays graphs showing the inhibition of PDGF-induced cell proliferation in human pulmonary artery smooth muscle cells (HPASMCs) by anti-PDGF-B antibodies. [Figure 29] This is a panel of graphs showing the fluorescence imaging plate reader (FLIPR) receptor internalization assay in pulmonary artery smooth muscle cells (PASMCs) pretreated with an anti-PDGF-B antibody-ligand complex. [Figure 30] This graph shows a study in monoclotaline-treated rats for catheter-based assessment of right ventricular pressure, demonstrating that anti-PDGF-B antibody treatment reduces right ventricular systolic pressure compared to isotype-controlled rats. [Figure 31] This graph shows that prophylactic treatment with anti-PDGF-B antibodies reduced right ventricular hypertrophy compared to isotype control rats. [Figure 32]This graph shows that the combination of the endothelin receptor antagonist macitentan and anti-PDGF-B treatment did not demonstrate any further survival benefit compared to monotherapy with an anti-PDGF-B antibody. [Modes for carrying out the invention]

[0043] Before describing the methods of the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and such methods and conditions may vary. Furthermore, since the scope of the present invention is limited only by the appended claims, it should be understood that the terms used herein are merely for describing specific embodiments and are not intended to be limiting.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the present invention, but preferred examples of methods and materials are described herein. All publications referenced herein are incorporated herein in their entirety by reference.

[0045] definition The terms “PDGF-B,” “PDGFB,” and “platelet-derived growth factor B” are synonymous with the human PDGF-B protein having the amino acid sequence of SEQ ID NO: 41 (see also UniProt acceptance number P01127). All references to proteins, polypeptides, and protein fragments herein are intended to refer to the respective human versions of the protein, polypeptide, or protein fragment unless explicitly identified as being of non-human origin (e.g., “mouse PDGF-B,” “monkey PDGF-B,” etc.). The circulation and pulmonary expression of PDGF-B, a potent mitogen for pulmonary smooth muscle cells and fibroblasts, are increased in both patients exhibiting idiopathic PAH and hypoxia / sugen PAH mouse models. The 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 beyond PDGFRβ by targeting signaling mediated by PDGFRαα, PDGFRαβ, and PDGFRββ, which work together to promote abnormal 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 its polymer (e.g., IgM) or its antigen-binding fragment. 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 hypervariable regions called complementarity-determining regions (CDRs), which are dotted with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FR of an antibody (or its antigen-binding fragment) may be identical to a human germline sequence or may be naturally or artificially modified. The amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0047] Substitution of one or more CDR residues, or elimination of one or more CDRs, is also possible. The scientific literature describes antibodies that can remove one or two CDRs for binding. Padlan et al. (FASEB J.1995,9:133-139), based on publicly available crystal structures, analyzed the contact region between antibodies and their antigens and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies where 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 are not in contact with the antigen can be identified by molecular modeling and / or empirically, based on previous studies. If a CDR or its residue is missing, it is usually replaced by an amino acid that occupies the corresponding position in another human antibody sequence or a consensus of such a sequence. The position for substitution within the CDR and the amino acid to be substituted can also be selected empirically. Empirical substitutions may be conserved or non-conserved.

[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 region of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. This disclosure includes antibodies and antigen-binding fragments derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in the framework and / or CDR region are mutated to corresponding residues in the germline sequence from which the antibody originated, or to corresponding residues in another human germline sequence, or to conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily produce many antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof starting from the heavy and light chain variable domain sequences disclosed herein. In certain embodiments, V H and / or V LIn other embodiments, all of the framework and / or CDR residues within the domain are mutated to revert to residues found in the original germline sequence from which the antibody originated. In other embodiments, only certain residues are mutated to revert to the original germline sequence, for example, only the mutated residues are found within the first eight amino acids of FR1 or within the last eight amino acids of FR4, or only the mutated residues are found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to corresponding residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally originated). Furthermore, the antibodies of this disclosure may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, certain individual residues are mutated to corresponding residues in a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Once obtained, antibody 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 (if applicable), or reduced immunogenicity. Antibodies and antigen-binding fragments obtained in this general manner are included in this disclosure.

[0050] The Disclosure also includes fully human anti-PDGF-B monoclonal antibodies comprising any variant of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the Disclosure includes anti-PDGF-B antibodies comprising HCVR, LCVR, and / or CDR amino acid sequences having any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions.

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

[0052] The term "pulmonary hypertension" ("PH") is used to describe high blood pressure in the lungs from any cause. On the other hand, the term "hypertension" or "high blood pressure" refers to high blood pressure in the arteries throughout the body.

[0053] The term "pulmonary 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 higher and pulmonary capillary or left atrial pressure of 15 mmHg or lower. These pressures are typically measured in resting subjects using right heart catheterization. If left untreated, PAH leads to death (on average) within 2.8 years of diagnosis.

[0054] The World Health Organization (WHO) provides a clinical classification of five groups of PAH (as described in Simonneau, et al. J Am Coll Cardiol. 2013; 62(25_S), the full content of which is incorporated herein by reference): 1. Pulmonary hypertension (PAH) 1.1. Idiopathic 1.2. Hereditary 1.2.1.BMPR2 1.2.2.ALK1, ENG, SMAD9, CAV1, KCNK3 1.2.3. Unknown 1.3. Drug and Toxin Induction 1.4. Related to the following: 1.4.1. Connective tissue diseases 1.4.2.HIV infection 1.4.3. 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 heart 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 3.2. Interstitial Lung Disease 3.3. Other lung diseases with a mixture of restrictive and obstructive patterns 3.4. Sleep-disordered breathing 3.5. Alveolar hypoventilation impairment 3.6. Chronic exposure to high altitudes 3.7. Developmental abnormalities 4. Chronic thromboembolic pulmonary hypertension (CTEPH) 5. Pulmonary hypertension with unknown multifactorial 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. Other: Tumor obstruction, fibrous mediastinitis, chronic renal failure due to dialysis, segmental PH.

[0055] In one embodiment, the subjects who may benefit from the method of this disclosure are subjects having a Group I (WHO) PAH.

[0056] At baseline (e.g., at diagnosis), PAH can be mild, moderate, or severe, as measured, for example, by the WHO functional class, a measure of disease severity in patients with PAH. The WHO functional class is an adaptation of the New York Heart Association (NYHA) system and is routinely used, for example, to qualitatively assess activity tolerance when 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 without limitations on physical activity; Normal physical activity does not cause excessive shortness of breath or fatigue, chest pain, or dizziness; Class II: Pulmonary hypertension that causes slight limitations in physical activity; The patient is comfortable at rest; normal physical activity causes excessive shortness of breath or fatigue, chest pain or dizziness; Class III: Pulmonary hypertension resulting in significant limitation of physical activity; the patient is comfortable at rest; less activity than usual causes excessive shortness of breath or fatigue, chest pain or dizziness, and Class IV: Pulmonary hypertension in which no physical activity can be performed without symptoms; the patient shows signs of right heart failure; shortness of breath and / or fatigue may be present even at rest; discomfort is exacerbated by any physical activity.

[0057] In one embodiment, subjects who may benefit from the method of the disclosure are subjects who, at baseline, have a WHO class I PAH (e.g., a group I (WHO) PAH). In another embodiment, subjects who may benefit from the method of the disclosure are subjects who, at baseline, have a WHO class II PAH (e.g., a group I (WHO) PAH). In yet another embodiment, subjects who may benefit from the method of the disclosure are subjects who, at baseline, have a WHO class III PAH (e.g., a group I (WHO) PAH).

[0058] As used herein, “subjects” refers to animals such as primates (humans, non-human primates, e.g., monkeys and chimpanzees), non-primates (cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses and whales), and mammals.

[0059] In one embodiment, the subjects are, as described herein, people being treated or evaluated for PAH, e.g., Group I (WHO) PAH; people at risk for PAH, e.g., Group I (WHO) PAH; people having PAH, e.g., Group I (WHO) PAH; and / or people being treated for PAH, e.g., Group I (WHO) PA).

[0060] As used herein, the terms “treat” or “cure” refer to a beneficial or desired outcome, including but not limited to the alleviation or improvement of one or more symptoms associated with PAH, e.g., Group I (WHO) PAH. “Cure” may also mean, in the absence of treatment, delaying the course of the disease, reducing the onset of symptoms of the disease, reducing the severity of late-onset disease, or extending survival compared to expected survival. For example, a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., at least about 10% on a clinically acceptable scale of the disease or disorder), or a delayed onset of symptoms (e.g., days, weeks, months, or years) would be considered an effective treatment.

[0061] Terms such as "specifically binds" or "binds specifically to" mean that an antibody or its antigen-binding fragment forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least approximately 1 × 10⁻⁶. -6 It can be characterized by an equilibrium dissociation constant less than or equal to M (for example, a smaller K). D(This shows a stronger binding.) Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis and surface plasmon resonance. As described herein, antibodies that specifically bind to PDGF-B are identified by surface plasmon resonance, e.g., BIACORE®. Furthermore, as used herein, a polyspecific antibody that binds to one domain of PDGF-B and one or more additional antigens, or a bispecific antibody that binds to two different regions of PDGF-B, is nevertheless considered a “specifically binding” antibody.

[0062] The term "high affinity" antibody is defined as having at least 10 when measured by surface plasmon resonance, e.g., BIACORE® or solution affinity ELISA. -7 M, preferably 10 -8 M, more comfortable 10 -9 M, even more more 10 -10 M, even more more 10 -11 M's K D This refers to mAbs that have binding affinity to PDGF-B, which is represented as follows.

[0063] The terms "slow dissociation rate," "Koff," or "kd" refer to a surface plasmon resonance, such as 1 × 10⁻¹⁶, as determined by BIACORE®. -3 s -1 The following is preferably 1 × 10 -4 s -1 This means that the antibody that dissociates from PDGF-B at the following rate constants should be described.

[0064] The “antigen-binding portion” of an antibody, the “antigen-binding fragment” of an antibody, and similar terms, as used herein, include natural, enzymatically available, synthetic, or genetically modified polypeptides or glycoproteins that specifically bind to an antigen to form a complex. The term “antigen-binding fragment” or “antibody fragment” of an antibody, 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 antibody or antibody fragment of the Disclosure may be conjugated with a therapeutic portion such as an antibiotic ("immunoconjugate"), 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 portion useful for the treatment of pulmonary arterial hypertension.

[0066] As used herein, “isolated antibody” is intended to mean an antibody that substantially does not contain other antibodies (Abs) with different antigen specificities (for example, an isolated antibody or fragment thereof that specifically binds to PDGF-B substantially does not contain any Abs that specifically bind to antigens other than PDGF-B).

[0067] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using BIACORE® systems (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0068] "K D When used herein, the term "equilibrium dissociation constant" 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 a paratope. A single antigen may have more than one epitope. Therefore, 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 the antigen to which an antibody binds. Epitopes can 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 can also be three-dimensional, i.e., they may consist of nonlinear 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, they may have specific three-dimensional structural features and / or specific charge features.

[0070] The terms “substantial identity” or “substantial identity,” when referring to a nucleic acid or fragment thereof, indicate that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), it has 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 with a reference nucleic acid molecule may, in some cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0071] When applied to polypeptides, the terms “substantial similarity” or “substantially identical” mean that two peptide sequences, when optimally aligned using predefined gap weights, such as by programmed GAP or BESTFIT, share at least 90% sequence identity, and more preferably at least 95%, 98%, or 99% sequence identity. Preferably, the non-identical residue positions 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 intended to substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the proportion or degree of similarity can be adjusted upward to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of amino acid groups 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 conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation 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” permutation 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 similarity measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between wild-type proteins and their mutant proteins. 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 best overlapping region between the query and search sequences (Pearson (2000) above). Another preferred algorithm for comparing the sequences of this disclosure with a database containing numerous sequences from different organisms is the computer program BLAST, in particular BLASTP or TBLASTN, with default parameters. For example, see 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, the antibody or antibody fragment for use in the methods of the present disclosure may be monospecific, bispecific, or polyspecific. A polyspecific antibody may be specific to a different epitope of one target polypeptide, or it may contain antigen-binding domains specific to epitopes of more than one target polypeptide.

[0074] When used herein, the term “therapeutic dose” is intended to include the amount of anti-PDGF-B antibody or its antigen-binding fragment that, when administered to a subject with PAH, e.g., Group I (WHO) PAH, is sufficient to achieve treatment of the disease (e.g., by reducing, improving, or maintaining one or more symptoms of the existing disease or disease) or to control the disease. “Therapeutic dose” may vary depending on the anti-PDGF-B antibody or its antigen-binding fragment, the method of administration of the anti-PDGF-B antibody or its antigen-binding fragment, the disease and its severity, as well as the patient’s 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] "Therapeutic dose" is also intended to include the amount of anti-PDGF-B antibody or its antigen-binding fragment that, when administered to a subject, is sufficient to improve the disease or one or more symptoms of the disease. Improving the disease includes delaying the course of the disease or reducing the severity of late-onset disease.

[0076] The “therapeutic effective dose” also includes the amount of anti-PDGF-B antibody or its antigen-binding fragment that produces several desired local or systemic effects with a reasonable benefit / risk ratio applicable to any treatment. The anti-PDGF-B antibody or its antigen-binding fragment used in the method 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 distinct 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 between the corresponding individual PDGF monomers. PDGF ligands exert their biological effects through interaction with the PDGF receptor (PDGFR). PDGFR is a single-pass, transmembrane, tyrosine kinase receptor consisting of a heterodimer or homodimer association of an alpha (a) receptor chain (PDGFR-alpha) and / or a beta (β) receptor chain (PDGF-B). Therefore, active PDGFR can consist of αα, ββ, or αβ receptor chain pairs. PDGFR shares a common domain structure containing five extracellular immunoglobulin (Ig) loops, a transmembrane domain, and a split intracellular tyrosine kinase (TK) domain. The interaction between dimeric PDGF ligands and PDGFR leads to receptor chain dimerization, receptor autophosphorylation, and intracellular signaling. It has been demonstrated in vitro that ββ receptors are activated by PDGF-BB and -DD, while 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 with pulmonary hypertension. They may be used alone or as adjunct therapies in combination with other known treatment sites or modes in the art for treating pulmonary hypertension, such as iron supplementation, dietary modifications to promote serum iron, and / or intravenous delivery of iron, blood transfusions, and iron-promoting agents, but not limited to these. They may be used in combination with additional antibodies specific to antigens other than PDGF-B, or in combination with other types of therapy.

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

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

[0081] The immunogen may be an immunogenic fragment of PDGF-B or DNA encoding that fragment. The immunogen may be PDGF-B bound to a fragment of the antibody's histidine tag and / or Fc region.

[0082] The amino acid sequence of full-length human PDGF-B is shown as sequence number 41.

[0083] In certain embodiments, antibodies that specifically bind to PDGF-B may be prepared using a peptide that extends by about 5 to about 20 amino acid residues from one or both of the N-terminus and C-terminus of the region described herein, beyond the specified region or beyond. In certain embodiments, any combination of the region or its fragments may be used to prepare PDGF-B specific antibodies. In certain embodiments, one or more of the regions of PDGF-B, or its fragments, may be used to prepare monospecific, bispecific, or multispecific antibodies.

[0084] Antibody antigen-binding fragments Unless otherwise specified, the term “antibody” as used herein should be understood to encompass an antibody molecule containing two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a “complete antibody molecule”), as well as its antigen-binding fragment. The “antigen-binding portion” of an antibody, the “antigen-binding fragment” of an antibody, and similar terms, as used herein, include naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. The term “antigen-binding fragment” or “antibody fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to PDGF-B. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. Antigen-binding fragments of an antibody may be derived from a complete antibody molecule using any suitable standard technique, such as proteolytic techniques or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding the antibody variable domain and (optionally) the constant domain. Such DNA is known and / or readily available from, for example, commercially available 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 biological techniques, for example, by arranging one or more variable and / or constant domains in appropriate positions, introducing codons, creating cysteine ​​residues, modifying, adding, or deleting amino acids.

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

[0086] The 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 related V H In antigen-binding fragments having a domain, V H and V L The domains can be arranged relative to each other in any preferred configuration. For example, the variable region is a dimer, V H -V H , V H -V L , or V L -V L It may contain dimers. Alternatively, the antigen-binding fragment of the antibody may be a monomer V. H or V L It may contain a domain.

[0087] In certain embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently bound to at least one constant domain. Non-limiting exemplary arrangements of variable and constant domains that may be found within the antigen-binding fragment of the antibody of this disclosure include (i)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 This includes. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may bind directly to each other or may be bound 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 result in mobile or semi-mobile binding between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies of this disclosure may bind to each other and / or to one or more monomers. H Or V L This may include any homodimer or heterodimer (or other polymer) among the variable and constant domain configurations listed above in non-covalent association with the domain (e.g., via disulfide bonds).

[0088] Similar to fully antibody molecules, antigen-binding fragments can be monospecific or polyspecific (e.g., bispecific). A polyspecific antigen-binding fragment of an antibody typically comprises at least two distinct variable domains, each capable of specifically binding to a distinct antigen or a different epitope on the same antigen. Any polyspecific antibody form, including the exemplary bispecific antibody forms disclosed herein, can be adapted for use in the context of the antigen-binding fragments of antibodies of this disclosure using common techniques available in the art.

[0089] This disclosure includes anti-PDGF-B antibodies and antigen-binding fragments having immunoglobulin chains containing the amino acid sequences described herein, as well as variants having cellular and / or in vitro post-translational modifications. For example, this disclosure includes antibodies and antigen-binding fragments thereof that specifically bind to PDGF-B containing the heavy and / or light chain amino acid sequences described herein (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3), 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 lack a C-terminal lysine.

[0090] This disclosure includes a recombinant method for producing an anti-PDGF-B antibody or its antigen-binding fragment or its immunoglobulin chain, wherein, for example, a polynucleotide is contained in a vector and / or operably linked to a promoter, (i) the antibody or antigen-binding fragment (e.g., heavy chain or its V H or immunoglobulins including HCDR1, HCDR2, and HCDR3, and / or light chains or their V LThe method comprises (ii) introducing one or more polynucleotides encoding the light and / or heavy immunoglobulin chains of an immunoglobulin (including its LCDR1, LCDR2, and LCDR3), (ii) culturing host cells (e.g., Chinese hamster ovary (CHO) cells, Pichia cells, or Pichia pastoris cells) under conditions favorable for polynucleotide expression, and (iii) optionally isolating an antibody, fragment, or chain from the host cells and / or the culture medium in which the host cells are growing. When producing an antibody or antigen-binding fragment containing one or more immunoglobulin chains, for example, an antibody containing two immunoglobulin heavy chains and two immunoglobulin light chains, co-expression of the chains in a single host cell results in the association of the chains intracellularly, on the cell surface, or extracellularly, for example, when such chains are secreted to form an antibody or antigen-binding fragment molecule. The method includes expressing only immunoglobulin heavy chains or only immunoglobulin light chains (e.g., any of those discussed herein, including mature fragments and / or their variable domains). Such chains are useful, for example, as intermediates in the expression of antibodies or antigen-binding fragments containing such chains. This disclosure relates to the products of such expression methods (e.g., antibodies, antigen-binding fragments, V H , or V L ) includes.

[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 this disclosure to produce human antibodies that specifically bind to PDGF-B.

[0092] A high-affinity chimeric antibody against PDGF-B, having a human variable region and a mouse constant region, is first isolated using VELOCIMMUNE® technology (see, for example, US6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies. VELOCIMMUNE® technology involves generating a transgenic mouse having a genome containing human heavy and light chain variable regions operably ligated to an endogenous mouse constant region locus, so that the mouse produces an antibody containing the human variable region and the mouse constant region in response to antigen stimulation. The DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably ligated to the DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing a fully human antibody.

[0093] Generally, VELOCIMMUNE® mice are challenged with the target antigen, and lymphocytes (such as B cells) are recovered from mice expressing antibodies. Lymphocytes are fused with myeloma cell lines to prepare immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the target antigen. DNA encoding the variable regions of the heavy and light chains can be isolated and ligated 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, antigen-specific chimeric antibodies or DNA encoding the variable domains of the light and heavy chains 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 features, including affinity, selectivity, and epitopes. The mouse constant region is replaced with a desired human constant region to produce a fully human antibody of this disclosure, e.g., wild-type or modified IgG1 or IgG4. The selected constant region may vary depending on the specific application, but the high-affinity antigen-binding and target-specific features reside in the variable region.

[0095] Generally, the antibodies of this disclosure have very high affinity when measured by binding to an antigen immobilized in either a solid or solution phase, typically about 10 -12 ~about 10 -7 M's K D The mouse constant region is replaced with a desired human constant region to generate the fully human antibody of this disclosure. The selected constant region may vary depending on the specific application, but the high affinity antigen binding and target specificity features reside in the variable region.

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

[0097] Two antigen-binding proteins or antibodies are considered bioequivalents if, for example, they are pharmaceutically equivalent or pharmaceutically equivalent, or pharmaceutically equivalent, when administered under similar experimental conditions, at the same molar dose, and in single or multiple doses, they do not exhibit significant differences in the rate and extent of absorption. Some antibodies are considered equivalent or pharmaceutically equivalent if their degree of absorption is equivalent but their absorption rates are not. Furthermore, they may be considered bioequivalents if such differences in absorption rates are intentional, reflected in the labeling, and are not considered medically significant for the particular drug product studied, for example, because they are not essential for achieving effective bodily drug concentrations in chronic use.

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

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

[0100] In one embodiment, two antigen-binding proteins are biologically equivalent if, under certain conditions or usage conditions, they both act by a common mechanism or mechanism of action, to a certain extent that such mechanism is known.

[0101] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Methods for measuring bioequivalence include, for example, (a) in vivo studies in humans or other mammals in which the concentration of an antibody or its metabolite is 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 appropriate acute pharmacological effect of an antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials to establish the safety, efficacy, or bioavailability or bioequivalence of an antibody.

[0102] Bioequivalent variants of the antibodies disclosed herein may be constructed, for example, by various substitutions of residues or sequences, or by deletions of terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity may be deleted or substituted with other amino acids to prevent the formation of unnecessary or inaccurate intramolecular disulfide crosslinks during regeneration. In other contexts, bioequivalent antibodies may include antibody variants that include amino acid changes that modify the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation.

[0103] Anti-PDGF-B antibody containing Fc variant According to certain embodiments of the present disclosure, an anti-PDGF-B antibody is provided, comprising an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, the present disclosure provides an Fc domain with C H 2 regions or C H The antibody contains an anti-PDGF-B antibody with mutations in three regions, which increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes where the pH is in the range of approximately 5.5 to 6.0). Such mutations may result in an increased serum half-life of the antibody when administered to animals.

[0104] All possible combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are contemplated within the scope of the present disclosure.

[0105] The present disclosure also includes anti-PDGF-B antibodies comprising a chimeric heavy chain constant (C H ) region, wherein the chimeric C H region comprises segments derived from the C H regions of more than one immunoglobulin isotype. For example, the antibodies of the present disclosure may comprise a chimeric C H region that combines a part or all of the C H 3 domain derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule with a part or all of the C H 2 domain derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule. According to certain embodiments, the antibodies of the present disclosure comprise a chimeric C H region having a chimeric hinge region. 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 hinge region, a human IgG2 hinge region, or a 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 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. Antibodies comprising the chimeric C H regions described herein may exhibit modified Fc effector functions in certain embodiments 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 the antibody In general, the antibodies of this disclosure may function by binding to PDGF-B. In some embodiments, the antibodies of this disclosure may bind to another antigen (cross-reactive antibody).

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

[0108] In one embodiment, the disclosure provides a fully human monoclonal antibody or an 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 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (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 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 at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. (iv) an HCDR1 domain having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an HCDR2 domain having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an HCDR2 domain having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an HCDR1 domain having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an HCDR2 domain having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an HCDR1 domain having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an HCDR1 domain having a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an HCDR2 domain having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and (v) 10 -7 The following K D It then binds to PDGF-B.

[0109] Certain anti-PDGF-B antibodies disclosed herein can bind to and neutralize 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 skilled in the art, including binding assays or activity assays described herein.

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

[0111] Antibodies specific to PDGF-B may or may not contain additional labels or moieties, or they may contain labels or moieties at the N-terminus or C-terminus. In one embodiment, the label or moiety is biotin. In a binding assay, the position of the label (if any) can 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 N-terminal biotin will be oriented so that the C-terminus 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 technologies This 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 continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids located within any of the aforementioned regions of the PDGF-B molecule (e.g., a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of discontinuous amino acids (or amino acid sequences) located in any 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, for example, the routine cross-blocking assay 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 cleavage 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 the amino acids in the polypeptide with which the antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, hydrogen / deuterium exchange involves deuterizing the protein of interest and then binding the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, and the exchangeable protons within the amino acids protected by the antibody complex undergo reverse exchange from deuterium to hydrogen at a slower rate than the exchangeable protons within the amino acids not part of the interface. As a result, the amino acids that form part of the protein / antibody interface can retain deuterium and therefore exhibit a relatively higher mass compared to amino acids not included in the interface. After antibody dissociation, protease cleavage and mass spectrometry are performed on the target protein to identify 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 and 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 from both consecutive amino acids or non-contiguous amino acids arranged in parallel by tertiary folding of proteins. Epitopes formed from consecutive amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five, or eight to ten amino acids in their own 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) targeting the same antigen according to the similarity of their binding profiles to chemically or enzymatically modified antigen surfaces (see US2004 / 0101920, which is incorporated herein by reference in its entirety). Each category may reflect unique epitopes that are distinctly different from or partially overlapping with those represented by other categories. This technique enables rapid filtering of genetically identical antibodies so that characterization can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired characteristics. Using MAP, the antibodies of this disclosure can be sorted into groups of antibodies that bind to different epitopes.

[0116] In a particular embodiment, an anti-PDGF-B antibody or its antigen-binding fragment binds to an epitope or fragment within one or more of the regions exemplified in human PDGF-B, as illustrated in SEQ ID NO: 41.

[0117] This disclosure includes human anti-PDGF-B antibodies that bind to the same epitope or portion of the same epitope as any of the specific exemplary antibodies described herein, or antibodies having the CDR sequence of any of the exemplary antibodies described herein. Similarly, this disclosure also includes anti-PDGF-B antibodies that compete with any of the specific exemplary antibodies described herein for binding to PDGF-B or PDGF-B fragments, or antibodies having the CDR sequence of any of the exemplary antibodies described herein.

[0118] Using common methods known in the art, it is readily possible to determine whether an antibody binds to the same epitope as the reference anti-PDGF-B antibody, or whether it competes for binding to it. For example, to determine whether a test antibody binds to the same epitope as the reference anti-PDGF-B antibody of this disclosure, the reference antibody can be conjugated to the PDGF-B protein or peptide under saturated conditions. The ability of the test antibody to bind to the PDGF-B molecule is then evaluated. If the test antibody can bind to PDGF-B after saturated 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 cannot bind to the PDGF-B chain after saturated binding with the reference anti-PDGF-B antibody, it may bind to the same epitope as the reference anti-PDGF-B antibody of this disclosure.

[0119] To determine whether an antibody competes for binding with a reference anti-PDGF-B antibody, the binding method described above is performed in two orientations. In the first orientation, the reference antibody is bound to the PDGF-B protein under saturated conditions, and then the binding of the test antibody to the PDGF-B molecule is evaluated. In the second orientation, the test antibody is bound to the PDGF-B molecule under saturated conditions, and then the binding of the reference antibody to the PDGF-B molecule is evaluated. In both orientations, if only the first (saturated) 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 competing for binding with a reference antibody does not necessarily bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

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

[0121] Next, further standard experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed deletion of the test antibody binding is indeed due to binding to the same epitope as the reference antibody, or whether stereoblocking (or another phenomenon) is the cause of the observed deletion of binding. These types of experiments 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] Immunoconjugate This disclosure encompasses human anti-PDGF-B monoclonal antibodies conjugated to a therapeutic portion ("immune conjugate"), such as an agent capable of reducing the severity of pulmonary arterial hypertension or improving at least one symptom associated with pulmonary arterial hypertension. As used herein, the term "immune conjugate" refers to an antibody chemically or biologically conjugated to a radiopharmaceutical, cytokine, interferon, target or reporter portion, enzyme, toxin, or therapeutic agent. An antibody may be conjugated to a radiopharmaceutical, cytokine, interferon, target or reporter portion, enzyme, toxin, or therapeutic agent at any position along the molecule, as long as it can bind to its target. An example of an immunoconjugate is an antibody-drug conjugate. In some embodiments, the agent may be PDGF-B, or a second different antibody against a cytokine such as IL-1, IL-6, or a chemokine such as TGF-β. The type of therapeutic portion 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 immune conjugates are known in the art (see, for example, WO05 / 103081). The preparation of immune conjugates and immunotoxins is generally well known in the art (see, for example, U.S. Patent No. 4,340,535). Immune conjugates are described in detail, for example, U.S. 7250,492, U.S. 7420,040, and U.S. 741,1046, each of which is incorporated herein by reference in whole.

[0123] Multispecific antibody The antibodies of the present disclosure can be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The antibodies of the present disclosure can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical bonding, 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 having 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 conjugated to a therapeutic moiety. Exemplary formats of bispecific antibodies that can be used in the context of the present disclosure include the use of a first immunoglobulin (Ig) C H3 domain and a second Ig C H3 domain, wherein the first and second Ig C H3 domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces the binding of the bispecific antibody to PDGF-B as compared to a bispecific antibody lacking the amino acid difference. Variations of the above-described bispecific antibody formats are intended to be within the scope of the present disclosure.

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

[0125] Therapeutic administration and formulations This disclosure provides therapeutic compositions comprising anti-PDGF-B antibodies or their antigen-binding fragments as discussed herein. Therapeutic compositions according to this disclosure may be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Numerous suitable formulations can be found in the prescription collection known to all pharmacists: 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 absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0126] The antibody dose may vary depending on the age and size of the recipient, the target disease, the pathological condition, and the route of administration. When the antibody disclosed herein is used to prevent or treat pulmonary arterial hypertension, it is advantageous to administer the antibody disclosed herein intravenously as a single dose of approximately 0.1 to approximately 100 mg per kg of body weight, more preferably as a single dose of approximately 5 to approximately 100 mg, approximately 10 to approximately 90 mg, or approximately 20 to approximately 70 mg per kg of body weight. The frequency and duration of treatment can be adjusted depending on the severity of the pathological condition. In certain embodiments, the antibody disclosed herein or its antigen-binding fragment may be administered as an initial dose of at least approximately 0.1 mg to approximately 800 mg, approximately 1 to approximately 500 mg, approximately 5 to approximately 300 mg, or approximately 10 to approximately 200 mg, approximately 10 to approximately 100 mg, or approximately 10 to approximately 50 mg. In certain embodiments, a second or more subsequent doses of the antibody or its antigen-binding fragment may be administered following an initial dose, in an amount that may be approximately the same as or less than the initial dose, with the subsequent doses spaced 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 apart.

[0127] Various delivery systems are known and may be used to administer the pharmaceutical compositions of this disclosure, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of delivery include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transnasal, epidural, and oral. The compositions may be administered by any convenient route, for example, by injection or bolus injection, by absorption through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and may be administered together with other bioactive agents. Administration may be systemic or topical. The pharmaceutical compositions may also be delivered in vesicles, specifically liposomes (see, e.g., Langer (1990) Science 249:1527-1533).

[0128] The use of nanoparticles for delivering the antibodies of this disclosure is also contemplated herein. Antibody-conjugated nanoparticles may be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles, as well as methods for their preparation and use, are described in detail in Arruebo, M., et al. 2009 ("Antibody-conjugated nanoparticles for biomedical applications" in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi:10.1155 / 2009 / 439389) (incorporated herein by reference). Nanoparticles for drug delivery are also described, for example, in US8277812, US8258256, US8257740, US8246995, and US8236330, each of which, as a whole, is incorporated herein by reference.

[0129] In certain circumstances, pharmaceutical compositions can be delivered by a controlled-release system. In one embodiment, a pump can be used. In another embodiment, a polymer material can be used. In yet another embodiment, the release control system can be positioned near the target of the composition, so that only a fraction of the systemic dose is required.

[0130] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, intravenous infusion, etc. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibodies or salts thereof in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, glucose-containing isotonic solutions, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injections thus prepared are preferably filled into suitable ampoules.

[0131] The pharmaceutical compositions of this disclosure can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, with respect to subcutaneous delivery, pen-type delivery devices readily find applications in the delivery of the pharmaceutical compositions of this disclosure. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there are no replaceable cartridges. Rather, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is emptied from the reservoir, the entire device is discarded.

[0132] Numerous reusable pen-type and auto-injector-type delivery devices have applications in subcutaneous delivery of the pharmaceutical compositions of this disclosure. Examples include AUTOPEN® (Owen Mumford, Inc., Woodstock, UK), DISETRONIC® pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), and BD pen (Becton Dickinson, Franklin). Examples of disposable pen delivery devices for use in subcutaneous delivery of the pharmaceutical compositions of this disclosure include, but are not limited to, OPTIPEN®, OPTIPEN® PRO, OPTIPEN® STARLET, and OPTICLIK® (Sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices for use in subcutaneous delivery of the pharmaceutical compositions of this disclosure include, but are not limited to, SOLOSTAR® pen (Sanofi-aventis), FLEXPEN® (Novo Nordisk), and KWIKPEN® (HUMALOG®), SURECLICK® Autoinjector (PENLET (Haselmeier, Stuttgart, Germany)), EPIPEN® (Mylan®), and HUMIRA® pen (Abbott Labs, Abbott Park, IL).

[0133] Beneficially, the above-described pharmaceutical compositions for oral or parenteral use are prepared into suitable unit dose formulations to accommodate the dose of the active ingredient. Such formulations in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antibody contained is generally about 5 to about 500 mg per dosage form in unit doses, and in particular, 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 disclosure includes injection devices (e.g., pre-filled syringes or pre-filled autoinjectors) or vials (e.g., glass or plastic vials) containing the antibody or antigen-binding fragment of the disclosure or the pharmaceutical composition thereof, including a pharmaceutically acceptable carrier.

[0134] Therapeutic use 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 intended for prophylactic use in patients at risk of developing pulmonary arterial hypertension. These patients include elderly or immunocompromised individuals due to disease or treatment with immunosuppressive therapy. The antibody of the present disclosure is intended to 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 may be delivered simultaneously with the antibody of the present disclosure, or they may be administered separately either before or after the antibody of the present disclosure. Patients who may receive the antibody or antigen-binding fragment or its pharmaceutically active composition include, for example, humans (e.g., elderly, e.g., 65 years of age or older), animals such as rabbits, mice, rats, cattle, pigs, dogs, primates, horses, or sheep.

[0135] In further embodiments of this disclosure, the antibody is used to prepare a pharmaceutical composition for treating patients suffering from pulmonary hypertension.

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

[0137] The anti-PDGF-B antibody of this disclosure may be formulated with and / or administered in combination with, for example, a VEGF antagonist, such as aflibercept described in US7,087,411, or other VEGF inhibitory fusion proteins, an anti-VEGF antibody or its antigen-binding fragment (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 can 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 from Fovista®, Opthotech Corp., Princeton, NJ, etc.], antisense molecules, ribozymes, siRNA, peptide bodies, nanobodies or antibody fragments directed towards PDGF ligands). In other embodiments, the anti-PDGF-B antibody of this disclosure may be 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 EGFRvlll-specific antagonist (e.g., an antibody that specifically binds to EGFRvlll), a cMET antagonist (e.g., an anti-cMET antibody), or an IGF1 R antagonist (e.g., an anti-IGF1 It may be formulated with and / or administered in combination with a R antibody or a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720).In some cases, the anti-PDGF-B antibodies of this disclosure may be combined, co-formulated, and / or administered in combination with PDGFR-alpha inhibitors (e.g., anti-PDGFR-alpha antibodies), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US2009 / 0142354 such as REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US2011 / 0027286 such as H1 H685P), etc. Other agents that may be beneficially administered in combination with the anti-PDGF-B antibodies of this disclosure include small molecule cytokine inhibitors and cytokine inhibitors that include 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, or their respective receptors.

[0138] The anti-PDGF-B antibodies of this disclosure may also be administered together with and / or co-formulated with antiviral agents, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, metal chelators, IFN-gamma, and / or NSAIDs. The anti-PDGF-B antibodies of this disclosure may also be administered as part of a treatment regimen, including radiotherapy and / or conventional chemotherapy (for example, in the context of methods to treat cancer or inhibit tumor growth).

[0139] Any of the additional therapeutic active ingredients described herein may be administered in combination with any of the anti-PDGF-B antibodies of this disclosure for the treatment of any disease or disorder for which administration of an anti-PDGFR-beta antibody would be beneficial, including, for example, any of the ocular diseases, fibrous diseases, vascular diseases, and / or cancers mentioned herein. For example, in the context of the treatment of an ocular disease (e.g., wet AMD, diabetic retinopathy, CRVO, or any of the other ocular diseases described herein), the anti-PDGF-B antibody of this disclosure may be formulated with and / or administered in combination with a VEGF antagonist, such as aflibercept described in US7,087,411 or other VEGF inhibitory fusion proteins, or an anti-VEGF antibody or its antigen-binding fragment (e.g., bevacizumab or ranibizumab).

[0140] In exemplary embodiments in which the anti-PDGF-B antibody of this disclosure is administered in combination with a VEGF antagonist (e.g., a VEGF trap such as aflibercept), including administration of a co-formulation comprising the anti-PDGF-B antibody and a VEGF antagonist, the individual components may be administered to a subject using various dosage combinations and / or co-formulated. For example, the anti-PDGF-B antibody may be administered to a subject 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 / or may be contained in a co-formulation containing a VEGF antagonist (e.g., For example, aflibercept (a VEGF trap) may be administered to the subject 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, and / or may be included in a co-formulation. Examples of anti-PDGF-B antibody / aflibercept dosage combinations provided herein include, for example, (i) 0.2 mg anti-PDGF-B antibody + 2 mg aflibercept, (ii) 0.5 mg anti-PDGF-B antibody + 2 mg aflibercept, (iii) 1 mg anti-PDGF-B antibody + 2 mg aflibercept, (iv) 3 mg anti-PDGF-B antibody + 2 mg aflibercept, and (v) 4 mg anti-PDGFR-beta antibody + 2 mg aflibercept. These combinations / co-formulations may be administered to subjects 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] Additional therapeutic active ingredients may be administered to the subject prior to the administration of the anti-PDGF-B antibody of this disclosure. For example, if the first component is administered one 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 the administration of the second component, the first component may be considered administered "before" the second component. In other embodiments, additional therapeutic active ingredients may be administered to the subject after the administration of the anti-PDGF-B antibody of this disclosure. For example, 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, and 72 hours after the administration of the second component, the first component may be considered to have been administered "after" the second component. In yet another embodiment, an additional therapeutic active ingredient may be administered to the subject simultaneously with the administration of the anti-PDGF-B antibody.

[0142] For the purposes of this disclosure, “simultaneous” administration includes, for example, the administration of an anti-PDGF-B antibody and an additional therapeutic active ingredient to a subject in a single dosage form (e.g., co-formulated) or in separate dosage forms administered to the subject within approximately 30 minutes of each other. When administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-PDGFR-beta antibody and the additional therapeutic active ingredient may be administered intravitreously, subcutaneously, etc.), or alternatively, each dosage form may be administered via different routes (e.g., the anti-PDGF-B antibody may be administered intravitreously and the additional therapeutic active ingredient may be administered systemically). In any event, administration of this ingredient in a single dosage form, in separate dosage forms via the same route, or in separate dosage forms via different routes is all considered “simultaneous administration” for the purposes of this disclosure. For the purposes of this disclosure, administration of an anti-PDGF-B antibody "before," "concurrently with," or "after" the administration of an additional therapeutic active ingredient is considered to be administration of an anti-PDGF-B antibody "in combination with" the additional therapeutic active ingredient.

[0143] This disclosure includes a pharmaceutical composition in which the anti-PDGFR-beta antibody of this disclosure is co-formulated with one or more additional therapeutically active ingredients described elsewhere in this specification.

[0144] The disclosure also includes additional therapeutic compositions comprising combinations of PDGF antagonists and VEGF antagonists. 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] Additional therapeutically active components may be administered before, concurrently with, or after the administration of the anti-PDGF-B antibody of this disclosure. For the purposes of this disclosure, such administration regimens are considered to be administration of the anti-PDGF-B antibody "combined" with one or more additional therapeutically active components.

[0146] Diagnostic use of antibodies The anti-PDGF-B antibodies of this disclosure may also be used, for example, to detect and / or measure PDGF-B in a sample for diagnostic purposes. An exemplary diagnostic assay for PDGF-B may include, for example, contacting a sample obtained from a patient with the anti-PDGF-B antibody of this disclosure, which may be labeled with a detectable label or reporter molecule, or used as a capture ligand for selectively isolating 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 is, 3 H, 14 C, 32 P, 35 S, or 125This may be a radioactive isotope such as 1, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure PDGF-B in a sample include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and fluorescence-activated cell sorting (FACS).

[0147] Samples that can be used in the PDGF-B diagnostic assay according to this disclosure include any tissue or body fluid sample obtainable from a patient that contains a detectable amount of PDGF-B or fragments thereof under normal or pathological conditions. Generally, a baseline or standard level of PDGF-B is first established by measuring the level of PDGF-B in a specific sample obtained from a healthy patient (e.g., a patient without pulmonary hypertension). 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 hypertension or symptoms associated with such a condition.

[0148] Antibodies specific to PDGF-B may or may not contain additional labels or moieties, or they may contain labels or moieties at the N-terminus or C-terminus. In one embodiment, the label or moiety is biotin. In a binding assay, the position of the label (if any) can 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 N-terminal biotin will be oriented so that the C-terminus of the peptide is distal to the surface. In some embodiments, the label may be a radionuclide, a fluorescent dye, or a detectable label such as an MRI-detectable label. The detectable label may be linked to an antibody that can be used in an imaging assay.

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

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

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

[0152] In further embodiments, administration of an anti-PDGF-B antibody or its antigen-binding fragment increases right ventricular output and / or cardiac index (CI) in a subject. Cardiac output ("CO") is defined as the amount of blood pumped out by the ventricles per unit time. Cardiac index ("CI") is a hemodynamic parameter that relates left ventricular output (CO) per minute to body surface area ("BSA"), and thus relates cardiac performance to the size of the individual. Using echocardiography and radionuclide imaging techniques, real-time changes in ventricular dimensions can be estimated, thereby calculating stroke volume, which, when multiplied by heart rate, yields cardiac output. BSA can be calculated using 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 approximately 4.0–8.0 L / min and a cardiac index of approximately 2.6–4.2 L / min per square meter. Subjects with PAH have a cardiac index of approximately 1.9–2.3 L / min per square meter (Ryan and Archer (2016) Circ Res 115:176-188).

[0153] Administration of an anti-PDGF-B antibody or its antigen-binding fragment to a subject with PAH in the method of this disclosure may improve other hemodynamic measurements in a subject with PAH, such as right atrial pressure, pulmonary artery pressure, terminal 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, terminal 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 skilled in the art.

[0154] Subjects without PAH have a right atrial pressure of approximately 1 mmHg to 5 mmHg, while subjects with PAH have a right atrial pressure of approximately 11 mmHg to 13 mmHg.

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

[0156] Subjects without PAH have pulmonary capillary wedge pressure in the presence of a terminal expiratory pressure of approximately 4 mmHg to 12 mmHg, while subjects with PAH have pulmonary capillary wedge pressure in the presence of a terminal expiratory pressure of approximately 9 mmHg to 11 mmHg.

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

[0158] Subjects without PAH have a heart rate of approximately 60 beats / min (bpm) to approximately 90 bpm, while subjects with PAH have a systemic arterial pressure of approximately 84 bpm to 88 bpm.

[0159] For objects without PAH, the rate is approximately 20 dyne seconds / cm². 5 ~Approximately 130 dynes / cm 5 Subjects with pulmonary vascular resistance of (or approximately 0.25 to 1.625 wood units) and PAH should have a pulmonary vascular resistance of approximately 1200 dyne-seconds / cm². 5 ~Approximately 1360 dynes / cm 5 It has pulmonary vascular resistance of (or approximately 15 to 17 wood units).

[0160] For objects without PAH, the rate is approximately 700 dyne seconds / cm². 5 ~Approximately 1600 dynes / seconds / cm 5 Subjects with systemic vascular resistance (or approximately 9 to 20 wood units) and PAH have a blood pressure of approximately 1840 dyne seconds / cm². 5 ~Approximately 2000 dyne seconds / cm 5 (Or approximately 23 to 25 wood units) of systemic vascular resistance.

[0161] The method of this disclosure may also improve other clinical parameters, such as lung function, in the subject being treated. For example, during or after treatment, the subject may have increased exercise capacity or activity, or a reduced Borg dyspnea index (BDI), as measured, for example, by the 6-minute walk distance (6MWD) test or an activity scale.

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

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

[0164] Any preferred measure of quality of life may be used. For example, the SF-36® Health Survey provides a self-report multi-item scale that measures eight health parameters: physical functioning, role limitation due to physical health problems, bodily pain, general health, vitality (energy and fatigue), social functioning, role limitation due to emotional problems, and mental health (psychological distress and psychological well-being). The survey also provides summaries of physical components and summaries of mental components. In one embodiment, the method of this disclosure targets 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 methods of this disclosure may also improve the prognosis of subjects being treated. For example, the methods of this disclosure may provide subjects with a reduced probability of clinical exacerbation events during the treatment period and / or a reduction from baseline in serum brain diuretic peptide (BNP) or NT pro-BNP or its N-terminal prohormone, NT-pro-BNP concentration, where the time from the initial diagnosis of the condition in the subject is approximately 2 years or less at baseline.

[0166] In various contexts, the time from initial diagnosis may be, for example, approximately 1.5 years or less, approximately 1 year or less, approximately 0.75 years or less, or approximately 0.5 years or less. Clinical exacerbation events (CWEs) include death, lung transplantation, hospitalization for PAH, atrial septal resection, initiation of additional pulmonary hypertension therapy, or a combination thereof. The time to clinical exacerbation of PAH is defined as the time from the initiation of treatment to the first occurrence of a CWE.

[0167] In one embodiment, the method of the present disclosure provides a reduction of at least about 15%, for example, at least about 25%, at least about 50%, or at least about 75% in BNP or NT-pro-BNP concentration from baseline.

[0168] In one embodiment, the method of the present disclosure provides a reduction of at least about 25%, for example, at least about 50%, less than at least about 75%, or at least about 80% in the probability of death during the treatment period, lung transplantation, hospitalization for pulmonary arterial hypertension, atrial septal resection, and / or initiation of additional pulmonary hypertension therapy.

[0169] The method of this disclosure may also extend the lifespan (extension of survival time) of subjects with PAH from the start of treatment, for example, to at least about 30 days.

[0170] A therapeutically effective dose of anti-PDGF-B antibody or its antigen-binding fragment for use in the method of this disclosure, approximately 0.05 mg to approximately 600 mg, for example, approximately 0.05 mg, approximately 0.1 mg, approximately 1.0 mg, approximately 1.5 mg, approximately 2.0 mg, approximately 10 mg, approximately 20 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 110 mg, approximately 120 mg, approximately 130 mg, approximately 140 mg, approximately 150 mg of each 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 It may be 0 mg, approximately 740 mg, approximately 750 mg, approximately 760 mg, approximately 770 mg, approximately 780 mg, approximately 790 mg, approximately 800 mg, approximately 810 mg, approximately 820 mg, approximately 830 mg, approximately 840 mg, approximately 850 mg, approximately 860 mg, approximately 870 mg, approximately 880 mg, approximately 890 mg, approximately 900 mg, approximately 910 mg, approximately 920 mg, approximately 930 mg, approximately 940 mg, approximately 950 mg, approximately 960 mg, approximately 970 mg, approximately 980 mg, approximately 990 mg, or approximately 1000 mg.

[0171] The amount of anti-PDGF-B antibody or antigen-binding fragment thereof contained in each 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 to a patient at a dose 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, 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, can be administered to a subject over a defined time course. The methods according to this aspect of the disclosure include continuously administering multiple doses of the active ingredient of the disclosure to a subject. As used herein, "administering sequentially" means that each dose of the active ingredient is administered to the subject at different time points, e.g., on different days, separated by a predetermined interval (e.g., time, day, week, or month). The disclosure includes methods that include continuously administering to a patient a single initial dose of the active ingredient, followed by one or more secondary doses of the active ingredient, and optionally followed by one or more tertiary doses of the active ingredient.

[0173] The terms “initial dose,” “secondary dose,” and “tertiary dose” refer to a time series of administrations of anti-PDGF-B antibody or its antigen-binding fragment or the combination therapy of the present disclosure. Thus, the “initial dose” is the dose administered at the start of the treatment regimen (also referred to as the “baseline dose”), the “secondary dose” is the dose administered after the initial dose, and the “tertiary dose” is the dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-PDGF-B antibody or its antigen-binding fragment, but may differ in terms of administration frequency. However, in certain embodiments, the amounts of anti-PDGF-B antibody or its antigen-binding fragment contained in the initial, secondary, and / or tertiary doses differ 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 “loading doses” at the start of the treatment regimen, with subsequent doses administered on a lower frequency basis (e.g., “maintenance doses”).

[0174] In certain exemplary embodiments of this disclosure, each secondary and / or tertiary dose is 1 to 26 doses from the immediately preceding dose (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 Administer after 3, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 / 2, or more weeks. The phrase "immediately preceding dose," as used herein, means a dose of anti-PDGF-B antibody or its antigen-binding fragment in a series of multiple doses, which is administered to the patient before the administration of the immediate following dose, which does not contain an intervention dose.

[0175] Methods according to this aspect of the Disclosure may include administering any number of secondary and / or tertiary doses to a patient. For example, in one particular embodiment, only a single secondary dose is administered to the patient. In another embodiment, two or more secondary doses (e.g., two, three, four, five, six, seven, eight, or more) are administered to the patient. Similarly, in one particular embodiment, only a single tertiary dose is administered to the patient. In another embodiment, two or more tertiary doses (e.g., two, three, four, five, six, seven, eight, or more) 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 to 2 weeks or 1 to 2 months after the most recent dose. 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 to 12 weeks after the most recent dose. In certain embodiments of this disclosure, the frequency at which secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment according to the individual patient's needs after clinical examinations.

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

[0178] In a combination therapy involving the administration of an anti-PDGF-B antibody or its antigen-binding fragment and at least one additional therapeutic agent to a subject, the antibody and the additional therapeutic agent may be administered to the subject simultaneously or substantially simultaneously, for example, in a single therapeutic dose, simultaneously with each other, or in two separate doses administered within approximately 5 minutes. Alternatively, the antibody and the additional therapeutic agent may be administered to the subject sequentially, for example, in separate therapeutic doses temporally separated from each other by more than approximately 5 minutes.

[0179] Accordingly, 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 additional therapeutic antibody or antibody, or antigen-binding fragment or fragment thereof. In one embodiment, the one or more additional antibodies or antibodies are selected from the group consisting of anti-Grem 1 antibody or antibody, anti-PDGFRβ antibody or antibody, anti-TLR4 antibody or antibody, anti-TLR2 antibody or antibody, anti-EDN1 antibody or antibody, and anti-ASIC1 antibody or antibody.

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

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

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

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

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

[0185] Suitable prostacyclin analogs include, but are not limited to, 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 therapies of this disclosure include, but are not limited to, chlorothalidone, indapamide, bendroflumethiazide, metrazone, cyclopentiazide, polythiazide, mefluside, chimapide, chlorothiazide, and hydrochlorothiazide.

[0188] Other examples of 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, omapatrilate, oxygen, and / or digoxin.

[0189] The method may also involve combinations of kinase inhibitors (e.g., BMS-354825, canertinib, erlotinib, gefitinib, imatinib, lapatinib, restautinib, ronafarnib, pegaptanib, peritinib, semaxanib, tandutinib, tipifarnib, batalanib, ronidamine, fasudil, leflunomide, bortezomib, imatinib, erlotinib, and gleevec) and / or elastase inhibitors.

[0190] Additional therapeutic active ingredients may be administered to the subject before the administration of the anti-PDGF-B antibody. For example, if the first component is administered one 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 the administration of the second component, the first component may be considered administered "before" the second component. In other embodiments, additional therapeutic active ingredients may be administered to the subject after the administration of the anti-PDGF-B antibody or its antigen-binding fragment. For example, 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, and 72 hours after the administration of the second component, the first component may be considered to have been administered "after" the second component.

[0191] In further embodiments, additional therapeutic active ingredients may be administered to a subject concurrently with the administration of the anti-PDGF-B antibody or its antigen-binding fragment of the Disclosure. For the purposes of the Disclosure, “concurrent” administration includes, for example, the administration of the anti-PDGF-B antibody and the additional therapeutic active ingredient to a subject in a single dosage form, or in separate dosage forms administered to the subject within approximately 30 minutes of each other. When administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-PDGF-B antibody and the additional therapeutic active ingredient may be administered intravenously, subcutaneously, intravitreally, etc.), or alternatively, each dosage form may be administered via a different route (e.g., the anti-PDGF-B antibody may be administered locally (e.g., intravitreally), and the additional therapeutic active ingredient may be administered systemically). In any event, administration of the component in a single dosage form, in separate dosage forms via the same route, or in separate dosage forms via different routes is all considered “concurrent” administration for the purposes of the Disclosure. For the purposes of this disclosure, the administration of an anti-PDGF-B antibody "before," "concurrently with," or "after" the administration of an additional therapeutic active ingredient is considered to be the administration of an anti-PDGF-B antibody or its antigen-binding fragment "in combination with" the additional therapeutic active ingredient. [Examples]

[0192] The following examples are provided to those skilled in the art to provide a complete disclosure and description of the methods and compositions of the present invention for preparation and use, and are not intended to limit the scope of what the inventors consider to be the invention. Unless otherwise specified, parts are parts by weight, molecular weight is the average molecular weight, temperature is in °C, and pressure is atmospheric pressure or near thereon.

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

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

[0195] B cells sorted by PES were isolated from 12 mice. Using Adam6 / VI-3, ULC1633, and ULC1635 mice, the antigen biotin-hPDGFBB or mPDGFBB was sorted, and 6387 B cells were collected. Eleven 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 the hIgG1 BST plasmid. Primary screening of Ag+ samples was performed by ELISA. A total of 854 Ag+ (81%) samples were identified (over 1000 MFIs were hPDFGBB (Peprotech)). Secondary screening of Ag+ samples was performed using blocking ELISA, blocking Luminex, Biacore, and bioassays. Mating with mice was analyzed using mouse Biacore and bioassays. The screening results for monoclonal antibodies derived from VI-3 mice are summarized in Figure 1 and Table 1 below. [Table 1]

[0196] The equilibrium dissociation constant (K) of human and mouse PDGF-B conjugated to the purified anti-PDGF-B monoclonal antibody of this disclosure. DThe values ​​were determined using a real-time surface plasmon resonance (SPR) biosensor instrument, MASS-1. All coupling studies were performed at 25°C and 37°C in 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) electrophoresis buffer (Figure 2). The HCA sensor surface was first derivatized with an amine coupling monoclonal mouse anti-human Fc antibody (GE, #BR100839) to individually capture anti-PDGF-B monoclonal antibodies. 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) at different concentrations, prepared in HBS-EHT electrophoresis buffer, were injected onto captured anti-PDGF-B monoclonal antibodies at a flow rate of 50 μL / min for 4 minutes. The dissociation of the PDGF-B reagent bound to the captured anti-PDGF-B monoclonal antibody was monitored in HBS-T electrophoresis buffer for 10 minutes. Using Scrubber 2.0c software, kinetic binding (k) was determined by fitting the real-time binding sensorgram to a 1:1 binding model with mass transport limitations. a ) and dissociation (k d The rate constant was determined. The binding-dissociation equilibrium constant (K) of different anti-PDGF-B monoclonal antibodies was determined. D The dissociation half-life (t1 / 2) was calculated from the dynamic rate constant as follows:

number

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

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

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

[0200] Example 2. Amino acid sequences of the heavy chain 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 to 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 antibody names used herein indicates a specific Fc region of the antibody. For example, an "H4H" antibody has a human IgG4 Fc. [Table 7]

[0201] Example 3. Cross-competition among 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 was captured by immersing an α-hFc-coated Octet biosensor in a well containing 50 μg / mL of anti-human PDGF-BB mAb for 3 minutes (Figure 3A). H4H hFc (isotype control) was used as a negative control. Unoccupied α-hFc Octet sensors were saturated by immersing them in a well containing a blocking mAb solution (200 μg / mL of H4H human Fc (isotype control) 1) for 4 minutes, and 100 nM of human PDGF-BB (R&D) was pre-incubated with 1 μM of anti-PDGF-BB mAb for at least 2 hours. Blocking mAb-saturated Octet biosensors were immersed in a well containing a pre-mixture of anti-PDGF-BB mAb and human PDGF-BB for 4 minutes. At the end of each cycle, the α-hFc Octet sensor was regenerated in 10 mM HCl. During analysis, self-background binding signals caused by mAb binding to the capture surface were 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 assays were performed using an Octet HTX instrument at 25°C with HBST + 0.1 mg / mL BSA electrophoresis buffer. The sensor type was anti-His, the capture flow rate / time was 3 minutes at 1000 rpm, and the sample injection flow rate / time was 1000 rpm for various durations. Figure 3B shows a matrix illustrating the binding response results of the antibody cross-competition assay. The affinity of the purified antibodies ranged from 0.002 nM to 2 nM.

[0203] Example 4. Characterization of anti-PDGF-B antibodies using ELISA. Anti-PDGF-B antibodies were characterized by blocking PDGF-BB and PDGF-AB, which bind to plate-captured PDGFR-B, in two blocking ELISA formats. To conduct 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 points of 3-fold serial dilutions of Abs from 100 nM + 100 pM final concentration PDGF-AB (R&D, 222-AB) or 60 pM final concentration PDGF-BB (R&D, 220-BB). 1 hour at room temperature. Detection: 1:10,000 SA-HRP.

[0205] As shown in Figures 4A-C, five potent anti-PDGF-B antibodies were identified that blocked more than 70%, and three of these anti-PDGF-B antibodies showed IC50 of 0.23-1.8 nM PDGF-B. 50 Both PDGF-BB and PDGF-AB, which have a range of values, are blocked, and the two anti-PDGF-B antibodies have an IC50 of 0.55-0.64 nM. 50 Only PDGF-BB with a range of values ​​was blocked; six moderate anti-PDGF-B antibodies blocked more than 45%; and two anti-PDGF-B antibodies blocked IC50 of PDGF-BB between 2.8 and 13 nM. 50 The four anti-PDGF-B antibodies block both PDGF-BB and PDGF-AB within a specified range, and exhibit IC50 levels of 0.64-2.8 nM. 50 Only PDGF-BB with a specified value range was blocked, while six anti-PDGF-B antibodies were non-blockers.

[0206] Example 5. Anti-PDGF-B antibody inhibits human PDGF-BB. To determine the activity of the anti-PDGF-B antibody, a bioassay was performed using PDGF-BB. For 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 using human PDGF-BB (Peprotech, catalog number 100-14B, lot number 111004, derived from E. coli) with 1:3 serial dilutions starting at 100 nM. Inhibition was determined by adding anti-PDGF-B, anti-PDGFRb (REGN2176, 08-R120731), and purified antibody (1:3 serial dilutions starting at 100 nM) to cells containing 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 reacted to 500 pM of hPDGF-BB, and IC 50 The values ​​ranged from 88 pM to 7.2 nM, and the maximum inhibition rate was 42-99%. Furthermore, as shown in Figure 5B, two anti-PDGF-B antibodies were activated by human PDGF-BB. Figure 5C shows that 8 out of 17 anti-hPDGF-B antibodies activated 5 nM human PDGF-AB in IC50. 50 The values ​​range from 99 pM to 50 nM, indicating that inhibition occurred with a maximum inhibition rate of 32% to 99%. H4H13132P and H4H13145P are ICs. 50 Values ​​of 8.8 and 2.6 nM blocked the baseline. Finally, Figure 5D shows that 10 of the 17 anti-hPDGF-B antibodies blocked 600 pM mouse PDGF-BB at IC50. 50 The values ​​ranged from 450 pM to 6.4 nM, indicating that the maximum inhibition rate was 39-98%. These results suggest that the six anti-PDGF-B antibodies were effective in increasing IC50. 50We demonstrate complete inhibition of human PDGF-BB at values ​​of 310 pM to 2 nM. Specifically, H4H13145P inhibits all three ligands—hPDGF BB, hPDGF AB, and mPDGF BB—up to baseline. The inhibition of PDGF-B activation by using exemplary anti-hPDGF-B antibodies in HEK293 / SRE-luc / hPDGFRβ cells is summarized in Tables 8-9 below. [Table 8]

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

[0209] H4H13145P is an IC for 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 scattering (SEC-MALLS) was used to evaluate 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 (equomolar ratio) was prepared in 1×PBS at pH 7.4 for each anti-PDGF-B mAb tested and incubated at room temperature for 3 hours before fractionation of total protein by SEC-MALLS. Under SEC-MALLS conditions, 100 mg (total protein) of each sample was double-injected into a GE Healthcare Superose 6 10 / 300 GL column in 1×PBS, pH 7.4 (mobile phase) with a run time of 90 minutes per injection.

[0211] H4H13145P (peak 1) formed a large complex (peak 2) distinct from human PDGF-BB, consistent with the 2:2 mAb:human PDGF-BB species (Figure 6A). Furthermore, H4H13145P (peak 1) formed a large complex (peak 2) distinct from human PDGF-BB, consistent with the 2:2 mAb:human PDGF-BB species (Figure 6B). Figure 6C shows an overlaid chromatogram from a sample forming the most distinct 2:2 mAb:human PDGF-BB species, where no detectable higher-order complexes were observed.

[0212] Generally, most anti-PDGF mAbs appeared to form complexes significantly different from human PDGF-BB, consistent with the 2:2 mAb:hPDGF-BB species, with little to no higher-order complexes ("paper dolls") observed. Smaller amounts of higher-order complexes, larger than those of the 2:2 mAb:hPDGF-BB species, were observed in samples H4H13132P, H4H13145P, and H4H13162P, but it is unclear whether these complexes formed as a result of interactions between hPDGF-BB and the monomeric or multimeric (HMW species) forms of the antibody present in varying amounts in each mAb sample. Alternatively, the higher-order complexes in these samples could be attributed to interactions between the monomeric antibody and the small amounts of multimeric hPDGF-BB detected. Samples H4H13148P and H4H13169P appeared to form the most distinct 2:2 mAb:hPDGF-BB species, in which no detectable higher-order complexes were present. Interestingly, although the complexes formed by these two samples had similar calculated molar masses, 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 was inherently more compact. A broad distribution 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 dissociated during the fractionation process, or that the sample did not reach equilibrium under the experimental conditions tested.

[0213] Example 7. Validation of PDGF-B monoclonal antibodies H4H13132P and H4H13145P in cynomolgus monkey PDGF-BB protein bioassays. 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 divided into 2.5 × 10⁶ wells. 580 μl / well of PDGF-BB at a cell / ml (20,000 cells / well) was added overnight at 37°C and 5% CO2. The dose-response of PDGF-BB was determined at 1–3 dilutions starting at 20 nM. To determine inhibition (detrmening), sequentially diluted 1:3 Ab samples were analyzed, starting at 500 nM with 500 pM PDGF-BB as a constant. The plates were incubated at 37°C and 5% CO2 for 5.5 hours. The plates were removed from the incubator and equilibrated at room temperature (RT) for approximately 30 minutes. 100 μl of One-glo substrate (equilibriumized at RT) was added to all wells of the plate and mixed on a plate shaker at RT for 10 minutes. Luminescence was measured using SpectraMaxi3X (PerkinElmer®). As shown in Figure 7, in this assay system, H4H13145P was identified as being more potent than H4H13132P for both human and cynomolgus monkey PDGF-BB protein.

[0214] The following assays were used to determine the efficacy of anti-PDGF-B antibodies in blocking cynomolgus monkey PDGF-BB-induced signaling in cells expressing the extracellular domain of monkey PDGFRβ.

[0215] Luciferase Bioassay The 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 monkey 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 was starved overnight (37°C, 5.0% CO2).

[0216] Generation of dose-response curves The dose-response curves for cynomolgus monkey PDGF-BB were determined by adding the 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, with wells without ligand added serving as negative controls. After adding cynomolgus monkey PDGF-BB, cells were incubated at 37°C, 5.0% CO2 for 5.5 hours, followed by equilibration at room temperature for 30 minutes. Equivolent volumes of ONE-Glo luciferase substrate were added to each well, and the plates were incubated at room temperature for a further 5 minutes. Relative light units (RLU) were measured using a SpectraMaxi3X plate reader, and the values ​​were analyzed by a four-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 monkey PDGF-BB-mediated mfPDGFRβ signaling in the HEK293 / SRE-Luc / mfPDGFRβ-ecto / hPDGFRb-cyto cell line. H4H13145P, H4H13132P, or the negative control antibody (REGN1945) was added to cells in a dual dose at concentrations ranging from 228 pM to 500 nM, followed by the addition of cynomolgus monkey PDGF-BB at 500 pM. The plates were incubated at 37°C in 5.0% CO2 for 5.5 hours and then equilibrated at room temperature for 30 minutes. Equivolutes of ONE-Glo luciferase substrate were added to each well, and the plates were incubated at room temperature for a further 5 minutes. Relative luminal units (RLU) were measured using a SpectraMaxi3X plate reader, and the values ​​were analyzed using a four-parameter logistic equation on a 10-point dose-response curve (GraphPad Prism).

[0218] result Ligand-mediated blockade of cynomolgus monkey PDGF-BB-mediated mfPDGFRβ signaling. 50% of the maximum activity level (EC2) in the HEK293 / SRE-Luc / mfPDGFRβ-ecto / hPDGFRb-cyto cell line50 The concentration of Sino Biologics cynomolgus monkey PDGF-BB Fc tag required to stimulate mfPDGFRβ signaling up to 167 pM (Figure 7). In the presence of a fixed concentration of cynomolgus monkey PDGF-BB, 50% of the maximum activity (IC) was achieved. 50 The antibody concentrations required to reduce PDGF-BB signaling up to IC5 were determined for H4H13132P, H4H13145P, and the isotype control antibody REGN1945 (an antibody raised against feline Fel d 1 and not bound to mfPDGFRβ). As shown in Figure 7, H4H13132P and H4H13145P respectively... 50 The values ​​of 781 pM and 16 pM effectively blocked cynomolgus monkey PDGF-BB signaling induced by a fixed concentration of 500 pM. In contrast, REGN1945, an IgG4 isotype control antibody, was ineffective in blocking cynomolgus monkey PDGF-BB signaling induced by cynomolgus monkey PDGF-BB.

[0219] Example 8. Verification of Biacore binding of commercially available monkey PDGF-BB reagent to anti-PDGF-B monoclonal antibodies H4H13132P and H4H13145P. To determine the binding kinetics of commercially available monkey PDGF-BB (Kingfisher, Sino Biological) to anti-PDGF-B mAbs H4H13132P and H4H13145P, approximately 250–350 RU of anti-PDGF-B mAbs were captured on a negative control (REGN1945) on an anti-hFc HCA chip of MASS-2 at 25°C. 90 nM human and monkey PDGF-BB were prepared and sequentially diluted 3-fold. Samples were injected at 25 uL / min for 3 minutes, and dissociation was monitored for 10 minutes. Reaction kinetic parameters were evaluated by fitting real-time data using a 1:1 binding model with mass transport constraints. 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-conjugated epitopes on PDGF-BB A key to understanding the various properties of antibodies is determining 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 major amino acid residues come together through protein folding. Linear epitopes may be preferred in applications where the protein target is completely or partially denatured during sample preparation prior to immunoassays such as Western blotting (WB). Therefore, Western blotting analysis of PDGF-BB with H4H13145P and H4H13132P was performed to determine whether these mAbs bind to linear epitopes.

[0221] Human recombinant PDGF-BB dimer protein (CF) from 1 μg of the R&D system was degraded on SDS-PAGE gradient gels at 5–20% under reducing (R) and non-reducing (NR) conditions. The following steps were used to detect PDGF-BB bands under both R and NR conditions using PDGF-BB mAb H4H13145P and a commercially available anti-PDGF-BB ab (R&D anti-human PDGF-BB ab: human PDGF-BB antibody AF-220-NA). For nonspecific blocking: PVDF membranes were blocked in 5% BSA at room temperature for 1 hour. For primary ab blotting: PVDF membranes were diluted overnight with 4C in 5% BSA TBST with (1) PDGF-BB mAb H4H13145P or (2) R&D anti-PDGF-BB mAb at 1.0 ug / ml. The membranes were washed in TBST wash buffer at room temperature for 10 minutes (3 times). Secondary antibody incubation involved incubating the membrane of the HRP-conjugated anti-mouse secondary antibody in 5% BSA TBST for 1 hour at room temperature. Membrane washing involved using TBST buffer for 10 minutes (3 times) at room temperature. The signal was expressed using Pierce® Western blot signal enhancer.

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

[0223] Example 10. Evaluation of PAH efficacy in a preclinical trial of H4H13145P Lead mAb-mediated PDGF-B blockade (H4H13145P) demonstrated robust therapeutic benefits 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 offer an effective non-vasodilator-based PAH therapy.

[0224] To evaluate the effects of the anti-PDGF-B antibody H4H13145P on pulmonary arterial hypertension, separate studies were conducted using a mouse model of chronic hypoxia-induced pulmonary arterial hypertension and a monocothalin rat model.

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

[0226] The following materials and methods were used in these studies.

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

[0228] The first study involved 14-16 week old male humanized PDGFRβ hu-hu Mice (MAID#1639) were used. Mice were divided into treatment groups based on weight to ensure similarity between groups with different starting body weights. Cages were either kept at approximately 21% O2 (normal pressure, normal oxygen) or placed in a 10% O2 (normal pressure, hypoxia) chamber (modified 6' semi-rigid isolator unit, Charles River) with adjusted N2 flow rate for stable inhalation of room air to maintain low O2 levels. Mice in the hypoxic chamber were subcutaneously administered 20 mg / kg of the VEGF receptor inhibitor Sugen5146 once a week for 6 weeks. Mice were administered antibodies for 3 weeks, starting on day 21, as outlined in Table 11. By the end of week 6, right ventricular systolic pressure (RVSP) was measured by right heart catheterization, and RV hypertrophy was calculated using the Fulton 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 to ensure similarity between groups with different starting body weights. Cages were either kept at approximately 21% O2 (normal pressure normal oxygen) or placed in a 10% O2 (normal pressure hypoxia) chamber (modified 6' semi-rigid isolator unit, Charles River) with adjusted N2 flow rate for stable inhalation of room air to maintain a low O2 level. Mice in the hypoxic chamber were subcutaneously administered 20 mg / kg of the VEGF receptor inhibitor Sugen5146 once a week for 6 weeks. Mice were administered antibodies to start on day 21 for 3 weeks, as outlined in Table 12. By the end of week 6, right ventricular systolic pressure (RVSP) was measured by right heart catheterization, and RV hypertrophy was calculated using the Fulton index as the weight ratio of RV to (LV + septum). Mouse serum was collected on day 42 for evaluation of the human antibody IgG.

[0230] Right heart catheterization and right ventricular systolic pressure Mice were anesthetized with isoflurane and maintained at approximately 37°C using a heated 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 portion of 5-0 silk suture was placed beneath the isolated jugular vein to retract the vessel cranially, and then the jugular vein was punctured with a 30-gauge needle. A pressure catheter (microchip catheter transducer SPR-1000, Millar Instruments, Inc.) was inserted into the opening of the jugular vein and advanced through the right atrium to the right ventricle. The catheter was connected to a pressure / volume meter (MPVS-300, Millar Instruments, Inc.) that measured both heart rate and 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 tracing (after 2 minutes of recording to allow pressure stabilization). The parameters analyzed were right ventricular systolic pressure (RVSP), heart rate (HR), and right ventricular pressure elevation rate (dP / dt max).

[0231] Right ventricular hypertrophy assessment After in vivo hemodynamic measurements, the animals were euthanized by bleeding under anesthesia, and the free wall of the rheic heart (RV), left ventricle (LV), and septal tissue were collected and weighted. RV hypertrophy was calculated using the Fulton index as the weight ratio of RV to (LV + septum). RV tissue was then cryopreserved (-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 Gyrolab Bioaffy® 200 CD by biotinylated mouse anti-human IgG1 / IgG4 monoclonal antibody (REGN2567) diluted in glycerol and detected by Alexa Fluor 647-conjugate mouse anti-human kappa antibody diluted in glycerol. Serum samples are processed as follows: 1) Dilute serum samples 1:50 with dilution buffer, and dilute a total of 8 standards with dilution buffer to a starting concentration of 2 μg / mL. 2) Dilute biotinylated mouse anti-human IgG1 / IgG4 to 100 ug / mL during washing. 3) Dilute Alexa Fluor 647-conjugate mouse anti-human kappa antibody to 10 ug / mL in washing buffer. 4) Centrifuge the captured and detected antibodies at 15,000 RCF for 5 minutes. 5) Further dilute the detection antibody in detection buffer until a final concentration of 0.5 ug / mL is reached (use only the top layer of the centrifuged detection antibody). 6) Using the plate map generated by GYROS, pack the sample, standard material, and wash buffer, capture the antibody on a 96-well PCR plate, and detect it.

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

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

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

[0236] Ultrasonic evaluation and analysis On the final day of each study, the pulmonary artery size and right ventricular function and dimensions of each mouse were evaluated using a high-frequency ultrasound system (Vevo 2100, VisualSonics). For evaluation, mice were anesthetized (with 1.5% isoflurane at a rate of 1.0 cc / mL of medical-grade air), their temperature was monitored with a rectal thermometer probe, and they were maintained at approximately 37°C using a heating platform (MouseMonitorS, Indus Instruments) and a heating lamp. Both brightness-mode (B-mode) and motion-mode (M-mode) imaging were used. B-mode imaging of the mouse heart 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 tracking of blood flow through the pulmonary artery. Right ventricular stroke volume (RV SV) was calculated from the product of PA CSA and VTI. Right ventricular output (RVCO) was calculated from the product of spheric volume (SV) and heart rate (HR). M-mode imaging was used to determine the right ventricular free wall (RVFW) thickness during diastole and systole. Before right ventricular pressure assessment, the animals were returned to their original cages.

[0237] Right ventricular pressure assessment Subsequently, right ventricular pressure was assessed in all treatment groups. Mice were anesthetized with isoflurane and maintained at approximately 37°C using a heated 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 portion of 5-0 silk suture was placed beneath the isolated jugular vein to retract the vessel cranially, and then the jugular vein was punctured with a 30-gauge needle. A pressure catheter (microchip 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 meter (MPVS-300, Millar Instruments, Inc.) that measured both heart rate and 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 tracing (after 2 minutes of recording to allow pressure stabilization). The parameters analyzed were right ventricular systolic pressure (RVSP), heart rate (HR), and right ventricular pressure elevation rate (dP / dt max). Serum / tissue collection and evaluation of right ventricular hypertrophy were performed.

[0238] After completing the measurement of right ventricular pressure, the catheter was removed and each animal was euthanized. The abdomen was opened and blood was collected from the superior vena cava for hematocrit assessment and serum collection. Next, the thoracic cavity was opened, and the middle lobe of the right lung was ligated and excised with 5-0 silk sutures, then placed in RNA (Sigma-Aldrich, catalog number R0901) and frozen at -80°C after 24 hours. The heart was excised from each animal, and the right ventricle (RV) was carefully excised 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 solution (PBS, pH 7.4) at 20–25 mmHg, followed by fixation with 10% neutral buffered formalin (NBF). The lungs were immersed in 10% NBF for 24 hours, then immersed in 70% ethanol for at least 48 hours, followed by tissue treatment and paraffin embedding. For animals that did not undergo lung perfusion fixation, the right lower lobe was ligated with 5-0 silk sutures, excised, weighed, and frozen in liquid nitrogen.

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

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

[0242] The administration 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 heated 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 portion of 5-0 silk suture was placed beneath the isolated jugular vein to retract the vessel cranially, and then the jugular vein was punctured with a 23-gauge needle. A pressure catheter (microchip 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 meter (MPVS-300, Millar Instruments, Inc.) that measured both heart rate and 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 tracing (after 2 minutes of recording to allow pressure stabilization). The parameters analyzed were right ventricular systolic pressure (RVSP), heart rate (HR), and right ventricular pressure elevation rate (dP / dt max).

[0244] Right ventricular hypertrophy assessment Following in vivo hemodynamic measurements, the animals were euthanized by bleeding under anesthesia, and the free wall of the rheic heart (RV), left ventricle (LV), and septal tissue were collected and weighted. RV hypertrophy was calculated using the Fulton index as the weight ratio of RV to (LV + septum). The RV tissue was then cryopreserved (-80°C) for biochemical analysis.

[0245] statistical analysis Quantitative data are expressed as mean ± standard deviation. Statistical analyses performed between two groups were conducted using t-tests. Comparisons over time were analyzed using two-way analysis of variance (ANOVA). Comparisons of treatment effects among multiple drug treatment groups were analyzed using 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 demonstrated superior efficacy in two clinically relevant endpoints of PAH when directly compared to anti-PDGFRβ. Body weight changes in hypoxic / sugen mice Mice exposed to hypoxia showed slower weight gain at 6 weeks compared to animals under normal oxygen conditions. Treatment with anti-PDGF-B antibody significantly attenuated weight loss compared to the isotype control hIgG4 treatment group. Treatment with anti-PDGFRβ antibody showed no effect on weight changes caused by hypoxia exposure (Figure 10). A significant difference was observed only between Hy / Su+ isotype control IgG and Hy / Su+ anti-PDGF-B at day 42 (**p<0.01).

[0247] Increased right ventricular pressure induced in hypoxia / sugen mice Catheter-based assessment of right ventricular pressure revealed a significant increase in right ventricular systolic pressure in isotype antibody-treated hypoxic / sugen mice at 6 weeks. Anti-PDGF-B antibody treatment significantly reduced the increase in right ventricular systolic pressure by 9 mmHg, while anti-PDGFRβ antibody also reduced right ventricular systolic pressure to a small 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 right ventricular weight in mice revealed significant changes in mice exposed to hypoxia / sugen (Figure 10). The ratio of right ventricular weight to left ventricle + septum weight provided an index of right ventricular hypertrophy (i.e., the Fulton index), and the 6-week hypoxia / sugen group showed a larger ratio compared to normal 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. The use of anti-PDGFRβ antibody did not reduce right ventricular hypertrophy in hypoxia / sugen exposed animals. Significant differences were observed between Hy / Su+ isotype control IgG, Hy / Su+ anti-PDGF-B (****p<0.0001), and Hy / Su+ anti-PDGFRβ (*p<0.05). A significant difference was also observed between Hy / Su + PDGFRβ and Hy / Su + anti-PDGF-B (*p<0.05).

[0249] In summary, anti-PDGF-B antibodies demonstrated superior efficacy in two clinically relevant endpoints of PAH (right ventricular pressure and hypertrophy) when directly compared with anti-PDGFRβ in hypoxia / 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. Elevated right ventricular pressure induced in monocotallin rats In Study 1, catheter-based assessment of right ventricular pressure revealed a significant increase in right ventricular systolic pressure in monocrotaline rats treated with isotype antibodies at 4 weeks. 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, monocrotaline + macitentan (***p<0.001), and monocrotaline + anti-PDGF-B (*p<0.05) (Figure 11).

[0251] Right ventricular hypertrophy induced in monochromatal rats Postmortem analysis of right ventricular weight in rats revealed significant changes in monocotalin rats treated with isotype control IgG. The ratio of right ventricular weight to left ventricle + septum weight provided an index of right ventricular hypertrophy (i.e., the Fulton index), and the monocotalin-treated rat group showed a larger ratio at 4 weeks compared to animals treated with saline, indicating the presence of right ventricular hypertrophy. (Prophylactic treatment with anti-PDGF-B antibody reduced right ventricular hypertrophy by approximately 40% compared to monocotalin in isotype control-treated rats (Figure 11). The use of the vasodilator macitentan did not reduce right ventricular hypertrophy in monocotalin rats. A significant difference was observed between monocotalin + isotype control IgG and monocotalin + anti-PDGF-B (*p<0.05).

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

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

[0254] Anti-PDGF-B has demonstrated efficacy in low-dose therapeutic treatment. Increased right ventricular pressure induced in hypoxia / sugen mice Catheter-based assessment of right ventricular pressure revealed a significant increase in right ventricular systolic pressure by week 6 in the isotype antibody-treated hypoxic / sugen mice. 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 group and the Hy / Su+ anti-PDGF-B group 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 right ventricular weight in mice showed significant changes in mice exposed to hypoxia / sugen (Figure 13A). The ratio of right ventricular weight to left ventricle + septum weight provides an index of right ventricular hypertrophy (i.e., Fulton index), and the 6-week hypoxia / sugen group showed a larger ratio compared to normal 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 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 analysis of circulating PDGF-B protein and human antibodies. Circulating levels of PDGF-B were significantly elevated in the hypoxic / sugen mouse group (Figure 13B). Measurement of human IgG in mouse serum confirmed a dose-dependent increase in antibody levels in the 1–25 mg / kg anti-PDGF-B treatment groups. Repeated subcutaneous administration of 1–25 mg / kg achieved nM levels of serum antibody, several thousand times higher than circulating PDGF-B (Figure 13B).

[0257] In summary, circulating PDGF-B 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 hypoxic / sugen mouse mode.

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

[0259] Targeting of 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 a rodent model. These effects were more impressive than any previous treatment or benchmark, including standard therapy, comparative clinical trial agents, and in-house PDGFRb blockers. PDGF-B blockers may offer an effective non-vasodilator-based PAH therapy.

[0260] Example 11. Safety evaluation of PDGFR-β blockade PDGF-B / PDGFRb signaling is involved in pericyte proliferation, migration, survival, and adhesion. Sprouting endothelial cells secrete PDGF-B, which binds to the pericyte-specific receptor PDGFRb, leading to pericyte recruitment and adhesion. Impairment of PDGF-B / PDGFRb signaling results in pericyte recruitment failure and reduced microvascular pericyte coverage, ultimately leading to endothelial hyperplasia, abnormal vascular morphogenesis, and microaneurysm formation. Mice ablated with endothelium-derived PDGF-B showed less than 52% normal pericyte density, which varied capillary and vein diameters, and exhibited receding capillary branching. Specific overexpression of PDGF-B in photoreceptor cells resulted in increased pericyte proliferation, but also increased astrocytes and endothelial cells. Long-term treatment with PDGF-B versus PDGF-Rb antagonists may potentially cause pericyte loss / vascular damage and subsequent edema and hemorrhage in humans. Furthermore, systemic toxicity of anti-PDGFRβ pegylated diFab (Celltech / Zymogenetics) has been reported in humans (peripheral edema in cancer patients), and the safety of long-term anti-PDGFR-B therapy remains unknown.

[0261] The inventors hypothesized that specific PDGF-B ligand blockade would allow the continuation of receptor signaling through the remaining PDGF ligands, resulting in less adverse effects on pericyte homeostasis. Systematic administration of anti-PDGF-B abs at 3 mg / kg sc did not cause loss of retinal pericytes in neonatal mice, but anti-PDGFRb abs were found to completely deplete retinal pericytes. Adult rodents were observed to be resistant to high-dose anti-PDGF-B and anti-PDGFRb treatments. Furthermore, high-dose anti-PDGFRb treatment for 8 months in adult mice did not induce any vascular damage, hemorrhage, or apparent edema.

[0262] Analysis of the effects of anti-PDGF-B antibodies on the retinal vascular system in P2 wild-type mice revealed no effect on pericyte coverage in anti-PDGF-BB treated retinas at a dose of 3 mg / kg. Systemic delivery of anti-PDGF-Rβ antibodies (2C5, REGN764) effectively depleted pericytes, but anti-PDGF-BB antibodies (H4H13132P and H4H13145P) showed no clear effect at 3 mg / kg in a retinal angiogenesis (RVD) model (Figure 14).

[0263] To determine the effect of anti-PDGF-B antibodies on the retinal vascular system of P2 WT mice, anti-PDGF-B antibodies were injected in P2 mice, and tissue was collected in P5 mice. All retinas were incubated with anti-NG2 antibodies to detect pericytes. Higher doses of anti-PDGF-B antibody (H4H13145P) were observed to have a moderate effect on pericyte depletion in the RVD model (Figure 15).

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

[0265] Example 12. Pharmacokinetic profile of anti-PDGF-B antibody The pharmacokinetic (PK) profiles of anti-PDGF-B antibodies were determined at three different doses in C57BL / 6 WT mice. Both anti-PDGF-B monoclonal antibodies used in this study cross-reacted with mouse PDGF-BB at sub-nanomolecular affinity at 37°C. Single subcutaneous doses of 0.1, 1, or 10 mg / kg were administered to 26-week-old female mice (total = 36 mice; 100% C57BL / 6 WT in a background) and analyzed at bleeding times of 6 hours, day 1, day 2, day 3, day 4, day 7, day 10, day 15, day 22, and day 30. 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 pharmacokinetic studies are listed in Table 16. [Table 16]

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

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

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

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

[0271] Example 13. Study on the vascular permeability of anti-PDGF-B antibodies The pathobiological mechanism of angioedema in response to 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 can 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, including ACE. Captopril, an ACE inhibitor, increases bradykinin levels by blocking the degradation of bradykinin, leading to vasodilation and increased vascular permeability.

[0272] A study was conducted to evaluate whether chronic treatment of healthy adult C57 mice with high doses of PDGF-BB antibody has any additional effects on gastrointestinal (GI), pulmonary, or cerebral vascular permeability. 11–16-week-old Taconic C57BL male mice were administered subcutaneously at 25 mg / kg control IgG, anti-PDGF-B, and anti-PDGFRβ antibodies twice weekly for 4 weeks, as shown in Figure 17. The Evans Blue (EB) assay was used to evaluate the effects of the 4-week treatment with 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. EB binding to albumin is used to quantify protein leakage as an indicator of increased vascular permeability. Under physiological conditions, endothelium is impermeable to albumin, so Evans blue-bound albumin remains restricted within 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 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 weight gain in the animals. Furthermore, no changes in the animals' behavior were observed.

[0274] Furthermore, as shown in Figure 19, in healthy mice, there was no significant change in GI fluid retention / edema in the small intestine after treatment with isotype control IgG, anti-PDGF-B, and anti-PDGFRβ antibodies. 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 alter the tissue edema in the small intestine caused by captopril. Similarly, Figure 20 shows that treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not significantly alter vascular permeability in the small intestine in mice. Treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not exacerbate captopril but caused acute increased vascular permeability in the small intestine.

[0275] Furthermore, as shown in Figure 21, treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not significantly alter gastric GI fluid retention / edema in healthy mice and captopril-treated mice. Captopril did not cause a statistically significant increase in hydrated body weight in the stomach. Similarly, Figure 22 shows that treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not significantly alter gastric vascular permeability in mice. Treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not exacerbate captopril but caused acute increased gastric vascular permeability.

[0276] Figure 23 shows results indicating that treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not significantly alter GI fluid retention / edema in the lungs of healthy and captopril-treated mice. Captopril did not cause a significant increase in lung weight. Similarly, as shown in Figure 24, treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not significantly alter vascular permeability in the lungs of healthy and captopril-treated mice. Acute captopril treatment did not cause vascular hyperpermeability in the lungs.

[0277] Furthermore, Figure 25 shows results indicating that treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not cause significant changes in glycemic acid (GI) fluid retention / edema in the brain in healthy and captopril-treated mice. Captopril did not cause a significant increase in brain weight. Similarly, as shown in Figure 26, treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not cause significant changes in vascular permeability in the brain in healthy and captopril-treated mice. Acute captopril treatment did not cause vascular permeability in the brain.

[0278] Overall, treatment with anti-PDGF-B and anti-PDGFRβ antibodies did not induce any significant tissue edema or changes in GI vascular permeability in mice. Therefore, 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 vascular pericytes in mice.

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

[0280] Using Scrubber 2.0c curve fitting software, the real-time coupling sensor gram is fitted to a 1:1 coupling model with mass transport constraints, thereby determining the association rate (k a ) and dissociation rate (k d The bond dissociation equilibrium constant (K) was determined. D The dissociation half-life (t1 / 2) was calculated from the dynamic velocity as follows:

number

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

[0282] Example 15. Antibody inhibition study of human PDGF-BB, PDGF-AB, or calcium flux combinations induced by PDGF-BB / AB using FLIPR with primary human pulmonary artery smooth muscle cells and primary human lung fibroblasts. For the calcium flux assay, primary human lung smooth muscle cells (Lonza, catalog no. CC-2581) and human lung fibroblasts (Lonza, catalog no. 2512) were seeded at a cell density of 10k / well on 96-well assay plates. Smooth muscle cells were cultured in the supplier-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] Once 100% confluence was reached in the wells, the PDGF-driven calcium flux was evaluated using the FLIPR® Calcium 5 Assay Kit (Molecular Devices, catalog no. R8185) on the FLIPR Tetra system (Molecular Devices) according to the manufacturer's protocol. Briefly, the complete medium was replaced with 50 μL of assay medium (DMEM-0.1% BSA-1×penicillin / streptomycin / L-glutamine). The calcium 5 dye was then reconstituted in buffer supplied in the presence of probenecid (Thermofisher, catalog no. P36400). 50 μL of the reconstituted calcium 5 dye was then added to the cells and incubated at 37°C in 5% CO2 for 1 hour before being placed on the FLIPR Tetra system. Individual dosing plates were prepared using human PDGF-BB (R&D Systems, catalog no. 220-GMP), human PDGF-AB (R&D Systems, catalog no. 222-AB), and PDGF-AB / PDGF-BB in a 3:1 ratio. To evaluate antibody inhibition, 1 nM concentrations of PDGF-AB, PDGF-BB, and the combined treatment PDGF-AB / BB were selected as constants. The antibodies were tested in the presence of 1 nM of PDGF protein H4H13145P or H4H13132P in the range of 500 nM to 0.06 nM (1:6 serial dilutions). Antibodies were pre-incubated with PDGF protein for 30 minutes before being added to cells via the FLIPR tetra system. Calcium flux traces were evaluated in both 60-second and 5-minute 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 in human lung smooth muscle cells for 60 seconds. Both antibodies exhibited IC50 in the range of 0.18 nM to 4.5 nM.50 The values ​​demonstrated inhibition of 1 nM PDGF treatment. H4H13145P inhibited 0.18 nM and 0.64 nM ICs, respectively. 50 These antibodies demonstrated greater efficacy against PDGF-BB than H4H13132P. These antibodies are more potent inhibitors of PDGF-BB alone than PDGF-AB and the PDGF-AB / BB (3:1) combination.

[0285] When recording calcium flux over a full 5-minute course, H4H13145P demonstrated a higher ability to neutralize PDGF-BB and prevent PDGF-driven calcium flux compared to H4H13132P. H4H13145P achieved IC50 of 0.36 nM for PDGF-BB inhibition, 16 nM for PDGF-AB inhibition, and 5.7 nM for the PDGF-AB / BB (3:1) combination. 50 Therefore, potent inhibition of all PDGF treatments was maintained. Isotyped control antibodies did not demonstrate 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 exhibited IC50 in the range of 0.59 nM to 1.6 nM. 50 The values ​​demonstrated inhibition of 1 nM PDGF treatment. H4H13145P inhibited 0.59 nM and 0.99 nM ICs, respectively. 50 Compared to H4H13132P, which has the same properties, H4H13145P demonstrated slightly higher efficacy in neutralizing PDGF-BB. H4H13145P also demonstrated slightly higher efficacy in neutralizing ICs of 0.16 nM and 0.54 nM, respectively. 50These antibodies were slightly more potent in neutralizing the PDGF-AB / BB treatment combination than H4H13132P, which has a value. These antibodies are more potent inhibitors of PDGF-AB alone than of PDGF-AB and the PDGF-AB / BB treatment combination.

[0287] When recording calcium flux over a full 5-minute course, H4H13145P demonstrated significantly higher ability to neutralize PDGF-BB, PDGF-AB, and concomitant treatment PDGF-AB / BB compared to H4H13132P. H4H13132P demonstrated lower ability to maximally inhibit the 1 nM constant of all PDGF treatments. H4H13145P neutralized 0.63 nM and 3.8 nM ICs, respectively. 50 Compared to H4H13132P, which possesses [specific trait], it showed greater efficacy in inhibiting PDGF-BB-driven calcium flux. The isotype control antibody did not demonstrate inhibition of PDGF-mediated calcium flux in human lung fibroblasts. [Table 21]

[0288] Example 16: Structural analysis of the antibody-PDGF-BB complex by cryo-electron microscopy (Cryo-EM). method Preparation of Fab fragments H4H13145P, H4H13132P, and H4H13127P IgG were processed using Fabricator enzyme (Genovis) according to the manufacturer's standard protocol, resulting in F(ab') 2 And it was cut into Fc fragments. F(ab') 2The Fab was 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 into a size exclusion chromatography (SEC) column (Superdex 200 Increase 15 / 300 GL, GE Healthcare) connected to an AKTA Avant 25 chromatography system (GE Healthcare) containing a run buffer with 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The peak fraction was pooled and concentrated in a 30 kDa cutoff centrifuge filter (Millipore Sigma) and subsequently used for complex preparation.

[0289] Complex preparation Recombinant human PDGF-BB protein (R&D) was mixed with either H4H13145P Fab or H4H13132P Fab in a molar ratio of 1:2.2. Excess, non-competitive H4H13127P Fab was further added to both 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 complex particles on the EM grid. After incubation at 4°C for 30 minutes, the samples were concentrated in a 30 kDa cutoff centrifuge filter (Millipore Sigma) until a concentration of 2.5 mg / mL was measured using a Nanodrop instrument (ThermoFisher).

[0290] Cryo-EM sample preparation and data acquisition The newly prepared PDGFBB-H4H13145P Fab-H4H13127 Fab complex or PDGFBB-H4H13132P Fab-H4H13127 Fab complex was mixed with approximately 0.15% Amphipole PMAL-C8 (Anatrace) in a 3.5 μL mixture immediately before pipetting onto an UltrAufoil R1.2 / 1.3, 300-mesh grid (Quantifoil). Excess liquid was absorbed using filter paper, and the mixture was rapidly frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (ThermoFisher) operated at 4°C and 100% humidity. The grid was then loaded into a Titan Krios G3i microscope (ThermoFisher) equipped with a K3 camera (Gatan). Using EPU software (ThermoFisher), approximately 7,000 videos were collected for both complexes in counting mode at a nominal magnification of 105,000 × (0.86 Å pixel size). Each video contained 46 dose fractions over a 2-second exposure. 2 The total amount obtained per unit was approximately 40 electrons.

[0291] Cryo-EM data processing and map generation Cryo-EM data were processed using Cryosparc v2.14.2. Motion correction was performed on the videos using patch motion correction, and CTF parameters were estimated using patch CTF estimation. Particles were initially picked using a Blob picker to generate a 2D class mean, which was then used as a template for subsequent template picking. After multiple rounds of 2D classification to remove junk particles, 249,832 and 429,948 particles remained in the PDGFBB-H4H13145P Fab-H4H13127 Fab complex and the PDGFBB-H4H13132P Fab-H4H13127 Fab complex, respectively. 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 purified to reconstructions at 3.4 Å and 3.5 Å resolution for the two complexes, respectively.

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

[0293] result The PDGFBB ligand is a homodimer containing 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 to two H4H13145P or H4H13132P Fab molecules and two H4H13127P Fab molecules. Both complexes exhibit 2x symmetry with two H4H13127P Fab molecules that bind to the outer edge of the β-sheet region. Both H4H13145P Fab and H4H13132P Fab bind to three interchain loops that also engage with PDGFRβ in the PDGFBB-PDGFRβ signaling complex (PDB:3MJG), which have similar binding sites. The data suggest that H4H13145P and H4H13132P antibodies bind to the terminals 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] Both the heavy and light chains of H4H13145P interact with the PDGFBB dimer via three interchain loops.

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

[0297] Dimer-versus-antibody interactions: 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. Loops 1 and 3 of chain 1 play a major role in the interactions.

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

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

[0300] Dimer-versus-antibody interactions: 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. Loops 1 and 3 of chain 1 play major roles in the interactions.

[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). The interactions between chain 2 of PDGFBB and the antibodies are fewer (two for H4H13145P and one for H4H13132P), but both interactions share the common R56 residue.

[0302] Example 17: Testing of antibody or small molecule inhibition of human PDGF-BB, PDGF-AA, or PDGF-DD-induced calcium flux using FLIPR or cell proliferation in primary human pulmonary artery smooth muscle cells. reagent Reagents used for 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 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; cerartinib (GB002), MedChemExpress, catalog number HY-109190; and imatinib mesylate, MedChemExpress, catalog number HY-50946.

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

[0304] Once 100% confluence was reached in each well, the PDGF-driven calcium flux was evaluated using the FLIPR® Calcium 5 Assay Kit on a FLIPR Tetra system (Molecular Devices) according to the manufacturer's protocol. In short, the complete medium was replaced with 50 μL of assay medium (DMEM-0.1% BSA-1×penicillin / streptomycin / L-glutamine). The calcium 5 dye was then reconstituted in buffer supplied in the presence of probenecid. 50 μL of the reconstituted calcium 5 dye was then added to the cells and incubated at 37°C in 5% CO2 for 1 hour before being placed on the FLIPR Tetra system. Serial dilutions and inhibition constants of human PDGF-AA, PDGF-BB, and PDGF-DD were generated on separate dosing plates. 0.5 nM PDGF-BB, 1 nM PDGF-DD, and 3 nM PDGF-AA were selected 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 the PDGF constants. Antibodies were pre-incubated with PDGF protein for 30 minutes and then added to cells via the FLIPR tetra system. Small molecule cerartinib and imatinib were pre-incubated for 30 minutes using cells in assay medium and calcium dyes in a concentration range of 10,000 nM to 8 nM (serial dilution 1:6). These calcium traces were analyzed for their maximum and minimum values ​​using SoftMax Pro Software (Molecular Devices).

[0305] Cell proliferation was evaluated using the 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 at PDGF constant for 30 minutes, while cells were pre-treated with the small molecule compounds imatinib and cerartinib before PDGF addition. Plates were imaged every 3 hours for 7 days, and proliferation was determined by measuring cell confluence in the wells on day 6 using IncuCyte-based analysis software (Sartorius).

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

[0307] Both cerartinib and imatinib completely inhibited PDGF-AA, PDGF-DD, and PDGF-BB-driven calcium flux and proliferation. This included PDGF-AA signaling via PDGFRa, PDGF-DD signaling via PDGFRb, and PDGF-BB signaling via both dimeric receptors. As shown in Table 22, cerartinib inhibited IC50 in the range of 6–27 nM. 50 In this cell-based assay with a value, it is more potent compared to imatinib. Imatinib is IC 50 H4H13145P was able to completely inhibit PDGF signaling with values ​​in the range of 110-650 nM. 50 However, H4H13145P was able to completely inhibit PDGF-BB-driven calcium flux and proliferation at concentrations of 0.11 nM and 0.14 nM, respectively. H4H13145P is more potent against PDGF-BB compared to the small molecule PDGFR inhibitors, cerartinib and imatinib (Table 22 and Figures 27-28). [Table 22]

[0308] Example 18: Testing of receptor-mediated antibody internalization in primary human pulmonary artery smooth muscle cells and primary human lung fibroblasts. reagent Reagents used for testing 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 no. CC-2581 lot no. 0000559495; normal human lung fibroblasts (NHLF), Lonza catalog no. CC-2512 lot no. 0000494609; DMEM medium, Thermofisher, catalog no. 11965092; bovine serum albumin solution, Millipore-Sigma, catalog no. A9576; smooth muscle cell medium, Lonza, CC-3182; pHrodo® Deep Red Antibody labeling kit, catalog no. P35355 Thermo Fisher; CellTrkr® Violet cell proliferation kit, catalog no. C34571 Thermo Fisher; PDGF-BB-R&D Systems, catalog no. 220-GMP; PDGF-AB-R&D Systems, catalog number 222-AB; Calcium 5 Assay Kit, Molecular Devices, catalog number R8185; Probenecid, Thermofisher, catalog number P36400; PDGFRa-APC, Abcam, catalog number AB119838; PDGFRb-APC, R&D Systems, catalog number FAB1263A; Isotyped Antibody-APC, Abcam, catalog number AB37391; DAPI Survivability Dye, Thermofisher, catalog number 62248; and TrypLE® Express Enzyme, Thermofisher, catalog number 12604013.

[0309] Experimental Procedure Antibody labeling: pHrodo Deep Red Antibody labeling was completed according to the user guide provided with the Invitrogen pHrodo® Deep Red Antibody Labeling Kit (catalog numbers P35355 and P35356). Antibody endocrine integration assays were performed using the following procedure:

[0310] Antibody internalization assay 1: (1) Seed 20,000 PASMCs per well in a 96-well plate and incubated overnight in complete medium at 37°C in 5% CO2; (2) Remove complete medium and wash once with PBS, then label cells with CellTrkr Violet dye for 15 minutes; (3) Remove CellTrkr solution; (4) Pre-conjugate (pHrodo) anti-PDGF-B mAb labeled with PDGF-BB or PDGF-AB to cells for 30 minutes at room temperature before processing; (5) Add antibody ligand solution to cells without pre-conjugation as a control; (6) Live cell imaging was acquired hourly for up to 21 hours using the Opera Phenix Imaging System; (7) Data analysis using Harmony 4.9 software.

[0311] Antibody internalization assay 2 (with receptor blocking): (1) Seed 20,000 PASMCs per well in a 96-well plate and incubated overnight in complete medium at 37°C in 5% CO2; (2) Remove complete medium and wash once with PBS, then label cells with CellTrkr Violet dye for 15 minutes; (3) Remove CellTrkr solution; (4) Add anti-PDGFRα (REGN1574) and anti-PDGFRβ (REGN764) at 500 nM each, or in combination with cells for 30 minutes; (5) Pre-conjugate labeled (pHrodo) anti-PDGF-B mAbs with PDGF-BB or PDGF-AB, respectively, at room temperature for 30 minutes before cell processing; (6) Add antibody ligand solution to cells; (7) Live cell imaging was acquired hourly for up to 72 hours using the Opera Phenix Imaging System; (8) Data analysis using Harmony 4.9 software.

[0312] FLIPR (Fluorescence Imaging Plate Reader) Antibody Endointernalization Assay: (1) Seed 10,000 PASMC cells per well in a 96-well plate and incubated cells overnight in complete medium with 5% CO2 at 37°C. (2) Pre-incubated a mixture of antibodies against PDGF-BB in assay medium (DMEM-0.1% BSA-1x penicillin / streptomycin / L-glutamine) for 10 minutes at a ratio of 10⁻¹ nM. (3) Replaced the complete medium with REGN1945 and H4H13145P against PDGF-BB at a ratio of 10⁻¹ nM. (4) Treated cells with the antibody-ligand mixture for 3, 24, and 48 hours. (5) Washed the cell monolayer three times with PBS. (6) Added 50 μL of assay medium and 50 μL of calcium 5 dye. (7) FLIPR Before reading the plate using the Tetra imager, the plate is incubated for 1 hour after adding the dye, and the treated 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), and the calcium trace for maximum and minimum values ​​is analyzed using SoftMax Pro software (Molecular Devices).

[0313] Flow cytometry antibody internalization assay: (1) Seed 300,000 PASMCs per well in a 6-well plate and incubated cells overnight in complete medium at 37°C with 5% CO2. (2) Pre-incubated a mixture of antibodies against PDGF-BB in assay medium (DMEM-0.1% BSA-1x penicillin / streptomycin / L-glutamine) for 10 minutes at a ratio of 10⁻¹ nM. (3) Replaced the complete medium with a ratio of 10⁻¹ nM of antibodies against PDGF-BB. (4) Treated cells with the antibody-ligand mixture for 24 hours. (5) Washed the cell monolayer three times with PBS. (6) Dissociated cells from the 6-well plate using TrypLE® Express Enzyme. (7) Allowed to settle and stained with cell staining buffer (10% DMEM, 5% (8) Resuspend in FBS, stain cells on ice for 1 hour with APC-conjugated antibodies against PDGFRa and PDGFRb according to the manufacturer's protocol, (9) rotate and wash cells three times, (10) add DAPI viability dye to 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 and perform gating of living single cells, and (12) analyze the flow cytometer data using FlowJo software (BD Biosciences).

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

[0315] Antibody Internalization: Labeling anti-PDGF-B antibodies with pHrodo Deep Red enabled the evaluation 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 levels, no light is emitted. This allows for the evaluation of time-dependent internalization using confocal imaging. As shown in Tables 23-24, co-treatment with labeled H4H13145P-PDGFBB resulted in a 108-127-fold increase in fluorescence signal in both PASMCs and NHLFs compared to the labeled isotype control REGN1945. A similar trend, albeit at a lower magnitude, was observed for PDGF-AB. This increase in fluorescence signal, which is emitted only when the labeled antibody is internalized in cells, decreases when 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, inducing 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 calcium flux induced by subsequent PDGF-BB treatment. 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 could not completely prevent PDGF-BB desensitization. H4H13145P-PDGFBB treatment showed a slight rightward shift in potency and a slight decrease in maximum and minimum calcium flux responses at 5 nM of PDGF-BB treatment. This can be explained by the antibody-ligand complex that binds to the PDGFR receptor and induces receptor internalization, albeit at a significantly slower rate and magnitude than unbound PDGF-BB.

[0317] Flow cytometry: PASMCs express both PDGFRa and PDGFRb. In the REGN1945 ligand-treated group, no fluorescence signals were detected for either PDGFRa or PDGFRb. This finding confirms rapid ligand-dependent internalization of the receptor in response to PDGF-BB. H4H13145P prevented the observed loss in the PDGFRb fluorescence signal. In the H4H13145P-treated group, there was a slight shift and loss of PDGFR cell surface expression compared to the medium control showing receptor internalization, although the rate and magnitude were reduced 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 a rat monoclotaline model of pulmonary arterial hypertension. reagent The reagents used to evaluate the effects of two inhibitory anti-PDGF-B antibodies in a rat monoclotaline model of pulmonary arterial hypertension include: monoclotaline (crotaline) Sigma catalog number PHL89251 - CAS number: 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 number HY-14184 - Oral active endothelin receptor antagonist, CAS number: 441798-33-0, Formula: C 19 H 20 Br2N6O4S, formula weight: 588.27 g / mol, lot number: 10673, storage temperature: 3 years -20°C, purity: 98%; and PEG-400 (50:50 v / v, Affymetrix Inc., #19957).

[0319] Experimental Procedure Two studies were conducted to evaluate the effects of two inhibitory anti-PDGF-B antibodies in a rat monoclotaline model of pulmonary arterial hypertension: Study 1 was conducted in monoclotaline PAH rats for 4 weeks with prophylactic drug treatment, and Study 2 was conducted in the survival of monoclotaline severe PAH rats for 5 weeks with therapeutic drug treatment (Tables 26-27).

[0320] Six-to-seven-week-old male Sprague Dawley rats were used. The rats were divided into treatment groups to ensure similarity between groups with different body weights. In Study 1, one day before monocrotaline injection, rats in the antibody treatment group were subcutaneously administered either anti-PDGF-B antibody or isotype control IgG at a dose of 10 mg / kg, and administration continued twice a week for 28 days. On day 1, the rats were subcutaneously administered either 40 mg / kg of monocrotaline or 5 mL / kg of saline as a control. On day 28, right ventricular systolic pressure (RVSP) was measured by right heart catheterization, and RV hypertrophy was calculated using the Fulton index as the weight ratio of RV to (LV + septum). In Study 2, the rats were subcutaneously administered either 60 mg / kg of monocrotaline or 5 mL / kg of saline. Starting on day 14, rats in the antibody treatment group were subcutaneously administered either 25 mg / kg of anti-PDGF-B antibody or isotype control IgG twice a week. The low molecular weight group received oral administration of 30 mg / kg of macitentan daily. Body weight changes from days 0 to 35 were used for general toxicity assessment, and animal mortality and median survival time were calculated up to 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 heated 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 portion of 5-0 silk suture was placed beneath the isolated jugular vein to retract the vessel cranially, and then a hole was made in the jugular vein with a 23-gauge needle. A pressure catheter (microchip catheter transducer SPR-1000, Millar Instruments, Inc.) was inserted into the opening of the jugular vein and advanced through the right atrium to the right ventricle. The catheter was connected to a pressure / volume meter (MPVS-300, Millar Instruments, Inc.) that measured both heart rate and 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 tracing (after 2 minutes of recording to allow pressure stabilization). The parameters analyzed were right ventricular systolic pressure (RVSP) and heart rate (HR).

[0322] Right ventricular hypertrophy assessment: After in vivo hemodynamic measurements, animals were euthanized by bleeding under anesthesia, and the free wall of the right ventricular cavity (RV), left ventricle (LV), and septal tissue were collected and weighted. RV hypertrophy was calculated using the Fulton index as the weight ratio of RV to (LV + septum).

[0323] result Elevated right ventricular pressure induced in monocothalin rats: In study number 1 of monocothalin-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. Treatment with two anti-PDGF-B antibodies significantly reduced right ventricular systolic pressure (Figure 30 and Table 28).

[0324] Right ventricular hypertrophy induced in monocothalline rats: Increased right ventricular cardiac weight was observed in monocothalline rats treated with isotype control IgG. The ratio of right ventricular weight to left ventricle + septal weight provides an index of right ventricular hypertrophy (i.e., Fulton index). An increased Fulton index was observed in monocothalline-treated rats compared to controls treated with saline, 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 number 2, rats injected with 60 mg / kg of monocrotaline developed severe pulmonary hypertension and showed a high mortality rate. In particular, 15 out of 16 monocrotaline rats in the isotype control IgG treatment group died by day 35. Anti-PDGF-B antibody H4H13145P, initiated 14 days after monocrotaline injection, significantly extended survival time and improved mortality. The standard treatment drug, endothelin receptor antagonist macitentan, did not extend survival time. The combination of macitentan and anti-PDGF-B treatment did not show any further survival benefit compared to anti-PDGF-B antibody monotherapy (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 monocothalline rat models of PAH. Therapeutic treatment with Regeneron anti-PDGF-B antibody showed superior survival benefits compared to the vasodilator endothelin receptor antagonist macitentan in severe monocothalline rat PAH models. The anti-PDGF-B antibody H4H13145P significantly rescued animals from mortality and extended survival time. [Table 28] [Table 29]

[0327] Equivalents Those skilled in the art will be able to identify or determine, using experiments not exceeding the usual scope, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the following claims.

[0328] Unofficial sequence list H4H13145P Sequence ID 1; HCVR CAGGTTCAGCTGGTGCAGTCTGGAACTGAGGTGAAGAAGCCTGGGGCCTCAGTAAAGGTCTCCTGCAAGGCCTCTGGTTATACTTATGGTGCCTATGCAATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACA AACTATGCACAGAAATTCCAGGACAGAGTCACCATGACCACAGACACATCCACGAACACAGCCTACATGGAACTGAGGGGCCTAAAATCTGACGACACGGCCGTGTATTTCTGTGCGAGGGCCTGGAACTCCTTTGACTACTGGGGCCAGGGCACCCTGGTCACTGTCTCCTCA Sequence ID 2; HCVR QVQLVQSGTEVKKPGASVKVSCKASGYTYGAYAISWVRQAPGQGLEWMGWISAYNGNTNYAQKFQDRVTMTTDTSTNTAYMELRGLKSDDTAVYFCARAWNSFDYWGQGTLVTVSS Sequence ID 3; HCDR1 GGT TAT ACT TAT GGT GCC TAT GCA Sequence ID 4; HCDR1 GYTYGAYA Sequence ID 5; HCDR2 ATC AGC GCT TAC AAT GGT AAC ACA Sequence ID 6; HCDR2 ISAYNGNT Sequence ID 7; HCDR3 GCG AGG GCC TGG AAC TCC TTT GAC TAC Sequence ID 8; HCDR3 ARAWNSFDY Sequence ID 9; LCVR GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCGTCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGGAAAAATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCCGATGCATCCACTT TAGAAACAGGGGTCCCATCAAGATTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAAAATATTACTGTCAACAATATTATAATCTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA Sequence ID 10; LCVR DIQMTQSPSSLSASVGDRVTITCQASQDIRKNLNWYQQKPGKAPKLLISDASTLETGVPSRFSGSGSGTDFTFTISSLQPEDIAKYYCQQYYNLPFTFGPGTKVDIK Sequence ID 11;LCDR1 CAG GAC ATT AGG AAA AAT Sequence ID 12;LCDR1 QDIRKN Sequence ID 13; LCDR2 GAT GCA TCC Sequence ID 14; LCDR2 DAS Sequence ID 15; LCDR3 CAA CAA TAT TAT AAT CTC CCA TTC ACT Sequence ID 16; LCDR3 QQYYNLPFT Sequence ID 17;HC Sequence ID 18;HC QVQLVQSGTEVKKPGASVKVSCKASGYTYGAYAISWVRQAPGQGLEWMGWISAYNGNTNYAQKFQDRVTMTTDTSTNTAYMELRGLKSDDTAVYFCARAWNSFDYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* Sequence ID 19; LC GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCGTCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGGAAAAATTTAAATTGGTATCAACAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCCGATGCATCCACTTTAGAAACAGGGGTCCCATCAAGATTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAAAATATTACTGTCAACAATATTATAATCTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG Accession No. 20; LC DIQMTQSPSSLSASVGDRVTITCQASQDIRKNLNWYQQKPGKAPKLLISDASTLETGVPSRFSGSGSGTDFTFTISSLQPEDIAKYYCQQYYNLPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* [[ID=�]]H4H13132P Accession No. {21}; HCVR Note: There seems to be a typo in the original text where "配列番号21;HCVR" should probably be something like "Accession No. 21; HCVR". I've made this correction in the translation for clarity. Also, the "H4H13132P" in the original might be a misspelling or something specific to the context that's not clear. I've left it as is in the translation. If you have any additional context or corrections regarding these, please let me know.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 Sequence ID 30; LCVR DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPDQSPRRLIYKISNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIK Sequence ID 31;LCDR1 CAA AGC CTC GTA TAC AGT GAT GGA AAC ACC TAC Sequence ID 32; LCDR1 QSLVYSDGNTY Sequence ID 33; LCDR2 AAG ATT TCT Sequence ID 34; LCDR2 KIS Sequence ID 35; LCDR3 ATG CAA GGT ACA CAC TGG CCT CCC ACT Sequence ID 36; LCDR3 MQ GTHWPPT Sequence ID 37;HC Sequence ID 38;HC QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITTDESTSTAYMELSSLRSEDTAVYYCAREGYGDYYFGMDVWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK* Sequence ID 39; LC GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACAGTGATGGAAACACCTACTTGAATTGGTTTCAGCAGAGGCCAGACCAATCTCCAAGGCGCCTAATTTATAAGATTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCTCCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG SEQ ID NO: 40; LC DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPDQSPRRLIYKISNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* hPDGF - B Sequence ID 41; P01127 | PDGFB_Human Platelet-Derived Growth Factor Subunit BOS=Homo sapiens OX=9606 GN=PDGFB PE=1 SV=1 MNRCWALFLSLCCYLRLVSAEGDPIPEELYEMLSDHSIRSFDDLQRLLHGDPGEEDGAELDLNMTRSHSGGELESLARGRRSLGSLTIAEPAMIAECKTRTEVFEISRRLIDRTNANFLV WPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVTRSPGGSQEQRAKTPQTRVTIRTVRVRRPPKGKHRKFKHTHDKTALKETLGA

Claims

1. An isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human platelet-derived growth factor subunit B (PDGF-B), wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), and the antibody or antigen-binding fragment is (i) an HCDR1 domain containing the amino acid sequence described in SEQ ID NO: 4, an HCDR2 domain containing the amino acid sequence described in SEQ ID NO: 6, an HCDR3 domain containing the amino acid sequence described in SEQ ID NO: 8, an LCDR1 domain containing the amino acid sequence described in SEQ ID NO: 12, an LCDR2 domain containing the amino acid sequence described in SEQ ID NO: 14, and an LCDR3 domain containing the amino acid sequence described in SEQ ID NO: 16, or (ii) HCDR1 domain containing the amino acid sequence described in SEQ ID NO: 24, HCDR2 domain containing the amino acid sequence described in SEQ ID NO: 26, HCDR3 domain containing the amino acid sequence described in SEQ ID NO: 28, LCDR1 domain containing the amino acid sequence described in SEQ ID NO: 32, LCDR2 domain containing the amino acid sequence described in SEQ ID NO: 34, and LCDR3 domain containing the amino acid sequence described in SEQ ID NO: 36 The antibody or antigen-binding fragment is (a) When measured by surface plasmon resonance, the coupling dissociation equilibrium constant (K) is less than 1.84 pM. Ｄ ) and bind to human PDGF-subunit B homodimer (PDGF-BB) at 37°C. (b) K1.36 pM when measured by surface plasmon resonance Ｄ And it binds to human PDGF-BB at 37°C. (c) When measured by surface plasmon resonance, it binds to human PDGF-BB at 37°C with a t1 / 2 of 1155 minutes or more. (d) K2.79 pM when measured by surface plasmon resonance Ｄ And it binds to human PDGF-BB at 25°C. (e) When measured by surface plasmon resonance, it binds to human PDGF-BB at 25°C with a t1 / 2 of 1155 minutes or more. (f) IC < 1.9 nM when measured by competitive ELISA assay at 25°C ５０ Therefore, inhibiting PDGF-B activation in human PDGF-BB, (g) IC < 8.8 nM when measured by competitive ELISA assay at 25°C ５０ Therefore, inhibiting PDGF-B activation in human PDGF-subunit A and subunit B heterodimer (PDGF-AB), and (h) Blocking the interaction between human PDGF-BB and one or more of human PDGFR-αα, PDGFR-αβ, and PDGFR-ββ. An isolated human monoclonal antibody or its antigen-binding fragment exhibiting one or more characteristics selected from the group consisting of the following.

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 a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR), and the antibody or antigen-binding fragment is (i) an HCDR1 domain containing the amino acid sequence described in SEQ ID NO: 4, an HCDR2 domain containing the amino acid sequence described in SEQ ID NO: 6, an HCDR3 domain containing the amino acid sequence described in SEQ ID NO: 8, an LCDR1 domain containing the amino acid sequence described in SEQ ID NO: 12, an LCDR2 domain containing the amino acid sequence described in SEQ ID NO: 14, and an LCDR3 domain containing the amino acid sequence described in SEQ ID NO: 16, or (ii) HCDR1 domain containing the amino acid sequence described in SEQ ID NO: 24, HCDR2 domain containing the amino acid sequence described in SEQ ID NO: 26, HCDR3 domain containing the amino acid sequence described in SEQ ID NO: 28, LCDR1 domain containing the amino acid sequence described in SEQ ID NO: 32, LCDR2 domain containing the amino acid sequence described in SEQ ID NO: 34, and LCDR3 domain containing the amino acid sequence described in SEQ ID NO: 36 Isolated human antibodies or their antigen-binding fragments, including [the specified substance].

3. The isolated human antibody or antigen-binding fragment according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment comprises (a) an HCVR having an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 2 and an LCVR having an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 10, or (b) an HCVR having an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 22 and an LCVR having an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO:

30.

4. The isolated human antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 and 22 / 30.

5. The antibody or antigen-binding fragment is (i) Light chain immunoglobulin containing the amino acid sequence described in Sequence ID No. 20, and Heavy chain immunoglobulin containing the amino acid sequence described in SEQ ID NO: 18, And / or (ii) Light chain immunoglobulin containing the amino acid sequence described in SEQ ID NO: 40, and Heavy chain immunoglobulin containing the amino acid sequence described in Sequence ID No. 38, An isolated human antibody or antigen-binding fragment according to any one of claims 1 to 4, comprising:

6. The antigen-binding fragment is the Fab fragment, F(ab') ２ The antigen-binding fragment according to any one of claims 1 to 5, which is a fragment, an Fd fragment, an Fv fragment, a single-chain Fv (scFv) molecule, or a dAb fragment.

7. A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 6, (i) Introducing one or more polynucleotides encoding the light chain immunoglobulin of the antibody or fragment and the heavy chain immunoglobulin of the antibody or fragment into a host cell, (ii) Culturing the host cells in a growth medium under conditions favorable for the expression of the polynucleotide, (iii) Selectively isolating the antibody or fragment from the host cells and / or the culture medium in which the host cells are growing, Methods that include...

8. An antibody or antigen-binding fragment produced by the method described in claim 7.

9. An injection device or container comprising an antibody or antigen-binding fragment according to any one of claims 1 to 6 and 8.

10. A pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof that binds to human PDGF-B as described in any one of claims 1 to 6 and 8, a pharmaceutically acceptable carrier or diluent, and optionally one or more additional therapeutic agents.

11. The pharmaceutical composition according to claim 10, wherein the one or more additional therapeutic agents include an iron supplement.

12. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and 8, or a pharmaceutical composition according to any one of claims 10 to 11, for preventing or treating pulmonary arterial hypertension (PAH) in subjects requiring prevention or treatment of PAH.

13. The composition according to claim 12, characterized in that the composition is administered subcutaneously, intravenously, intradermally, or orally, or intramuscularly.

14. The PAH causes a condition selected from the group consisting of thickening of the pulmonary artery of the subject, a decrease in the stroke volume of the subject, a decrease in the right ventricular output of the subject, and a decrease in the survival time of the subject. The composition according to any one of claims 12 to 13, wherein administration of the composition treats the condition or reduces the severity of one or more symptoms of the condition.

15. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and 8, for use in the treatment of a patient having PAH.

16. A composition for use in the treatment of PAH, comprising one or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 6 and 8.

17. Use of an isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and 8 in the manufacture of a pharmaceutical product for treating a patient having PAH.

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